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The PDF version of the Atlas of Genetics and Cytogenetics in Oncology and Haematology is a reissue of the original articles published in collaboration with the Institute for Scientific and Technical Information (INstitut de l’Information Scientifique et Technique - INIST) of the French National Center for Scientific Research (CNRS) on its electronic publishing platform I-Revues. Online and PDF versions of the Atlas of Genetics and Cytogenetics in Oncology and Haematology are hosted by INIST-CNRS.

Atlas of Genetics and Cytogenetics in Oncology and Haematology

OPEN ACCESS JOURNAL AT INIST-CNRS

Volume 15 - Number 9 September 2011

The PDF version of the Atlas of Genetics and Cytogenetics in Oncology and Haematology is a reissue of the original articles published in collaboration with the Institute for Scientific and Technical Information (INstitut de l’Information Scientifique et Technique - INIST) of the French National Center for Scientific Research (CNRS) on its electronic publishing platform I-Revues. Online and PDF versions of the Atlas of Genetics and Cytogenetics in Oncology and Haematology are hosted by INIST-CNRS.



Scope

The Atlas of Genetics and Cytogenetics in Oncology and Haematology is a peer reviewed on-line journal in open access, devoted to genes, cytogenetics, and clinical entities in cancer, and cancer-prone diseases. It presents structured review articles ("cards") on genes, leukaemias, solid tumours, cancer-prone diseases, more traditional review articles on these and also on surrounding topics ("deep insights"), case reports in hematology, and educational items in the various related topics for students in Medicine and in Sciences.

Editorial correspondance

Jean-Loup Huret Genetics, Department of Medical Information, University Hospital F-86021 Poitiers, France tel +33 5 49 44 45 46 or +33 5 49 45 47 67 [email protected] or [email protected]

Staff Mohammad Ahmad, Mélanie Arsaban, Marie-Christine Jacquemot-Perbal, Maureen Labarussias, Vanessa Le Berre, Anne Malo, Catherine Morel-Pair, Laurent Rassinoux, Alain Zasadzinski. Philippe Dessen is the Database Director, and Alain Bernheim the Chairman of the on-line version (Gustave Roussy Institute – Villejuif – France).

The Atlas of Genetics and Cytogenetics in Oncology and Haematology (ISSN 1768-3262) is published 12 times a year by ARMGHM, a non profit organisation, and by the INstitute for Scientific and Technical Information of the French National Center for Scientific Research (INIST-CNRS) since 2008. The Atlas is hosted by INIST-CNRS (http://www.inist.fr)

http://AtlasGeneticsOncology.org

© ATLAS - ISSN 1768-3262

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9)







Editor

Jean-Loup Huret (Poitiers, France)

Editorial Board Sreeparna Banerjee (Ankara, Turkey) Solid Tumours Section Alessandro Beghini (Milan, Italy) Genes Section Anne von Bergh (Rotterdam, The Netherlands) Genes / Leukaemia Sections Judith Bovée (Leiden, The Netherlands) Solid Tumours Section Vasantha Brito-Babapulle (London, UK) Leukaemia Section Charles Buys (Groningen, The Netherlands) Deep Insights Section Anne Marie Capodano (Marseille, France) Solid Tumours Section Fei Chen (Morgantown, West Virginia) Genes / Deep Insights Sections Antonio Cuneo (Ferrara, Italy) Leukaemia Section Paola Dal Cin (Boston, Massachussetts) Genes / Solid Tumours Section Louis Dallaire (Montreal, Canada) Education Section Brigitte Debuire (Villejuif, France) Deep Insights Section François Desangles (Paris, France) Leukaemia / Solid Tumours Sections Enric Domingo-Villanueva (London, UK) Solid Tumours Section Ayse Erson (Ankara, Turkey) Solid Tumours Section Richard Gatti (Los Angeles, California) Cancer-Prone Diseases / Deep Insights Sections Ad Geurts van Kessel (Nijmegen, The Netherlands) Cancer-Prone Diseases Section Oskar Haas (Vienna, Austria) Genes / Leukaemia Sections Anne Hagemeijer (Leuven, Belgium) Deep Insights Section Nyla Heerema (Colombus, Ohio) Leukaemia Section Jim Heighway (Liverpool, UK) Genes / Deep Insights Sections Sakari Knuutila (Helsinki, Finland) Deep Insights Section Lidia Larizza (Milano, Italy) Solid Tumours Section Lisa Lee-Jones (Newcastle, UK) Solid Tumours Section Edmond Ma (Hong Kong, China) Leukaemia Section Roderick McLeod (Braunschweig, Germany) Deep Insights / Education Sections Cristina Mecucci (Perugia, Italy) Genes / Leukaemia Sections Yasmin Mehraein (Homburg, Germany) Cancer-Prone Diseases Section Fredrik Mertens (Lund, Sweden) Solid Tumours Section Konstantin Miller (Hannover, Germany) Education Section Felix Mitelman (Lund, Sweden) Deep Insights Section Hossain Mossafa (Cergy Pontoise, France) Leukaemia Section Stefan Nagel (Braunschweig, Germany) Deep Insights / Education Sections Florence Pedeutour (Nice, France) Genes / Solid Tumours Sections Elizabeth Petty (Ann Harbor, Michigan) Deep Insights Section Susana Raimondi (Memphis, Tennesse) Genes / Leukaemia Section Mariano Rocchi (Bari, Italy) Genes Section Alain Sarasin (Villejuif, France) Cancer-Prone Diseases Section Albert Schinzel (Schwerzenbach, Switzerland) Education Section Clelia Storlazzi (Bari, Italy) Genes Section Sabine Strehl (Vienna, Austria) Genes / Leukaemia Sections Nancy Uhrhammer (Clermont Ferrand, France) Genes / Cancer-Prone Diseases Sections Dan Van Dyke (Rochester, Minnesota) Education Section Roberta Vanni (Montserrato, Italy) Solid Tumours Section Franck Viguié (Paris, France) Leukaemia Section José Luis Vizmanos (Pamplona, Spain) Leukaemia Section Thomas Wan (Hong Kong, China) Genes / Leukaemia Sections




Volume 15, Number 9, September 2011

Table of contents

Gene Section

FAT1 (FAT tumor suppressor homolog 1 (Drosophila)) 717 Kunzang Chosdol, Bhawana Dikshit, Subrata Sinha

KSR1 (kinase suppressor of ras 1) 721 Mario Fernandez, Robert Lewis

PEG10 (paternally expressed 10) 724 Andreas Lux

PIK3CD (phosphoinositide-3-kinase, catalytic, delta polypeptide) 731 Emily Burns, Bart Vanhaesebroeck

S100A8 (S100 calcium binding protein A8) 735 Claus Kerkhoff, Saeid Ghavami

S100A9 (S100 calcium binding protein A9) 746 Claus Kerkhoff, Saeid Ghavami

TRIAP1 (TP53 regulated inhibitor of apoptosis 1) 758 Veruska Alves, Roberta Felix, Andre Vettore, Gisele Colleoni

Leukaemia Section

t(X;11)(q13;q23) 761 Adriana Zamecnikova

t(X;11)(q22;q23) 763 Adriana Zamecnikova

t(X;11)(q24;q23) MLL-SEPTIN6 765 Adriana Zamecnikova

Deep Insight Section

S100 Protein Family and Tumorigenesis 768 Geetha Srikrishna, Hudson H Freeze

Visualize Dynamic Chromosome 777 Eisuke Gotoh

NK cell receptors: evolution and diversity 787 Gwenoline Borhis, Salim I Khakoo

Somatostatin (SS), SS receptors and SS analog treatment in tumorigenesis 797 Liliana Steffani, Luca Passafaro, Diego Ferone, Paolo Magni, Massimiliano Ruscica

t(11;14)(q13;q32) in multiple myeloma Huret JL, Laï JL




Case Report Section

A new case of translocation t(14;14)(q11;q32) in B lineage ALL 806 Elvira D Rodrigues Pereira Velloso, Priscila Pereira dos Santos Teixeira, Karina Prandi Melillo, Luciana J Rodrigues da Silva, Cristina Alonso Ratis, Daniela Borri, Cristóvão LP Mangueira

Gene Section Review

Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9) 717



FAT1 (FAT tumor suppressor homolog 1 (Drosophila)) Kunzang Chosdol, Bhawana Dikshit, Subrata Sinha

Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi-110029, India (KC, BD, SS)

Published in Atlas Database: February 2011

Online updated version : http://AtlasGeneticsOncology.org/Genes/FAT1ID40533ch4q35.html DOI: 10.4267/2042/46017

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity Other names: CDHF7; CDHR8; FAT; ME5; hFat1

HGNC (Hugo): FAT1

Location: 4q35.2

Note FAT1 is an ortholog of the Drosophila tumor suppressor gene 'fat'. In Drosophila, it is essential for controlling cell proliferation during development. The gene product is a member of the cadherin superfamily, characterized by the presence of cadherin-type repeats. In addition to containing 34 tandem cadherin-type repeats, the gene product has five epidermal growth factor (EGF)-like repeats and one laminin A-G domain. This gene is expressed at high levels in a number of fetal epithelia. Its product probably functions as an adhesion molecule and/or signaling receptor, and is likely to be important in developmental processes and cell-cell communication.

DNA/RNA Description FAT1 gene is located on the chromosome 4q35.2 (Accession: NC_000004.11). The total length of the gene is 136050 bases (187509746 bp to 187630981 bp from pter) of reverse strand. There are 27 exons. An alternate assembly suggested to be starting from 187745931 bp to 187881981 bp from pter.

Transcription The length of the transcript is 14773 bps made from 27 exons (Accession: NM_005245.3).

Pseudogene FAT tumor suppressor homolog 1 (Drosophila) pseudogene 1 (FAT1P1). Other name: dJ697P8.1; sequence accession ID: AL050403; location chromosome: 20p12.2.

Protein Note Known protein coding gene. Protein names Recommended name: protocadherin Fat 1. Alternative names: Cadherin-related tumor suppressor homolog, Protein fat homolog, Cadherin family member 7.

Description 4588 aa (Accession: NP_005236.2).

Expression Expressed in epithelial, endothelial and smooth muscle cells.

Localisation Cell membrane; single-pass type I membrane protein.

Function Could function as a cell-adhesion molecule, cell signalling molecule, and have a role in cell migration. Fat in Drosophila acts via SWH signalling pathway as tumour suppressor gene. Homolog of SWH pathway molecules are present in human, so there is a possibility of acting FAT1 as an upstream regulator of SWH pathway in human.

FAT1 (FAT tumor suppressor homolog 1 (Drosophila)) Chosdol K, et al.


Salvador-Warts-Hippo pathway. Mammalian hippo signaling pathway shows homology with Drosophila pathway proteins (depicted in similar color and shape). In Drosophila fat (ft) interacts with core kinase cascade via Expanded (Ex). The core kinase cascade includes kinase Hippo (hpo), adaptor proteins mats and Salvador (Sav) and kinase Warts. The core kinase cascade inhibits phosphorylation of transcriptional co-activator Yorkie (Yki) causing its translocation to nucleus where it binds to transcriptional activator Scalloped (Sd) and modulates gene expression. In mammals, whether FAT1 is involved in hippo pathway regulation is not clear. The effector molecule, phospho-YAP, is reported to interact with p73 in the nucleus and promotes cell death. There is no p73 homolog known to be reported in Drosophila. YAP is also found to interact with other transcription factors and modulate gene expression, thus, the outcome of hippo pathway is context dependent.

Organism Gene Locus Description Similarity to human FAT1

Dog (Canis familiaris)

FAT1 Chr. 16 FAT tumor suppressor homolog 1 (Drosophila)

86.95(n), 91.17(a)

Pig (Sus scrofa)


86(n), 90(a)

Cow (Bos Taurus)


84.18(n) 89.93(a)

Rat (Rattus norvegicus)

Fat1 Chr. 16q11 FAT tumor suppressor homolog 1 (Drosophila)

82.93(n) 88.16(a)

Mouse (Mus musculus)

Fat1 Chr. 8 (25.00 cM)

FAT tumor suppressor homolog 1 (Drosophila)

82.51(n) 88.14(a)

Chicken (Gallus gallus)

FAT Chr. 4 FAT tumor suppressor homolog 1 (Drosophila)

76.35(n) 81.43(a)

Zebrafish fat1 Chr. 1 FAT tumor suppressor 64.68(n) 64.82(a)



(Danio rerio) homolog 1

Fruit fly (Drosophila melanogaster)

ft and fat2 Chr. 2L (ft) Chr. 3L (fat2)

fat and fat2 ft - 42.8(n) 42(a), fat2 - 47.99(n) 39.02(a)

Worm (Caenorhabditis elegans)

cdh-4 Chr. III Cadherin family 44.19(n) 30.89(a)

African malaria mosquito (Anopheles gambiae)

AgaP_AGAP011526

Chr. 3L AGAP011526-PA 48.06(n) 39.5(a)

Table. Orthologs for FAT1 gene from other species. In human, FAT1 expression is highest at the embryonic stages and diminishes later in adult life. In human fetal tissues, high levels of FAT1 transcripts were found in kidney, lungs, and eye epithelia, and the expression was found to be down regulated in the corresponding adult tissues, indicating the role of FAT1 in organ development. FAT1 also has a role in cell migration (Moeller et al., 2004; Tanoue and Takeichi, 2004) and found to be up-regulated in migrating cells, also crucial for efficient wound healing (Braun et al., 2007). In Drosophila, fat is an upstream regulator of the Salvador-Wart-Hippo (SWH) signaling pathway (Cho et al., 2006; Bennett and Harvey, 2006). The signalling molecules of SWH pathway are conserved in mammals (figure below) but the role of FAT1 as an apical regulator of SWH pathway in human has not yet been established.

Homology Paralogs for FAT1 gene: FAT2, FAT3, FAT4. See table above.

Mutations Note No known mutations. Single nucleotide polymorphism (SNPs): gene: FAT1 (ENSG00000083857).

Implicated in Various cancers Note FAT1, a member of the cadherin gene family, is homologue of Drosophila tumour suppressor gene fat. In Drosophila, fat gene is important in controlling cell proliferation during development and any defect in the expression of fat would lead to tumor development (Bryant et al., 1988). Dunne et al. (1995) have identified the human homologue and studied the tissue distribution of FAT transcripts in adult and fetal tissues. Loss of heterozygosity and altered expression of FAT1 has been found in human glial tumors (Chosdol et al., 2009). Homozygous deletion of FAT1 gene was detected in oral cancer (Nakaya et al., 2007). Kwaepila et al. (2006) found higher FAT1 expression in more

malignant form of breast cancer tissues by immunohistochemistry (IHC). There are studies showing LOH and/or deletion of the chromosome 4q34-35 region (which harbors FAT gene) in many tumors including gliomas. LOH was found in grade IV gliomas using microsatellite markers (Hu et al., 2002), though the gene itself has not been implicated. Other tumors like small cell lung carcinoma (Cho et al., 2002), hepatocellular carcinoma (Zhang et al., 2005; Chang et al., 2002) and cervical carcinoma (Backsch et al., 2005) etc showed alterations/LOH in the chromosomal 4q34-q35 locus and significant association of 4q34-q35 region with increased risk of progression of these tumors was suggested. Since the FAT gene is located in this region it may have an important role to play in the development and progression of these tumors.

Astrocytic tumour Note Loss of heterozygosity and altered expression of FAT1 in astrocytic tumors (Chosdol et al., 2009).

Breast cancer Note Increased FAT1 expression contributes to loss of duct formation, and increased cell migration and invasion in breast cancer (Kwaepila et al., 2006).

Oral cancer Note Homozygous deletion of FAT in the cell lines and in primary oral cancers was studied. Homozygous deletion hot spots were observed in exon 1 (9/20, 45%) and exon 4 (7/20, 35%). The methylation status of the FAT CpG island in squamous cell carcinomas correlated negatively with its expression. Mutations in FAT is suggested as an important factor in the development of oral cancer. Moreover, loss of gene expression was identified in other types of squamous cell carcinoma (Nakaya et al., 2007).

Psychiatric disorders Note Bipolar disorder: a positional cloning strategy, combined with association analysis have provided



evidence that a cadherin gene, FAT, confers susceptibility to bipolar disorder (Blair et al., 2006).

Cell migration Note FAT1 is known to play role in cell migration. FAT1 knockdown decreases cell migration in vascular smooth muscle cells (Hou et al., 2006; Hou and Sibinga, 2009). FAT1 plays an integrative role in regulating cell migration by participating in Ena/VASP-dependent regulation of cytoskeletal dynamics (Moeller et al., 2004).

References Bryant PJ, Huettner B, Held LI Jr, Ryerse J, Szidonya J. Mutations at the fat locus interfere with cell proliferation control and epithelial morphogenesis in Drosophila. Dev Biol. 1988 Oct;129(2):541-54

Dunne J, Hanby AM, Poulsom R, Jones TA, Sheer D, Chin WG, Da SM, Zhao Q, Beverley PC, Owen MJ. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics. 1995 Nov 20;30(2):207-23

Ponassi M, Jacques TS, Ciani L, ffrench Constant C. Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech Dev. 1999 Feb;80(2):207-12

Cox B, Hadjantonakis AK, Collins JE, Magee AI. Cloning and expression throughout mouse development of mfat1, a homologue of the Drosophila tumour suppressor gene fat. Dev Dyn. 2000 Mar;217(3):233-40

Chang J, Kim NG, Piao Z, Park C, Park KS, Paik YK, Lee WJ, Kim BR, Kim H. Assessment of chromosomal losses and gains in hepatocellular carcinoma. Cancer Lett. 2002 Aug 28;182(2):193-202

Cho ES, Chang J, Chung KY, Shin DH, Kim YS, Kim SK, Kim SK. Identification of tumor suppressor loci on the long arm of chromosome 4 in primary small cell lung cancers. Yonsei Med J. 2002 Apr;43(2):145-51

Hu J, Jiang C, Ng HK, Pang JC, Tong CY. Chromosome 14q may harbor multiple tumor suppressor genes in primary glioblastoma multiforme. Chin Med J (Engl). 2002 Aug;115(8):1201-4

Moeller MJ, Soofi A, Braun GS, Li X, Watzl C, Kriz W, Holzman LB. Protocadherin FAT1 binds Ena/VASP proteins and is necessary for actin dynamics and cell polarization. EMBO J. 2004 Oct 1;23(19):3769-79

Tanoue T, Takeichi M. Mammalian Fat1 cadherin regulates actin dynamics and cell-cell contact. J Cell Biol. 2004 May 24;165(4):517-28

Backsch C, Rudolph B, Kühne-Heid R, Kalscheuer V, Bartsch O, Jansen L, Beer K, Meyer B, Schneider A, Dürst M. A region on human chromosome 4 (q35.1-->qter) induces senescence in cell hybrids and is involved in cervical carcinogenesis. Genes Chromosomes Cancer. 2005 Jul;43(3):260-72

Down M, Power M, Smith SI, Ralston K, Spanevello M, Burns GF, Boyd AW. Cloning and expression of the large zebrafish

protocadherin gene, Fat. Gene Expr Patterns. 2005 Apr;5(4):483-90

Magg T, Schreiner D, Solis GP, Bade EG, Hofer HW. Processing of the human protocadherin Fat1 and translocation of its cytoplasmic domain to the nucleus. Exp Cell Res. 2005 Jul 1;307(1):100-8

Zhang SH, Cong WM, Xian ZH, Wu MC. Clinicopathological significance of loss of heterozygosity and microsatellite instability in hepatocellular carcinoma in China. World J Gastroenterol. 2005 May 28;11(20):3034-9

Bennett FC, Harvey KF. Fat cadherin modulates organ size in Drosophila via the Salvador/Warts/Hippo signaling pathway. Curr Biol. 2006 Nov 7;16(21):2101-10

Blair IP, Chetcuti AF, Badenhop RF, Scimone A, Moses MJ, Adams LJ, Craddock N, Green E, Kirov G, Owen MJ, Kwok JB, Donald JA, Mitchell PB, Schofield PR. Positional cloning, association analysis and expression studies provide convergent evidence that the cadherin gene FAT contains a bipolar disorder susceptibility allele. Mol Psychiatry. 2006 Apr;11(4):372-83

Cho E, Feng Y, Rauskolb C, Maitra S, Fehon R, Irvine KD. Delineation of a Fat tumor suppressor pathway. Nat Genet. 2006 Oct;38(10):1142-50

Hou R, Liu L, Anees S, Hiroyasu S, Sibinga NE. The Fat1 cadherin integrates vascular smooth muscle cell growth and migration signals. J Cell Biol. 2006 May 8;173(3):417-29

Katoh Y, Katoh M. Comparative integromics on FAT1, FAT2, FAT3 and FAT4. Int J Mol Med. 2006 Sep;18(3):523-8

Kwaepila N, Burns G, Leong AS. Immunohistological localisation of human FAT1 (hFAT) protein in 326 breast cancers. Does this adhesion molecule have a role in pathogenesis? Pathology. 2006 Apr;38(2):125-31

Schreiner D, Müller K, Hofer HW. The intracellular domain of the human protocadherin hFat1 interacts with Homer signalling scaffolding proteins. FEBS Lett. 2006 Oct 2;580(22):5295-300

Braun GS, Kretzler M, Heider T, Floege J, Holzman LB, Kriz W, Moeller MJ. Differentially spliced isoforms of FAT1 are asymmetrically distributed within migrating cells. J Biol Chem. 2007 Aug 3;282(31):22823-33

Nakaya K, Yamagata HD, Arita N, Nakashiro KI, Nose M, Miki T, Hamakawa H. Identification of homozygous deletions of tumor suppressor gene FAT in oral cancer using CGH-array. Oncogene. 2007 Aug 9;26(36):5300-8

Chosdol K, Misra A, Puri S, Srivastava T, Chattopadhyay P, Sarkar C, Mahapatra AK, Sinha S. Frequent loss of heterozygosity and altered expression of the candidate tumor suppressor gene 'FAT' in human astrocytic tumors. BMC Cancer. 2009 Jan 7;9:5

Hou R, Sibinga NE. Atrophin proteins interact with the Fat1 cadherin and regulate migration and orientation in vascular smooth muscle cells. J Biol Chem. 2009 Mar 13;284(11):6955-65

This article should be referenced as such:

Chosdol K, Dikshit B, Sinha S. FAT1 (FAT tumor suppressor homolog 1 (Drosophila)). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):717-720.

Gene Section Mini Review




KSR1 (kinase suppressor of ras 1) Mario Fernandez, Robert Lewis

Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE 68198, USA (MF, RL)


Online updated version : http://AtlasGeneticsOncology.org/Genes/KSR1ID41107ch17q11.html DOI: 10.4267/2042/46024


Identity Other names: KSR; RSU2

HGNC (Hugo): KSR1

Location: 17q11.1

Local order: From centromere to telomere on 17q11.2, the KSR1 gene is flanked by: EOE1, EVI2A, JJAZ1, KNO3, KRT24, KSR1, LGALS9, LYZL6, MIR144, MIR451 (NCBI).

DNA/RNA Description The KSR1 gene was discovered in D. melanogaster and is highly conserved along the evolutionary tree through H. sapiens.

Transcription Transcription can produce two different mRNA transcripts. The KSR1 is expressed in the thymus, bone marrow, brain, heart, kidney, lung, liver, pancreas, mammary gland, ovary, testis and muscle.

Pseudogene No known pseudogenes.

Protein Note Belongs to the KSR family of proteins.

Description The KSR1 protein is 921 kDa. There have been 15 phosphorylation sites identified.

Expression Brain, kidney, lung, pancreas, ovaries, testis, breast.

Localisation Sequestered in the cytoplasm in quiescents cells. Upon stimulation, KSR1 protein localizes to the plasma membrane.

Function Acts as a scaffold for the Raf/MEK/ERK kinase cascade.

Homology R. norvegicus KSR1 (91.85%), M. musculus KSR1 (91.3%), C. familians KSR1 (93.62%), G. gallus KSR1 (76.39%), D. reno KSR1 (67.85%), D. Melanogaster KSR (41.06%), C. elegans KSR1 (35.37%), X. laevis KSR1 (76.06%).

KSR1 contains 17 exons, 154426 base pairs.

KSR1 (kinase suppressor of ras 1) Fernandez M, Lewis R


The KSR1 protein contains five conserved areas (CA). The CA1 region is unique to KSR1 protein; the CA2 region is a proline-rich region; the CA3 region is implicated in the Ras-induced plasma membrane localization; the CA4 region is a serine/threonine rich region and contains the MAPK docking site (FXFP); and the putative kinase domain (CA5), which contains an amino acid variation in subdomain II that suggests that this protein is catalytically inert. The KSR1 protein is composed of 921 amino acids.

Mutations Note There have been no prominent germinal or somatic mutations identified.

Implicated in Cancer Note KSR1 has not been found to be upregulated or downregulated in tumors. However, studies in KSR1 knockout mice showed that the mice were less susceptible to papilloma virus driven tumors, indicating that KSR1 is required for Ras-mediated oncogenesis.

To be noted Note KSR1 is primarily recognized as a scaffold for the Raf/MEK/ERK kinase cascade. However, there are several published studies that sustain that KSR1 has catalytic activity. KSR1 is recognized as a pseudokinase, since mammalian KSR1 does not possess the lysine responsible for ATP orientation and hydrolysis in the putative kinase domain. This lysine is present in C. elegans and D. melanogaster, but mutation of this site did not affect activation of the Raf/MEK/ERK pathway. A recent publication (Rajakulendran et al., 2009) showed that KSR1 dimerization via its kinase domain to Raf facilitated the activation of Raf without KSR1 catalytic activity. It remains possible that KSR1 proteins have catalytic activity based on a yet to be identified novel mechanism.

References Kornfeld K, Hom DB, Horvitz HR. The ksr-1 gene encodes a novel protein kinase involved in Ras-mediated signaling in C. elegans. Cell. 1995 Dec 15;83(6):903-13

Sundaram M, Han M. The C. elegans ksr-1 gene encodes a novel Raf-related kinase involved in Ras-mediated signal transduction. Cell. 1995 Dec 15;83(6):889-901

Therrien M, Chang HC, Solomon NM, Karim FD, Wassarman DA, Rubin GM. KSR, a novel protein kinase required for RAS signal transduction. Cell. 1995 Dec 15;83(6):879-88

Therrien M, Michaud NR, Rubin GM, Morrison DK. KSR modulates signal propagation within the MAPK cascade. Genes Dev. 1996 Nov 1;10(21):2684-95

Michaud NR, Therrien M, Cacace A, Edsall LC, Spiegel S, Rubin GM, Morrison DK. KSR stimulates Raf-1 activity in a kinase-independent manner. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12792-6

Müller J, Cacace AM, Lyons WE, McGill CB, Morrison DK. Identification of B-KSR1, a novel brain-specific isoform of KSR1 that functions in neuronal signaling. Mol Cell Biol. 2000 Aug;20(15):5529-39

Müller J, Ory S, Copeland T, Piwnica-Worms H, Morrison DK. C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1. Mol Cell. 2001 Nov;8(5):983-93

Giblett SM, Lloyd DJ, Light Y, Marais R, Pritchard CA. Expression of kinase suppressor of Ras in the normal adult and embryonic mouse. Cell Growth Differ. 2002 Jul;13(7):307-13

Nguyen A, Burack WR, Stock JL, Kortum R, Chaika OV, Afkarian M, Muller WJ, Murphy KM, Morrison DK, Lewis RE, McNeish J, Shaw AS. Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated protein kinase activation in vivo. Mol Cell Biol. 2002 May;22(9):3035-45

Roy F, Laberge G, Douziech M, Ferland-McCollough D, Therrien M. KSR is a scaffold required for activation of the ERK/MAPK module. Genes Dev. 2002 Feb 15;16(4):427-38

Lozano J, Xing R, Cai Z, Jensen HL, Trempus C, Mark W, Cannon R, Kolesnick R. Deficiency of kinase suppressor of Ras1 prevents oncogenic ras signaling in mice. Cancer Res. 2003 Jul 15;63(14):4232-8

Ory S, Zhou M, Conrads TP, Veenstra TD, Morrison DK. Protein phosphatase 2A positively regulates Ras signaling by dephosphorylating KSR1 and Raf-1 on critical 14-3-3 binding sites. Curr Biol. 2003 Aug 19;13(16):1356-64

Xing HR, Cordon-Cardo C, Deng X, Tong W, Campodonico L, Fuks Z, Kolesnick R. Pharmacologic inactivation of kinase suppressor of ras-1 abrogates Ras-mediated pancreatic cancer. Nat Med. 2003 Oct;9(10):1266-8

Kortum RL, Lewis RE. The molecular scaffold KSR1 regulates the proliferative and oncogenic potential of cells. Mol Cell Biol. 2004 May;24(10):4407-16

Laurent MN, Ramirez DM, Alberola-Ila J. Kinase suppressor of Ras couples Ras to the ERK cascade during T cell development. J Immunol. 2004 Jul 15;173(2):986-92

Razidlo GL, Kortum RL, Haferbier JL, Lewis RE. Phosphorylation regulates KSR1 stability, ERK activation, and cell proliferation. J Biol Chem. 2004 Nov 12;279(46):47808-14

Yan F, John SK, Wilson G, Jones DS, Washington MK, Polk DB. Kinase suppressor of Ras-1 protects intestinal epithelium from cytokine-mediated apoptosis during inflammation. J Clin Invest. 2004 Nov;114(9):1272-80

Kim M, Yan Y, Kortum RL, Stoeger SM, Sgagias MK, Lee K, Lewis RE, Cowan KH. Expression of kinase suppressor of Ras1 enhances cisplatin-induced extracellular signal-regulated kinase activation and cisplatin sensitivity. Cancer Res. 2005 May 15;65(10):3986-92

KSR1 (kinase suppressor of ras 1) Fernandez M, Lewis R


Kortum RL, Costanzo DL, Haferbier J, Schreiner SJ, Razidlo GL, Wu MH, Volle DJ, Mori T, Sakaue H, Chaika NV, Chaika OV, Lewis RE. The molecular scaffold kinase suppressor of Ras 1 (KSR1) regulates adipogenesis. Mol Cell Biol. 2005 Sep;25(17):7592-604

Salerno M, Palmieri D, Bouadis A, Halverson D, Steeg PS. Nm23-H1 metastasis suppressor expression level influences the binding properties, stability, and function of the kinase suppressor of Ras1 (KSR1) Erk scaffold in breast carcinoma cells. Mol Cell Biol. 2005 Feb;25(4):1379-88

Fusello AM, Mandik-Nayak L, Shih F, Lewis RE, Allen PM, Shaw AS. The MAPK scaffold kinase suppressor of Ras is involved in ERK activation by stress and proinflammatory cytokines and induction of arthritis. J Immunol. 2006 Nov 1;177(9):6152-8

Kortum RL, Johnson HJ, Costanzo DL, Volle DJ, Razidlo GL, Fusello AM, Shaw AS, Lewis RE. The molecular scaffold kinase suppressor of Ras 1 is a modifier of RasV12-induced and replicative senescence. Mol Cell Biol. 2006 Mar;26(6):2202-14

McKay MM, Morrison DK. Caspase-dependent cleavage disrupts the ERK cascade scaffolding function of KSR1. J Biol Chem. 2007 Sep 7;282(36):26225-34

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Gene Section Review




PEG10 (paternally expressed 10) Andreas Lux

Institute of Molecular and Cell Biology, Mannheim University of Applied Sciences, Mannheim, Germany (AL)


Online updated version : http://AtlasGeneticsOncology.org/Genes/PEG10ID44104ch7q21.html DOI: 10.4267/2042/46025


Identity Other names: EDR; HB-1; KIAA1051; MEF3L; Mar2; Mart2; RGAG3

HGNC (Hugo): PEG10

Location: 7q21.3

Local order: Located next to the sarcoglycan epsilon gene SGCE in a head-to-head orientation. The transcription start sites of these two genes are separated by 130 bp. Both genes belong to a maternally imprinted gene cluster in humans as well as in the syntenic chromosomal regions of several other mammalian species and are expressed from the paternal allele.

DNA/RNA Description The PEG10 gene is comprised of two exons separated by a 6753 bp long intron for transcript variant 1 (PEG10-A) or by a 6742 bp long intron for transcript variant 2 (PEG10-B). Transcript variant 2 is the result of alternative splicing where splicing occurs 11 nucleotides after the major splice site of exon 1 (Lux et

al., 2010) (see also figure 1). PEG10-A, has a length of 6573 bp and PEG10-B of 6584 bp. The analysis of four different cell lines (HepG2, HEK293, HL60 and SH-SY5Y) suggest that variant 1 is the major transcript. How alternative splicing for PEG10 is regulated and why two alternative splice products exist in parallel is not known.

Transcription The major transcription start site (mTSS) was determined to be at position 19.519.958 of reference sequence NT_007933|Hs7_8090 (Lux et al., 2010). This TSS is preceded by a typical TATA-box element in the ideal distance of 24-30 nucleotides. It appears as if there is at least one additional may be cell type dependent but less frequently used TSS further upstream. Several studies attempted to analyse the PEG10 promoter and how PEG10 expression is regulated but the results of these studies do not provide a coherent picture. For example, it was reported that c-MYC upregulates PEG10 expression in pancreatic and hepatic carcinoma cells as well as in a B-lymphocyte cell line (Li et al., 2006).

Figure 1. PEG10 splice variants PEG10-A and PEG10-B. Shown is the sequence around the splice junctions for the two PEG10 splice variants. The different start codons, translation initiation sites (TIS), in exon 1 and 2 are in red and underlined.

PEG10 (paternally expressed 10) Lux A


This effect appears to be mediated by c-MYC binding to an E-box sequence in the proximal region of the PEG10 intron, thereby influencing PEG10 promoter activity. In reporter assays, analysing just the promoter sequence upstream of the mTSS, overexpression of c-MYC showed an inhibitory effect (Lux et al., 2010). By bioinformatic analysis of the PEG10 promoter region +1 to -220, binding sites for transcription factors like TBP, Sp1 or E2F were identified (Lux et al., 2010). Binding of E2F members E2F-1 and E2F-4 to this region was experimentally proven and it was demonstrated that both factors positively regulate PEG10 expression (Wang C et al., 2008). A previous report showed that PEG10 expression is also positively regulated by E2F-2 and E2F-3 in the U2OS osteosarcoma cell line (Müller et al., 2001). These data suggest that PEG10 expression can be controled by the E2F/Rb pathway that involves the cyclin D/CDK4 complex, which phosphorylates pocket proteins like the retinoblastoma protein Rb and releases E2Fs. Thus, it can be expected that overexpression of cyclin D and CDK4 does also increase PEG10 expression, which indeed is the case (Wang C et al., 2008). In contrast, the presence of TGF-beta leads to a dephosphorylation of Rb, therefore repressing the expression of E2F target genes like PEG10 (Wang C et al., 2008). The repressive activity of TGF-beta on PEG10 expression is in agreement with our own unpublished results with PEG10 promoter-reporter constructs. Previous results showed that the PEG10-RF1 protein inhibits TGF-beta3 signalling in a TGF-beta-specific luciferase reporter assay (Lux et al., 2005), may be representing a self-protecting mechanism from down-regulation. Furthermore, it was reported that increased PEG10 expression is subjected to hormonal regulation by the male hormon androgen (Jie et al., 2007). In this study, three androgen receptor binding sites (ARE) were identified for PEG10. Two sites were reported for exon 2 and one for the promoter region. Unfortunately, no exact specifications were given about these sites. However, using the in the publication given primer sets for a Blast search against the PEG10 sequence reveals that ARE-1 is not located in the promoter but in exon 1. Therefore, all three PEG10-specific ARE sites are distal to the promoter. Transcript processing. Northern blot analyses have shown that for humans depending on the tissue PEG10 transcripts of different size exist. One between 6 and 7 kb, corresponding to the major 6.6 kb PEG10 transcript, as well as minor sized transcripts (Ono et al., 2001; Smallwood et al., 2003; Lux et al., 2005). The major 6.6 kb PEG10 transcript is polyadenylated and at the distal end of exon 2 there are two canonical polyadenylation sequences, AATAAA . In a recent study, minor sized alternatively polyadenylated transcripts were isolated but none of these transcripts

contained the typical polyadenylation signal motif at their 3'-end nor any known alternative polyadenylation signal sequence motifs (Lux et al., 2010). If PEG10 transcripts are truly subjected to alternative polyadenylation then future studies have to address the question whether this influences PEG10 expression, mRNA stability, mRNA localisation or translation and if it might be related to pathological processes. Because PEG10 is most likely derived from a retrotransposon it is interesting to note that non-conserved poly(A) sites are associated with transposable elements to a much greater extent than conserved ones (Lee et al., 2008).

Protein Description Transcription of PEG10 results in several protein isoforms due to alternative splicing, alternative translation initiation sites, posttranslational proteolytic cleavage and -1 ribosomal frameshift translation. The most prominent feature of PEG10 is its -1 ribosomal frameshift translation mechanism that hints at his retroviral/retrotransposon origin. The existence of PEG10 was reported by three different groups in 2001 (Ono et al., 2001; Shigemoto et al., 2001; Volff et al., 2001). In their search for novelle patternally expressed imprinted genes for an imprinted region on mouse chromosome 6, syntenic to a human imprinting cluster on chromosome 7q21 containing the imprinted SGCE gene, Ono and colleagues performed a database search for EST sequences mapping to this region. Three entries were identified, HB-1 (GenBank Accession No. AF216076), KIAA1051 (GenBank Accession No. AB028974), and 23915 mRNA (GenBank Accession No. AF038197) that were identical and mapped near SGCE. Sequence analysis of these clones predicted two open reading frames with homology to Gag and Pol proteins of some vertebrate retrotransposons, respectively. The deduced gene was named Paternally Expressed Gene 10 (PEG10). Similar results were obtained by Volff and colleagues, when they analysed public sequence databases for long-terminal-repeat (LTR) retrotransposon-like sequences of the Ty3/Gypsy retrotransposon family in mammals. They identified KIAA1051, which showed significant similarities to the Gag structural core protein of some Ty3/Gypsy retrotransposons from the Ty3 family, including Sushi from the pufferfish Fugu rubripes (42.5% similarities), Skippy, Maggy, and Cft1 from different fungi. No significant similarity to other families of Ty3/Gypsy retrotransposons and retroviruses was found and no LTR-like sequences flanking KIAA1051 were identified. It was further reported that the KIAA1051 cDNA contains a partial pol-like sequence (1.5 kb in length),



Figure 3. Schematic view of the PEG10-RF1b/2 protein. Shown are the locations of different predicted and proven protein domains.

which overlaps the gag-like sequence (1 kb in length) over approximately 250 bp. Its conceptual translation product displayed protease and truncated reverse transcriptase (RT) regions, including well-conserved first and second of the seven RT domains and showed the highest similarity to the Pol protein of again the retrotransposon Sushi (43.7% similarity). Further evidence for the existence of PEG10 came from the work by Shigemoto and colleagues. They analysed the mouse gene Edr, which was identified initially by differential screening of an embryonal carcinoma cDNA library for genes expressed at a reduced level following retinoic acid induced differentiation (Gorman et al., 1985). Their study identified in the Edr gene two open reading frames that overlapped and were set appart by a frameshift of one nucleotide. Subsequent analysis demonstrated that both reading frames are translated by -1 frameshifting (Shigemoto et al., 2001; Lux et al., 2005; Manktelow et al., 2005; Clark et al., 2007). Translation. In order to perform the -1 frameshift, the reading frame 1 (RF1) - reading frame 2 (RF2) overlap sequence contains a seven nucleotide "slippery" sequence with typical consecutive homopolymeric triplets. The underlined PEG10 "slippery" heptanucleotide sequence G GGA AAC TC follows the general pattern of X XXY YYZ where the A- and P-site tRNAs detach from the zero frame codons XXY YYZ and re-pair after shifting back one nucleotide to XXX YYY and restart translation with the codon after the YYY triplet. Thus, the deduced amino acid sequence of the frameshift site after frameshift translation is GNL. The heptanucleotide "slippery" sequence is completely conserved in all species and the sequence of the downstream pseudoknot is completely conserved in the mammalian species. except for one nucleotide change in the rodent sequence. A detailed analysis of the PEG10 frameshift sequence was done by Manktelow and colleagues (2005).

Due to alternative splicing two transcript variants exist, PEG10-A and PEG10-B, leading to several protein isoforms. These isoforms are first, the result of different translation initiation sites in reading frame 1 (RF1) (Lux et al., 2010). Second, due to the fact, whether the reading frames 1 and 2 (RF2) are translated into an RF1 protein or into an RF1/2 protein by succesfull -1 frameshift translation, and third, in the RF1/2 translation products, shortly after the frameshift site, there is a retroviral typical functional aspartic protease motif usually for Gag-Pol protein processing leading to proteolytic cleavage products (Clark et al., 2007). It was demonstrated that upstream of the originaly predicted ATG translation initiation site (TIS) a second in frame non-ATG exists. For clarification, the non-ATG translation site will be named TIS-1a and the previous ATG start site TIS-2. This non-ATG start codon, a CTG, is 102 nucleotides upstream of the ATG start codon. The alternative splice event that leads to transcript PEG10-B introduces an additional in frame ATG start codon even further upstream of the previous two, which will be named TIS-1b. Their might be an additional TIS further downstream of TIS-2 (Lux et al., 2010). Transcript PEG10-A codes for PEG10-RF1 (using TIS-2), PEG10-RF1a (using TIS-1a), PEG10-RF1/2 and PEG10-RF1a/2. While in theory, transcript PEG10-B can lead to all six isoforms, PEG10-RF1, PEG10-RF1a, PEG10-RF1b (using TIS-1b), PEG10-RF1/2, PEG10-RF1a/2 and PEG10-RF1b/2. Figure 1 shows in a more schematic way the different TIS for transcripts PEG10-A and PEG10-B. The deduced amino acid sequences of the different isoforms are listed in figure 2. Investigation of PEG10 translation in mouse placenta during gestation and human placenta showed that in vivo both reading frames are translated as an RF1 protein and an RF1/2 fusion protein (Clark et al., 2007). The mouse RF1/2 protein is about 40 kDa larger than the corresponding human protein due to an in frame



insertion of approximately 600 nucleotides into the RF2 sequence. Interestingly, the size of RF1 and RF1/2 proteins and the translational frame shift efficiency varies during gestation. From 9.5 dpc when PEG10 expression in mice is first detectable, the 150 kDa frameshift protein is dominant. By using an RF1-specific antibody the frameshift efficiency was estimated and showed an apparent decrease from 68% at 9.5 dpc to 43% by 21.5 dpc. At 15.5 dpc an additional protein of 105 kDa was detected at about equal amounts as the 150 kDa protein. At late gestation, 21.5 dpc, it was present in greater amounts than the 150 kDa PEG10-RF1/2 protein. Mass spectrometry analysis identified the 105 kDa protein as a PEG10 product consisting primarily of PEG10 RF2 but containing peptides from both reading frames. PEG10 protein analysis for amniotic membrane showed a similar profile to that of placenta. Starting with a low expression at 9.5 dpc and then an increased and continued expression throughout gestation. The RF1/2 fusion protein again was the dominant band. Surprisingly, at 10.5 dpc a transient RF1 protein of increased mass of about 50 kDa was detected that disappeared at later time points and only the slightly smaller 47-kDa band identical in size to that in placenta was present again. Furthermore, in this report three RF1 protein populations were detected for HepG2 cells ranging from 47 to 55 kDa. Whether these different PEG10 protein masses are the result of post-translational modifications or due to the use of different TIS or a mixture of both is not clear and awaits further investigations. In addition, western blot analysis with an RF1-specific antibody of adult mouse heart, spleen and brain tissue extracts showed a weak, single protein band but of different mass, around 50 kDa, for each tissue (Clark et al., 2007). No RF1/2 proteins were detected. The authors concluded based on their further analysis that these proteins do not represent Peg10 proteins. Protein domains/motifs. By bioinformatic analyses using different programmes like the Simple Modular Architecture Research Tool (SMART), the SUPERFAMILY Sequence Search (SCOP domains) and the Eukaryotic Linear Motif (ELM) resource for functional site prediction, several domains and motifs were predicted. Some are exemplarily shown schematicaly in figure 3 for the 784 amino acid long PEG10-RF1b/2 protein. The Zink-finger domain was consistently identified although the size of the domain varies from amino acid 357-389 or a core region from 370-386 for a ZNF-C2HC (CX2CX4HX4C) consensus sequence, which is highly conserved in Gag proteins in most retroviruses and some retrotransposons. There are two proline rich regions, one at the N-terminus and one at the C-terminus. Proline-rich regions are recognized as presenting binding motifs to, for example, Src homology 2 (SH2) and SH3 domains. The ELM programme predicted the C-terminal proline stretch to be a possible binding site for SH3 domain containing

proteins. As already reported, PEG10 contains a retroviral typical aspartyl protease consensus sequence, AMIDSGA. In order to test whether this motif is catalytic active the aspartate was mutated to an alanine (Clark et al., 2007). This change disrupted the protease activity and proved that the aspartyl protease is responsible for the cleavage of the full length PEG10 frameshift protein in to the RF1 and RF2 parts. Taken the protease activity into account the previously estimated PEG10 frameshift efficiency of 15-30% (Shigemoto et al., 2001; Lux et al., 2005) was reestimated to be 60% (Clark et al., 2007). Interacting proteins. Aside from the protease motif, for none of the other domains it is known whether they are functional nor if they bind to other proteins. The only known binding partners for PEG10 are currently the SIAH1 and SIAH2 proteins (Okabe et al., 2003) and the TGF-beta type I receptor ALK1 (Lux et al., 2005). All three proteins were identified by a yeast two-hybrid screen with the PEG10-RF1 protein and the interactions were confirmed by co-immunoprecipitation experiments. The exact SIAH1/SIAH2 binding region was not determined, but the ELM programme predicted a potential SIAH1 binding site (figure 3, PEG10-RF1b amino acids 329-337). Co-immunoprecipitation experiments by overexpressing PEG10-RF1 and several other type I and II receptors of the TGF-beta superfamily in COS-1 cells showed that PEG10 does also interact with other members of this receptor group (Lux et al., 2005). Nevertheless, when specifically investigated in the two-hybrid assay under stringent conditions none of these receptors reacted with PEG10-RF1 to activate the reporter system. Thus, the most specific interaction appears to be with ALK1.

Expression Based on data obtained from mice, Peg10 is predominantly expressed during embryonic development, whereas later on expression in most tissues ceases or is low except for testis and brain of adult animals (Shigemoto et al., 2001). However, significant induction of Peg10 expression was detected in hepatocellular carcinomas (HCC) and for the regenerating livers of mice after partial hepatectomy (Tsou et al., 2003). Studies with mice by RNA in situ hybridisation showed a high expression during embryonic development especially from day 9.5 to 16.5, specifically in bone and cartilage forming tissues as well as in extra embryonic tissues at all stages between E7.5 and E17.5. For a detailed description see Shigemoto et al. (2001). In humans, expression of PEG10 in adult tissues was seen in brain, kidney, lung, testis and only weak to very weak expression in spleen, liver, colon, small intestine and muscle, but no expression for heart and stomach (Ono et al., 2001). Furthermore, strong expression was also reported for mouse and human placenta. In humans PEG10 expression is low at the early hypoxic phase of placental growth and increases at 11-12 weeks of



gestation. This high level of expression is maintained and is significantly increased in term placenta compared with that in early pregnancy (Smallwood et al., 2003). The authors hypothesize that the gene product might be essential for trophoblast differentiation and uterine implantation. Peg10 expression was observed in relation to adipocyte differentiation in mice. Peg10 was identified as one of the genes expressed early in adipogenesis (Hishida et al., 2007). Expression of Peg10 was elevated after the addition of differentiation inducers in adipocyte differentiable 3T3-L1 cells, but not in the non-adipogenic cell line NIH-3T3. The knockdown of Peg10 by RNA interference inhibited the differentiation of 3T3-L1 cells. Moreover, Peg10 siRNA treatment impaired mitotic clonal expansion (MCE), necessary for adipocyte differentiation, and the crucial expression of C/EBPbeta and C/EBPdelta at the immediate early stage of the differentiation process was inhibited by the knock-down. These results indicate that Peg10 plays an important role at the immediate early stage of adipocyte differentiation. Aside from bone and cartilage tissue differentiation and adipocyte cell differentiation PEG10 might also be involved in neuronal cell differentiation. In a preliminary experiment with the neuroblastoma cell line SH-SY5Y increasing PEG10 expression was reported over a period of 14 days after treatment with all-trans retinoic acid for differentiation (Lux et al., 2010).

Localisation Data regarding PEG10's cellular localisation do only exist for PEG10-RF1. Okabe and colleagues (2003) report for the hepatoma cell lines HepG2, Huh7 and Alexander as well as for hepatocellular carcinoma (HCC) tissues nuclear and cytoplasmic staining of PEG10 with a PEG10-RF1-specific antibody. In experiments overexpressing PEG10-RF1 in HEK293T cells, only cytoplasmic localisation was reported (Tsou et al., 2003), which was also shown by immunofluorescence analysis with a PEG10-RF1-specific antibody for endogenous PEG10 in HepG2 and B-CLL cells (Lux et al., 2005; Kainz et al., 2007).

Function The knowledge regarding PEG10's protein function is sparse and all direct evidence that exists so far was gained by experiments with the PEG10-RF1 isoform only. No data exists for the recently identified isoform PEG10-RF1a, most likely the major RF1 isoform, or the PEG10-RF1b protein. Functional data regarding the different RF1/2 isoforms are lacking too. Nevertheless, PEG10-RF1 appears to enhance cell proliferation and blocks apoptosis. Evidence for PEG10's role in cell proliferation were reported by Tsou et al. (2003). Induced PEG10 expression was found during G2/M phase of regenerating mouse liver and elevated expression of PEG10 was found in HCC.

The authors state that both, HCC and regenerating mouse livers, represent the two proliferative states of the otherwise quiescent liver tissue. In addition, ectopic expression of PEG10 in 293T cells enhanced cell cycle progression. Complementary data were presented by Okabe and colleagues (2003). The hepatoma cell line, SNU423 that has no endogenous PEG10, was stable transfected with an expression construct for PEG10-RF1 to test the effect of PEG10 on cell growth. The PEG10 stable transfectant cells revealed significant growth promotion compared with the parental or mock cells. Under conditions of serum starvation (0.1% FBS), the mock cells rapidly underwent growth arrest, but stable PEG10-expressing cells continued to proliferate. In the same report, in a yeast two-hybrid screen, the apoptosis inducing protein SIAH1 was identified as a PEG10-RF1 interactor. Overexpression of SIAH1 increased cell death in different hepatoma cell lines, whereas co-expression of PEG10-RF1 in tested SNU423 revealed a partial but significant protection from apoptosis. Anti-apoptotic activity of PEG10 was also reported by Yoshibayashi et al. (2007). The finding that PEG10 enhances cell proliferation was further confirmed by PEG10-specific siRNA knock-down experiments in a series of carcinoma cell lines, i.e. Panc1, HepG2, and Hep3B, which led to a significantly reduced cell proliferation (Li et al., 2006; Yoshibayashi et al., 2007). Anti-apoptotic activity by PEG10 were not only seen for HCC/hepatoma but also reported for B-cell acute and chronic lymphocytic leukemia, B-ALL and B-CLL. It was observed that B-ALL and B-CLL CD19+CD34+ B cells expressed elevated levels of PEG10, regulated by the chemokines CXCL13 and CCL19 and that these cells were resistant to TNF-alpha induced apoptosis. Treatment of the cells with PEG10 antisense constructs reversed this effect (Hu et al., 2004; Chunsong et al., 2006; Wang et al., 2007). Kainz and colleagues (2007) reported that PEG10 overexpression is associated with high-risk B-CLL. Expression levels in CD19+ B-CLL cells were up to 100-fold higher than in B-cells from healthy donors and expression levels in B-CLL patient samples remained stable over time even after chemotherapy. The intensity of intracellular staining of PEG10 protein corresponded to mRNA levels. Further analysis of PEG10's anti-apoptotic potential showed that short term knock-down (2 days after transfection) of PEG10 in B-CLL cells was not associated with changes in cell survival but long term inhibition (4 days after transfection of PEG10) led to a significant effect on the induction of apoptosis and late apoptosis/necrosis in B-CLL cells. As described above, during gestation PEG10 is highly expressed in the placenta. Mouse placenta is positive for Peg10 transcripts as well as for proteins from reading frame 1 and reading frame 1/2 (Clark et al., 2007). Peg10 knock-out experiments in mice demonstrated that Peg10 and therefore the Peg10 proteins have an important role during placenta



development. Heterozygous knock-outs for the patternal allele died at 10.5 dpc. Their placentas were severely depleted and the labyrinth layer was not developed and the spongiotrophoblast cells were missing (Ono et al., 2006). As discussed by the authors, parthenogenetic embryos die before 9.5 d.p.c. and show early embryonic lethality with poorly developed extraembryonic tissues. Morphological defects of the most developed parthenotes are very similar to those of Peg10-Pat KO embryos; they lack the diploid trophoblast cells of the labyrinth layer and the spongiotrophoblast. However, the majority of parthenotes show more severe phenotypes; suggesting that other genes could also contribute to the parthenogenetic phenotypes. Nevertheless, the result for mouse Peg10 suggests that one of the Peg10 isoforms could be critical for parthenogenetic development in mice and therefore also in man.

Homology PEG10 orthologous sequences can be found in several other eutherian species: Pan troglodytes (chimpanzee), Papio anubis (baboon), Macaca mulatta (Rhesus monkey), Callithrix jacchus (marmoset), Sus scrofa (pig), Canis familiaris (dog), Felis catus (cat), Bos taurus (bovine), Ovis aries (sheep), Mus musculus (mouse), Rattus norvegicus (rat), Rhinolophus ferrumequinum (bat), Sorex araneus (shrew), Monodelphis domestica (opossum) as well as in the metatherian tammar wallaby (Macropus eugenii) (Ono et al., 2001; Brandt et al., 2005a; Brandt et al., 2005b; Suzuki et al., 2007; Clark et al., 2007) and there is a high degree of amino acid conservation. The PEG10 sequence is not conserved in reptiles or birds. Hence, during evolution the PEG10 gene was introduced into the therian mammal genome after the split of birds about 300 million years (myr) ago and after the split of prototherian mammals (monotremes) 166 myr ago, but before the divergence between placental mammals and marsupials about 148 myr ago.

Implicated in Various cancers Note Two major pathological conditions are reported in which PEG10 plays a role, hepato cellular carcinomas and B-cell acute and chronic lymphocytic leukemia. As already mentioned above, the presence of PEG10 is linked to resistance of apoptosis and increased cell growth. In addition, further malignancies in which an over expression or prolonged expression of PEG10 was seen are the embryonic kidney malignancy Wilms tumor (Dekel et al., 2006), pancreatic cancer (Li et al., 2006) and the embryonic form of biliary atresia (Zhang et al., 2004). Widespread DNA copy number alterations are well recognized in HCC and concurrent genomic gains within the chromosome region 7q21 has been

implicated in the progression of HCC. In a study by Ip et al. (2007), it was suggested that PEG10 may be a potential biomarker in the progressive development of HCC. Quantitative PCR and qRT-PCR showed the chromosomal gain of 7q21 as well as over expression of PEG10 in HCC cell lines and primary tumors. In addition, qRT-PCR demonstrated a significant progressive trend of increasing PEG10 expression from the putative pre-malignant adjacent livers to early resectable HCC tumors, and to late inoperable HCCs. The authors concluded that genomic gain represents one of the major mechanisms in the induction of PEG10 over expression. This conclusion is further supported by independent data from Tsuji et al. (2010). Increased PEG10 expression might also serve as a biomarker for nephropathy in peripheral blow cells of type 2 diabetes (T2DN) (Guttula et al., 2010).

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Li CM, Margolin AA, Salas M, Memeo L, Mansukhani M, Hibshoosh H, Szabolcs M, Klinakis A, Tycko B. PEG10 is a c-MYC target gene in cancer cells. Cancer Res. 2006 Jan 15;66(2):665-72

Ono R, Nakamura K, Inoue K, Naruse M, Usami T, Wakisaka-Saito N, Hino T, Suzuki-Migishima R, Ogonuki N, Miki H, Kohda T, Ogura A, Yokoyama M, Kaneko-Ishino T, Ishino F. Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality. Nat Genet. 2006 Jan;38(1):101-6

Clark MB, Jänicke M, Gottesbühren U, Kleffmann T, Legge M, Poole ES, Tate WP. Mammalian gene PEG10 expresses two reading frames by high efficiency -1 frameshifting in embryonic-associated tissues. J Biol Chem. 2007 Dec 28;282(52):37359-69

Hishida T, Naito K, Osada S, Nishizuka M, Imagawa M. peg10, an imprinted gene, plays a crucial role in adipocyte differentiation. FEBS Lett. 2007 Sep 4;581(22):4272-8

Ip WK, Lai PB, Wong NL, Sy SM, Beheshti B, Squire JA, Wong N. Identification of PEG10 as a progression related biomarker for hepatocellular carcinoma. Cancer Lett. 2007 Jun 8;250(2):284-91

Jie X, Lang C, Jian Q, Chaoqun L, Dehua Y, Yi S, Yanping J, Luokun X, Qiuping Z, Hui W, Feili G, Boquan J, Youxin J, Jinquan T. Androgen activates PEG10 to promote

carcinogenesis in hepatic cancer cells. Oncogene. 2007 Aug 23;26(39):5741-51

Kainz B, Shehata M, Bilban M, Kienle D, Heintel D, Krömer-Holzinger E, Le T, Kröber A, Heller G, Schwarzinger I, Demirtas D, Chott A, Döhner H, Zöchbauer-Müller S, Fonatsch C, Zielinski C, Stilgenbauer S, Gaiger A, Wagner O, Jäger U. Overexpression of the paternally expressed gene 10 (PEG10) from the imprinted locus on chromosome 7q21 in high-risk B-cell chronic lymphocytic leukemia. Int J Cancer. 2007 Nov 1;121(9):1984-93

Suzuki S, Ono R, Narita T, Pask AJ, Shaw G, Wang C, Kohda T, Alsop AE, Marshall Graves JA, Kohara Y, Ishino F, Renfree MB, Kaneko-Ishino T. Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting. PLoS Genet. 2007 Apr 13;3(4):e55

Wang X, Yuling H, Yanping J, Xinti T, Yaofang Y, Feng Y, Ruijin X, Li W, Lang C, Jingyi L, Zhiqing T, Jingping O, Bing X, Li Q, Chang AE, Sun Z, Youxin J, Jinquan T. CCL19 and CXCL13 synergistically regulate interaction between B cell acute lymphocytic leukemia CD23+CD5+ B Cells and CD8+ T cells. J Immunol. 2007 Sep 1;179(5):2880-8

Yoshibayashi H, Okabe H, Satoh S, Hida K, Kawashima K, Hamasu S, Nomura A, Hasegawa S, Ikai I, Sakai Y. SIAH1 causes growth arrest and apoptosis in hepatoma cells through beta-catenin degradation-dependent and -independent mechanisms. Oncol Rep. 2007 Mar;17(3):549-56

Lee JY, Ji Z, Tian B. Phylogenetic analysis of mRNA polyadenylation sites reveals a role of transposable elements in evolution of the 3'-end of genes. Nucleic Acids Res. 2008 Oct;36(17):5581-90

Wang J, Tai LS, Tzang CH, Fong WF, Guan XY, Yang M. 1p31, 7q21 and 18q21 chromosomal aberrations and candidate genes in acquired vinblastine resistance of human cervical carcinoma KB cells. Oncol Rep. 2008 May;19(5):1155-64

Wang C, Xiao Y, Hu Z, Chen Y, Liu N, Hu G. PEG10 directly regulated by E2Fs might have a role in the development of hepatocellular carcinoma. FEBS Lett. 2008 Aug 6;582(18):2793-8

Guttula SV, Rao AA, Sridhar GR, Chakravarthy MS, Nageshwararo K, Rao PV. Cluster analysis and phylogenetic relationship in biomarker identification of type 2 diabetes and nephropathy. Int J Diabetes Dev Ctries. 2010 Jan;30(1):52-6

Lux H, Flammann H, Hafner M, Lux A. Genetic and molecular analyses of PEG10 reveal new aspects of genomic organization, transcription and translation. PLoS One. 2010 Jan 13;5(1):e8686

Tsuji K, Yasui K, Gen Y, Endo M, Dohi O, Zen K, Mitsuyoshi H, Minami M, Itoh Y, Taniwaki M, Tanaka S, Arii S, Okanoue T, Yoshikawa T. PEG10 is a probable target for the amplification at 7q21 detected in hepatocellular carcinoma. Cancer Genet Cytogenet. 2010 Apr 15;198(2):118-25


Lux A. PEG10 (paternally expressed 10). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):724-730.

Gene Section Review




PIK3CD (phosphoinositide-3-kinase, catalytic, delta polypeptide) Emily Burns, Bart Vanhaesebroeck

Centre of Cell Signalling, Barts Cancer Institute, Queen Mary University of London, London, EC1M 6BQ, UK (EB, BV)


Online updated version : http://AtlasGeneticsOncology.org/Genes/PIK3CDID46261ch1p36.html DOI: 10.4267/2042/46026


Identity Other names: P110DELTA; PI3K; p110D

HGNC (Hugo): PIK3CD

Location: 1p36.22

Local order: Telomere-SPSB1-SLC25A33-TMEM201-PIK3CD-C1orf200-CR604408-KIAAO00911-centromere.

DNA/RNA Description The PIK3CD gene spans a genomic region of 24 coding exons and over 77.17 kb.

Transcription The dominant transcript contains two upstream untranslated exons, -2a and -1, as well as an additional first untranslated exon (-2b) identified in the human PIK3CD gene (Kok et al., 2009).

Protein Description The PI3K enzymes are a family of activation dependent lipid kinases, which have been divided into three classes (Vanhaesebroeck et al., 1999a). PIK3CD encodes p110delta which belongs to the class I PI3Ks alongside p110alpha, p110beta and p110gamma. The PI3K enzymes are heterodimeric molecules which consist of a catalytic p110 subunit, and a constitutively bound p85 regulatory unit of which there are five isoforms encoded by three genes (PIK3R1, PIK3R2 and PIK3R3) (Vanhaesebroeck and Waterfield, 1999; Geering et al., 2007). The regulatory subunit has a dual function of recruiting the p110 catalytic subunit to an activated upstream tyrosine kinase receptor, and inhibiting catalytic activity in the absence of this stimulatory interaction (Yu et al., 1998).

Location of PIK3CD on human chromosome 1.

PIK3CD (phosphoinositide-3-kinase, catalytic, delta polypeptide) Burns E, Vanhaesebroeck B


The illustration represents the exon structure of the most prevalent transcript in leukocytes, which contains two untranslated exons upstream of the start site (-2a and -1) and 24 coding exons.

p110delta protein domains. p110delta interacts with its associated p85 regulatory subunit via the adaptor binding domain (ABD). The C Lobe and N Lobe together form the catalytic domain. Unlike p110alpha, p110delta does not phoshorylate its associated p85 regulatory subunit and instead auto-phosphorylates (Vanhaesebroeck et al., 1997; Vanhaesebroeck et al., 1999a).

Expression p110delta has a restricted expression pattern; it is highly expressed in leukocytes (Seki et al., 1997; Vanhaesebroeck et al., 1997) and to a lesser extent expressed in neurons (Eickholt et al., 2007). In addition p110delta expression has also been observed in a number of cancerous tissues including breast cancer and melanoma cells (Sawyer et al., 2003). A transcription factor binding cluster located immediately upstream of the untranslated exon -2a in the human gene, has been identified and found to have enhanced promotor activity in leukocytes. This promoter region may therefore facilitate the enhanced expression of p110delta in leukocytes (Kok et al., 2009).

Localisation p110 delta localises to the cytosol and is recruited to the periplasmic region upon stimulation of upstream activators.

Function Cell signalling and lipid kinase activity. PI3K signalling has been found to play a crucial role in the regulation of numerous cellular processes including proliferation, metabolism (Foukas et al., 2006; Engelman et al., 2006), and migration (Papakonstanti et al., 2007). Active p110delta initiates signalling cascades by phosphorylating phosphoinositide (PI) lipids such as PIP2 producing pools of PIP3 in the periplasmic region (Vanhaesebroeck et al., 1997). A wide range of effector proteins recognise and interact with the PI species produced, and the resulting alteration in location and

activities of these effectors initiates a number of signalling cascades. The classical effector of PI3K signalling is the serine/threonin kinase AKT (aka protein kinase B), which contains a pleckstrin homology (PH) domain that interacts with PIP3 (Franke et al., 1997). The affinity of Akt for PIP3facilitates activation of Akt's protein kinase activity, resulting in activity-modifying phosphorylation of a range of downstream Akt targets. Roles of p110delta in immune function. The role of p110delta in vivo has been studied in a p110delta null mouse (Clayton et al., 2002; Jou et al., 2002) and in a p110delta kinase-dead mouse known as p110deltaD910A/D910A (Okkenhaug et al., 2002). Phenotypic analysis of the p110deltaD910A/D910A mouse revealed an important role for p110delta in immunity; the mice have notable defects in their T and B cell responses, as well as several other immunological abnormalities (Okkenhaug et al., 2002). In vitro and ex vivo studies suggest that p110delta is the predominant PI3K class IA isoform regulating the phenotype and responses of many leukocyte cell types (Vanhaesebroeck et al., 1999b; Puri et al., 2004; Papakonstanti et al., 2008). In mast cells derived from the p110delta kinase-dead mice, ninety percent of the total PI3K lipid kinase activity was dependent on p110delta (Ali et al., 2004). p110delta has been found to be a key player in the induction of amongst other things, proliferation, chemotaxis, and cytokine/chemokine release in response to both physiological stimuli and pathological stimuli, in many leukocytes (Okkenhaug and Vanhaesebroeck, 2003; Dos Santos et al., 2007; Papakonstanti et al., 2008; Dil et al., 2009; Low et al., 2010).

Homology p110delta is most homologous to p110beta (Clayton et al., 2001).



Mutations Note No prevalent p110delta mutations have been observed in primary samples or cell lines.

Implicated in Haematological malignancy Note Constitutive phosphorylation of Akt (indicating constitutive activity) has been observed in many haematological malignancies, and appears to be responsible for or contributing to growth factor-independent proliferation (Min et al., 2003, Ikeda et al., 2010). p110delta appears to be upstream of the constitutively active Akt in these transformed cells; p110delta-selective inhibitors significantly reduce the abberent Akt activation and also results in a reduction in proliferation (Billottet et al., 2006; Ikeda et al., 2010; Herman et al., 2010; Lannutti et al., 2011). Despite the dominance of p110delta in the regulation of proliferation in leukocytes and the abberant activity of it's major downstream target Akt, no mutations or amplifications of p110delta have been observed in these or other haematological malignancies. A series of clinical trials are now underway to assess the effects of selective inhibition of p110delta on refractory haematological malignancies, and preliminary results are promising (Furman, 2010).

Disease CLL, T-ALL, B-ALL, DLBCL, AML, and multiple myeloma.

Allergic disease (Ali et al., 2004; Lee et al., 2006; Medina-Tato et al., 2007; Park et al., 2008)

Note Mast cells derived from p110deltaD910A/D910A mice have a degranulation defect, as do wild-type mast cells treated with a p110delta-selective inhibitor. Specifically cells lacking active p110delta showed a 45-55% reduction in degranulation induced by an antigen-IgE complex, compared to the degranulation observed in WT mast cells (Ali et al., 2004). Furthermore inactivation of p110delta has been shown to attenuate anaphylactic responses in mice (Ali et al., 2004). p110delta-selective inhibitors have been shown to attenuate OVA-induced influx into lungs of total leukocytes, eosinophils, neutrophils, and lymphocytes, as well as reducing the production of a number of pro-inflammatory mediators (Lee et al., 2006).

Disease Asthma, allergic rhinitis.

Chronic inflammatory conditions Note p110delta has been identified as a major regulator of inflammatory responses in a wide range of leukocytes

(including macrophages, neutrophils, mast cells as well as the adaptive immune cells). It it is considered an excellent potential therapeutic target for reducing inflammation both in the acute allergic context but also in the context of chronic inflammatory disorders (Ji et al., 2007; Rommel et al., 2007). The importance of p110delta in the regulation of inflammatory responses is highlighted by the development of spontaneous chronic colonic inflammation in the p110deltaD910A/D910A mice, which has been attributed to a failure to repress inflammatory responses to commensal bacteria in the gut (Uno et al., 2010). Thus p110delta-selective drugs are currently under investigation as a treatment for rheumatoid arthritis, asthma and other inflammatory conditions.

Disease Rheumatoid arthritis, inflammatory bowel disease (IBD).

References Franke TF, Kaplan DR, Cantley LC, Toker A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science. 1997 Jan 31;275(5300):665-8

Seki N, Nimura Y, Ohira M, Saito T, Ichimiya S, Nomura N, Nakagawara A. Identification and chromosome assignment of a human gene encoding a novel phosphatidylinositol-3 kinase. DNA Res. 1997 Oct 31;4(5):355-8

Vanhaesebroeck B, Welham MJ, Kotani K, Stein R, Warne PH, Zvelebil MJ, Higashi K, Volinia S, Downward J, Waterfield MD. P110delta, a novel phosphoinositide 3-kinase in leukocytes. Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4330-5

Yu J, Zhang Y, McIlroy J, Rordorf-Nikolic T, Orr GA, Backer JM. Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. Mol Cell Biol. 1998 Mar;18(3):1379-87

Vanhaesebroeck B, Higashi K, Raven C, Welham M, Anderson S, Brennan P, Ward SG, Waterfield MD. Autophosphorylation of p110delta phosphoinositide 3-kinase: a new paradigm for the regulation of lipid kinases in vitro and in vivo. EMBO J. 1999a Mar 1;18(5):1292-302

Vanhaesebroeck B, Jones GE, Allen WE, Zicha D, Hooshmand-Rad R, Sawyer C, Wells C, Waterfield MD, Ridley AJ. Distinct PI(3)Ks mediate mitogenic signalling and cell migration in macrophages. Nat Cell Biol. 1999b May;1(1):69-71

Vanhaesebroeck B, Waterfield MD. Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res. 1999 Nov 25;253(1):239-54

Clayton E, McAdam S, Coadwell J, Chantry D, Turner M. Structural organization of the mouse phosphatidylinositol 3-kinase p110d gene. Biochem Biophys Res Commun. 2001 Feb 9;280(5):1328-32

Clayton E, Bardi G, Bell SE, Chantry D, Downes CP, Gray A, Humphries LA, Rawlings D, Reynolds H, Vigorito E, Turner M. A crucial role for the p110delta subunit of phosphatidylinositol 3-kinase in B cell development and activation. J Exp Med. 2002 Sep 16;196(6):753-63

Jou ST, Carpino N, Takahashi Y, Piekorz R, Chao JR, Carpino N, Wang D, Ihle JN. Essential, nonredundant role for the phosphoinositide 3-kinase p110delta in signaling by the B-cell receptor complex. Mol Cell Biol. 2002 Dec;22(24):8580-91



Okkenhaug K, Bilancio A, Farjot G, Priddle H, Sancho S, Peskett E, Pearce W, Meek SE, Salpekar A, Waterfield MD, Smith AJ, Vanhaesebroeck B. Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice. Science. 2002 Aug 9;297(5583):1031-4

Min YH, Eom JI, Cheong JW, Maeng HO, Kim JY, Jeung HK, Lee ST, Lee MH, Hahn JS, Ko YW. Constitutive phosphorylation of Akt/PKB protein in acute myeloid leukemia: its significance as a prognostic variable. Leukemia. 2003 May;17(5):995-7

Okkenhaug K, Vanhaesebroeck B. PI3K in lymphocyte development, differentiation and activation. Nat Rev Immunol. 2003 Apr;3(4):317-30

Sawyer C, Sturge J, Bennett DC, O'Hare MJ, Allen WE, Bain J, Jones GE, Vanhaesebroeck B. Regulation of breast cancer cell chemotaxis by the phosphoinositide 3-kinase p110delta. Cancer Res. 2003 Apr 1;63(7):1667-75

Ali K, Bilancio A, Thomas M, Pearce W, Gilfillan AM, Tkaczyk C, Kuehn N, Gray A, Giddings J, Peskett E, Fox R, Bruce I, Walker C, Sawyer C, Okkenhaug K, Finan P, Vanhaesebroeck B. Essential role for the p110delta phosphoinositide 3-kinase in the allergic response. Nature. 2004 Oct 21;431(7011):1007-11

Puri KD, Doggett TA, Douangpanya J, Hou Y, Tino WT, Wilson T, Graf T, Clayton E, Turner M, Hayflick JS, Diacovo TG. Mechanisms and implications of phosphoinositide 3-kinase delta in promoting neutrophil trafficking into inflamed tissue. Blood. 2004 May 1;103(9):3448-56

Billottet C, Grandage VL, Gale RE, Quattropani A, Rommel C, Vanhaesebroeck B, Khwaja A. A selective inhibitor of the p110delta isoform of PI 3-kinase inhibits AML cell proliferation and survival and increases the cytotoxic effects of VP16. Oncogene. 2006 Oct 26;25(50):6648-59

Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006 Aug;7(8):606-19

Foukas LC, Claret M, Pearce W, Okkenhaug K, Meek S, Peskett E, Sancho S, Smith AJ, Withers DJ, Vanhaesebroeck B. Critical role for the p110alpha phosphoinositide-3-OH kinase in growth and metabolic regulation. Nature. 2006 May 18;441(7091):366-70

Lee KS, Lee HK, Hayflick JS, Lee YC, Puri KD. Inhibition of phosphoinositide 3-kinase delta attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model. FASEB J. 2006 Mar;20(3):455-65

Dos Santos S, Delattre AI, De Longueville F, Bult H, Raes M. Gene expression profiling of LPS-stimulated murine macrophages and role of the NF-kappaB and PI3K/mTOR signaling pathways. Ann N Y Acad Sci. 2007 Jan;1096:70-7

Eickholt BJ, Ahmed AI, Davies M, Papakonstanti EA, Pearce W, Starkey ML, Bilancio A, Need AC, Smith AJ, Hall SM, Hamers FP, Giese KP, Bradbury EJ, Vanhaesebroeck B. Control of axonal growth and regeneration of sensory neurons by the p110delta PI 3-kinase. PLoS One. 2007 Sep 11;2(9):e869

Geering B, Cutillas PR, Nock G, Gharbi SI, Vanhaesebroeck B. Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers. Proc Natl Acad Sci U S A. 2007 May 8;104(19):7809-14

Ji H, Rintelen F, Waltzinger C, Bertschy Meier D, Bilancio A, Pearce W, Hirsch E, Wymann MP, Rückle T, Camps M, Vanhaesebroeck B, Okkenhaug K, Rommel C. Inactivation of PI3Kgamma and PI3Kdelta distorts T-cell development and

causes multiple organ inflammation. Blood. 2007 Oct 15;110(8):2940-7

Medina-Tato DA, Ward SG, Watson ML. Phosphoinositide 3-kinase signalling in lung disease: leucocytes and beyond. Immunology. 2007 Aug;121(4):448-61

Papakonstanti EA, Ridley AJ, Vanhaesebroeck B. The p110delta isoform of PI 3-kinase negatively controls RhoA and PTEN. EMBO J. 2007 Jul 11;26(13):3050-61

Rommel C, Camps M, Ji H. PI3K delta and PI3K gamma: partners in crime in inflammation in rheumatoid arthritis and beyond? Nat Rev Immunol. 2007 Mar;7(3):191-201

Papakonstanti EA, Zwaenepoel O, Bilancio A, Burns E, Nock GE, Houseman B, Shokat K, Ridley AJ, Vanhaesebroeck B. Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages. J Cell Sci. 2008 Dec 15;121(Pt 24):4124-33

Park SJ, Min KH, Lee YC. Phosphoinositide 3-kinase delta inhibitor as a novel therapeutic agent in asthma. Respirology. 2008 Nov;13(6):764-71

Dil N, Marshall AJ. Role of phosphoinositide 3-kinase p110 delta in TLR4- and TLR9-mediated B cell cytokine production and differentiation. Mol Immunol. 2009 Jun;46(10):1970-8

Kok K, Nock GE, Verrall EA, Mitchell MP, Hommes DW, Peppelenbosch MP, Vanhaesebroeck B. Regulation of p110delta PI 3-kinase gene expression. PLoS One. 2009;4(4):e5145

Furman RR. New agents in early clinical trials for CLL therapy. Clin Adv Hematol Oncol. 2010 Jul;8(7):475-6

Herman SE, Gordon AL, Wagner AJ, Heerema NA, Zhao W, Flynn JM, Jones J, Andritsos L, Puri KD, Lannutti BJ, Giese NA, Zhang X, Wei L, Byrd JC, Johnson AJ. Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals. Blood. 2010 Sep 23;116(12):2078-88

Ikeda H, Hideshima T, Fulciniti M, Perrone G, Miura N, Yasui H, Okawa Y, Kiziltepe T, Santo L, Vallet S, Cristea D, Calabrese E, Gorgun G, Raje NS, Richardson P, Munshi NC, Lannutti BJ, Puri KD, Giese NA, Anderson KC. PI3K/p110{delta} is a novel therapeutic target in multiple myeloma. Blood. 2010 Sep 2;116(9):1460-8

Low PC, Misaki R, Schroder K, Stanley AC, Sweet MJ, Teasdale RD, Vanhaesebroeck B, Meunier FA, Taguchi T, Stow JL. Phosphoinositide 3-kinase δ regulates membrane fission of Golgi carriers for selective cytokine secretion. J Cell Biol. 2010 Sep 20;190(6):1053-65

Uno JK, Rao KN, Matsuoka K, Sheikh SZ, Kobayashi T, Li F, Steinbach EC, Sepulveda AR, Vanhaesebroeck B, Sartor RB, Plevy SE. Altered macrophage function contributes to colitis in mice defective in the phosphoinositide-3 kinase subunit p110δ. Gastroenterology. 2010 Nov;139(5):1642-53, 1653.e1-6

Lannutti BJ, Meadows SA, Herman SE, Kashishian A, Steiner B, Johnson AJ, Byrd JC, Tyner JW, Loriaux MM, Deininger M, Druker BJ, Puri KD, Ulrich RG, Giese NA. CAL-101, a p110delta selective phosphatidylinositol-3-kinase inhibitor for the treatment of B-cell malignancies, inhibits PI3K signaling and cellular viability. Blood. 2011 Jan 13;117(2):591-4


Burns E, Vanhaesebroeck B. PIK3CD (phosphoinositide-3-kinase, catalytic, delta polypeptide). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):731-734.

Gene Section Review




S100A8 (S100 calcium binding protein A8) Claus Kerkhoff, Saeid Ghavami

Dept VAC / IMCI, Helmholtz Centre for Infection Research, Inhoffenstr 7, D-38124 Braunschweig, Germany (CK), Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada (SG)


Online updated version : http://AtlasGeneticsOncology.org/Genes/S100A8ID46446ch1q21.html DOI: 10.4267/2042/46027


Identity Other names: 60B8AG; CAGA; CFAG; CGLA; CP-10; L1Ag; MA387; MIF; MRP8; NIF; P8

HGNC (Hugo): S100A8

Location: 1q21.3

Local order: Distal to LOC645900, proximal to S100A7A (S100 calcium binding protein A7A).

DNA/RNA Note S100A8 belongs to the S100/calgranulin family of small non-ubiquitous cytoplasmic Ca2+-binding proteins of EF-hand type. The proteins were referred to "S100" because of their solubility in saturated ammonium sulphate solution. Sixteen of 21 members are localised in a cluster on human chromosome 1q21. The clustered organization of these S100 genes is conserved during evolution (Ridinger et al., 1998). A comparison between man and mouse has shown that during evolution, the colinearity of the S100 gene cluster has been destroyed by some inversions. However, the colocalization of the myeloid expressed S100 genes such as S100A8, S100A9, and S100A12 is conserved. It has been speculated, that the structural integrity of that part of the locus is necessary for the coordinated expression of these genes (Nacken et al., 2001). Remarkably, the S100 gene cluster is located in close proximity to a region which has been frequently rearranged in human cancer (Carlsson et al., 2005) and to the epidermal differentiation complex (EDC) (Mischke et al., 1996). EDC is a cluster of genes on chromosome 1q21 encoding proteins that fulfil important functions in terminal differentiation in the

human epidermis, including filaggrin, loricrin and others. In addition, linkage analyses have identified a psoriasis susceptibility region, the PSORS4 locus, that is close to the S100 gene cluster (Hardas et al., 1996; Semprini et al., 2002). These data are important indications for the involvement of S100 genes in inflammatory as well as neoplastic disorders. It has been speculated that the rearrangements result in a deregulated expression of S100 genes associated with neoplasia.

Description The S100 gene structure has been structurally conserved during evolution. Similar to most S100 genes S100A8 consists of three exons that are separated by two introns.

Transcription In the S100A8 gene, exon 1 encodes the untranslated region. The protein is encoded by sequences in exon 2 and exon3, encoding a N-terminal and a C-terminal EF-hand motif, respectively. The sequence of human S100A8 cDNA has an open reading frame of 279 nucleotides predicting a protein of 93 amino acids. S100A8 expression appears to be restricted to a specific stage of myeloid differentiation. The protein is present in circulating neutrophils and monocytes, but not in resting tissue macrophages. In peripheral blood monocytes it is down regulated during maturation to macrophages. Despite a number of distinct regulatory regions are located upstream of the transcription initiation site, the corresponding nuclear factors as well as the underlying molecular mechanisms still remain unclear. Transcription factors such as PU.1 (Henkel et al., 2002), C/EBP-alpha and C/EBP-beta (Kuruto-Niwa et al., 1998) have been shown to drive S100 gene expression within the myeloid lineage.

S100A8 (S100 calcium binding protein A8) Kerkhoff C, Ghavami S


Beside its expression in myeloid cells S100A8 is expressed in epithelia under specific conditions. Its expression is transiently induced in keratinocytes after epidermal injury and UVB irradiation, and the protein is expressed at extremely high levels in psoriatic keratinocytes. Furthermore, its expression is induced by pro-inflammatory cytokines such as TNFalpha and IL1beta. Recently, a complex of Poly (ADP-ribose) polymerase (PARP-1) and Ku70/Ku80 has been demonstrated to drive the stress response-specific S100 gene expression (Grote et al., 2006). The stress response-induced expression of the S100 proteins points to an important role in skin pathology.

Pseudogene Not known.

Protein Description The sequence of human S100A8 cDNA has an open reading frame of 279 nucleotides predicting a protein of 93 amino acids and a calculated Mr of 10835 Da. The theoretical isoelectric point is 6.6. S100A8 is composed of two helix-loop-helix EF-hand motifs. The C-terminal EF-hand contains a canonical Ca2+-binding loop of 12 amino acids. Conversely, the N-terminal EF-hand contains a Ca2+-binding loop of 14 residues that binds Ca2+ mostly through main-chain carbonyl groups that which is specific to S100 proteins. Consequently, S100 proteins have a weaker Ca2+ affinity than typical Ca2+ sensors such as calmodulin (Donato, 2003). In vivo and in vitro experiments have shown that S100 proteins form homo-, hetero- and oligomeric assemblies (Hunter and Chazin, 1998; Osterloh et al., 1998; Pröpper et al., 1999; Moroz et al., 2003). Together with their specific cell- and tissue-expression patterns, the structural variations, and the different metal ion binding properties (Ca2+, Zn2+ and Cu2+) the S100 protein complexes might be functionally diversified. S100A8 preferentially interacts with S100A9. It is worthwhile mentioning that the murine analogs display a stronger tendency to form homodimeric protein complexes. In view of the formation of different tertiary structures with putative distinct functions it is tempting to speculate that S100A8 and/or A9 have different functions in mouse and man.

Expression S100A8 is mainly expressed in cells of the myeloid lineage, however, its gene expression is induced in

epithelial cells in response to stress, in specific conditions such as wound healing, UV exposition, abundant in psoriasis keratinocytes, differentially expressed in several cancers.

Localisation Mostly cytoplasmic, but also at membranes and cytoskeleton. In resting phagocytes the S100A8/A9 protein complex is mainly located in the cytosol. Upon cellular activation the protein complex is either translocated to cytoskeleton and plasma membrane or released into the extracellular environment. The translocation pathways occur upon the elevation of the intracellular calcium level (Roth et al., 1993). At a later time point, the S100A8/A9 heterodimers can be detected on the surface of monocytes (Bhardwaj et al., 1992). The mechanism by which the S100A8/A9 heterodimer penetrates the plasma membrane remains unclear since the S100 proteins lack a transmembrane signaling region. The secretion pathway relies on the activation of protein kinase C. This pathway differs from the classical as well as the alternative secretion pathways of cytokines (Moqbel and Coughlin, 2006). It has been demonstrated that this novel secretion pathway is energy-consuming and depends on an intact microtubule network (Murao et al., 1990; Rammes et al., 1997). Recent investigations give evidence that interaction of S100A8/A9 with annexin-6 is involved in surface expression and release of S100A8/A9 (Bode et al., 2008). Annexins are another class of Ca2+-regulated proteins. They are characterized by the unique architecture of their Ca2+-binding sites, which enables them to peripherally dock onto negatively charged membrane surfaces in their Ca2+-bound conformation. This property links annexins to many membrane-related events such as certain exocytic and endocytic transport steps. This is an interesting finding since S100A8 and S100A9 are expressed in cancerous cells of secretory tissues as breast and prostate. Cells originating from such glandular tissues are rich in membrane structures, suggesting that membrane-associated molecular targets for the S100A8/A9 proteins could be potentially found in these cells. Recent investigations also demonstrated the association of S100A8/A9 with cholesterol-enriched membrane microdomains (lipid rafts) (Nacken et al., 2004). This observation is in agreement with the enhancing effect of S100A8/A9 on NADPH oxidase since the formation of the oxidase complex takes place at lipid rafts.



Gene: Box = exon (light blue = 5'UTR, yellow = CDS, red = 3'UTR); Line = intron. Protein: Upper boxes, alternating colours: exons (coding part only). Lower boxes: protein domains. Green box = not structure; blue box = helix; violett box = calcium-binding domain. Function Intra- as well as extracellular roles have been proposed for the S100 proteins. Intracellular activities of S100A8/A9 In the intracellular milieu, S100 proteins are considered as calcium sensors changing their conformation in response to calcium influx and then mediating calcium signals by binding to other intracellular proteins. In a mouse knock-out model chemokine-induced down regulation of the cytosolic Ca2+-level was detected (Nacken et al., 2005). After calcium binding, the S100A8/A9 protein complex binds specifically polyunsaturated fatty acids. S100A8/A9 represents the exclusive arachidonic acid-binding capacity in the neutrophil cytosol (Kerkhoff et al., 1999), and participates in NADPH oxidase activation by transferring arachidonic acid to membrane-bound gp91phox during interactions with two cytosolic oxidase activation factors, p67phox and Rac-2. The functional relevance of S100A8/A9 in the phagocyte NADPH oxidase activation was demonstrated by the impairment of NADPH oxidase activity in neutrophil-like NB4 cells, after specifically blocking S100A9 expression, and employing bone marrow-derived PMNs from S100A9-/- mice (Kerkhoff et al., 2005). In accordance to their role in myeloid cells, S100A8/A9 enhances epithelial NADPH oxidases (Benedyk et al., 2007). As a consequence of enhanced ROS levels, NF-kB activation and subsequently TNF-alpha and IL-8 mRNA levels are increased in S100A8/A9-HaCaT keratinocytes, consistent with the view that NF-kB is a redox-sensitive transcription factor. Further consequences of S100A8/A9-mediated NF-kB activation are reduced cell growth, increased expression of differentiation markers, and enhanced

PARP cleavage as an indicator of increased cell death (Voss et al., 2011). In view of the stress response-induced expression of the two S100 proteins in keratinocytes these findings have great implications for tissue remodeling and repair. For example, keratinocytes acquire an activated state after cutaneous wounding in which proliferation is favored over differentiation in order to replenish the lost material and rapidly close the site of injury. Thus, it is likely to hypothesize that S100A8/A9-mediated growth reduction is required for the upcoming cell fate decision of damaged cells, i.e. for a survival phase to be followed by differentiation, proliferation, or apoptosis. These data have also an impact on tumorigenesis since S100 gene expression is associated with neoplastic disorders. In migrating monocytes the S100A8/A9 complex has been found to be associated with cytoskeletal tubulin and to modulate transendothelial migration (Vogl et al., 2004). Investigations using two different mouse knock-out models demonstrated no obvious phenotype (Manitz et al., 2003; Hobbs et al., 2003). However, reduced migration of S100A9-deficient neutrophils and decreased surface expression of CD11b, which belongs to the integrin family, were observed upon in vitro stimulation. Extracellular activities of S100A8/A9 The S100/calgranulins display antimicrobial activity by depriving bacterial pathogens of essential trace metals such as Zn2+ and Mn2+ (Steinbakk et al., 1990; Murthy et al., 1993; Clohessy and Golden, 1995; Sohnle et al., 2000). In the context of inflammation, it has been proposed that S100A8/A9 is massively released when neutrophils die to provide a growth-inhibitory type of host defense that is adjunctive to the usual microbicidal functions by binding metals other than Ca2+ (Corbin et al., 2008).



In addition, S100/calgranulins serve as leukocyte chemoattractants (Lackmann et al., 1992; Lackmann et al., 1993; Kocher et al., 1996; Lim et al., 2008). Murine S100A8 has potent chemotactic activity for neutrophils and monocytes in vitro and in vivo (Lackmann et al., 1992). In contrast, human S100A8 displays only weak leukocyte chemotactic activity in vitro and in vivo (Lackmann et al., 1993). Detailed analysis revealed that the hinge region contributes to the chemotactic activity of murine, but not human S100A8. These data questioned whether the proteins are orthologs since there is a high degree of homology between murine and human S100A8 but a functional divergence. Intriguingly, human S100A12 is chemotactic and the hinge region of human S100A12 has been implicated herein (Yang et al., 2001). Thus, the functional and sequence divergence suggested complex evolution of the S100 family in mammals. The putative pro-inflammatory functions of S100A8 and S100A9 have recently been investigated in two different mouse knock-out models. S100A9 deficiency did not result in an obvious phenotype (Manitz et al., 2003; Hobbs et al., 2003). However, reduced migration of S100A9-deficient neutrophils and decreased surface expression of CD11b, which belongs to the integrin family, were observed upon in vitro stimulation. In addition, chemokine-induced down regulation of the cytosolic Ca2+-level was detected. Obviously, these in vitro effects are compensated by alternative pathways in vivo. Remarkably, cancer cells utilize S100A8 and S100A9 as guidance for the adhesion and invasion of disseminating malignant cells (Hiratsuka et al., 2006). In the context of malignancy it was reported that S100A8/A9 attracts Mac-1+ myeloid cells to the lung tissue. Recruited Mac-1+ myeloid cells in lung in turn produce S100A8/A9 in response to primary malignant cells in a so called "premetastatic phase". This phase shows the general characteristics of an inflammation state which facilitates the micro-environmental changes required for the migration and implantation of primary tumor cells to lung tissue. After preparation of the target tissue for accepting the malignant cells, tumor cells mimic Mac-1+ myeloid cells in response to S100A8/A9 chemotactic signaling and migrate to lung. So, it seems that tumor cells and Mac-1+ myeloid cells utilize a common pathway for migration to lung which involves the activation of mitogen-activated protein kinase pathway (Hiratsuka et al., 2006). These findings suggest S100A8/A9 as an attractive target for the development of strategies counteracting tumor metastasizing to certain organs. S100A8 and S100A9 have been identified as important endogenous damage-associated molecular pattern (DAMP) proteins. Although receptors for S100A8/A9 are still largely uncharacterized, more recent findings support the notion that they function as potent ligands of pattern-recognition receptors, such as the toll-like receptor 4 (TLR4) (Vogl et al., 2007) and the receptor

for advanced glycation end products (RAGE) (Srikrishna and Freeze, 2009). The S100/calgranulins display cytokine-like functions, including activation of the receptor for advanced glycation endproducts (RAGE) (Hofmann et al., 1999; Herold et al., 2007). RAGE is a member of the immunoglobulin superfamily and present on numerous cell types. It has been shown to play crucial roles in a variety of pathophysiological situations, such as wound healing, atherosclerotic lesion development, tumor growth and metastasis, systemic amyloidosis, and Alzheimer disease (Bierhaus et al., 2005). RAGE/S100 interaction has been considered a very attractive model to explain how RAGE and its proinflammatory ligand contribute to the pathophysiology of several inflammatory diseases. Beside the above mentioned receptors a number of other cell surface binding sites specific for S100A8/A9 have been reported, such as novel carboxylated glycans (Srikrishna et al., 2001), heparan sulfate glycosaminoglycans (Robinson et al., 2002), beta2-integrin (Newton and Hogg, 1998), and the fatty acid transporter FAT/CD36 (Kerkhoff et al., 2001). Therefore, the cell surface receptor of S100A8/A9 is still in debate. Interestingly, the growth-stimulatory activity of S100A8/A9 has been demonstrated to be mediated by binding to the receptor of advanced glycation end products (RAGE) (Ghavami et al., 2008b; Turovskaya et al., 2008; Gebhardt et al., 2008). It is likely to speculate that the selective up-regulation of S100 proteins may be of importance for survival and proliferation of metastasizing cancer cells. S100A8/A9 complexes that are secreted from phorbolester-stimulated neutrophil-like HL-60 cells have been shown to carry the eicosanoid precursor arachidonic acid (Kerkhoff et al., 1999). The S100A8/A9-arachidonic acid complex is recognized by the fatty acid transporter FAT/CD36, and the fatty acid is rapidly taken up (Kerkhoff et al., 2001). Endothelial cells as well as neutrophils themselves utilize both endogenous and exogenous arachidonic acid for transcellular production of eicosanoids (Sala et al., 1999). Therefore, the secreted S100A8/A9-AA complex may serve as a transport protein to move AA to its target cells. This may represent a mechanism by which AA-derived eicosanoids are synthesized in a cooperative manner between different cell species due to environmental cues. S100A8/A9 displays apoptosis-inducing activity against various tumor cells (Yui et al., 1995; Yui et al., 2002; Ghavami et al., 2004; Ghavami et al., 2008a; Kerkhoff and Ghavami, 2009; Ghavami et al., 2009; Ghavami et al., 2010). It was speculated that this activity was due to the ability to bind divalent metal ions including Zn2+, Mn2+ and Cu2+ at sites that are distinct from Ca2+-binding sites. However, a number of recent reports now indicate that S100A8/A9 exerts its activity by both chelation of trace



metal ions such as Zn2+ and cell surface receptor mediated pathways. Although a number of receptors have been shown to bind S100A8/A9, the nature of the receptor involved in S100A8/A9-induced cell death remains to be elucidated. Experiments with certain cell lines either deficient for or over expressing components of the death signaling machinery as well as RAGE gene silencing and blocking RAGE-specific antibody approaches excluded both RAGE and the classical death receptor to be involved in S100A8/A9-induced cell death, even though S100A8/A9 can specifically bind to cancer cells and RAGE mediates the growth-promoting activity obvious at low micromolar concentrations of S100A8/A9. Clearly, investigations to identify the receptor involved in S100A8/A9-induced cell death are critical.

Homology Overall, the S100 proteins share significant sequential homology in the EF-hand motifs, but are least conserved in the hinge region. This region is proposed to provide for specific interaction with target proteins (Groves et al., 1998; Zimmer et al., 2003; Santamaria-Kisiel et al., 2006; Fernandez-Fernandez et al., 2008; van Dieck et al., 2009). The availability of high-resolution S100-target structures has highlighted important structural features that contribute to S100 protein functional specificity (Bhattacharya et al., 2003). The functional diversification of S100 proteins is achieved by their specific cell- and tissue-expression patterns, structural variations, different metal ion binding properties (Ca2+, Zn2+ and Cu2+) as well as their ability to form homo-, hetero- and oligomeric assemblies (Hunter and Chazin, 1998; Osterloh et al., 1998; Pröpper et al., 1999; Tarabykina et al., 2001; Moroz et al., 2003; Fritz et al., 2010). Although the function of S100 proteins in cancer cells in most cases is still unknown, the specific expression patterns of these proteins are a valuable diagnostic tool.

Implicated in General note Note Comparative and functional genomics have revealed that a number of S100 proteins are found to be differentially expressed in cancer cells. Several of these have been associated with tumor development, cancer invasion or metastasis in recent studies (for review see Salama et al., 2008). S100A8 and S100A9 are abundant in cells of the myeloid lineage, are released from activated phagocytes and display intra- and extracellular functions. Their expression is ubiquitously observed in the squamous epithelia under normal, inflammatory and cancerous conditions. Immunohistochemical investigations have shown that the S100 proteins are

over expressed in skin cancers, pulmonary adenocarcinoma, pancreatic adenocarcinoma, bladder cancers, ductal carcinoma of the breast, and prostate adenocarcinoma. In contrast, S100A8 and S100A9 are down-regulated in esophageal squamous cell carcinomas. Furthermore, plasma levels of S100A8/A9 are elevated in patients suffering from various cancers. Insofar, S100A8 and S100A9 might represent novel diagnostic markers for some carcinomas. S100A8 and S100A9 have been suggested to have potential roles in carcinogenesis and tumor progression. However, the biological role of S100A8/A9 remains to be elucidated. It is conceivable that S100A8 and S100A9 modulate signal pathways to directly promote invasion, migration and metastasis, probably via activation of NF-kB, Akt or MAP kinases. In the last decade the concept of the functional relationship between inflammation and cancer has been developed that is based on numerous findings, ranging from epidemiological studies to molecular analyses of mouse models (Coussens and Werb, 2002). In this concept, the generation of an inflammatory microenvironment supports tumorigenesis by promoting cancer cell survival, proliferation, migration, and invasion. Although it is clear that inflammation alone does not cause cancer, it is evident that an environment that is rich in inflammatory cells, growth factors, activated stroma, and DNA-damage-promoting agents certainly potentiates and/or promotes neoplastic risk. In addition, many cancers arise from sites of infection, chronic irritation and inflammation. Recent data have expanded our knowledge demonstrating that specific soluble factors released from primary tumors induce the S100A8 and S100A9 gene expression in the target tissue. After secretion S100A8 and S100A9 might display chemokine- and cytokine-like properties that promote invasion, migration and metastasis. These data indicate that tumor cells are able to reprogram some of the signaling molecules of the innate immune system. These insights are fostering new anti-inflammatory therapeutic approaches to cancer development.

Skin cancer Note The expression of S100A8 and S100A9 in epithelial cells was first detected in the squamous epithelia (Gabrielsen et al., 1986). Normal S100A8 and S100A9 are expressed at minimal levels in the epidermis. However, their expression is induced in inflammatory and cancerous conditions, and pro-inflammatory cytokines such as TNF-alpha and IL1 beta are involved herein. Gene expression analysis in a mouse model of chemically induced skin carcinogenesis identified a large set of novel tumor-associated genes including S100A8 (Hummerich et al., 2006). The data was confirmed by in situ hybridization and immunofluorescence analysis on mouse tumor sections,



in mouse keratinocyte cell lines that form tumors in vivo, and in human skin tumor specimens. However, conflicting results have been published concerning S100 expression in skin cancer. For instance, esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. DNA microarray data analysis revealed that S100A8 and S100A9 were significantly down regulated in human ESCC versus the normal counterparts (Zhi et al., 2003). Interestingly, among the 42 genes either up regulated or down regulated in tumors, as compared to normal esophageal squamous epithelia, nine of the altered expression genes were related to arachidonic acid (AA) metabolism, suggesting that AA metabolism pathway and its altered expression may contribute to esophageal squamous cell carcinogenesis. Similar data were obtained by Ji et al. (2004). They investigated the differential expression of the S100 gene family at the RNA level in human ESCC. Eleven out of 16 S100 genes were significantly down regulated in ESCC versus the normal counterparts. Only the S100A7 gene was found to be markedly up regulated. Another study demonstrated that poorly differentiated ESCC displayed a stronger decrease in S100A8 and S100A9 expression than well and moderately differentiated tumors, with a correlation between protein level and histopathological grading (Kong et al., 2004). These findings suggest that decreased expression of S100A8 and S100A9 might play an important role in the ESCC pathogenesis, being particularly associated with poor differentiation of tumor cells.

Lung adenocarcinomas Note S100A9 over expression has been detected in various carcinomas of glandular cell origin, and its expression has been associated with poor tumor differentiation. Similarly, S100A9 immunopositivity was also detected in pulmonary adenocarcinoma cell lines and resected pulmonary adenocarcinoma (Arai et al., 2001). Examination of the relation of S100A9 expression to tumor differentiation showed that the expression rate in pulmonary adenocarcinoma showed higher correlation in poorly differentiated carcinomas. Another study confirmed these data (Su et al., 2010). Immunohistochemical staining of both S100 proteins showed a significant up-regulation in lung cancer tissue, and quantitative PCR revealed significantly higher levels of S100A8 and S100A9 mRNA transcripts in lung cancer tissues. Moreover, this study correlates S100A9 expression with inflammation and other clinical features (Su et al., 2010). Primary tumors influence the environment in the lungs before metastasis. They release specific soluble factors that prepare the premetastatic niche for the engraftment of tumor cells. In several studies it has been shown that tumor cells induce both expression and secretion of S100A8 and S100A9 in the target organ that display a promoting role in cancer cell survival, proliferation,

migration, and invasion (Hiratsuka et al., 2002; Hiratsuka et al., 2006; Hiratsuka et al., 2008; Saha et al., 2010). Microarray analysis of lungs from tumor-bearing and non-bearing mice revealed the strong up-regulation of a number of genes including S100A8 and S100A9 (Hiratsuka et al., 2002). Their expression in Mac 1+-myeloid cells and endothelial cells was induced by factors such as vascular endothelial growth factor A (VEGF-A), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta), both in vitro and in vivo (Hiratsuka et al., 2006). Remarkably, anti-S100A8 neutralizing antibody treatment blocked metastasis. S100A8 and S100A9 were shown to induce the expression of serum amyloid A (SAA) that attracted Mac 1+-myeloid cells in the premetastatic lung (Hiratsuka et al., 2008). These studies demonstrated that lung cancer cells utilize S100A8 and S100A9 as guidance for the adhesion and invasion of disseminating malignant cells.

Pancreatic adenocarcinoma Note Patients with ductal adenocarcinoma of the pancreas have a dismal prognosis. Thus, there is an urgent need for early detection markers and the development of immunotherapeutical approaches concentrating on the induction and enhancement of immune responses against tumors. Proteomic analyses of pancreatic adenocarcinoma, normal adjacent tissues, pancreatitis, and normal pancreatic tissues revealed a number of differentially expressed genes (Shen et al., 2004). S100A8 was found to be specifically over expressed in tumors compared with normal and pancreatitis tissues. These data are in accordance with another study (Sheikh et al., 2007). Strong expression of S100A8 and S100A9 was found in tumor-associated stroma but not in benign or malignant epithelia. Further analyses identified stromal CD14+ CD68- monocytes/macrophages as source for S100 expression. Interestingly, the number of S100A8-positive cells in the tumor microenvironment negatively correlated with the expression of the tumor suppressor protein, Smad4. The number of S100A9-positive cells was not altered in Smad4-negative or Smad4-positive tumors. A similar correlation was found in colorectal cancer tumors (Ang et al., 2010). The number of stromal S100A8- and S100A9-positive cells was associated with the presence or absence of Smad4. Smad4-negative tumors showed enhanced numbers of S100A8/A9 stroma cells, and the corresponding patients had a poor survival prognosis. Investigation of the underlying molecular mechanisms revealed that both migration and proliferation was enhanced in response to exogenous S100A8 and S100A9, irrespective of Smad4-presence. However, depletion of Smad4 resulted in loss of responsiveness to exogenous S100A8, but not S100A9. Vice versa, Smad4 expression in Smad4-negative cells enhanced the



responsive-ness to S100A8 and S100A9. Further analyses give evidence that similar to TGF-beta, S100A8 and S100A9 induce the phosphorylation of both Smad2 and Smad3 that was blocked by a RAGE-specific antibody. These data point to a functional relationship between inflammation and tumorigenesis.

Bladder cancers Note Gene expression profiles revealed that thirteen members of the S100 gene family were differentially expressed in human bladder cancers. S100A8 and S100A9 were found to be over expressed (Yao et al., 2007). Another study investigated S100A9 expression and DNA methylation in urothelial cancer cell lines and cancer tissue (Dokun et al., 2008). Expression of S100A9 was found to be generally elevated in the tumor tissues but S100A9 was weakly expressed in most cancer cell lines. The S100A9 promoter contains 6 CpG sites, and its methylation state was unrelated to the variable expression. It has been hypothesized that over expression of genes is the consequence of DNA hypomethylation, however, DNA methylation and gene expression are less strictly related for those genes having promoters within CpG-islands. Alternatively, the increased S100A9 gene expression may be related to that of other immune-related genes in the carcinoma cell cultures. This is sustained by the facts that S100A9 is secreted by epithelial and other cell types to modulate inflammatory reactions as well as to promote cancer proliferation and metastasis. Two recent studies propose S100A8 and S100A9 gene expression as prognostic value for bladder cancer (Minami et al., 2010; Ha et al., 2010). By proteomic analysis of pre- and postoperative sera from bladder cancer patients S100A8 and S100A9 were identified as tumor-associated proteins (Minami et al., 2010). Interestingly, S100A8 expression was associated with bladder wall muscle invasion of the tumor and cancer-specific survival while S100A9 expression was associated with the tumor grade. In addition, the expression of both proteins S100A8/A9 was correlated with recurrence-free survival. In another study it was evaluated whether S100A8 is a prognostic value for non-muscle-invasive bladder cancer (NMIBC) (Ha et al., 2010). S100A8 expression was evaluated in a total of 103 primary NMIBC samples by quantitative PCR. The mRNA expression levels of S100A8 were significantly related to the progression of NMIBC, suggesting that S100A8 might be a useful prognostic marker for disease progression of NMIBC.

Breast cancers Note S100A8 and S100A9 are expressed in breast cancers (Cross et al., 2005), especially in invasive breast carcinoma (Arai et al., 2004). By immunohistochemical

analyses a strong S100A9 immunoreactivity has been demonstrated in invasive as well as non-invasive ductal carcinoma. No immunopositive reaction was observed in invasive lobular carcinomas, and no significant differences were detected in the number of myelomonocytic cells expressing S100A9. These data give evidence that S100A9 in glandular epithelial cells is newly expressed under cancerous conditions and is over-expressed in poorly differentiated adenocarcinoma (Arai et al., 2004). Further analyses target on the relationship between S100A8/A9 expression and pathological parameters that reflect the aggressiveness of carcinoma. The immunopositivity for S100A8/A9 correlated with poor tumor differentiation, mitotic activity, HER2/neu over expression, poor pT categories, node metastasis, and poor pStage, but not with vessel invasion. These data may indicate that S100A8 and S100A9 over expression should be considered marker of poor prognosis in invasive breast ductal carcinoma (Arai et al., 2008). By analyses of ductal carcinoma in situ and invasive ductal carcinoma of the breast S100A9 has been demonstrated to be most abundantly expressed in the invasive tumor (Seth et al., 2003). Therefore, the expression of S100A8 and S100A9 has been correlated with the degree of noninvasive / invasive behavior. There are conflicting data concerning this correlation. For instance, non-invasive MCF-7 breast cancer cells do not express S100A9, and its gene expression is induced by cytokine oncostatin M through the STAT3 signaling cascade (Li et al., 2004). However, non-invasive MDA-MB-468 cells are abundant for both S100 proteins (Bode et al., 2008) and invasive breast cancer cells MDA-MB-231 show low transcript level of S100A9 (Nagaraja et al., 2006).

Thyroid carcinoma Note Similar to other carcinomas of glandular cell origin, expression of S100A8 and S100A9 is significantly linked to dedifferentiation of thyroid carcinoma (Ito et al., 2005; Ito et al., 2009). S100A8 and S100A9 immunreactivity was found in all undifferentiated carcinomas examined, while papillary carcinoma, follicular carcinoma, follicular adenoma and medullary carcinoma and normal follicules were negative for both proteins. Further analyses revealed that S100A9 is a useful marker for discriminating intrathyroid epithelial tumor from squamous cell carcinoma or undifferentiated carcinoma with squamoid component (Ito et al., 2006).

Prostate cancer Note Increased levels of S100A8, S100A9, and RAGE have been reported in prostatic intra epithelial neoplasia and preferentially in high-grade adenocarcinomas, whereas benign tissue was negative or showed weak expression of the proteins. The three proteins showed a strong



overlap in the expression pattern. S100A9 serum level was significantly elevated in cancer patients compared with benign prostatic hyperplasia patients or healthy individuals. Therefore, S100A8 and S100A9 might represent novel diagnostic markers for prostate cancer and benign prostate hyperplasia (Hermani et al., 2005). In further analyses it has been demonstrated that S100A8 and S100A9 are secreted by prostate cancer cells, and extracellular S100A8/A9 stimulates migration of benign prostatic cells in vitro by activation of NF-kB and increased phosphorylation of p38 and p44/p42 MAP kinases. Immunofluorescence analyses give evidence for a RAGE-mediated response (Hermani et al., 2006). The significance of being diagnostic markers for prostate cancer has been questioned by Ludwig et al. (2007). Their re-evaluation study has shown that S100A8/A9 did not improve the differentiation between patients with and without prostate cancer. The data give no evidence for the replacement of the established marker PSA by S100A8/A9.

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Hofmann MA, Drury S, Fu C, Qu W, Taguchi A, Lu Y, Avila C, Kambham N, Bierhaus A, Nawroth P, Neurath MF, Slattery T, Beach D, McClary J, Nagashima M, Morser J, Stern D, Schmidt AM. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell. 1999 Jun 25;97(7):889-901

Kerkhoff C, Klempt M, Kaever V, Sorg C. The two calcium-binding proteins, S100A8 and S100A9, are involved in the metabolism of arachidonic acid in human neutrophils. J Biol Chem. 1999 Nov 12;274(46):32672-9

Klempt M, Melkonyan H, Hofmann HA, Sorg C. Identification of epithelial and myeloid-specific DNA elements regulating MRP14 gene transcription. J Cell Biochem. 1999 Apr 1;73(1):49-55

Pröpper C, Huang X, Roth J, Sorg C, Nacken W. Analysis of the MRP8-MRP14 protein-protein interaction by the two-hybrid system suggests a prominent role of the C-terminal domain of S100 proteins in dimer formation. J Biol Chem. 1999 Jan 1;274(1):183-8

Sala A, Zarini S, Folco G, Murphy RC, Henson PM. Differential metabolism of exogenous and endogenous arachidonic acid in human neutrophils. J Biol Chem. 1999 Oct 1;274(40):28264-9

Sohnle PG, Hunter MJ, Hahn B, Chazin WJ. Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14). J Infect Dis. 2000 Oct;182(4):1272-5

Arai K, Teratani T, Nozawa R, Yamada T. Immunohistochemical investigation of S100A9 expression in pulmonary adenocarcinoma: S100A9 expression is associated with tumor differentiation. Oncol Rep. 2001 May-Jun;8(3):591-6

Hiratsuka S, Maru Y, Okada A, Seiki M, Noda T, Shibuya M. Involvement of Flt-1 tyrosine kinase (vascular endothelial growth factor receptor-1) in pathological angiogenesis. Cancer Res. 2001 Feb 1;61(3):1207-13

Kerkhoff C, Sorg C, Tandon NN, Nacken W. Interaction of S100A8/S100A9-arachidonic acid complexes with the scavenger receptor CD36 may facilitate fatty acid uptake by endothelial cells. Biochemistry. 2001 Jan 9;40(1):241-8

Nacken W, Lekstrom-Himes JA, Sorg C, Manitz MP. Molecular analysis of the mouse S100A9 gene and evidence that the myeloid specific transcription factor C/EBPepsilon is not required for the regulation of the S100A9/A8 gene expression in neutrophils. J Cell Biochem. 2001;80(4):606-16

Srikrishna G, Panneerselvam K, Westphal V, Abraham V, Varki A, Freeze HH. Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells. J Immunol. 2001 Apr 1;166(7):4678-88

Tarabykina S, Scott DJ, Herzyk P, Hill TJ, Tame JR, Kriajevska M, Lafitte D, Derrick PJ, Dodson GG, Maitland NJ, Lukanidin EM, Bronstein IB. The dimerization interface of the metastasis-associated protein S100A4 (Mts1): in vivo and in vitro studies. J Biol Chem. 2001 Jun 29;276(26):24212-22

Yang Z, Tao T, Raftery MJ, Youssef P, Di Girolamo N, Geczy CL. Proinflammatory properties of the human S100 protein S100A12. J Leukoc Biol. 2001 Jun;69(6):986-94

Coussens LM, Werb Z. Inflammation and cancer. Nature. 2002 Dec 19-26;420(6917):860-7

Henkel GW, McKercher SR, Maki RA. Identification of three genes up-regulated in PU.1 rescued monocytic precursor cells. Int Immunol. 2002 Jul;14(7):723-32

Kerkhoff C, Hofmann HA, Vormoor J, Melkonyan H, Roth J, Sorg C, Klempt M. Binding of two nuclear complexes to a novel regulatory element within the human S100A9 promoter drives the S100A9 gene expression. J Biol Chem. 2002 Nov 1;277(44):41879-87

Robinson MJ, Tessier P, Poulsom R, Hogg N. The S100 family heterodimer, MRP-8/14, binds with high affinity to heparin and heparan sulfate glycosaminoglycans on endothelial cells. J Biol Chem. 2002 Feb 1;277(5):3658-65

Semprini S, Capon F, Tacconelli A, Giardina E, Orecchia A, Mingarelli R, Gobello T, Zambruno G, Botta A, Fabrizi G, Novelli G. Evidence for differential S100 gene over-expression in psoriatic patients from genetically heterogeneous pedigrees. Hum Genet. 2002 Oct;111(4-5):310-3

Yui S, Nakatani Y, Hunter MJ, Chazin WJ, Yamazaki M. Implication of extracellular zinc exclusion by recombinant human calprotectin (MRP8 and MRP14) from target cells in its apoptosis-inducing activity. Mediators Inflamm. 2002 Jun;11(3):165-72

Bhattacharya S, Large E, Heizmann CW, Hemmings B, Chazin WJ. Structure of the Ca2+/S100B/NDR kinase peptide complex: insights into S100 target specificity and activation of the kinase. Biochemistry. 2003 Dec 16;42(49):14416-26

Donato R. Intracellular and extracellular roles of S100 proteins. Microsc Res Tech. 2003 Apr 15;60(6):540-51

Hobbs JA, May R, Tanousis K, McNeill E, Mathies M, Gebhardt C, Henderson R, Robinson MJ, Hogg N. Myeloid cell function in MRP-14 (S100A9) null mice. Mol Cell Biol. 2003 Apr;23(7):2564-76

Manitz MP, Horst B, Seeliger S, Strey A, Skryabin BV, Gunzer M, Frings W, Schönlau F, Roth J, Sorg C, Nacken W. Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro. Mol Cell Biol. 2003 Feb;23(3):1034-43

Moroz OV, Dodson GG, Wilson KS, Lukanidin E, Bronstein IB. Multiple structural states of S100A12: A key to its functional diversity. Microsc Res Tech. 2003 Apr 15;60(6):581-92

Seth A, Kitching R, Landberg G, Xu J, Zubovits J, Burger AM. Gene expression profiling of ductal carcinomas in situ and invasive breast tumors. Anticancer Res. 2003 May-Jun;23(3A):2043-51

Zhi H, Zhang J, Hu G, Lu J, Wang X, Zhou C, Wu M, Liu Z. The deregulation of arachidonic acid metabolism-related genes in human esophageal squamous cell carcinoma. Int J Cancer. 2003 Sep 1;106(3):327-33

Zimmer DB, Wright Sadosky P, Weber DJ. Molecular mechanisms of S100-target protein interactions. Microsc Res Tech. 2003 Apr 15;60(6):552-9

Arai K, Teratani T, Kuruto-Niwa R, Yamada T, Nozawa R. S100A9 expression in invasive ductal carcinoma of the breast: S100A9 expression in adenocarcinoma is closely associated with poor tumour differentiation. Eur J Cancer. 2004 May;40(8):1179-87

Ghavami S, Kerkhoff C, Los M, Hashemi M, Sorg C, Karami-Tehrani F. Mechanism of apoptosis induced by S100A8/A9 in colon cancer cell lines: the role of ROS and the effect of metal ions. J Leukoc Biol. 2004 Jul;76(1):169-75

Ji J, Zhao L, Wang X, Zhou C, Ding F, Su L, Zhang C, Mao X, Wu M, Liu Z. Differential expression of S100 gene family in



human esophageal squamous cell carcinoma. J Cancer Res Clin Oncol. 2004 Aug;130(8):480-6

Kong JP, Ding F, Zhou CN, Wang XQ, Miao XP, Wu M, Liu ZH. Loss of myeloid-related proteins 8 and myeloid-related proteins 14 expression in human esophageal squamous cell carcinoma correlates with poor differentiation. World J Gastroenterol. 2004 Apr 15;10(8):1093-7

Li C, Zhang F, Lin M, Liu J. Induction of S100A9 gene expression by cytokine oncostatin M in breast cancer cells through the STAT3 signaling cascade. Breast Cancer Res Treat. 2004 Sep;87(2):123-34

Nacken W, Sorg C, Kerkhoff C. The myeloid expressed EF-hand proteins display a diverse pattern of lipid raft association. FEBS Lett. 2004 Aug 13;572(1-3):289-93

Shen J, Person MD, Zhu J, Abbruzzese JL, Li D. Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry. Cancer Res. 2004 Dec 15;64(24):9018-26

Vogl T, Ludwig S, Goebeler M, Strey A, Thorey IS, Reichelt R, Foell D, Gerke V, Manitz MP, Nacken W, Werner S, Sorg C, Roth J. MRP8 and MRP14 control microtubule reorganization during transendothelial migration of phagocytes. Blood. 2004 Dec 15;104(13):4260-8

Bierhaus A, Humpert PM, Stern DM, Arnold B, Nawroth PP. Advanced glycation end product receptor-mediated cellular dysfunction. Ann N Y Acad Sci. 2005 Jun;1043:676-80

Boniface K, Bernard FX, Garcia M, Gurney AL, Lecron JC, Morel F. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes. J Immunol. 2005 Mar 15;174(6):3695-702

Carlsson H, Petersson S, Enerbäck C. Cluster analysis of S100 gene expression and genes correlating to psoriasin (S100A7) expression at different stages of breast cancer development. Int J Oncol. 2005 Dec;27(6):1473-81

Cross SS, Hamdy FC, Deloulme JC, Rehman I. Expression of S100 proteins in normal human tissues and common cancers using tissue microarrays: S100A6, S100A8, S100A9 and S100A11 are all overexpressed in common cancers. Histopathology. 2005 Mar;46(3):256-69

Hermani A, Hess J, De Servi B, Medunjanin S, Grobholz R, Trojan L, Angel P, Mayer D. Calcium-binding proteins S100A8 and S100A9 as novel diagnostic markers in human prostate cancer. Clin Cancer Res. 2005 Jul 15;11(14):5146-52

Ito Y, Arai K, Ryushi, Nozawa, Yoshida H, Tomoda C, Uruno T, Miya A, Kobayashi K, Matsuzuka F, Kuma K, Kakudo K, Miyauchi A. S100A9 expression is significantly linked to dedifferentiation of thyroid carcinoma. Pathol Res Pract. 2005;201(8-9):551-6

Kerkhoff C, Nacken W, Benedyk M, Dagher MC, Sopalla C, Doussiere J. The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2. FASEB J. 2005 Mar;19(3):467-9

Lominadze G, Rane MJ, Merchant M, Cai J, Ward RA, McLeish KR. Myeloid-related protein-14 is a p38 MAPK substrate in human neutrophils. J Immunol. 2005 Jun 1;174(11):7257-67

Nacken W, Mooren FC, Manitz MP, Bode G, Sorg C, Kerkhoff C. S100A9 deficiency alters adenosine-5'-triphosphate induced calcium signalling but does not generally interfere with calcium and zinc homeostasis in murine neutrophils. Int J Biochem Cell Biol. 2005 Jun;37(6):1241-53

Grote J, König S, Ackermann D, Sopalla C, Benedyk M, Los M, Kerkhoff C. Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression. BMC Mol Biol. 2006 Dec 22;7:48

Hermani A, De Servi B, Medunjanin S, Tessier PA, Mayer D. S100A8 and S100A9 activate MAP kinase and NF-kappaB signaling pathways and trigger translocation of RAGE in human prostate cancer cells. Exp Cell Res. 2006 Jan 15;312(2):184-97

Hiratsuka S, Watanabe A, Aburatani H, Maru Y. Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis. Nat Cell Biol. 2006 Dec;8(12):1369-75

Hummerich L, Müller R, Hess J, Kokocinski F, Hahn M, Fürstenberger G, Mauch C, Lichter P, Angel P. Identification of novel tumour-associated genes differentially expressed in the process of squamous cell cancer development. Oncogene. 2006 Jan 5;25(1):111-21

Ito Y, Miyauchi A, Arai K, Nozawa R, Miya A, Kobayashi K, Nakamura Y, Kakudo K. Usefulness of S100A9 for diagnosis of intrathyroid epithelial thymoma (ITET)/carcinoma showing thymus-like differentiation (CASTLE). Pathology. 2006 Dec;38(6):541-4

Moqbel R, Coughlin JJ. Differential secretion of cytokines. Sci STKE. 2006 Jun 6;2006(338):pe26

Nagaraja GM, Othman M, Fox BP, Alsaber R, Pellegrino CM, Zeng Y, Khanna R, Tamburini P, Swaroop A, Kandpal RP. Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics. Oncogene. 2006 Apr 13;25(16):2328-38

Santamaria-Kisiel L, Rintala-Dempsey AC, Shaw GS. Calcium-dependent and -independent interactions of the S100 protein family. Biochem J. 2006 Jun 1;396(2):201-14

Benedyk M, Sopalla C, Nacken W, Bode G, Melkonyan H, Banfi B, Kerkhoff C. HaCaT keratinocytes overexpressing the S100 proteins S100A8 and S100A9 show increased NADPH oxidase and NF-kappaB activities. J Invest Dermatol. 2007 Aug;127(8):2001-11

Herold K, Moser B, Chen Y, Zeng S, Yan SF, Ramasamy R, Emond J, Clynes R, Schmidt AM. Receptor for advanced glycation end products (RAGE) in a dash to the rescue: inflammatory signals gone awry in the primal response to stress. J Leukoc Biol. 2007 Aug;82(2):204-12

Ludwig S, Stephan C, Lein M, Loening SA, Jung K. S100A8, S100A9, and the S100A8/A9 complex in circulating blood are not associated with prostate cancer risk-A re-evaluation study. Prostate. 2007 Sep 1;67(12):1301-7

Sheikh AA, Vimalachandran D, Thompson CC, Jenkins RE, Nedjadi T, Shekouh A, Campbell F, Dodson A, Prime W, Crnogorac-Jurcevic T, Lemoine NR, Costello E. The expression of S100A8 in pancreatic cancer-associated monocytes is associated with the Smad4 status of pancreatic cancer cells. Proteomics. 2007 Jun;7(11):1929-40

Vogl T, Tenbrock K, Ludwig S, Leukert N, Ehrhardt C, van Zoelen MA, Nacken W, Foell D, van der Poll T, Sorg C, Roth J. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock. Nat Med. 2007 Sep;13(9):1042-9

Yao R, Lopez-Beltran A, Maclennan GT, Montironi R, Eble JN, Cheng L. Expression of S100 protein family members in the pathogenesis of bladder tumors. Anticancer Res. 2007 Sep-Oct;27(5A):3051-8



Arai K, Takano S, Teratani T, Ito Y, Yamada T, Nozawa R. S100A8 and S100A9 overexpression is associated with poor pathological parameters in invasive ductal carcinoma of the breast. Curr Cancer Drug Targets. 2008 Jun;8(4):243-52

Bode G, Lüken A, Kerkhoff C, Roth J, Ludwig S, Nacken W. Interaction between S100A8/A9 and annexin A6 is involved in the calcium-induced cell surface exposition of S100A8/A9. J Biol Chem. 2008 Nov 14;283(46):31776-84

Corbin BD, Seeley EH, Raab A, Feldmann J, Miller MR, Torres VJ, Anderson KL, Dattilo BM, Dunman PM, Gerads R, Caprioli RM, Nacken W, Chazin WJ, Skaar EP. Metal chelation and inhibition of bacterial growth in tissue abscesses. Science. 2008 Feb 15;319(5865):962-5

Dokun OY, Florl AR, Seifert HH, Wolff I, Schulz WA. Relationship of SNCG, S100A4, S100A9 and LCN2 gene expression and DNA methylation in bladder cancer. Int J Cancer. 2008 Dec 15;123(12):2798-807

Fernandez-Fernandez MR, Rutherford TJ, Fersht AR. Members of the S100 family bind p53 in two distinct ways. Protein Sci. 2008 Oct;17(10):1663-70

Gebhardt C, Riehl A, Durchdewald M, Németh J, Fürstenberger G, Müller-Decker K, Enk A, Arnold B, Bierhaus A, Nawroth PP, Hess J, Angel P. RAGE signaling sustains inflammation and promotes tumor development. J Exp Med. 2008 Feb 18;205(2):275-85

Ghavami S, Kerkhoff C, Chazin WJ, Kadkhoda K, Xiao W, Zuse A, Hashemi M, Eshraghi M, Schulze-Osthoff K, Klonisch T, Los M. S100A8/9 induces cell death via a novel, RAGE-independent pathway that involves selective release of Smac/DIABLO and Omi/HtrA2. Biochim Biophys Acta. 2008 Feb;1783(2):297-311

Ghavami S, Rashedi I, Dattilo BM, Eshraghi M, Chazin WJ, Hashemi M, Wesselborg S, Kerkhoff C, Los M. S100A8/A9 at low concentration promotes tumor cell growth via RAGE ligation and MAP kinase-dependent pathway. J Leukoc Biol. 2008 Jun;83(6):1484-92

Hiratsuka S, Watanabe A, Sakurai Y, Akashi-Takamura S, Ishibashi S, Miyake K, Shibuya M, Akira S, Aburatani H, Maru Y. The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase. Nat Cell Biol. 2008 Nov;10(11):1349-55

Lim SY, Raftery M, Cai H, Hsu K, Yan WX, Hseih HL, Watts RN, Richardson D, Thomas S, Perry M, Geczy CL. S-nitrosylated S100A8: novel anti-inflammatory properties. J Immunol. 2008 Oct 15;181(8):5627-36

Salama I, Malone PS, Mihaimeed F, Jones JL. A review of the S100 proteins in cancer. Eur J Surg Oncol. 2008 Apr;34(4):357-64

Turovskaya O, Foell D, Sinha P, Vogl T, Newlin R, Nayak J, Nguyen M, Olsson A, Nawroth PP, Bierhaus A, Varki N, Kronenberg M, Freeze HH, Srikrishna G. RAGE, carboxylated glycans and S100A8/A9 play essential roles in colitis-associated carcinogenesis. Carcinogenesis. 2008 Oct;29(10):2035-43

Ghavami S, Chitayat S, Hashemi M, Eshraghi M, Chazin WJ, Halayko AJ, Kerkhoff C. S100A8/A9: a Janus-faced molecule in cancer therapy and tumorgenesis. Eur J Pharmacol. 2009 Dec 25;625(1-3):73-83

Ito Y, Arai K, Nozawa R, Yoshida H, Hirokawa M, Fukushima M, Inoue H, Tomoda C, Kihara M, Higashiyama T, Takamura Y, Miya A, Kobayashi K, Matsuzuka F, Miyauchi A. S100A8

and S100A9 expression is a crucial factor for dedifferentiation in thyroid carcinoma. Anticancer Res. 2009 Oct;29(10):4157-61

Kerkhoff C, Ghavami S.. Innate immunity molecules S100A8/A9 involved in stress response and cancer biology. Anti-Inflammatory and Anti-Allergy Agents in Medicinal Chemistry, 2009; (8)4:279-281.

Srikrishna G, Freeze HH.. Endogenous damage-associated molecular pattern molecules at the crossroads of inflammation and cancer. Neoplasia. 2009 Jul;11(7):615-28. (REVIEW)

van Dieck J, Fernandez-Fernandez MR, Veprintsev DB, Fersht AR.. Modulation of the oligomerization state of p53 by differential binding of proteins of the S100 family to p53 monomers and tetramers. J Biol Chem. 2009 May 15;284(20):13804-11. Epub 2009 Mar 18.

Ang CW, Nedjadi T, Sheikh AA, Tweedle EM, Tonack S, Honap S, Jenkins RE, Park BK, Schwarte-Waldhoff I, Khattak I, Azadeh B, Dodson A, Kalirai H, Neoptolemos JP, Rooney PS, Costello E.. Smad4 loss is associated with fewer S100A8-positive monocytes in colorectal tumors and attenuated response to S100A8 in colorectal and pancreatic cancer cells. Carcinogenesis. 2010 Sep;31(9):1541-51. Epub 2010 Jul 9.

Fritz G, Botelho HM, Morozova-Roche LA, Gomes CM.. Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity. FEBS J. 2010 Nov;277(22):4578-90. doi: 10.1111/j.1742-4658.2010.07887.x. (REVIEW)

Ghavami S, Eshragi M, Ande SR, Chazin WJ, Klonisch T, Halayko AJ, McNeill KD, Hashemi M, Kerkhoff C, Los M.. S100A8/A9 induces autophagy and apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes that involves BNIP3. Cell Res. 2010 Mar;20(3):314-31. Epub 2009 Nov 24.

Ha YS, Kim MJ, Yoon HY, Kang HW, Kim YJ, Yun SJ, Lee SC, Kim WJ.. mRNA Expression of S100A8 as a Prognostic Marker for Progression of Non-Muscle-Invasive Bladder Cancer. Korean J Urol. 2010 Jan;51(1):15-20. Epub 2010 Jan 21.

Minami S, Sato Y, Matsumoto T, Kageyama T, Kawashima Y, Yoshio K, Ishii J, Matsumoto K, Nagashio R, Okayasu I.. Proteomic study of sera from patients with bladder cancer: usefulness of S100A8 and S100A9 proteins. Cancer Genomics Proteomics. 2010 Jul-Aug;7(4):181-9.

Saha A, Lee YC, Zhang Z, Chandra G, Su SB, Mukherjee AB.. Lack of an endogenous anti-inflammatory protein in mice enhances colonization of B16F10 melanoma cells in the lungs. J Biol Chem. 2010 Apr 2;285(14):10822-31. Epub 2010 Jan 29.

Su YJ, Xu F, Yu JP, Yue DS, Ren XB, Wang CL.. Up-regulation of the expression of S100A8 and S100A9 in lung adenocarcinoma and its correlation with inflammation and other clinical features. Chin Med J (Engl). 2010 Aug;123(16):2215-20.

Voss A, Bode G, Sopalla C, Benedyk M, Varga G, Bohm M, Nacken W, Kerkhoff C.. Expression of S100A8/A9 in HaCaT keratinocytes alters the rate of cell proliferation and differentiation. FEBS Lett. 2011 Jan 21;585(2):440-6. Epub 2010 Dec 28.


Kerkhoff C, Ghavami S. S100A8 (S100 calcium binding protein A8). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):735-745.

Gene Section Review




S100A9 (S100 calcium binding protein A9) Claus Kerkhoff, Saeid Ghavami

Dept VAC / IMCI, Helmholtz Centre for Infection Research, Inhoffenstr 7, D-38124 Braunschweig, Germany (CK), Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada (SG)


Online updated version : http://AtlasGeneticsOncology.org/Genes/S100A9ID45569ch1q21.html DOI: 10.4267/2042/46028


Identity Other names: 60B8AG; CAGB; CFAG; CGLB; L1AG; LIAG; MAC387; MIF; MRP14; NIF; P14

HGNC (Hugo): S100A9

Location: 1q21.3

Local order: Distal to PGLYRP4 peptidoglycan recognition protein 4, proximal to S100A12 (S100 calcium binding protein A12).

DNA/RNA Note S100A9 belongs to the S100/calgranulin family of small non-ubiquitous cytoplasmic Ca2+-binding proteins of EF-hand type. The proteins were referred to "S100" because of their solubility in saturated ammonium sulphate solution. Sixteen of 21 members are localised in a cluster on human chromosome 1q21. The clustered organization of these S100 genes is conserved during evolution (Ridinger et al., 1998). A comparison between man and mouse has shown that during evolution, the colinearity of the S100 gene cluster has been destroyed by some inversions. However, the colocalization of the myeloid expressed S100 genes such as S100A8, S100A9, and S100A12 is conserved. It has been speculated, that the structural integrity of that part of the locus is necessary for the coordinated expression of these genes (Nacken et al., 2001). Remarkably, the S100 gene cluster is located in close proximity to a region which has been frequently rearranged in human cancer (Carlsson et al., 2005) and to the epidermal differentiation complex (EDC) (Mischke et al., 1996). EDC is a cluster of genes on chromosome 1q21 encoding proteins that fulfil

important functions in terminal differentiation in the human epidermis, including filaggrin, loricrin and others. In addition, linkage analyses have identified a psoriasis susceptibility region, the PSORS4 locus, that is close to the S100 gene cluster (Hardas et al., 1996; Semprini et al., 2002). These data are important indications for the involvement of S100 genes in inflammatory as well as neoplastic disorders. It has been speculated that the rearrangements result in a deregulated expression of S100 genes associated with neoplasia.

Description The S100 gene structure has been structurally conserved during evolution. Similar to most S100 genes S100A9 consists of three exons that are separated by two introns.

Transcription In the S100A9 gene, exon 1 encodes the untranslated region. The protein is encoded by sequences in exon 2 and exon 3, encoding a N-terminal and a C-terminal EF-hand motif, respectively. The sequence of human S100A9 cDNA has an open reading frame of 352 nucleotides. S100A9 expression appears to be restricted to a specific stage of myeloid differentiation. The protein is present in circulating neutrophils and monocytes, but not in resting tissue macrophages. In peripheral blood monocytes it is down regulated during maturation to macrophages. Despite a number of distinct regulatory regions are located upstream of the transcription initiation site, the corresponding nuclear factors as well as the underlying molecular mechanisms still remain unclear. Transcription factors such as PU.1 (Henkel et al., 2002), C/EBP-alpha and C/EBP-beta (Kuruto-Niwa et al., 1998) have been shown to drive S100 gene expression within the myeloid lineage.



For example, during differentiation of HL-60 cells into monocyte-like cells two still not identified factors were found to bind to the upstream regions of S100A9 gene; one adjacent to the TATA box and another in the region between -400 and -150 (Kuwayama et al., 1993). Another study revealed a CCAAT/enhancer binding protein (C/EBP)-binding motif located at position -81 upstream of the S100A9 gene. Both C/EBP-alpha and -beta bind to this motif in a myeloid/monocytic differentiation-dependent manner (Kuruto-Niwa et al., 1998). C/EBP was shown to be alone sufficient to drive S100A9 expression in otherwise negative cells. C/EBP up-regulation is antagonized by myb, a transcription factor active in differentiated myeloid/monocytic cells (Klempt et al., 1998). The presence of distinct epithelial and myeloid-specific regulatory regions upstream of the transcription initiation site has been demonstrated by detailed deletion analysis (Klempt et al., 1999). Besides the very specific action of particular upstream DNA elements, the S100A9 gene contains a potent enhancer, which is harbored within positions 153 to 361 of its first intron (Melkonyan et al., 1998). The functional relevance of this enhancer in S100A9 expression is supported by its conservation in human and murine S100A9 genes at almost identical positions. Promoter analyses revealed a regulatory element within the S100A9 promoter referred to as MRP regulatory element (MRE) that drives the S100A9 gene expression in a cell-specific and differentiation-dependent manner. This regulatory region is located at position -400 to -374 bp, and two distinct nuclear complexes were demonstrated to bind to this region. Interestingly, the formation of the nuclear protein complexes closely correlates with the myeloid-specific expression of the S100A9 gene and, were therefore referred to as MRE-binding complex A (MbcA) and MbcB, respectively. Analysis of one of the two nuclear complexes revealed a heterocomplex consisting of transcriptional intermediary factor 1 beta (TIF1 beta) and a yet unidentified protein with homology to KRAB domain-containing (Kruppel-related) zinc finger proteins (ZFP) (Kerkhoff et al., 2002). Beside its expression in myeloid cells S100A9 is expressed in epithelia under specific conditions. Its expression is transiently induced in keratinocytes after epidermal injury and UVB irradiation, and the protein is expressed at extremely high levels in psoriatic keratinocytes. Furthermore, its expression is induced by pro-inflammatory cytokines such as TNF alpha and IL1 beta. Recently, a complex of Poly (ADP-ribose) polymerase (PARP-1) and Ku70/Ku80 has been demonstrated to drive the stress response-specific S100 gene expression (Grote et al., 2006). The stress

response-induced expression of the S100 proteins points to an important role in skin pathology. In breast cancer cells S100A9 gene expression is induced by the cytokine oncostatin M (OM) through the STAT3-signaling cascade (Li et al., 2004). This finding is in accordance with another study showing that IL-22 up-regulates the expression of S100A7, S100A8, and S100A9 in keratinocytes. IL-22 has been demonstrated to induce STAT3 activation in keratinocytes (Boniface et al., 2005).

Pseudogene Not known.

Protein Description The sequence of human S100A9 cDNA has an open reading frame of 352 nucleotides predicting a protein of 114 amino acids and a calculated Mr of 13242 Da. Beside the full-length form of S100A9 there is a truncated isoform of S100A9 resulting from alternative translation. The full-length form of S100A9 lacks the first Met, and Thr at position 2 is acetylated leading to a calculated molecular mass of 13154 Da. The N-truncated isoform starts with Met at position 5. Posttranslational removing of Met at position 5 and consequent acetylation of Ser at position 6 leads to a calculated molecular mass of 12690 Da. The theoretical isoelectric point of the full length form is 5.7 and for the truncated form is 5.5, respectively. S100A9 is composed of two helix-loop-helix EF-hand motifs. The C-terminal EF-hand contains a canonical Ca2+-binding loop of 12 amino acids. Conversely, the N-terminal EF-hand contains a Ca2+-binding loop of 14 residues that binds Ca2+ mostly through main-chain carbonyl groups that which is specific to S100 proteins. Consequently, S100 proteins have a weaker Ca2+ affinity than typical Ca2+ sensors such as calmodulin (Donato, 2003). An important posttranslational modification of S100A9 represents the phosphorylation of threonine at position 113. It can be phosphorylated upon PMN activation, and phosphorylation of this residue is specifically regulated by the Ca2+-ionophore, ionomycin. Recent studies give evidence for S100A9 being a p38 MAPK substrate in human neutrophils (Lominadze et al., 2005). This phosphorylation is involved in translocation and functional events. In vivo and in vitro experiments have shown that S100 proteins form homo-, hetero- and oligomeric



Gene: Box = exon (light blue = 5'UTR, yellow = CDS, red = 3'UTR); Line = intron. Protein: Upper boxes, alternating colours: exons (coding part only). Lower boxes: protein domains. Green box = not structure; blue box = helix; violett box = calcium-binding domain. assemblies (Hunter and Chazin, 1998; Osterloh et al., 1998; Pröpper et al., 1999; Moroz et al., 2003). Together with their specific cell- and tissue-expression patterns, the structural variations, and the different metal ion binding properties (Ca2+, Zn2+ and Cu2+) the S100 protein complexes might be functionally diversified. S100A9 preferentially interacts with S100A8. It is worthwhile mentioning that the murine analogs display a stronger tendency to form homodimeric protein complexes. In view of the formation of different tertiary structures with putative distinct functions it is tempting to speculate that S100A8 and/or A9 have different functions in mouse and man.

Expression S100A9 is mainly expressed in cells of the myeloid lineage, however, its gene expression is induced in epithelial cells in response to stress, in specific conditions such as wound healing, UV exposition, abundant in psoriais keratinocytes, differentially expressed in several cancers.

Localisation Mostly cytoplasmic, but also at membranes and cytoskeleton. In resting phagocytes the S100A8/A9 protein complex is mainly located in the cytosol. Upon cellular activation the protein complex is either translocated to cytoskeleton and plasma membrane or released into the extracellular environment. The translocation pathways occur upon the elevation of the intracellular calcium level (Roth et al., 1993). At a later time point, the S100A8/A9 heterodimers can be detected on the surface of monocytes (Bhardwaj et al., 1992). The mechanism by which the S100A8/A9 heterodimer penetrates the plasma membrane remains unclear since the S100 proteins lack a transmembrane signaling region.

The secretion pathway relies on the activation of protein kinase C. This pathway differs from the classical as well as the alternative secretion pathways of cytokines (Moqbel and Coughlin, 2006). It has been demonstrated that this novel secretion pathway is energy-consuming and depends on an intact microtubule network (Murao et al., 1990; Rammes et al., 1997). Recent investigations give evidence that interaction of S100A8/A9 with annexin-6 is involved in surface expression and release of S100A8/A9 (Bode et al., 2008). Annexins are another class of Ca2+-regulated proteins. They are characterized by the unique architecture of their Ca2+-binding sites, which enables them to peripherally dock onto negatively charged membrane surfaces in their Ca2+-bound conformation. This property links annexins to many membrane-related events such as certain exocytic and endocytic transport steps. This is an interesting finding since S100A8 and S100A9 are expressed in cancerous cells of secretory tissues as breast and prostate. Cells originating from such glandular tissues are rich in membrane structures, suggesting that membrane-associated molecular targets for the S100A8/A9 proteins could be potentially found in these cells. Recent investigations also demonstrated the association of S100A8/A9 with cholesterol-enriched membrane microdomains (lipid rafts) (Nacken et al., 2004). This observation is in agreement with the enhancing effect of S100A8/A9 on NADPH oxidase since the formation of the oxidase complex takes place at lipid rafts.

Function Intra- as well as extracellular roles have been proposed for the S100 proteins. Intracellular activities of S100A8/A9 In the intracellular milieu, S100 proteins are considered as calcium sensors changing their



conformation in response to calcium influx and then mediating calcium signals by binding to other intracellular proteins. In a mouse knock-out model chemokine-induced down regulation of the cytosolic Ca2+-level was detected (Nacken et al., 2005). After calcium binding, the S100A8/A9 protein complex binds specifically polyunsaturated fatty acids. S100A8/A9 represents the exclusive arachidonic acid-binding capacity in the neutrophil cytosol (Kerkhoff et al., 1999), and participates in NADPH oxidase activation by transferring arachidonic acid to membrane-bound gp91phox during interactions with two cytosolic oxidase activation factors, p67phox and Rac-2. The functional relevance of S100A8/A9 in the phagocyte NADPH oxidase activation was demonstrated by the impairment of NADPH oxidase activity in neutrophil-like NB4 cells, after specifically blocking S100A9 expression, and employing bone marrow-derived PMNs from S100A9-/- mice (Kerkhoff et al., 2005). In accordance to their role in myeloid cells, S100A8/A9 enhances epithelial NADPH oxidases (Benedyk et al., 2007). As a consequence of enhanced ROS levels, NF-kB activation and subsequently TNF-alpha and IL-8 mRNA levels are increased in S100A8/A9-HaCaT keratinocytes, consistent with the view that NF-kB is a redox-sensitive transcription factor. Further consequences of S100A8/A9-mediated NF-kB activation are reduced cell growth, increased expression of differentiation markers, and enhanced PARP cleavage as an indicator of increased cell death (Voss et al., 2011). In view of the stress response-induced expression of the two S100 proteins in keratinocytes these findings have great implications for tissue remodeling and repair. For example, keratinocytes acquire an activated state after cutaneous wounding in which proliferation is favored over differentiation in order to replenish the lost material and rapidly close the site of injury. Thus, it is likely to hypothesize that S100A8/A9-mediated growth reduction is required for the upcoming cell fate decision of damaged cells, i.e. for a survival phase to be followed by differentiation, proliferation, or apoptosis. These data have also an impact on tumorigenesis since S100 gene expression is associated with neoplastic disorders. In migrating monocytes the S100A8/A9 complex has been found to be associated with cytoskeletal tubulin and to modulate transendothelial migration (Vogl et al., 2004). Investigations using two different mouse knock-out models demonstrated no obvious phenotype (Manitz et al., 2003; Hobbs et al., 2003). However, reduced migration of S100A9-deficient neutrophils and decreased surface expression of CD11b, which belongs to the integrin family, were observed upon in vitro stimulation. Extracellular activities of S100A8/A9 The S100/calgranulins display antimicrobial activity by

depriving bacterial pathogens of essential trace metals such as Zn2+ and Mn2+ (Steinbakk et al., 1990; Murthy et al., 1993; Clohessy and Golden, 1995; Sohnle et al., 2000). In the context of inflammation, it has been proposed that S100A8/A9 is massively released when neutrophils die to provide a growth-inhibitory type of host defense that is adjunctive to the usual microbicidal functions by binding metals other than Ca2+ (Corbin et al., 2008). In addition, S100/calgranulins serve as leukocyte chemoattractants (Lackmann et al., 1992; Lackmann et al., 1993; Kocher et al., 1996; Lim et al., 2008). Murine S100A8 has potent chemotactic activity for neutrophils and monocytes in vitro and in vivo (Lackmann et al., 1992). In contrast, human S100A8 displays only weak leukocyte chemotactic activity in vitro and in vivo (Lackmann et al., 1993). Detailed analysis revealed that the hinge region contributes to the chemotactic activity of murine, but not human S100A8. These data questioned whether the proteins are orthologs since there is a high degree of homology between murine and human S100A8 but a functional divergence. Intriguingly, human S100A12 is chemotactic and the hinge region of human S100A12 has been implicated herein (Yang et al., 2001). Thus, the functional and sequence divergence suggested complex evolution of the S100 family in mammals. The putative pro-inflammatory functions of S100A8 and S100A9 have recently been investigated in two different mouse knock-out models. S100A9 deficiency did not result in an obvious phenotype (Manitz et al., 2003; Hobbs et al., 2003). However, reduced migration of S100A9-deficient neutrophils and decreased surface expression of CD11b, which belongs to the integrin family, were observed upon in vitro stimulation. In addition, chemokine-induced down regulation of the cytosolic Ca2+-level was detected. Obviously, these in vitro effects are compensated by alternative pathways in vivo. Remarkably, cancer cells utilize S100A8 and S100A9 as guidance for the adhesion and invasion of disseminating malignant cells (Hiratsuka et al., 2006). In the context of malignancy it was reported that S100A8/A9 attracts Mac-1+ myeloid cells to the lung tissue. Recruited Mac-1+ myeloid cells in lung in turn produce S100A8/A9 in response to primary malignant cells in a so called "premetastatic phase". This phase shows the general characteristics of an inflammation state which facilitates the micro-environmental changes required for the migration and implantation of primary tumor cells to lung tissue. After preparation of the target tissue for accepting the malignant cells, tumor cells mimic Mac-1+ myeloid cells in response to S100A8/A9 chemotactic signaling and migrate to lung. So, it seems that tumor cells and Mac-1+ myeloid cells utilize a common pathway for migration to lung which involves the activation of mitogen-activated protein kinase pathway (Hiratsuka et al., 2006). These findings



suggest S100A8/A9 as an attractive target for the development of strategies counteracting tumor metastasizing to certain organs. S100A8 and S100A9 have been identified as important endogenous damage-associated molecular pattern (DAMP) proteins. Although receptors for S100A8/A9 are still largely uncharacterized, more recent findings support the notion that they function as potent ligands of pattern-recognition receptors, such as the toll-like receptor 4 (TLR4) (Vogl et al., 2007) and the receptor for advanced glycation end products (RAGE) (Srikrishna and Freeze, 2009). The S100/calgranulins display cytokine-like functions, including activation of the receptor for advanced glycation endproducts (RAGE) (Hofmann et al., 1999; Herold et al., 2007). RAGE is a member of the immunoglobulin superfamily and present on numerous cell types. It has been shown to play crucial roles in a variety of pathophysiological situations, such as wound healing, atherosclerotic lesion development, tumor growth and metastasis, systemic amyloidosis, and Alzheimer disease (Bierhaus et al., 2005). RAGE/S100 interaction has been considered a very attractive model to explain how RAGE and its proinflammatory ligand contribute to the pathophysiology of several inflammatory diseases. Beside the above mentioned receptors a number of other cell surface binding sites specific for S100A8/A9 have been reported, such as novel carboxylated glycans (Srikrishna et al., 2001), heparan sulfate glycosaminoglycans (Robinson et al., 2002), beta2-integrin (Newton and Hogg, 1998), and the fatty acid transporter FAT/CD36 (Kerkhoff et al., 2001). Therefore, the cell surface receptor of S100A8/A9 is still in debate. Interestingly, the growth-stimulatory activity of S100A8/A9 has been demonstrated to be mediated by binding to the receptor of advanced glycation end products (RAGE) (Ghavami et al., 2008b; Turovskaya et al., 2008; Gebhardt et al., 2008). It is likely to speculate that the selective up-regulation of S100 proteins may be of importance for survival and proliferation of metastasizing cancer cells. S100A8/A9 complexes that are secreted from phorbolester-stimulated neutrophil-like HL-60 cells have been shown to carry the eicosanoid precursor arachidonic acid (Kerkhoff et al., 1999). The S100A8/A9-arachidonic acid complex is recognized by the fatty acid transporter FAT/CD36, and the fatty acid is rapidly taken up (Kerkhoff et al., 2001). Endothelial cells as well as neutrophils themselves utilize both endogenous and exogenous arachidonic acid for transcellular production of eicosanoids (Sala et al., 1999). Therefore, the secreted S100A8/A9-AA complex may serve as a transport protein to move AA to its target cells. This may represent a mechanism by which AA-derived eicosanoids are synthesized in a cooperative manner between different cell species due to environmental cues.

S100A8/A9 displays apoptosis-inducing activity against various tumor cells (Yui et al., 1995; Yui et al., 2002; Ghavami et al., 2004; Ghavami et al., 2008a; Kerkhoff and Ghavami, 2009; Ghavami et al., 2009; Ghavami et al., 2010). It was speculated that this activity was due to the ability to bind divalent metal ions including Zn2+, Mn2+ and Cu2+ at sites that are distinct from Ca2+-binding sites. However, a number of recent reports now indicate that S100A8/A9 exerts its activity by both chelation of trace metal ions such as Zn2+ and cell surface receptor mediated pathways. Although a number of receptors have been shown to bind S100A8/A9, the nature of the receptor involved in S100A8/A9-induced cell death remains to be elucidated. Experiments with certain cell lines either deficient for or over expressing components of the death signaling machinery as well as RAGE gene silencing and blocking RAGE-specific antibody approaches excluded both RAGE and the classical death receptor to be involved in S100A8/A9-induced cell death, even though S100A8/A9 can specifically bind to cancer cells and RAGE mediates the growth-promoting activity obvious at low micromolar concentrations of S100A8/A9. Clearly, investigations to identify the receptor involved in S100A8/A9-induced cell death are critical.

Homology Overall, the S100 proteins share significant sequential homology in the EF-hand motifs, but are least conserved in the hinge region. This region is proposed to provide for specific interaction with target proteins (Groves et al., 1998; Zimmer et al., 2003; Santamaria-Kisiel et al., 2006; Fernandez-Fernandez et al., 2008; van Dieck et al., 2009). The availability of high-resolution S100-target structures has highlighted important structural features that contribute to S100 protein functional specificity (Bhattacharya et al., 2003). The functional diversification of S100 proteins is achieved by their specific cell- and tissue-expression patterns, structural variations, different metal ion binding properties (Ca2+, Zn2+ and Cu2+) as well as their ability to form homo-, hetero- and oligomeric assemblies (Hunter and Chazin, 1998; Osterloh et al., 1998; Pröpper et al., 1999; Tarabykina et al., 2001; Moroz et al., 2003; Fritz et al., 2010). Although the function of S100 proteins in cancer cells in most cases is still unknown, the specific expression patterns of these proteins are a valuable diagnostic tool.

Implicated in General note Note Comparative and functional genomics have revealed that a number of S100 proteins are found to be differentially expressed in cancer cells. Several of these have been associated with tumor development, cancer



invasion or metastasis in recent studies (for review see Salama et al., 2008). S100A8 and S100A9 are abundant in cells of the myeloid lineage, are released from activated phagocytes and display intra- and extracellular functions. Their expression is ubiquitously observed in the squamous epithelia under normal, inflammatory and cancerous conditions. Immunohistochemical investigations have shown that the S100 proteins are over expressed in skin cancers, pulmonary adenocarcinoma, pancreatic adenocarcinoma, bladder cancers, ductal carcinoma of the breast, and prostate adenocarcinoma. In contrast, S100A8 and S100A9 are down-regulated in esophageal squamous cell carcinomas. Furthermore, plasma levels of S100A8/A9 are elevated in patients suffering from various cancers. Insofar, S100A8 and S100A9 might represent novel diagnostic markers for some carcinomas. S100A8 and S100A9 have been suggested to have potential roles in carcinogenesis and tumor progression. However, the biological role of S100A8/A9 remains to be elucidated. It is conceivable that S100A8 and S100A9 modulate signal pathways to directly promote invasion, migration and metastasis, probably via activation of NF-kB, Akt or MAP kinases. In the last decade the concept of the functional relationship between inflammation and cancer has been developed that is based on numerous findings, ranging from epidemiological studies to molecular analyses of mouse models (Coussens and Werb, 2002). In this concept, the generation of an inflammatory microenvironment supports tumorigenesis by promoting cancer cell survival, proliferation, migration, and invasion. Although it is clear that inflammation alone does not cause cancer, it is evident that an environment that is rich in inflammatory cells, growth factors, activated stroma, and DNA-damage-promoting agents certainly potentiates and/or promotes neoplastic risk. In addition, many cancers arise from sites of infection, chronic irritation and inflammation. Recent data have expanded our knowledge demonstrating that specific soluble factors released from primary tumors induce the S100A8 and S100A9 gene expression in the target tissue. After secretion S100A8 and S100A9 might display chemokine- and cytokine-like properties that promote invasion, migration and metastasis. These data indicate that tumor cells are able to reprogram some of the signaling molecules of the innate immune system. These insights are fostering new anti-inflammatory therapeutic approaches to cancer development.

Skin cancer Note The expression of S100A8 and S100A9 in epithelial cells was first detected in the squamous epithelia (Gabrielsen et al., 1986). Normal S100A8 and S100A9 are expressed at minimal levels in the epidermis. However, their expression is induced in inflammatory

and cancerous conditions, and pro-inflammatory cytokines such as TNF-alpha and IL1 beta are involved herein. Gene expression analysis in a mouse model of chemically induced skin carcinogenesis identified a large set of novel tumor-associated genes including S100A8 (Hummerich et al., 2006). The data was confirmed by in situ hybridization and immunofluorescence analysis on mouse tumor sections, in mouse keratinocyte cell lines that form tumors in vivo, and in human skin tumor specimens. However, conflicting results have been published concerning S100 expression in skin cancer. For instance, esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. DNA microarray data analysis revealed that S100A8 and S100A9 were significantly down regulated in human ESCC versus the normal counterparts (Zhi et al., 2003). Interestingly, among the 42 genes either up regulated or down regulated in tumors, as compared to normal esophageal squamous epithelia, nine of the altered expression genes were related to arachidonic acid (AA) metabolism, suggesting that AA metabolism pathway and its altered expression may contribute to esophageal squamous cell carcinogenesis. Similar data were obtained by Ji et al. (2004). They investigated the differential expression of the S100 gene family at the RNA level in human ESCC. Eleven out of 16 S100 genes were significantly down regulated in ESCC versus the normal counterparts. Only the S100A7 gene was found to be markedly up regulated. Another study demonstrated that poorly differentiated ESCC displayed a stronger decrease in S100A8 and S100A9 expression than well and moderately differentiated tumors, with a correlation between protein level and histopathological grading (Kong et al., 2004). These findings suggest that decreased expression of S100A8 and S100A9 might play an important role in the ESCC pathogenesis, being particularly associated with poor differentiation of tumor cells.

Lung adenocarcinomas Note S100A9 over expression has been detected in various carcinomas of glandular cell origin, and its expression has been associated with poor tumor differentiation. Similarly, S100A9 immunopositivity was also detected in pulmonary adenocarcinoma cell lines and resected pulmonary adenocarcinoma (Arai et al., 2001). Examination of the relation of S100A9 expression to tumor differentiation showed that the expression rate in pulmonary adenocarcinoma showed higher correlation in poorly differentiated carcinomas. Another study confirmed these data (Su et al., 2010). Immunohistochemical staining of both S100 proteins showed a significant up-regulation in lung cancer tissue, and quantitative PCR revealed significantly higher levels of S100A8 and S100A9 mRNA transcripts in lung cancer tissues. Moreover, this study



correlates S100A9 expression with inflammation and other clinical features (Su et al., 2010). Primary tumors influence the environment in the lungs before metastasis. They release specific soluble factors that prepare the premetastatic niche for the engraftment of tumor cells. In several studies it has been shown that tumor cells induce both expression and secretion of S100A8 and S100A9 in the target organ that display a promoting role in cancer cell survival, proliferation, migration, and invasion (Hiratsuka et al., 2002; Hiratsuka et al., 2006; Hiratsuka et al., 2008; Saha et al., 2010). Microarray analysis of lungs from tumor-bearing and non-bearing mice revealed the strong up-regulation of a number of genes including S100A8 and S100A9 (Hiratsuka et al. 2002). Their expression in Mac 1+-myeloid cells and endothelial cells was induced by factors such as vascular endothelial growth factor A (VEGF-A), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta), both in vitro and in vivo (Hiratsuka et al., 2006). Remarkably, anti-S100A8 neutralizing antibody treatment blocked metastasis. S100A8 and S100A9 were shown to induce the expression of serum amyloid A (SAA) that attracted Mac 1+-myeloid cells in the premetastatic lung (Hiratsuka et al., 2008). These studies demonstrated that lung cancer cells utilize S100A8 and S100A9 as guidance for the adhesion and invasion of disseminating malignant cells.

Pancreatic adenocarcinoma Note Patients with ductal adenocarcinoma of the pancreas have a dismal prognosis. Thus, there is an urgent need for early detection markers and the development of immunotherapeutical approaches concentrating on the induction and enhancement of immune responses against tumors. Proteomic analyses of pancreatic adenocarcinoma, normal adjacent tissues, pancreatitis, and normal pancreatic tissues revealed a number of differentially expressed genes (Shen et al., 2004). S100A8 was found to be specifically over expressed in tumors compared with normal and pancreatitis tissues. These data are in accordance with another study (Sheikh et al., 2007). Strong expression of S100A8 and S100A9 was found in tumor-associated stroma but not in benign or malignant epithelia. Further analyses identified stromal CD14+ CD68- monocytes/macrophages as source for S100 expression. Interestingly, the number of S100A8-positive cells in the tumor microenvironment negatively correlated with the expression of the tumor suppressor protein, Smad4. The number of S100A9-positive cells was not altered in Smad4-negative or Smad4-positive tumors. A similar correlation was found in colorectal cancer tumors (Ang et al., 2010). The number of stromal S100A8- and S100A9-positive cells was associated with the presence or absence of Smad4. Smad4-negative tumors showed enhanced numbers of

S100A8/A9 stroma cells, and the corresponding patients had a poor survival prognosis. Investigation of the underlying molecular mechanisms revealed that both migration and proliferation was enhanced in response to exogenous S100A8 and S100A9, irrespective of Smad4-presence. However, depletion of Smad4 resulted in loss of responsiveness to exogenous S100A8, but not S100A9. Vice versa, Smad4 expression in Smad4-negative cells enhanced the responsive-ness to S100A8 and S100A9. Further analyses give evidence that similar to TGF-beta, S100A8 and S100A9 induce the phosphorylation of both Smad2 and Smad3 that was blocked by a RAGE-specific antibody. These data point to a functional relationship between inflammation and tumorigenesis.

Bladder cancers Note Gene expression profiles revealed that thirteen members of the S100 gene family were differentially expressed in human bladder cancers. S100A8 and S100A9 were found to be over expressed (Yao et al., 2007). Another study investigated S100A9 expression and DNA methylation in urothelial cancer cell lines and cancer tissue (Dokun et al., 2008). Expression of S100A9 was found to be generally elevated in the tumor tissues but S100A9 was weakly expressed in most cancer cell lines. The S100A9 promoter contains 6 CpG sites, and its methylation state was unrelated to the variable expression. It has been hypothesized that over expression of genes is the consequence of DNA hypomethylation, however, DNA methylation and gene expression are less strictly related for those genes having promoters within CpG-islands. Alternatively, the increased S100A9 gene expression may be related to that of other immune-related genes in the carcinoma cell cultures. This is sustained by the facts that S100A9 is secreted by epithelial and other cell types to modulate inflammatory reactions as well as to promote cancer proliferation and metastasis. Two recent studies propose S100A8 and S100A9 gene expression as prognostic value for bladder cancer (Minami et al., 2010; Ha et al., 2010). By proteomic analysis of pre- and postoperative sera from bladder cancer patients S100A8 and S100A9 were identified as tumor-associated proteins (Minami et al., 2010). Interestingly, S100A8 expression was associated with bladder wall muscle invasion of the tumor and cancer-specific survival while S100A9 expression was associated with the tumor grade. In addition, the expression of both proteins S100A8/A9 was correlated with recurrence-free survival. In another study it was evaluated whether S100A8 is a prognostic value for non-muscle-invasive bladder cancer (NMIBC) (Ha et al., 2010). S100A8 expression was evaluated in a total of 103 primary NMIBC samples by quantitative PCR. The mRNA expression levels of S100A8 were significantly related to the



progression of NMIBC, suggesting that S100A8 might be a useful prognostic marker for disease progression of NMIBC.

Breast cancers Note S100A8 and S100A9 are expressed in breast cancers (Cross et al., 2005), especially in invasive breast carcinoma (Arai et al., 2004). By immunohistochemical analyses a strong S100A9 immunoreactivity has been demonstrated in invasive as well as non-invasive ductal carcinoma. No immunopositive reaction was observed in invasive lobular carcinomas, and no significant differences were detected in the number of myelomonocytic cells expressing S100A9. These data give evidence that S100A9 in glandular epithelial cells is newly expressed under cancerous conditions and is over-expressed in poorly differentiated adenocarcinoma (Arai et al., 2004). Further analyses target on the relationship between S100A8/A9 expression and pathological parameters that reflect the aggressiveness of carcinoma. The immunopositivity for S100A8/A9 correlated with poor tumor differentiation, mitotic activity, HER2/neu over expression, poor pT categories, node metastasis, and poor pStage, but not with vessel invasion. These data may indicate that S100A8 and S100A9 over expression should be considered marker of poor prognosis in invasive breast ductal carcinoma (Arai et al., 2008). By analyses of ductal carcinoma in situ and invasive ductal carcinoma of the breast S100A9 has been demonstrated to be most abundantly expressed in the invasive tumor (Seth et al., 2003). Therefore, the expression of S100A8 and S100A9 has been correlated with the degree of noninvasive / invasive behavior. There are conflicting data concerning this correlation. For instance, non-invasive MCF-7 breast cancer cells do not express S100A9, and its gene expression is induced by cytokine oncostatin M through the STAT3 signaling cascade (Li et al., 2004). However, non-invasive MDA-MB-468 cells are abundant for both S100 proteins (Bode et al., 2008) and invasive breast cancer cells MDA-MB-231 show low transcript level of S100A9 (Nagaraja et al., 2006).

Thyroid carcinoma Note Similar to other carcinomas of glandular cell origin, expression of S100A8 and S100A9 is significantly linked to dedifferentiation of thyroid carcinoma (Ito et al., 2005; Ito et al., 2009). S100A8 and S100A9 immunreactivity was found in all undifferentiated carcinomas examined, while papillary carcinoma, follicular carcinoma, follicular adenoma and medullary carcinoma and normal follicules were negative for both proteins. Further analyses revealed that S100A9 is a useful marker for discriminating intrathyroid epithelial tumor from squamous cell carcinoma or

undifferentiated carcinoma with squamoid component (Ito et al., 2006).

Prostate cancer Note Increased levels of S100A8, S100A9, and RAGE have been reported in prostatic intra epithelial neoplasia and preferentially in high-grade adenocarcinomas, whereas benign tissue was negative or showed weak expression of the proteins. The three proteins showed a strong overlap in the expression pattern. S100A9 serum level was significantly elevated in cancer patients compared with benign prostatic hyperplasia patients or healthy individuals. Therefore, S100A8 and S100A9 might represent novel diagnostic markers for prostate cancer and benign prostate hyperplasia (Hermani et al., 2005). In further analyses it has been demonstrated that S100A8 and S100A9 are secreted by prostate cancer cells, and extracellular S100A8/A9 stimulates migration of benign prostatic cells in vitro by activation of NF-kB and increased phosphorylation of p38 and p44/p42 MAP kinases. Immunofluorescence analyses give evidence for a RAGE-mediated response (Hermani et al., 2006). The significance of being diagnostic markers for prostate cancer has been questioned by Ludwig et al. (2007). Their re-evaluation study has shown that S100A8/A9 did not improve the differentiation between patients with and without prostate cancer. The data give no evidence for the replacement of the established marker PSA by S100A8/A9.

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Mischke D, Korge BP, Marenholz I, Volz A, Ziegler A. Genes encoding structural proteins of epidermal cornification and S100 calcium-binding proteins form a gene complex ("epidermal differentiation complex") on human chromosome 1q21. J Invest Dermatol. 1996 May;106(5):989-92

Rammes A, Roth J, Goebeler M, Klempt M, Hartmann M, Sorg C. Myeloid-related protein (MRP) 8 and MRP14, calcium-binding proteins of the S100 family, are secreted by activated monocytes via a novel, tubulin-dependent pathway. J Biol Chem. 1997 Apr 4;272(14):9496-502

Groves P, Finn BE, Kuźnicki J, Forsén S. A model for target protein binding to calcium-activated S100 dimers. FEBS Lett. 1998 Jan 16;421(3):175-9

Hunter MJ, Chazin WJ. High level expression and dimer characterization of the S100 EF-hand proteins, migration inhibitory factor-related proteins 8 and 14. J Biol Chem. 1998 May 15;273(20):12427-35

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Kuruto-Niwa R, Nakamura M, Takeishi K, Nozawa R. Transcriptional regulation by C/EBP alpha and -beta in the

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Kerkhoff C, Sorg C, Tandon NN, Nacken W. Interaction of S100A8/S100A9-arachidonic acid complexes with the scavenger receptor CD36 may facilitate fatty acid uptake by endothelial cells. Biochemistry. 2001 Jan 9;40(1):241-8

Nacken W, Lekstrom-Himes JA, Sorg C, Manitz MP. Molecular analysis of the mouse S100A9 gene and evidence that the myeloid specific transcription factor C/EBPepsilon is not required for the regulation of the S100A9/A8 gene expression in neutrophils. J Cell Biochem. 2001;80(4):606-16



Srikrishna G, Panneerselvam K, Westphal V, Abraham V, Varki A, Freeze HH. Two proteins modulating transendothelial migration of leukocytes recognize novel carboxylated glycans on endothelial cells. J Immunol. 2001 Apr 1;166(7):4678-88

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Bhattacharya S, Large E, Heizmann CW, Hemmings B, Chazin WJ. Structure of the Ca2+/S100B/NDR kinase peptide complex: insights into S100 target specificity and activation of the kinase. Biochemistry. 2003 Dec 16;42(49):14416-26

Donato R. Intracellular and extracellular roles of S100 proteins. Microsc Res Tech. 2003 Apr 15;60(6):540-51

Hobbs JA, May R, Tanousis K, McNeill E, Mathies M, Gebhardt C, Henderson R, Robinson MJ, Hogg N. Myeloid cell function in MRP-14 (S100A9) null mice. Mol Cell Biol. 2003 Apr;23(7):2564-76

Manitz MP, Horst B, Seeliger S, Strey A, Skryabin BV, Gunzer M, Frings W, Schönlau F, Roth J, Sorg C, Nacken W. Loss of S100A9 (MRP14) results in reduced interleukin-8-induced CD11b surface expression, a polarized microfilament system, and diminished responsiveness to chemoattractants in vitro. Mol Cell Biol. 2003 Feb;23(3):1034-43

Moroz OV, Dodson GG, Wilson KS, Lukanidin E, Bronstein IB. Multiple structural states of S100A12: A key to its functional diversity. Microsc Res Tech. 2003 Apr 15;60(6):581-92

Seth A, Kitching R, Landberg G, Xu J, Zubovits J, Burger AM. Gene expression profiling of ductal carcinomas in situ and invasive breast tumors. Anticancer Res. 2003 May-Jun;23(3A):2043-51

Zhi H, Zhang J, Hu G, Lu J, Wang X, Zhou C, Wu M, Liu Z. The deregulation of arachidonic acid metabolism-related genes in human esophageal squamous cell carcinoma. Int J Cancer. 2003 Sep 1;106(3):327-33

Zimmer DB, Wright Sadosky P, Weber DJ. Molecular mechanisms of S100-target protein interactions. Microsc Res Tech. 2003 Apr 15;60(6):552-9

Arai K, Teratani T, Kuruto-Niwa R, Yamada T, Nozawa R. S100A9 expression in invasive ductal carcinoma of the breast: S100A9 expression in adenocarcinoma is closely associated with poor tumour differentiation. Eur J Cancer. 2004 May;40(8):1179-87


Ji J, Zhao L, Wang X, Zhou C, Ding F, Su L, Zhang C, Mao X, Wu M, Liu Z. Differential expression of S100 gene family in human esophageal squamous cell carcinoma. J Cancer Res Clin Oncol. 2004 Aug;130(8):480-6

Kong JP, Ding F, Zhou CN, Wang XQ, Miao XP, Wu M, Liu ZH. Loss of myeloid-related proteins 8 and myeloid-related proteins 14 expression in human esophageal squamous cell carcinoma correlates with poor differentiation. World J Gastroenterol. 2004 Apr 15;10(8):1093-7

Li C, Zhang F, Lin M, Liu J. Induction of S100A9 gene expression by cytokine oncostatin M in breast cancer cells through the STAT3 signaling cascade. Breast Cancer Res Treat. 2004 Sep;87(2):123-34

Nacken W, Sorg C, Kerkhoff C. The myeloid expressed EF-hand proteins display a diverse pattern of lipid raft association. FEBS Lett. 2004 Aug 13;572(1-3):289-93

Shen J, Person MD, Zhu J, Abbruzzese JL, Li D. Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry. Cancer Res. 2004 Dec 15;64(24):9018-26

Vogl T, Ludwig S, Goebeler M, Strey A, Thorey IS, Reichelt R, Foell D, Gerke V, Manitz MP, Nacken W, Werner S, Sorg C, Roth J. MRP8 and MRP14 control microtubule reorganization during transendothelial migration of phagocytes. Blood. 2004 Dec 15;104(13):4260-8

Bierhaus A, Humpert PM, Stern DM, Arnold B, Nawroth PP. Advanced glycation end product receptor-mediated cellular dysfunction. Ann N Y Acad Sci. 2005 Jun;1043:676-80

Boniface K, Bernard FX, Garcia M, Gurney AL, Lecron JC, Morel F. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes. J Immunol. 2005 Mar 15;174(6):3695-702

Carlsson H, Petersson S, Enerbäck C. Cluster analysis of S100 gene expression and genes correlating to psoriasin (S100A7) expression at different stages of breast cancer development. Int J Oncol. 2005 Dec;27(6):1473-81

Cross SS, Hamdy FC, Deloulme JC, Rehman I. Expression of S100 proteins in normal human tissues and common cancers using tissue microarrays: S100A6, S100A8, S100A9 and S100A11 are all overexpressed in common cancers. Histopathology. 2005 Mar;46(3):256-69

Hermani A, Hess J, De Servi B, Medunjanin S, Grobholz R, Trojan L, Angel P, Mayer D. Calcium-binding proteins S100A8 and S100A9 as novel diagnostic markers in human prostate cancer. Clin Cancer Res. 2005 Jul 15;11(14):5146-52

Ito Y, Arai K, Ryushi, Nozawa, Yoshida H, Tomoda C, Uruno T, Miya A, Kobayashi K, Matsuzuka F, Kuma K, Kakudo K, Miyauchi A. S100A9 expression is significantly linked to dedifferentiation of thyroid carcinoma. Pathol Res Pract. 2005;201(8-9):551-6



Kerkhoff C, Nacken W, Benedyk M, Dagher MC, Sopalla C, Doussiere J. The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2. FASEB J. 2005 Mar;19(3):467-9

Lominadze G, Rane MJ, Merchant M, Cai J, Ward RA, McLeish KR. Myeloid-related protein-14 is a p38 MAPK substrate in human neutrophils. J Immunol. 2005 Jun 1;174(11):7257-67

Nacken W, Mooren FC, Manitz MP, Bode G, Sorg C, Kerkhoff C. S100A9 deficiency alters adenosine-5'-triphosphate induced calcium signalling but does not generally interfere with calcium and zinc homeostasis in murine neutrophils. Int J Biochem Cell Biol. 2005 Jun;37(6):1241-53

Grote J, König S, Ackermann D, Sopalla C, Benedyk M, Los M, Kerkhoff C. Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression. BMC Mol Biol. 2006 Dec 22;7:48



Hummerich L, Müller R, Hess J, Kokocinski F, Hahn M, Fürstenberger G, Mauch C, Lichter P, Angel P. Identification of novel tumour-associated genes differentially expressed in the process of squamous cell cancer development. Oncogene. 2006 Jan 5;25(1):111-21

Ito Y, Miyauchi A, Arai K, Nozawa R, Miya A, Kobayashi K, Nakamura Y, Kakudo K. Usefulness of S100A9 for diagnosis of intrathyroid epithelial thymoma (ITET)/carcinoma showing thymus-like differentiation (CASTLE). Pathology. 2006 Dec;38(6):541-4

Moqbel R, Coughlin JJ. Differential secretion of cytokines. Sci STKE. 2006 Jun 6;2006(338):pe26

Nagaraja GM, Othman M, Fox BP, Alsaber R, Pellegrino CM, Zeng Y, Khanna R, Tamburini P, Swaroop A, Kandpal RP. Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics. Oncogene. 2006 Apr 13;25(16):2328-38

Santamaria-Kisiel L, Rintala-Dempsey AC, Shaw GS. Calcium-dependent and -independent interactions of the S100 protein family. Biochem J. 2006 Jun 1;396(2):201-14

Benedyk M, Sopalla C, Nacken W, Bode G, Melkonyan H, Banfi B, Kerkhoff C. HaCaT keratinocytes overexpressing the S100 proteins S100A8 and S100A9 show increased NADPH oxidase and NF-kappaB activities. J Invest Dermatol. 2007 Aug;127(8):2001-11

Herold K, Moser B, Chen Y, Zeng S, Yan SF, Ramasamy R, Emond J, Clynes R, Schmidt AM. Receptor for advanced glycation end products (RAGE) in a dash to the rescue: inflammatory signals gone awry in the primal response to stress. J Leukoc Biol. 2007 Aug;82(2):204-12

Ludwig S, Stephan C, Lein M, Loening SA, Jung K. S100A8, S100A9, and the S100A8/A9 complex in circulating blood are not associated with prostate cancer risk-A re-evaluation study. Prostate. 2007 Sep 1;67(12):1301-7

Sheikh AA, Vimalachandran D, Thompson CC, Jenkins RE, Nedjadi T, Shekouh A, Campbell F, Dodson A, Prime W, Crnogorac-Jurcevic T, Lemoine NR, Costello E. The

expression of S100A8 in pancreatic cancer-associated monocytes is associated with the Smad4 status of pancreatic cancer cells. Proteomics. 2007 Jun;7(11):1929-40


Yao R, Lopez-Beltran A, Maclennan GT, Montironi R, Eble JN, Cheng L. Expression of S100 protein family members in the pathogenesis of bladder tumors. Anticancer Res. 2007 Sep-Oct;27(5A):3051-8

Arai K, Takano S, Teratani T, Ito Y, Yamada T, Nozawa R. S100A8 and S100A9 overexpression is associated with poor pathological parameters in invasive ductal carcinoma of the breast. Curr Cancer Drug Targets. 2008 Jun;8(4):243-52

Bode G, Lüken A, Kerkhoff C, Roth J, Ludwig S, Nacken W. Interaction between S100A8/A9 and annexin A6 is involved in the calcium-induced cell surface exposition of S100A8/A9. J Biol Chem. 2008 Nov 14;283(46):31776-84

Corbin BD, Seeley EH, Raab A, Feldmann J, Miller MR, Torres VJ, Anderson KL, Dattilo BM, Dunman PM, Gerads R, Caprioli RM, Nacken W, Chazin WJ, Skaar EP. Metal chelation and inhibition of bacterial growth in tissue abscesses. Science. 2008 Feb 15;319(5865):962-5

Dokun OY, Florl AR, Seifert HH, Wolff I, Schulz WA. Relationship of SNCG, S100A4, S100A9 and LCN2 gene expression and DNA methylation in bladder cancer. Int J Cancer. 2008 Dec 15;123(12):2798-807

Fernandez-Fernandez MR, Rutherford TJ, Fersht AR. Members of the S100 family bind p53 in two distinct ways. Protein Sci. 2008 Oct;17(10):1663-70


Ghavami S, Kerkhoff C, Chazin WJ, Kadkhoda K, Xiao W, Zuse A, Hashemi M, Eshraghi M, Schulze-Osthoff K, Klonisch T, Los M. S100A8/9 induces cell death via a novel, RAGE-independent pathway that involves selective release of Smac/DIABLO and Omi/HtrA2. Biochim Biophys Acta. 2008 Feb;1783(2):297-311


Hiratsuka S, Watanabe A, Sakurai Y, Akashi-Takamura S, Ishibashi S, Miyake K, Shibuya M, Akira S, Aburatani H, Maru Y. The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase. Nat Cell Biol. 2008 Nov;10(11):1349-55

Lim SY, Raftery M, Cai H, Hsu K, Yan WX, Hseih HL, Watts RN, Richardson D, Thomas S, Perry M, Geczy CL. S-nitrosylated S100A8: novel anti-inflammatory properties. J Immunol. 2008 Oct 15;181(8):5627-36


Turovskaya O, Foell D, Sinha P, Vogl T, Newlin R, Nayak J, Nguyen M, Olsson A, Nawroth PP, Bierhaus A, Varki N, Kronenberg M, Freeze HH, Srikrishna G. RAGE, carboxylated glycans and S100A8/A9 play essential roles in colitis-



associated carcinogenesis. Carcinogenesis. 2008 Oct;29(10):2035-43


Ito Y, Arai K, Nozawa R, Yoshida H, Hirokawa M, Fukushima M, Inoue H, Tomoda C, Kihara M, Higashiyama T, Takamura Y, Miya A, Kobayashi K, Matsuzuka F, Miyauchi A. S100A8 and S100A9 expression is a crucial factor for dedifferentiation in thyroid carcinoma. Anticancer Res. 2009 Oct;29(10):4157-61

Kerkhoff C, Ghavami S.. Innate immunity molecules S100A8/A9 involved in stress response and cancer biology. Anti-Inflammatory and Anti-Allergy Agents in Medicinal Chemistry, 2009; (8)4:279-281.

Srikrishna G, Freeze HH.. Endogenous damage-associated molecular pattern molecules at the crossroads of inflammation and cancer. Neoplasia. 2009 Jul;11(7):615-28. (REVIEW)

van Dieck J, Fernandez-Fernandez MR, Veprintsev DB, Fersht AR.. Modulation of the oligomerization state of p53 by differential binding of proteins of the S100 family to p53 monomers and tetramers. J Biol Chem. 2009 May 15;284(20):13804-11. Epub 2009 Mar 18.

Ang CW, Nedjadi T, Sheikh AA, Tweedle EM, Tonack S, Honap S, Jenkins RE, Park BK, Schwarte-Waldhoff I, Khattak I, Azadeh B, Dodson A, Kalirai H, Neoptolemos JP, Rooney PS, Costello E.. Smad4 loss is associated with fewer S100A8-positive monocytes in colorectal tumors and attenuated response to S100A8 in colorectal and pancreatic cancer cells. Carcinogenesis. 2010 Sep;31(9):1541-51. Epub 2010 Jul 9.

Fritz G, Botelho HM, Morozova-Roche LA, Gomes CM.. Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity. FEBS J. 2010 Nov;277(22):4578-90. doi: 10.1111/j.1742-4658.2010.07887.x. (REVIEW)

Ghavami S, Eshragi M, Ande SR, Chazin WJ, Klonisch T, Halayko AJ, McNeill KD, Hashemi M, Kerkhoff C, Los M.. S100A8/A9 induces autophagy and apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes that involves BNIP3. Cell Res. 2010 Mar;20(3):314-31. Epub 2009 Nov 24.

Ha YS, Kim MJ, Yoon HY, Kang HW, Kim YJ, Yun SJ, Lee SC, Kim WJ.. mRNA Expression of S100A8 as a Prognostic Marker for Progression of Non-Muscle-Invasive Bladder Cancer. Korean J Urol. 2010 Jan;51(1):15-20. Epub 2010 Jan 21.

Minami S, Sato Y, Matsumoto T, Kageyama T, Kawashima Y, Yoshio K, Ishii J, Matsumoto K, Nagashio R, Okayasu I.. Proteomic study of sera from patients with bladder cancer: usefulness of S100A8 and S100A9 proteins. Cancer Genomics Proteomics. 2010 Jul-Aug;7(4):181-9.

Saha A, Lee YC, Zhang Z, Chandra G, Su SB, Mukherjee AB.. Lack of an endogenous anti-inflammatory protein in mice enhances colonization of B16F10 melanoma cells in the lungs. J Biol Chem. 2010 Apr 2;285(14):10822-31. Epub 2010 Jan 29.

Su YJ, Xu F, Yu JP, Yue DS, Ren XB, Wang CL.. Up-regulation of the expression of S100A8 and S100A9 in lung adenocarcinoma and its correlation with inflammation and other clinical features. Chin Med J (Engl). 2010 Aug;123(16):2215-20.

Voss A, Bode G, Sopalla C, Benedyk M, Varga G, Bohm M, Nacken W, Kerkhoff C.. Expression of S100A8/A9 in HaCaT keratinocytes alters the rate of cell proliferation and differentiation. FEBS Lett. 2011 Jan 21;585(2):440-6. Epub 2010 Dec 28.


Kerkhoff C, Ghavami S. S100A9 (S100 calcium binding protein A9). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):746-757.

Gene Section Mini Review




TRIAP1 (TP53 regulated inhibitor of apoptosis 1) Veruska Alves, Roberta Felix, Andre Vettore, Gisele Colleoni

Universidade Federal de Sao Paulo - UNIFESP, Laboratory of Cancer Molecular Biology, Sao Paulo, Brazil (VA, RF, AV, GC)


Online updated version : http://AtlasGeneticsOncology.org/Genes/TRIAP1ID44577ch12q24.html DOI: 10.4267/2042/46029


Identity Other names: HSPC132; p53CSV; P53CSV; WF-1

HGNC (Hugo): TRIAP1

Location: 12q24.31

DNA/RNA Description 2452 bases, starts at 119366147 and ends at 119368598 bp from promoter with minus strand orientation.

Transcription This gene contains 2 introns which transcription gives 3

different mRNAs, 2 alternatively spliced variants and 1 unspliced form that encodes good proteins (see figure 1).

Protein Note The P53CSV protein is involved in programmed cell death. It contains a p53-binding site and it is induced when cells are at low genotoxic stress. It is probably involved in cell survival by interaction between Apaf-1 (apoptosis protease activating factor 1) and heat shock protein 70 (Hsp70) with subsequent inhibition of caspase-9 activation.

Figure 1.

TRIAP1 (TP53 regulated inhibitor of apoptosis 1) Alves V, et al.


Description This protein contains 76 amino acids and has 8786 (Da) of weight.

Figure 2.

Localisation The protein is localized in cytoplasm and perinuclear region.

Function P53CSV is a novel p53-target gene. This gene can modulate apoptotic pathways by interaction with heat shock protein 70 (HSP70), preventing the induction of apoptosis. When cells are submitted to low levels of genotoxic stress, it is an important player in P53-mediated cell survivor pathway (Park and Nakamura, 2005; Felix et al., 2009). P53CSV can inhibit apoptosis through interaction with APAF1 and HSP70 complex.

Mutations Note There are two identified alterations until now. One of them is located at position 270 of mRNA and the allele G (guanine) is switched to the allele C (cytosine) at position 77 of the amino acid sequence protein. The other one is a synonymous alteration localized at position 160 of mRNA involving the protein residue Leucine. The allele C (cytosine) is switched to the allele T (thymine) at position 40 of the amino acid sequence protein (NCBI).

Implicated in Multiple myeloma Note Felix et al. (2009), described that P53CSV gene was upregulated in multiple myeloma SAGE (serial analysis of gene expression) library when compared to normal/reactive plasma cells.

Figure 3. Hypothetical illustration about TRIAP1 (P53CSV) involvement in the p53-dependent cell survival pathway. The TRIAP1 mediates cell survival at low level of genotoxic stress by inhibiting activation of the complex APAF-1/caspase-9/cytochrome C preventing the apoptosis induction.

TRIAP1 (TP53 regulated inhibitor of apoptosis 1) Alves V, et al.


Figure 4. Hypothetical P53CSV mechanism of action in interaction with heat shock protein 70 in normal and tumor cells (Felix et al.,

2009)

They suggested that the interaction between P53CSV/Hsp70 should be evaluated as a potential target for multiple myeloma patients. Real time quantitative PCR analyses confirmed upregulation of P53CSV in 90% of multiple myeloma plasma samples cells.

Inflammatory stress Note Staib et al. (2005) reported P53CSV expression in colon carcinoma cells in the course of inflammatory responses associated with four microenvironmental components: nitric oxide, hydrogen peroxide, DNA replication arrest, and hypoxia.

Solid cancers Note Yu Kun et al. (2008), using a genome-wide computational strategy identified genes exhibiting precise transcriptional control in solid tumors and evaluated if they linked to multiple cancer-related pathways such as metastatic and invasive potential. siRNA knockdown of five genes supports the existence of precisely controlled genes in solid tumors, including P53CSV.

References Park WR, Nakamura Y. p53CSV, a novel p53-inducible gene involved in the p53-dependent cell-survival pathway. Cancer Res. 2005 Feb 15;65(4):1197-206

Staib F, Robles AI, Varticovski L, Wang XW, Zeeberg BR, Sirotin M, Zhurkin VB, Hofseth LJ, Hussain SP, Weinstein JN, Galle PR, Harris CC. The p53 tumor suppressor network is a key responder to microenvironmental components of chronic inflammatory stress. Cancer Res. 2005 Nov 15;65(22):10255-64

Yu K, Ganesan K, Tan LK, Laban M, Wu J, Zhao XD, Li H, Leung CH, Zhu Y, Wei CL, Hooi SC, Miller L, Tan P. A precisely regulated gene expression cassette potently modulates metastasis and survival in multiple solid cancers. PLoS Genet. 2008 Jul 18;4(7):e1000129

Felix RS, Colleoni GW, Caballero OL, Yamamoto M, Almeida MS, Andrade VC, Chauffaille Mde L, Silva WA Jr, Begnami MD, Soares FA, Simpson AJ, Zago MA, Vettore AL. SAGE analysis highlights the importance of p53csv, ddx5, mapkapk2 and ranbp2 to multiple myeloma tumorigenesis. Cancer Lett. 2009 Jun 8;278(1):41-8


Alves V, Felix R, Vettore A, Colleoni G. TRIAP1 (TP53 regulated inhibitor of apoptosis 1). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):758-760.

Leukaemia Section Mini Review




t(X;11)(q13;q23) Adriana Zamecnikova

Kuwait Cancer Control Center, Laboratory of Cancer Genetics, Department of Hematology, Shuwaikh, 70653 Kuwait (AZ)


Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0X11ID1127.html DOI: 10.4267/2042/46030

This article is an update of : Bojesen SE. t(X;11)(q13;q23). Atlas Genet Cytogenet Oncol Haematol 2001;5(4):291-292. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology

Identity

t(X;11)(q13;q23) G- banding - Courtesy Melanie Zenger and Claudia Haferlach.

Clinics and pathology Disease Described in infants and young children; 4 cases of acute myeloid leukemia (AML) (Pui et al., 1987; Raimondi et al., 1989; Pui et al., 1989; Harrison et al., 1998) and one case of acute lymphoblastic leukemia (ALL) (Smith et al., 1973). With one exception, the FAB types in cases of AML were M4. Peripheral leucocytes at diagnosis of this ALL case were cultured and are presently known as the KARPAS-45 cell line (Karpas et al., 1977). In addition, MLL/AFX1 fusion was confirmed in an AML case with highly complex change originally published involving the Xq22 locus (Nacheva et al., 1982; Parry et al., 1994; Borkhardtet al., 1997).

Note This translocation has also been found in 2 cases of CLL (Bentz et al., 1995; Kalla et al., 2005). In one case

a t(X;11)(q13;q23) was cloned revealing the involvement of BRWD3 gene recently located on Xq21.1 (Kalla et al., 2005). Phenotype/cell stem origin Suggested involvement of a pluripotent stem cell or a myeloid progenitor cell; very rarely in lymphoid lineage.

Etiology No known prior exposure; case of AML M2 developed in a 6 years old male previously treated by chemotherapy and radiotherapy for acute lymphoblastic leukemia (Harrison et al., 1998).

Epidemiology 6 cases to date, children aged 6 months to 5 years, male predominance; sex ratio 4M/2F.

Clinics From the known data: WBC: 21.6 to 91x109/L, case with a complex t(X;11) associated with fever, enlargement of the liver, spleen and parotid glands, blood in the stool (Karpas et al., 1977); mediastinal mass, dyspnoea, no hepatosplenomegaly, WBC: 5x109/L in T-ALL (Smith et al., 1973).

Prognosis Survival: poor prognosis; 3 patients died within a year after diagnosis, and one patient died after 24.5 months.

Genetics Note Breakpoints difficult to ascertain in suboptimal preparations.

t(X;11)(q13;q23) Zamecnikova A


Cytogenetics Probes Pooled cDNA FISH-probes from AFX1: AFX 12, 115, 106, 108, 114.

Additional anomalies Part of a highly complex change in one case; in KARPAS 45: Hypotetraploidy. -Y, -3, +6, -14, -18 t(1;5)(q21;q12.2)x2, del4(4)(q22), del(16)(q22).

Genes involved and proteins Note Cloning and characterization of AFX the gene that fuses to MLL in one case of AML and in the leukemic cell line.

AFX1 (All-1 fusion partner on chromosome X, MLLT7) Location Xq13

DNA/RNA AFX consists of two exons and encodes for a protein of 501 amino acids.

Protein Transcription factor; high degree of homology between AFXI and the forkhead protein family and highly homologous to the human FKHR protein.

MLL (Mixed lineage leukemia gene, ALL1, HRX, and Hrtx) Location 11q23

DNA/RNA The Mixed-Lineage Leukemia gene consists of at least 36 exons, encoding a 3969 amino-acid nuclear protein with a molecular weight of nearly 430 kDa.

Protein Multidomain molecule; shares homology with the Drosophila trithorax protein; function as a positive regulator of gene expression in embryonic development and hematopoiesis.

Result of the chromosomal anomaly Hybrid gene Note 5' MLL - AFX 3' as well as the 5' AFX - MLL 3'.

Fusion protein Note Chimeric proteins that contain the N-terminus of MLL; hybrid transcript MLL-AFX1 contains the code for the following domains: AT-hook + DNA methyltransferase (from MLL) + part, aa 147-187 of the DNA-binding domain (from AFX1).

References Smith JL, Clein GP, Barker CR, Collins RD. Characterisation of malignant mediastinal lymphoid neoplasm (Sternberg sarcoma) as thymic in origin. Lancet. 1973 Jan 13;1(7794):74-7

Karpas A, Hayhoe FGJ, Greenberger JS, Barker CR, Cawley JC, Lowenthal RM, Moloney WC.. The Establishment and Cytological, Cytochemical and Immunological Characterisation of Human Haemic Cell Lines: Evidence for Heterogeneity. Leukaemia Res 1977; 1 : 35-49.

Nacheva E, Fischer P, Haas O, Manolova Y, Manolov G, Levan A.. Acute myelogenous leukemia in a child with primary involvement of chromosomes 11 and X. Hereditas. 1982;97(2):273-88.

Pui CH, Raimondi SC, Murphy SB, Ribeiro RC, Kalwinsky DK, Dahl GV, Crist WM, Williams DL.. An analysis of leukemic cell chromosomal features in infants. Blood. 1987 May;69(5):1289-93.

Pui CH, Behm FG, Raimondi SC, Dodge RK, George SL, Rivera GK, Mirro J Jr, Kalwinsky DK, Dahl GV, Murphy SB.. Secondary acute myeloid leukemia in children treated for acute lymphoid leukemia. N Engl J Med. 1989 Jul 20;321(3):136-42.

Raimondi SC, Kalwinsky DK, Hayashi Y, Behm FG, Mirro J Jr, Williams DL.. Cytogenetics of childhood acute nonlymphocytic leukemia. Cancer Genet Cytogenet. 1989 Jul 1;40(1):13-27.

Parry P, Wei Y, Evans G.. Cloning and characterization of the t(X;11) breakpoint from a leukemic cell line identify a new member of the forkhead gene family. Genes Chromosomes Cancer. 1994 Oct;11(2):79-84.

Bentz M, Huck K, du Manoir S, Joos S, Werner CA, Fischer K, Dohner H, Lichter P.. Comparative genomic hybridization in chronic B-cell leukemias shows a high incidence of chromosomal gains and losses. Blood. 1995 Jun 15;85(12):3610-8.

Borkhardt A, Repp R, Haas OA, Leis T, Harbott J, Kreuder J, Hammermann J, Henn T, Lampert F.. Cloning and characterization of AFX, the gene that fuses to MLL in acute leukemias with a t(X;11)(q13;q23). Oncogene. 1997 Jan 16;14(2):195-202.

Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, Mugneret F, Moorman AV, Secker-Walker LM.. Ten novel 11q23 chromosomal partner sites. European 11q23 Workshop participants. Leukemia. 1998 May;12(5):811-22.

Kalla C, Nentwich H, Schlotter M, Mertens D, Wildenberger K, Dohner H, Stilgenbauer S, Lichter P.. Translocation t(X;11)(q13;q23) in B-cell chronic lymphocytic leukemia disrupts two novel genes. Genes Chromosomes Cancer. 2005 Feb;42(2):128-43.


Zamecnikova A. t(X;11)(q13;q23). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):761-762.





t(X;11)(q22;q23) Adriana Zamecnikova



Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0X11q22q23ID1573.html DOI: 10.4267/2042/46031


Identity

Partial karyotypes showing the chromosomal translocation t(X;11)(q22;q23).

Clinics and pathology Disease The chromosomal translocation t(X;11)(q22;q23) occurs very rarely, with only three cases of infants young children having been described in the literature; 2 AML cases: a 3 years old male, diagnosed with

AML-M2 (Harrison et al., 1998) and 2 years old female diagnosed with acute megakaryoblastic leukemia (FAB type M7) (Ribeiro et al., 1993). The one ALL case described in a 4 years old male had a complex karyotype with chromosomal translocation t(11;14)(q13;q32), and monosomy 22 (Soszynska et al., 2008). Of note, the fourth AML (FAB type M2) case reported by Slater in a 10 months old male was shown

t(X;11)(q22;q23) Zamecnikova A


to involve the SEPTIN6 gene located on Xq24 (Slater et al., 2002).

Phenotype/cell stem origin Suggested involvement of a pluripotent stem cell or a myeloid progenitor cell; myeloid lineage.

Etiology No known prior exposure.

Epidemiology Only 3 cases to date, sex ratio 2M/1F.

Prognosis From the known data, the 3 years old male, diagnosed with AML-M2 remained alive in complete remission at 97 months; the ALL patient was in complete remission after 39 months.

Cytogenetics Note Breakpoints difficult to ascertain; cytogenetic appearance may be similar to t(X;11)(q13;q23) involving the AFX gene that fuses to MLL in acute leukemias.

Cytogenetics morphological t(X;11)(q22;q23).

Additional anomalies Sole abnormality in AML-M2 case, part of a hyperploid karyotype associated with +6, +8, +19, +21, +21 in a child with acute megakaryoblastic leukemia and complex karyotype in ALL case associated with t(11;14)(q13;q32), and monosomy 22, indicating that the t(X;11)(q22;q23) is likely to be a secondary anomaly to t(11;14)(q13;q32) in ALL.

Genes involved and proteins Note The gene in Xq22 is yet unknown, it is therefore uncertain whether this translocation involve a new MLL partner.

MLL Location 11q23

Note The MLL gene is frequently disrupted by a variety of chromosomal rearrangements that occur in acute myeloblastic leukemia (AML) and in acute lymphoblastic leukemia (ALL), with a peak incidence in infant leukemia as well as in secondary, topoisomerase II inhibitor-related leukemia.

DNA/RNA The MLL genomic structure consists of 36 exons distributed over 100 kb, the mRNA of ~11.9 kb encodes a 3969 amino-acid nuclear protein with a molecular weight of of 430 kDa.

Protein The MLL protein is a multi-domain molecule with regions of homology to diverse proteins; a major regulator of class I homeobox (HOX) gene expression.

Result of the chromosomal anomaly Hybrid gene Note 5' MLL - PARTNER GENE 3'. MLL translocation breakpoints cluster within an 8.3-kb region spanning exons 5-11; genomic breakpoint junction usually created on the der(11) chromosome.

Fusion protein Oncogenesis Expression of a chimeric protein with actively transforming properties; altered patterns of MLL activity in hematopoietic stem cells resulting in blockage of hematopoietic maturation.

References Ribeiro RC, Oliveira MS, Fairclough D, Hurwitz C, Mirro J, Behm FG, Head D, Silva ML, Raimondi SC, Crist WM. Acute megakaryoblastic leukemia in children and adolescents: a retrospective analysis of 24 cases. Leuk Lymphoma. 1993 Jul;10(4-5):299-306

Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, Mugneret F, Moorman AV, Secker-Walker LM. Ten novel 11q23 chromosomal partner sites. European 11q23 Workshop participants. Leukemia. 1998 May;12(5):811-22

Slater DJ, Hilgenfeld E, Rappaport EF, Shah N, Meek RG, Williams WR, Lovett BD, Osheroff N, Autar RS, Ried T, Felix CA. MLL-SEPTIN6 fusion recurs in novel translocation of chromosomes 3, X, and 11 in infant acute myelomonocytic leukaemia and in t(X;11) in infant acute myeloid leukaemia, and MLL genomic breakpoint in complex MLL-SEPTIN6 rearrangement is a DNA topoisomerase II cleavage site. Oncogene. 2002 Jul 11;21(30):4706-14

Soszynska K, Mucha B, Debski R, Skonieczka K, Duszenko E, Koltan A, Wysocki M, Haus O. The application of conventional cytogenetics, FISH, and RT-PCR to detect genetic changes in 70 children with ALL. Ann Hematol. 2008 Dec;87(12):991-1002


Zamecnikova A. t(X;11)(q22;q23). Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):763-764.





t(X;11)(q24;q23) MLL-SEPTIN6 Adriana Zamecnikova



Online updated version : http://AtlasGeneticsOncology.org/Anomalies/t0X11q24q23ID1219.html DOI: 10.4267/2042/46018


Clinics and pathology Disease All the described cases were diagnosed as having acute myeloid leukemia (AML), classified as FAB- M2 (5 cases), M4 (4 cases), M1 (1 case) and M5 (1 case), indicating that AML with the MLL-SEPTIN6 fusion gene have a tendency to differentiate into the myeloid lineage. All the patients were infants and young children aged 0 to 29 months, suggesting that AML with t(X;11)(q24;q23) is a subgroup of infant leukemia.

Phenotype/cell stem origin Suggested involvement of a pluripotent stem cell or a myeloid progenitor cell.

Etiology No known prior exposure; putative association with in utero exposure to recurrent genetic insults.

Epidemiology Involvement of the SEPTIN6 gene on Xq24 in MLL rearrangements occurs very rarely, with only 13 cases (7 males, 6 females) having been documented in the literature. In addition, 3 AML cases with chromosomal translocation t(X;11)(q24;q23) (3 males aged 0 to 6 years), which also potentially could be found to involve MLL and SEPTIN6 genes have been described confirming the recurrent nature of this translocation.

Clinics Hepatosplenomegaly (3 cases), massive and diffuse adenopathy (2 cases), lympadenophaty (2 cases), CNS involvement in 2 cases as well as chloroma, scalp nodules, mucosal and cutaneous pallor, bluish cutaneous nodules and petecchiae were described. Notably, in 2 of the patients bilateral and right exophthalmus was described. Peripheral blood

leukocytosis (WBC 13.4x109/L to 608x109/L; mean 223x109/L), anemia and thrombocytopenia were reported in the majority of patients.

Prognosis From the 4 patients treated with chemotherapy one is alive (13+ months), 3 patients died 1 to 8 months from diagnosis; 8 patients received bone marrow transplantation, among them 2 of the patients died after 9 and 11 months, 6 patients are alive (one months to 7 years) indicating the prognosis is rather poor.

Cytogenetics Cytogenetics morphological Chromosomal rearrangements of 11q23 and Xq24 resulting in MLL-SEPT6 fusions are often complex and sometimes cryptic associated with 11q insertions. In addition, molecular detection of MLL-SEPTIN6 transcripts in cases with normal cytogenetics and in patients with chromosomal Xq22 breakpoints indicates the difficulty in precise chromosomal breakpoint definition.

Additional anomalies +6 (2 cases), del(11)(q13), i(10)(q10), add(X)(p11) described in single cases.

Variants At least four different types of chromosomal rearrangements have been described that can generate the MLL-SEPT6 fusion.

Genes involved and proteins Note MLL and SEPTIN6 reside on their respective chromosome loci in reverse orientation, that is, the orientation of the MLL gene is centromere-to-telomere

t(X;11)(q24;q23) MLL-SEPTIN6 Zamecnikova A


and the orientation of the SEPTIN6 gene is reversed, telomere to centromere at Xq24. This may explain why the MLL/SEPTIN6/Xq24 rearrangement is often associated with complex translocations and with 11q insertions.

MLL (Mixed lineage leukemia gene, ALL1, HRX, and HRTX) Location 11q23

DNA/RNA The MLL genomic structure consists of at least 36 exons spanning a region of ~89 kb. The mRNA of ~11.9 kb encodes a massive nuclear protein of 3969 amino acids with a molecular weight of nearly 430 kDa.

Protein Multi-domain protein characteristic of several domains with assigned activities including an N terminus with DNA binding motifs; AT-hook motifs, 4 cysteine-rich zinc fingers, a transactivation domain, and a highly conserved C-terminal domain with histone methyltransferase activity. Nuclear protein; a major regulator of class I homeobox (HOX) gene expression; functions as a positive regulator of gene expression in early embryonic development and hematopoiesis regulation.

SEPTIN6 Location Xq24

DNA/RNA The SEPT6 gene, belongs to the evolutionarily conserved family of genes of septins consisting of 12 exons. Four types of transcripts: 2.3 kb, 2.7 kb, 3.1 kb and 4.6 kb coding for three isoforms. SEPT6 is ubiquitously expressed in tissues; in the human, several alternatively spliced SEPTIN6 transcripts are differentially expressed in adult and fetal tissues.

Protein 434 amino acids; 49717 Da.

SEPT6 is a GTP-binding protein with a central conserved ATP-GTP binding motif, a lysin rich region, a variable N-terminal extension domain and a C-terminal coiled coil. May function in heteropolymeric complexes; roles in GTPase signaling, cell division, cytokinesis, cytoskeletal filament formation, cell polarity, and oncogenesis. Septins, a family of conserved GTP-binding proteins, are characteristically found in the heteropolymeric filaments and associate with cellular membranes, microtubules and actin filaments which are assembled from asymmetrical heterotrimers, composed of SEPT2, SEPT6 and SEPT7 that associate head-to-head to form a hexameric unit. Mammalian septins localize in the cytoplasm and assemble into heteromeric complexes composed of three or more septin subunits.

Result of the chromosomal anomaly Hybrid gene Note 5' MLL - SEPTIN6 3' The MLL genomic breakpoints in MLL-SEPT6 AML patients in all cases occurred in the MLL 8.3 kb breakpoint cluster region (BCR) and seem to occur preferentially in the telomeric half (between introns 7 and 11) of the MLL BCR. In the majority of reported cases 5' MLL sequences joined in-frame with SEPTIN6 downstream of SEPT6 exon 1. In rare cases, out-of-frame fusion between MLL exon 7 and SEPT6 exon 2, with splicing of MLL exon 6 have been described. The breakpoint junctions in the SEPT6 intron 1 mapped to the vicinity of GC-rich low-complexity repeats, Alu repeats, and a topoisomerase II recognition sequence raising the possibility that the non-homologous DNA end-joining pathway may be involved in the in the generation of MLL-SEPT6 rearrangements in infant acute myeloid leukemia and a putative association with in utero exposure to topoisomerase II inhibitors has been hypothesized.

Schematic representation of MLL-SEPTIN6 fusion protein.

t(X;11)(q24;q23) MLL-SEPTIN6 Zamecnikova A


Transcript 5'-MLL/SEPTIN6-3' chimeric transcript.

Fusion protein Note The MLL-SEPT6 chimeric protein consists of the AT-hook DNA-binding, the DNA methyltransferase, the and repression domains of MLL and almost the entire open reading frame of SEPT6 including the central conserved ATP-GTP binding motif.

Expression / Localisation MLL fusion genes express in- frame chimeric proteins residing in the nucleus.

Oncogenesis MLL is fused with a partner gene in MLL-related leukemias leading to the aberrant activation of target genes, including HOX genes. The phenotype depends on the fusion partner, indicating that each fusion partner is critical for the leukemogenesis. Among partner genes, septins are the protein family most frequently involved in rearrangements with MLL, suggesting that SEPTIN family members are particularly vulnerable to form MLL translocations. MLL fusions with several different SEPTIN family members (SEPT2, SEPT5, SEPT9, and SEPT11) are preferentially associated with myeoloblastic rather than lymphoblastic leukemogenesis suggesting an important common pathway to leukaemogenesis in AML with these translocations. The observation that overexpression of SEPT6 itself does not lead to the myeloid immortalization of murine hematopoietic progenitors in vitro, whereas the overexpression of MLL-SEPT6 does indicate that the fusion partner-mediated homooligomerization of MLL-SEPT6 through its intact GTP-binding domain and coiled-coil region in the nucleus is essential to immortalize hematopoietic progenitors. However, MLL-SEPT6 rearrangment induced lethal myeloproliferative disease with long latency in mice, but not acute leukemia in experimental models. These findings suggest that secondary genotoxic effects on DNA repair and/or cell-cycle regulation are required for oncogenesis in MLL-SEPT6 associated leukemias.

References Köller U, Haas OA, Ludwig WD, Bartram CR, Harbott J, Panzer-Grümayer R, Hansen-Hagge T, Ritter J, Creutzig U, Knapp W. Phenotypic and genotypic heterogeneity in infant acute leukemia. II. Acute nonlymphoblastic leukemia. Leukemia. 1989 Oct;3(10):708-14

Harrison CJ, Cuneo A, Clark R, Johansson B, Lafage-Pochitaloff M, Mugneret F, Moorman AV, Secker-Walker LM.

Ten novel 11q23 chromosomal partner sites. European 11q23 Workshop participants. Leukemia. 1998 May;12(5):811-22

Nakata Y, Mori T, Yamazaki T, Suzuki T, Okazaki T, Kurosawa Y, Kinoshita A, Ohyashiki K, Nakazawa S. Acute myeloid leukemia with hypergranular cytoplasm accompanied by t(X;11)(q24;q23) and rearrangement of the MLL gene. Leuk Res. 1999 Jan;23(1):85-8

Borkhardt A, Teigler-Schlegel A, Fuchs U, Keller C, König M, Harbott J, Haas OA. An ins(X;11)(q24;q23) fuses the MLL and the Septin 6/KIAA0128 gene in an infant with AML-M2. Genes Chromosomes Cancer. 2001 Sep;32(1):82-8

Ono R, Taki T, Taketani T, Kawaguchi H, Taniwaki M, Okamura T, Kawa K, Hanada R, Kobayashi M, Hayashi Y. SEPTIN6, a human homologue to mouse Septin6, is fused to MLL in infant acute myeloid leukemia with complex chromosomal abnormalities involving 11q23 and Xq24. Cancer Res. 2002 Jan 15;62(2):333-7

Slater DJ, Hilgenfeld E, Rappaport EF, Shah N, Meek RG, Williams WR, Lovett BD, Osheroff N, Autar RS, Ried T, Felix CA. MLL-SEPTIN6 fusion recurs in novel translocation of chromosomes 3, X, and 11 in infant acute myelomonocytic leukaemia and in t(X;11) in infant acute myeloid leukaemia, and MLL genomic breakpoint in complex MLL-SEPTIN6 rearrangement is a DNA topoisomerase II cleavage site. Oncogene. 2002 Jul 11;21(30):4706-14

Fu JF, Liang DC, Yang CP, Hsu JJ, Shih LY. Molecular analysis of t(X;11)(q24;q23) in an infant with AML-M4. Genes Chromosomes Cancer. 2003 Nov;38(3):253-9

Kim HJ, Ki CS, Park Q, Koo HH, Yoo KH, Kim EJ, Kim SH. MLL/SEPTIN6 chimeric transcript from inv ins(X;11)(q24;q23q13) in acute monocytic leukemia: report of a case and review of the literature. Genes Chromosomes Cancer. 2003 Sep;38(1):8-12

Ono R, Nakajima H, Ozaki K, Kumagai H, Kawashima T, Taki T, Kitamura T, Hayashi Y, Nosaka T. Dimerization of MLL fusion proteins and FLT3 activation synergize to induce multiple-lineage leukemogenesis. J Clin Invest. 2005 Apr;115(4):919-29

Kadkol SS, Bruno A, Oh S, Schmidt ML, Lindgren V. MLL-SEPT6 fusion transcript with a novel sequence in an infant with acute myeloid leukemia. Cancer Genet Cytogenet. 2006 Jul 15;168(2):162-7

Strehl S, König M, Meyer C, Schneider B, Harbott J, Jäger U, von Bergh AR, Loncarevic IF, Jarosova M, Schmidt HH, Moore SD, Marschalek R, Haas OA. Molecular dissection of t(11;17) in acute myeloid leukemia reveals a variety of gene fusions with heterogeneous fusion transcripts and multiple splice variants. Genes Chromosomes Cancer. 2006 Nov;45(11):1041-9

Cerveira N, Micci F, Santos J, Pinheiro M, Correia C, Lisboa S, Bizarro S, Norton L, Glomstein A, Asberg AE, Heim S, Teixeira MR. Molecular characterization of the MLL-SEPT6 fusion gene in acute myeloid leukemia: identification of novel fusion transcripts and cloning of genomic breakpoint junctions. Haematologica. 2008 Jul;93(7):1076-80


Zamecnikova A. t(X;11)(q24;q23) MLL-SEPTIN6. Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):765-767.





S100 Protein Family and Tumorigenesis Geetha Srikrishna, Hudson H Freeze

Sanford-Burnham Medical Research Institute, 10905 Road to the Cure, San Diego, CA 92121, USA (GS, HHF)

Published in Atlas Database: January 2011

Online updated version : http://AtlasGeneticsOncology.org/Deep/S100ProtFamilyTumorID20092.html DOI: 10.4267/2042/46019


1. Introduction S100 proteins are a family of 25 homologous intracellular calcium-binding proteins characterized by EF hand motifs, low molecular weights (9-13 kDa), ability to form homodimers, heterodimers and oligomeric assemblies, and are characterized by tissue and cell-specific expression (Donato, 2001; Heizmann et al., 2002; Marenholz et al., 2004; Roth et al., 2003) (Figure 1). They are solely present in vertebrates (Donato, 2001). While human S100B, S100P, S100Z and S100G are located at 21q22, 4p16, 5q14 and Xp22 respectively, 21 of the human S100 genes (S100A1-S100A18, trichohyalin, filaggrin and repetin) are clustered at the chromosomal region 1q21, a region that is frequently deleted, translocated or duplicated in epithelial tumors and tumors of soft tissues (Craig et al., 1994; Donato, 2001; Gebhardt et al., 2006; Heizmann et al., 2002) (Figure 2). There is growing evidence that expression of S100 proteins is altered in many tumors, often in association with tumor progression, and they are therefore potentially important tumor biomarkers and therapeutic targets. However, their precise roles in tumor progression are not completely understood.

Figure 1. Dimer structure of S100 proteins. S100 proteins form a large multi-gene family of low molecular weight proteins that are characterized by calcium-binding EF hand motifs and exhibit remarkable tissue and cell-specific expression. They exist as homo and heterodimers, and oligomers. Each monomer consists of two EF-hands connected by a hinge region. (Reproduced from Heizmann CW, Fritz G, Schafer BW. Frontiers in Bioscience. 2002 May 1;7:d1356-68).

S100 Protein Family and Tumorigenesis Srikrishna G, Freeze HH


Figure 2. The S100 gene cluster on human chromosome 1q21. Most human S100 genes are located in the epidermal differentiation complex on chromosome 1q21, a region prone to rearrangements. Genes located in the cluster region are indicated, as well as two commonly used genomic markers (D1S1664 and D1S2346). p and q indicate the short and the long arm of the chromosome, respectively. Human S100B, S100P, S100Z and S100G are located on chromosomes 21q22, 4p16, 5q14 and Xp22 respectively. (Reproduced from Heizmann CW, Fritz G, Schafer BW. Frontiers in Bioscience. 2002 May 1;7:d1356-68).

2. Structure and functions of S100 proteins S100 proteins have two distinct EF-hand (helix-loop-helix motif) calcium-binding domains connected by a hinge region (Fritz et al., 2010). The canonical C-terminal calcium-binding EF- hand is common to all EF-hand proteins, while the N-terminal EF-hand is non-canonical. There is variable degree of sequence identity among S100 proteins. S100 proteins exist as

anti-parallel hetero and homodimers within cells. Calcium binding causes conformational changes that exposes binding sites for target proteins. Intracellular functions of S100 proteins have been extensively studied. These include calcium homeostasis, cell cycle regulation, cell growth and migration, cytoskeletal interactions, membrane trafficking, protein phosphorylation, and regulation of transcriptional factors among others (Donato, 2001; Heizmann et al., 2002; Marenholz et al., 2004; Roth et al., 2003). S100 proteins can also be released into extracellular space in response to stimuli, or during cell damage, and they promote responses including neuronal survival and extension (S100B), apoptosis (S100A4 and S100A6), inflammation (S100B, S100A8/A9, S100A11 and S100A12), autoimmunity (S100A8/A9), chemotaxis (S100A8/A9) and cell proliferation and survival (S100P, S100A7), thus effectively functioning as paracrine and autocrine mediators. S100B, S100P, S100A4, S100A6, S100A8/A9, S100A11 and S100A12 are known to act via interaction with cell surface receptors, primarily the Receptor for Advanced Glycation End Products (RAGE) (Donato, 2007; Leclerc et al., 2009), while S100A8/A9 also bind Toll-like receptors or TLRs (Vogl et al., 2007). Multimeric forms of S100 proteins appear to be necessary for the extracellular functions of S100 proteins (Donato, 2007; Leukert et al., 2006). Multimeric assemblies have been reported for S100A12, S100A4, S100B and S100A8/A9 (Fritz et al., 2010). Elevated S100 protein levels are associated with chronic inflammation, neurodegeneration, cardiomyopathies, atherosclerosis and cancer (Pietzsch, 2010).

3. S100 proteins and tumorigenesis A number of S100 proteins are up-regulated in tumors (Salama et al., 2008). With the identification of binding proteins and signaling pathways for at least a few of its family members, S100 proteins promise to offer both functional biomarkers and therapeutic targets in cancers, and the case for considering their importance is evolving rapidly (summarized in Table 1).

Table 1. Expression of S100 proteins in tumors*.

S100 proteins

Tumors in which expression is up-regulated

Tumors in which expression is down-regulated

Known functions / interactions / mechanism of action

S100A1 Renal, clear cell and papillary Endometriod subtype of ovarian and endometrial

Known RAGE ligand.

S100A2

Lung, non-small cell Pancreatic Gastric Thyroid, papillary and anaplastic

Oral squamous cell Prostate

Promotes p53 transcriptional activity and reduces expression of Cox-2.

S100A4 Breast Promotes tumor migration, invasion and



Colorectal Gastric Prostate Lung, non-small cell Ovarian Pancreatic Melanoma

angiogenesis. Regulates matrix metalloproteinases and interacts with p53 and inhibits p53 phosphorylation.

S100A6

Colorectal Pancreatic Gastric Hepatocellular Lung Melanoma

Known RAGE ligand.

S100A7

Breast, ER negative invasive, DCIS Bladder Skin

Possible interaction with Jab-1.

S100A8/A9

Gastric Colon Pancreatic Bladder Ovarian Thyroid Breast Skin

Interaction with RAGE and TLR4; promote tumor proliferation, and migration, accumulation of myeloid derived suppressor cells, activation of protumorigenic genes, and formation of premetastatic niches in distal organs.

S100A11

Uterine, smooth muscle Lymphoma, anaplastic large cell Pancreatic

Bladder Esophageal, squamous cell

S100B Melanoma (biomarker) Astrocytoma, anaplastic Glioblastomas

Interacts with p53 and down-regulates p53-mediated apoptosis in melanoma. Well known RAGE ligand.

S100P

Ovarian Pancreatic Breast Gastric Colorectal Prostate Lung

Activation of RAGE dependent signaling pathways.

* Relevant references are provided in the text. 3.1. S100A1 S100A1 is predominantly expressed in the heart, and to a lesser extent in the skeletal muscle (Heizmann et al., 2007; Leclerc et al., 2009). S100A1 is a key modulator of calcium homeostasis in the heart and targets several key regulators of sarcoplasmic reticulum including Ca2+ ATPase, ryanodine receptors and other targets, thereby enhancing cardiomyocyte performance. Dysregulation of cardiomyocyte S100A1 protein, and diminished levels following myocardial infarction contributes to cardiac hypertrophy and heart failure. Extracellularly, S100A1 exists as both a homodimer and heterodimer with S100B, S100A4 and S100P and interacts with RAGE (Leclerc et al., 2009). Besides cardiac and skeletal muscle, expression of S100A1 is

low in most normal tissues, but up-regulated in cancers of the kidneys, skin and ovary. S100A1 expression helps to differentiate subtypes of renal carcinoma. S100A1 protein is expressed in renal oncocytomas, and in clear cell and papillary renal cell carcinomas but not in chromophobe renal cell carcinomas (Cossu-Rocca et al., 2009; Li et al., 2007). In addition, S100A1 is a specific and sensitive immunohistochemical marker to differentiate nephrogenic adenoma from prostatic adenocarcinoma where it is not expressed (Cossu-Rocca et al., 2009). S100A1 messenger RNA and protein are up-regulated in ovarian tumors and ovarian cancer metastasis compared with normal ovarian tissues. In the endometrioid subtype of ovarian and endometrial cancers, there is a negative correlation



between relapse-free survival and S100A1 expression, suggesting that S100A1 is a marker for poor prognosis of endometrioid subtypes of cancer (DeRycke et al., 2009). 3.2. S100A2 S100A2 protein is present in many organs or tissues, with high expression in lung and kidney. Unlike most other S100 proteins, S100A2 is markedly down-regulated in many tumors suggesting that it acts as a tumor suppressor gene. It promotes transcriptional activity of p53 (Mueller et al., 2005) and reduces expression of Cox-2 (Tsai et al., 2006). On the other hand, S100A2 is also up-regulated in some tumors (Salama et al., 2008; Wolf et al., 2010). Loss of S100A2 expression has been associated with poor prognosis and shorter survival. S100A2 expression decreases in epithelial cells from normal to tumor stages, with the decrease more pronounced in glandular than in squamous epithelial tissue (Nagy et al., 2002). S100A2 expression is reduced in early-stage oral squamous cell carcinoma and is significantly associated with tumor recurrence and metastasis (Suzuki et al., 2005; Tsai et al., 2005). Patients with S100A2 positive laryngeal squamous cell carcinoma had a better relapse-free overall survival than patients with S100A2-negative tumors (Almadori et al., 2009). Benign prostate hyperplasia and prostatitis are characterized by higher levels of S100A2 than low-grade cancer, and expression is lost in high-grade and metastatic cancer specimens (Gupta et al., 2003). S100A2 is up-regulated in some tumors such as non-small cell lung carcinoma (Smith et al., 2004), with higher expression found in early-stage carcinomas, associated with reduced overall survival, and higher propensity to metastasis (Bartling et al., 2007; Bulk et al., 2009; Feng et al., 2001; Wang et al., 2005; Zech et al., 2006). In pancreatic cancer tissues and cell lines, S100A2 expression is significantly higher than in normal pancreatic tissues, and higher expression co-related with poor disease outcome (Biankin et al., 2009; Ohuchida et al., 2007). S100A2 is also over-expressed in gastric tumors compared to normal gastric mucosa (Lee et al., 2006). Varied expression has been reported with oesophageal squamous carcinoma (Cao et al., 2009; Imazawa et al., 2005; Ji et al., 2004). In thyroid carcinomas, S100A2 is absent in follicular adenomas and carcinomas, but up-regulated in papillary and anaplastic carcinomas, providing a possible marker for distinguishing these two types of thyroid carcinoma (Ito et al., 2005). 3.3. S100A4 S100A4, also called Metastatin due to its well-established association with tumor metastasis (Tarabykina et al., 2007; Sherbet, 2009; Boye and Maelandsmo, 2010), is normally found in the nervous system and believed to be involved in neuritogenesis. S100A4 is also expressed at low levels in normal human fibroblasts, monocytes, macrophages, T cells, neutrophils, and endothelial cells. Although its

expression is low in normal tissues, it is highly expressed in many tumors such as breast, colorectal, gastric, prostate and non-small cell lung cancer, ovarian and pancreatic cancers, and malignant melanoma (Boye and Maelandsmo, 2010; Sherbet, 2009). Expression is a significant predictor of patient survival and metastatic disease. S100A4 was first cloned from highly metastatic breast cancer cells, and since then its involvement in cancer metastasis has been substantiated by several studies (Boye and Maelandsmo, 2010; Sherbet, 2009; Tarabykina et al., 2007). Studies show that extracellular, intracellular, tumor-derived, and stroma-derived S100A4 all contribute to the metastatic process, and influence several steps in the metastatic cascade, including migration, invasion, and angiogenesis. Much of this metastatic potential has been linked to the ability of S100A4 to regulate matrix metalloproteinases, modulate cell motility, promote angiogenesis and epithelial mesenchymal transition, and the association of S100A4 expression with reduced expression of tumor suppressor genes such as p53 (Garrett et al., 2006; Salama et al., 2008). S100A4 interacts with p53 and inhibits p53-mediated tumor suppression by inhibiting its phosphorylation (Grigorian et al., 2001). The extensive association of S100A4 with tumor progression and metastasis has positioned it to be a target for novel therapeutic strategies. 3.4. S100A6 S100A6 is highly expressed in various organs, and on fibroblasts, epithelial and other cells (Leclerc et al., 2009; Lesniak et al., 2009). S100A6 is predominantly cytoplasmic protein but can translocate in the presence of Ca2+ to plasma membrane and the nuclear envelope. S100A6 expression can be up-regulated by platelet-derived growth factor, epidermal growth factor, tumor necrosis factor, retinoic acid and estrogen, and upon stress conditions, and at the transcriptional level by NF-κB. S100A6 interacts with many proteins including CacyBP/SIP, annexins II and XI, tropomycin and RAGE (Leclerc et al., 2009; Lesniak et al., 2009). S100A6 is overexpressed in many cancers including colorectal, pancreatic, gastric, hepatocellular and lung cancers, and melanoma. Expression in melanoma, pancreatic and colorectal cancers has been shown to correlate with tumor growth and metastatic progression suggesting a potential role for S100A6 in the development of malignancy (Lesniak et al., 2009; Salama et al., 2008). It is however down-regulated in prostate cancer and medulloblastoma. 3.5. S100A7 S100A7 was first identified in inflamed psoriatic skin, hence is also called psoriasin (Watson et al., 1998). It is released from keratinocytes around wounds and believed to exert cytokine and anti bacterial effects, and is also chemotactic for granulocyes, monocytes and lymphocytes (Eckert et al., 2004). Contrary to other S100 proteins, calcium binding does not induce large conformational changes in S100A7 (Streicher et al.,



2010). S100A7 is up regulated in breast, bladder and skin cancers (Salama et al., 2008). Increasing evidence show that S100A7 is up-regulated in ductal carcinoma in situ and ER negative invasive breast cancer and expression correlates with aggressive phenotype and patient survival (Emberley et al., 2004b). S100A7 interacts with Jab1 (c-jun activation domain binding protein 1) a protein with highly recognizable role in tumorigenesis to elicit functional effects in breast tumor progression (Emberley et al., 2004a). 3.6. S100A8/A9 S100A8 and S100A9 are expressed predominantly by myeloid cells, including granulocytes, monocytes, myeloid derived suppressor cells (MDSC) and other immature cells of myeloid lineage (Cheng et al., 2008; Goyette and Geczy, 2010; Roth et al., 2003; Sinha et al., 2008). Although the proteins are products of distinct genes, they are often co-expressed and function mainly as heterodimer of S100A8/A9 (calprotectin). Expression is down-regulated during macrophage and dendritic cell differentiation (Cheng et al., 2008; Lagasse and Clerc, 1988; Odink et al., 1987), but can be induced in epithelial cells, osteoclasts and keratinocytes (Gebhardt et al., 2006). S100A8/A9 released into the extracellular medium in response to cell damage or activation become danger signals (Damage Associated Molecular Pattern molecules or DAMP), which alert the host of danger by triggering immune responses and activating repair mechanisms through interaction with pattern recognition receptors (Donato, 2007; Ehrchen et al., 2009; Foell et al., 2007; Leclerc et al., 2009; Srikrishna and Freeze, 2009). Elevated S100A8/A9 is the hallmark of inflammatory conditions such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, cystic fibrosis and psoriasis (Foell et al., 2007; Roth et al., 2001; Roth et al., 2003). Critical roles for these proteins in endotoxin-induced lethality and systemic autoimmunity have recently been recognized (Loser et al., 2010; Vogl et al., 2007). In addition to expression within inflammatory milieu, strong up-regulation of these proteins has also been observed in many tumors, including gastric, colon, pancreatic, bladder, ovarian, thyroid, breast and skin cancers (Gebhardt et al., 2006; Salama et al., 2008). S100A8/A9 exhibit concentration-dependent dichotomy of function in tumors. At high concentrations S100A8/A9 exert apoptotic effects on tumor cells (Ghavami et al., 2004), while at low concentrations promote tumor cell growth (Ghavami et al., 2008; Turovskaya et al., 2008). S100A8/A9 also stimulate tumor cell migration at low concentrations (Ang et al., 2010; Hermani et al., 2006; Hiratsuka et al., 2006; Moon et al., 2008; Saha et al., 2010). S100A8/A9 regulate the accumulation of MDSC (Cheng et al., 2008; Sinha et al., 2008), which are immature myeloid cells that expand during inflammation and in tumors, and are potent suppressors of T-cell mediated immune responses (Dolcetti et al., 2008; Gabrilovich and

Nagaraj, 2009; Ostrand-Rosenberg and Sinha, 2009). Tumor derived factors promote sustained STAT3 dependent upregulation of S100A9 in myeloid precursors which results in inhibition of differentiation to DC and accumulation of MDSC (Cheng et al., 2008). S100A8/A9 are not only synthesized and secreted by MDSC, but they also have binding sites for S100A8/A9, and activate intracellular signaling that promote their migration (Sinha, et al., 2008), suggesting that S100A8/A9 support an autocrine feedback loop that sustains accumulation of MDSC in tumors (Ostrand-Rosenberg, 2008). They also bind to tumor cells and activate MAPK and NF-κB signaling pathways and specific downstream genes that promote tumorigenesis (Ghavami et al., 2009; Ichikawa et al., 2011). S100A8/A9 are involved in early metastatic processes. Expression of S100A8/A9 in myeloid and endothelial cells in premetastatic organs in response to soluble factors such as VEGF, TGFβ and TNFα expressed by distal primary tumors promotes homing of tumor cells to premetastatic niches (Hiratsuka et al., 2006). Recent studies argue for prominent roles for two pattern recognition receptors, TLR4 and RAGE, in S100A8/A9 mediated pathological effects (Gebhardt et al., 2008; Loser et al., 2010; Sinha et al., 2008; Turovskaya et al., 2008; Vogl et al., 2007). 3.7. S100A11 S100A11, also called S100C or calgizzarin, is expressed in many tissues including the placenta, heart, lung, and kidney (He et al., 2009; Inada et al., 1999; Salama et al., 2008). Expression levels are low in skeletal muscle and liver (Inada et al., 1999). S100A11 is overexpressed in uterine smooth muscle tumors (Kanamori et al., 2004), anaplastic large cell lymphomas (Rust et al., 2005), and pancreatic tumors (Ohuchida et al., 2006b), while significantly down-regulated in esophageal squamous cell (Ji et al., 2004) and bladder tumors (Memon et al., 2005). In bladder carcinoma, down-regulation of S100A11 is associated with poor prognosis and decreased survival, suggesting that S100A11 functions as a tumor suppressor (Memon et al., 2005). In pancreatic cancer, S100A11 is over-expressed in early stages and down-regulated in advanced tumors, again supporting a tumor-suppressor role and suggesting that S100A11 expression could be valuable in detecting early pancreatic tumors (Ohuchida et al., 2006b). On the other hand, S100A11 expression in prostate cancer is associated with advanced disease (Rehman et al., 2004), showing that S100A11 plays opposing roles depending on the tumor involved. 3.8. S100B S100B is one of the best-studied proteins of the S100 family and its interaction with RAGE has been well characterized (Donato, 2007; Donato et al., 2008; Leclerc et al., 2009). S100B is particularly abundant in the brain and is highly expressed by astrocytes, oligodendrocytes and Schwann cells. It is considered as an intracellular regulator and extracellular signal,



exerting concentration-dependent trophic as well as toxic effects on neurons (Donato et al., 2008). It also activates microglia, and may have a role in the pathogenesis of neurodegenerative disorders. S100B is over-expressed in anaplastic astrocytomas and glioblastomas (Camby et al., 1999), and melanomas (Salama et al., 2008). S100B interacts with p53 in melanomas and down-regulates p53 mediated apopotosis (Lin et al., 2010). S100B is the best-studied biomarker for melanoma (Gogas et al., 2009; Salama et al., 2008). Serum levels of S100B increases in a stage-dependent manner in patients with melanoma, reflecting tumor load, with decline during therapy and remission, and correlate well with overall survival. Elevated levels following therapy has shown to correlate with melanoma recurrence. S100B is thus the first of the S100 proteins to be validated in a clinical setting. 3.9. S100P S100P was first purified from placenta, but it is also expressed in many normal tissues including the GI tract, and in prostate and leukocytes (Arumugam and Logsdon, 2010; Leclerc et al., 2009). S100P is also expressed in many tumors, including ovarian, pancreatic, breast, gastric, colorectal, prostate, and lung carcinomas, and has been shown to be associated with poor clinical outcomes (Arumugam and Logsdon, 2010; Leclerc et al., 2009). Recent studies implicate DNA hypomethylation, bone morphogenic protein and non-steroidal anti-inflammatory drugs in the regulation of S100P expression in tumors (Hamada et al., 2009; Namba et al., 2009; Sato et al., 2004). S100P is expressed in pancreatic cancer cells, but not in pancreatic inflammation, and is also elevated early in preneoplastic cells, suggesting that S100P may be a useful biomarker for pancreatic cancer (Logsdon et al., 2003; Ohuchida et al., 2006a). S100P is absent in normal breast tissue but detected in hyperplasia, as well as in situ and invasive ductal carcinoma (Guerreiro Da Silva et al., 2000). Immunochemical studies show correlation between S100P and estrogen receptor expression, and with high-risk lesions and decreased survival (Schor et al., 2006). S100P has been shown to induce breast cancer metastasis (Wang et al., 2006). S100P is also up-regulated in colon cancer cells (Bertram et al., 1998; Fuentes et al., 2007), and in flat adenomas in the colon (Kita et al., 2006). S100P can be secreted, and interacts with RAGE on pancreatic and colon tumor cells (Arumugam et al., 2005; Fuentes et al., 2007), activating NF-κB and MAPK pathways (Fuentes et al., 2007). Efforts are therefore underway to develop small molecule inhibitors that block the interaction of S100P and RAGE.

Summary There is growing evidence that many S100 proteins are altered in human tumors. Future studies are likely to reveal molecular mechanisms that define the multiple and specific roles that S100 proteins play in tumor

progression and metastasis, providing novel therapeutic targets and biomarkers.

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Visualize Dynamic Chromosome Eisuke Gotoh

Division of Genetic Resources, National Institute of Infectious Diseases Japan 1-23-1, Toyama, Shin-juku-ku, Tokyo, 162-8640, Japan (EG)

Published in Atlas Database: January 2011

Online updated version : http://AtlasGeneticsOncology.org/Deep/VisuDynChID20093.html DOI: 10.4267/2042/46020

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2011 Atlas of Genetics and Cytogenetics in Oncology and Haematology Key words: chromosome condensation/compaction, chromosome structure, DNA replication, cell cycle, mitosis, S-phase, premature chromosome condensation (PCC), prematurely condensed chromosomes (PCCs), calyculin A, beads loading method A most miracle mysterious and profound event in eukaryote cell is how DNA folds to chromosomes. In human diploid cell (2n), for example, the total DNA (~6x109 nucleotide base pairs, a meter of length when fully relaxed) is packed to 46 chromosomes (22 pairs of autosomes and 1 pair of sex chromosomes) and contained in nuclei size of ~5 µm in diameter (Alberts et al., 1989). It is quite difficult to imagine how such long length thin fibrous linear molecule is folded in small sized chromosomes without entangling in a narrow nucleus space. Very earlier, the concept about the chromosome architecture formation during cell cycling was conceived as follows: (1) chromosomes are diffused over nucleus as decondensed form in G1-phase (Gap 1 phase), (2) DNA synthesis starts and chromosome replicates in S-phase (Synthesis of DNA phase), (3) DNA synthesis finished, the resulted chromosomes are duplicated and ready for cell division in G2-phase (Gap 2 phase) and then (4) chromosomes condense: separation/segregation and cell division occurs in M-phase (Mitotic phase). This traditional concept seems to tell that DNA replication and chromosome condensation are independent events that proceed in S- and M- cell cycle stage, respectively. Recently, number of accumulated evidences suggests a close relationship between DNA replication and chromosome condensation. Premature chromosome condensation (PCC) technique was introduced in the 1970's as a useful technique that allows the interphase nuclei to be visualized as condensed mitotic chromosome (Johnson and Rao, 1970; Johnson et al.,

1970; Sperling and Rao, 1974). Since then, a lot of studies including DNA replication and chromosome packaging have been archived using the PCC method (Hittelman and Rao, 1976; Rao et al., 1977; Hanks and Rao, 1980; Mullinger and Johnson, 1980; Lau and Arrighi, 1981; Mullinger and Johnson, 1983). These studies seem to teach that the different DNA packaging appearance in different sub-phase of S-phase suggest that the degree of chromosome condensation might be tightly coupled with the progressing of DNA replication. However, the limited available methodologies at that time did not allow the precise mechanism to be cleared. More recently, accumulated evidences have further concrete that eukaryote DNA replication/transcription is involved in compaction of chromosomes (Zink et al., 1998; Manders et al., 1999; Samaniego et al., 2002, Pflumm, 2002). Molecular genetic studies have also provided supporting evidence for the idea that mutation (in genes as HIRA/Tuple1, XCDT1, cdt1, Orc2, Orc3, Orc5, MCM2, MCM4, MCM10, RECQL4, required for DNA replication) showed abnormal phenotype in chromosome condensation (Loupart et al., 2000; Maiorano et al., 2000; Nishitani et al., 2000; Pflumm and Botchan, 2001; Christensen and Tye, 2003; McHugh and Heck, 2003; Prasanth et al., 2004), inherited diseases (D'Antoni et al., 2004; Sangrithi et al., 2005), genomic instability or prone to cancer (Tatsumi et al., 2006; Pruitt et al., 2007; Shima et al., 2007), or aberrant replication timing causes abnormal chromosome condensation (Loupart et al., 2000; Marheineke and Hyrien, 2001; Pliss et al., 2009). For more detailed knowledge about the DNA replication, see also the following excellent reviews; Bell and Dutta, 2002 and Masai et al., 2010. In the present article, using drug-induced PCC technique and direct Cy3-dUTP fluorescent replicating DNA by beads loading method, we demonstrate the

Visualize Dynamic Chromosome Gotoh E


dynamics of chromosome structure, formation and transition during the S-phase progression in which tight-coupled relation between DNA replication and chromosome condensation /compaction. Possible hypothetical chromosome condensation/compaction model involving the role of DNA replication will be suggested. Chromosome dynamics: DNA replication, condensation, decondensation, cohesion, separation, segregation, cytokinesis etc. Under a quite stringent and higher ordered mechanism, chromosomes condense during mitosis within a very short lapse of time in the mitotic phase. Mitotic phase is further divided into several subphases (preprophase, prophase, prometaphase, metaphase, anaphase and telophase), followed by cytokinesis. In the course of Mitotic phase, number of sequential drastic conformational transactions are proceeded as following: (1) chromatins condense to well-defined visible chromosomes under the microscope (2) mitotic spindle assemble begin, nuclear envelope breakdown into membrane vesicles, centriole and mitotic spindle formation followed by spindle attaches to chromosome centromeres (3) kinetocore microtubules align the chromosomes at metaphase plate (4) chromosome separation segregates to spindle poles (5) separated daughter chromatids reach the poles followed by the nuclear envelope re-forms (6) formation of contractile ring and cleavage furrows which constrict the cell center, cytokinesis, cell dividing into two daughter cells, chromosome decondensation in divided cells and finally re-entering the cells in the G1 phase (Alberts et al., 1989). The detailed of the whole mechanism is still almost unclear. However, number of molecules which involved in the mitotic events have been identified such as SMC proteins, including condensin (chromosome condensation), cohesion (chromosome cohesion of replicated chromosomes) (Swedlow and Hirano, 2003), NuMA protein for spindle pole formation (Chang et al., 2009; Haren et al., 2009; Silk et al., 2009; Torres et al., 2010), nuclear lamins (Moir et al., 2000), aurora kinases in centromere function (Tanno et al., 2006; Meyer et al., 2010; Tanno et al., 2010), shugoshin and protein protein phosphatase 2A in chromosome cohesion (Kitajima et al., 2006; Tanno et al., 2010), cdk1 in chromosome condensation, chromosome bi-orientation (Tsukahara et al., 2010), cyclin B, cdc2, cdc25 in chromosome condensation (Masui, 1974; Draetta and Beach, 1988; Dunphy et al., 1988; Kumagai and Dunphy, 1992), Polo and Rho in cytokinesis (Burkard et al., 2009; Wolfe et al., 2009; Li et al., 2010) and many other proteins. Chromosome dynamic consists of such number of various elements. Regarding these dynamics, numerous visualizing studies reported or in progression are achieved through the mitosis events, relatively easy to observe under microscope. However, visualizing approaches in chromosome dynamics, coupled with DNA replication,

is still limited. This restraint is due to difficulties as to observe the chromosomes in the S-phase: chromosomes are usually invisible at this stage, since they are decondensed. In the present review, we simply focus on visualizing the chromosome dynamics coupled with DNA replication during the S-phase progression and we show how replicating DNA is folded into higher order chromosomes. Tools to visualize the dynamic chromosomes Drug-induced premature chromosome condensation (PCC) method Cytogenetic analysis studies are usually performed on chromosomes. As condensed in mitosis, chromosomes are usually visible, but as they decondensed in the interphase, they are invisible (Manders et al., 1996; Gotoh and Durante, 2006). Therefore, it is practically difficult or even impossible to analyze the dynamics of chromosome condensation during the interphase by conventional chromosome methods such as colcemid block. Premature chromosome condensation (PCC) is a useful and a unique technique that allows the interphase nuclei to be visualized as a condensed form of mitotic chromosome (Johnson and Rao, 1970; Rao and Johnson, 1970). Conventional PCC has been carried out by cell fusion using either fusogenic viruses (i.e. Sendai virus) (Johnson and Rao, 1970) or polyethylene glycol (PEG) (Pantelias and Maillie, 1983) (cell fusion-mediated PCC). But these protocols are usually technically demanding and keenly depend on the activity of the virus or PEG. Virus-mediated PCC might be also problematic because of infectious viruses use. Moreover, resulting chromosomes are mixture of those inducer and recipient cells (Gotoh and Durante, 2006). Due to these restrictions, conventional PCC has been used in limited institutions. These drawbacks of the conventional PCC technique have been recently overcome with a much easier and more rapid technique using calyculin A or okadaic acid, specific inhibitors of protein phosphatases (drug-induced PCC technique) (Gotoh et al., 1995; Gotoh and Asakawa, 1996; Asakawa and Gotoh, 1997; Durante et al., 1998; Gotoh and Durante, 2006). Drug-induced PCC is becoming now 'popular' and has been used in a wide range of cytogenetic applications (Gotoh and Asakawa, 1996; Asakawa and Gotoh, 1997; Gotoh et al., 1999; Ito et al., 2002; Terzoudi et al., 2003; El Achkar et al., 2005; Gotoh and Tanno, 2005; Gotoh et al., 2005; Srebniak et al., 2005; Terzoudi et al., 2005; Deckbar et al., 2007; Gotoh, 2007; Beucher et al., 2009; van Harn et al., 2010). Thus, drug-induced PCC technique is suitable to visualize dynamic chromosomes particularly in interphase nuclei. This technique will be also useful and applicable in many fields of cytogenetic approaches including traditional chromosome analysis study, because the technique is very simple and much easier even than the conventional colcemid blocking method (Gotoh, 2009). Beads loading method



Cytogenetically visualization of the replicating DNA is certainly a most direct approach to identify the DNA replication dynamics. In the very earlier studies, the fibre autoradiography of DNA had been labeled with 3H-thymidine (Fakan and Hancock, 1974; Edenberg and Huberman, 1975; Hand, 1978). The spatial resolution of fibre autoradiography is, however, limited because the location of the silver grains, developed in photosensitive emulsion layer and covered the specimens, do not correctly reflect the actual regions of the foci incorporating the 3H-thymidine and the size of grains are not enough tiny to determine the precise location of replicating regions. More precise localization and measure the replication foci were then done using thymidine analog BrdU (Bromodeoxy Uridine) labeling and its antibodies (Nakamura et al., 1986; Mills et al., 1989; Nakayasu and Berezney, 1989). However, the resolution is still limited presumably because it is based on accessibility problems or size of immunocomplex (antigen/antibodies). Recently, the replication regions and chromosome formation in living cells were visualized using Cy5-dUTP directly labeled fluorescent DNA (Manders et al., 1999) by beads loading methods (McNeil and Warder, 1987). The procedure facilitate the analogues (Cy3-dUTP or Cy5-dUTP) to be incorporated in the cell nucleus in a very short time whereby transiently permeabilizes the cell membranes. This method allows the replicating DNA to be Cy3 fluorescently imaged within very short lapse of time. The obtaining fluorescence signal reflects the real incorporated site of analogue replicating DNA with a very fine signal resolution. Combined with the beads loading method and drug-induced PCC, dynamic study of chromosome condensation, involving DNA replication, has been realized (Gotoh, 2007). Chemicals and Instruments Calyculin A to induce PCC in interphase nuclei; purchased from Wako Chemicals (Osaka, Japan), dissolved in 100% DMSO, 100 µM of stock solution was stored at -20°C. Cy3- or Cy5-dUTP or other fluorochrome conjugated dUTP for labelling replicating DNA. Fluorochrome choice is strictly dependent on the light source (laser) equipped on the microscope available in the individual institute. BrdU (Bromo-deoxy-Uridine) is commonly used for labeling replicating DNA and gives substantial high quality signals. But combined to laser confocal microscope, Cy3-dUTP gives much more fine signals. Glass beads for beads loading methods. Various particle size and various surface treatment glass beads are provided from the company. DNA labeling efficiency using beads loading method may be varies in different cell lines, cell conditions, beads size and surface treatment. The optimum choice of beads for individual cell lines should be determined prior to the experiment.

Microscope. Confocal laser microscope is ideal for visualize dynamic chromosome coupled with DNA replication, although the conventional microscope may substantially work. Visualize the dynamics of chromosome structure formation coupled with DNA replication during S-phase Many studies, for visualizing the dynamics of chromosome condensation during cell division in mitosis, have been achieved and well documented. However, the visualizing study on the relationship between chromosome condensation and DNA replication is still limited. Several studies tried to define fairly well the replication foci distribution in interphase nuclei (Nakamura et al., 1986), but little is yet known about how replicating DNA is folded to higher order chromosomes (since chromosomes are invisible in interphase stage as they decondensed). To visualize the chromosome compaction dynamics coupled with DNA replication, more precisely in S-phase nucleus, the drug-induced PCC method was used (Gotoh et al., 1995; Asakawa and Gotoh, 1997; Johnson et al., 1999; Ito et al., 2002). The cells were unsynchronized because cell synchronization using DNA synthesis inhibitor such as thymidine may give some bias in DNA replication and consequently all phases of replication can be observed. Individual substage of S-phase can be easily identified by typical diagnostic appearances seen in different phases of S-PCCs (Mullinger and Johnson, 1983; Gollin et al., 1984; Hameister and Sperling, 1984; Savage et al., 1984; Gotoh et al., 1995; Gotoh and Durante, 2006). A drastic conformational change of chromosome structure formation along with the proceed of DNA replication, as shown in Fig. 1 (reproduced from Chromosoma. Gotoh, 2007; 116(5):453-462), is clearly revealed in PCCs following Cy3-dUTP loading. Cy3-dUTP loading procedure takes 10 minutes followed by 10 minutes of PCC induction and fixation (for details, see Materials and Methods in Chromosoma. Gotoh, 2007; 116(5):453-462). Accordingly, only replicated DNA in this short lapse will be fluoresced. Thus, the observed S-PCCs in the present study reflected the replication stages at most 20 minutes before the cell fixation. (i) In early S-phase, PCCs showed a cloudy spreading mass of thin fibres like a 'nebula', where numerous fine granular foci homogeneously distributed on overall the fibres (Fig. 1I), showing 'beads on a string' or 'particles on a string': these structures are observed under an electron microscope (Olins and Olins, 1974; Thoma et al., 1979). (ii) In the middle of S-phase, typical 'pulverized' PCCs were recognized; the size of foci was increased while the number of foci, unevenly distributed on chromosomes, was decreased. As shown in Fig. 1J, the foci become brighter. (iii) In the late S-phase, chromosomes were mostly condensed like mitotic chromosomes. Cy3-dUTP incorporated regions were recognized as band arrays inserted in the



condensed chromosome (Fig. 1K, indicated by arrows). The similar appearance of replication foci along longitudinally on chromosomes were previously reported on metaphase of kangaroo-rat kidney PtK1 cells (Ma et al., 1998). The size of foci is still up and their number is still down to the point that they could

be easily scored. (iv) In the very late S-phase, the number of foci is further reduced and predominantly they are localized at centromeric or telomeric regions (Fig. 1L, indicated by arrows). These regions are actually known as satellite heterochromatic DNA regions where DNA replicates at very late S (O'Keefe et al., 1992).

Figure 1: (1) DNA replication regions on prematurely condensed chromosomes (PCCs) of different substages of S-phase. Ten minutes after Cy3-dUTP loading, cells were condensed prematurely using 50 nM of calyculin A (Gotoh et al., 1995). From left to right column, (A,B,C) early S-phase PCCs, (D,E,F) middle S-PCCs, (G,H,I) late S-PCCs and (J,K,L) very late S-PCCs. (A,D,G,J) DAPI counterstained DNA, (B,E,H,K) Cy3-dUTP labelled DNA replication region and (C,F,I,L) Merged image of DAPI and Cy3. (L) Centromeric region (arrow) or telomeric region (arrowhead) replicates in very-late S-phase are indicated. (I,L) Late S- and very late S-PCCs already condensed like as mitotic chromosomes, but these PCCs were actually S-phase chromosomes because they incorporated Cy3-dUTP. G2/M chromosomes are easily distinguished from late or very late S chromosomes as G2/M chromosomes do not incorporate Cy3-dUTP (data not shown). Inset in (C) is higher magnification of the boxed portion. Scale bar, 10 µm. (2) DNA replication regions seen on prominent fibre of PCCs. (M) early-S-phase and (N) middle S-phase. Replication foci are clearly seen as 'beads on a string' structure, some of these are indicated by arrowhead. Scale bar, 10 µm. (Figure reproduced from Figure 2 of Chromosoma 2007; 116(5):453-462. By Gotoh).



Chromosome condensation/compaction coupled with DNA replication In the present review, the dynamics of chromosomal conformation change, which is tightly coupled with DNA replication during S-phase, was clearly seen on PCCs of different sub S-phase using drug-induced PCC method (Gotoh and Durante, 2006) and on Cy3-dUTP direct labeling method (McNeil and Warder, 1987). Drug-induced PCC would be, therefore, a useful tool that provides new insights of the dynamics of chromosome formation and DNA replication. As described in the previous section, number of accumulated evidences suggested the role of DNA replication in chromosome condensation/compaction (Pflumm, 2002). As previously reported, evidence and results of this study show that: (i) The different appearance of condensation in different sub-phase of S-PCCs is thought to be depended on the different degrees of chromosome conformation at the time of PCC induction (Johnson and Rao, 1970; Rao, 1977; Rao et al., 1977). In the late or very late S phase, particularly, chromosome conformation already changes like mitotic chromosomes (Fig. 1F). (ii) Chromosomes condense asynchronous and the different degree of condensation depend on the time of chromatin replication (Kuroiwa, 1971). (iii) Chromosomes are not fully diffused nor nonrandomely positioned in the nucleus, but are separately compartmentalized in interphase nuclei (Cremer et al., 1993; Ferreira et al., 1997; Berezney et al., 2000). These chromosomes, occupying the 'territory', do not intermingle (Hadlaczky et al., 1986; Cremer et al., 1993; Swedlow and Hirano, 2003; Cremer et al., 2006; Heard and Bickmore, 2007). (iv) Late replication foci were prealigned during interphase. They moved subtly to generate recognizable chromosomes presumably due to shortening of the longitudinal chromosome axis (Manders et al., 1999). (v) The gross structure of an interphase chromosome territories is directly related to that of the prophase chromosomes (Manders et al., 1999). (vi) The structure of mitotic chromosomes and the nuclear chromosome territories are closely related (Manders et al., 1999) and the different bands of mitotic chromosomes are presented as distinct domains regarded subchromosomal foci within chromosome territories (Zink et al., 1999). (vii) During the cell cycling, the global chromosome territories are conserved. Although some conflicts still remains, several studies reported that chromosome territories are transmitted through mitosis (Manders et al., 1999; Gerlich et al., 2003; Gerlich and Ellenberg, 2003) whereas others reported that positional relations of chromosome territories are lost either at mitosis (Walter et al., 2003) or at early G1 (Essers et al., 2005). (viii) The spatio-temporal organization of DNA replication is determined by the specific nuclear order of these stable chromosomal units (Sadoni et al., 2004). (ix) Chromatin domains with the dimension of replication foci may be fundamental units of

chromosomal architecture (Berezney et al., 2000). (x) DNA replication occurs at fixed sites and replicated DNA move through replication center (Berezney and Coffey, 1975; Pardoll et al., 1980; Hozak et al., 1993). (xi) DNA replication contributes to a longitudinal contraction of the chromosome axis (Hearst et al., 1998). (xii) Functional replication origins are a critical requirement for longitudinal condensation of the chromosome axis (Pflumm and Botchan, 2001). The results presented in this review and previous findings strongly suggest that DNA replication, nuclear organization and chromosome condensation are mutually integrated to construct a higher ordered structure of eukaryote chromosomes. A hypothetical chromosome compaction model coupled with DNA replication Number of models for eukaryote chromosome architecture have been proposed (Marsden and Laemmli, 1979; Woodcock et al., 1984; Woodcock and Dimitrov, 2001; Swedlow and Hirano, 2003; Kireeva et al., 2004), but they are controversial and many aspects are still unclear. In addition, these models do not take account of the involvement of DNA replication/transcription in chromosome packaging. DNA/RNA polymerase are known to be tightly immobilized to the replication/transcription factories (Cook, 1999; Frouin et al., 2003). In the proposed model, DNA polymerase is thought to be a 'reel in DNA template and extrude replicated DNA' (Hozak et al., 1996; Cook, 1999) rather than an enzyme track along DNA template, which is proposed in many conventional models. In the context of Cook's model, some kinds of mechanical tension force should be generated in the DNA template along with DNA replication goes on because the factory is not freely suspended in the nucleus but attached to nucleoskeleton. Consequently, this force may pull and aggregate the replication foci of both sides as to release the tension in DNA strands, which may result in the formation of the chromosomes as seen in mitosis. Based on the above mechanism and the observed findings obtained from chromosome structure dynamics coupled with DNA replication, Fig. 2 shows a hypothetical model for the relationship between DNA replication and chromosomal conformation changes, and it shows too how the interphase chromatin is constructed into chromosomes (Figure reproduced from Chromosoma. Gotoh, 2007; 116(5): 453-462). During the S-phase, chromosomal conformation changes and the chromosome formation would be mostly completed at the end of DNA replication (Fig. 2A,B,C). From G2 to prophase, chromosomes are still more elastic, less condensed, folded only several times and prealigned in interphase nuclei (Manders et al., 1999). At these phases, the chromosomes would be observed as chromosome territories (Cremer et al., 1993; Berezney et al., 2000) (Fig. 2D).



Entering in mitosis, these chromosomes would condense even more as shortening the longitudinal axis

to form solid and rod shape appearance of recognizable mitotic chromosomes (Manders et al., 1999) (Fig. 2E).

Figure 2: A hypothetical two-dimensional model for chromosome conformational change involving DNA replication based on the models proposed by Cook (Cook, 1995) or Pflumm (Pflumm, 2002). (A) Early S-phase. DNA replication starts at multiple origins and proceeds bi-directionally. Early S-PCCs are seen as 'beads on a string' appearance. (B) Middle S-phase. As DNA replication proceeds, replicated DNA pass through replication factory and some tension are generated. The generated tension may pull back the replication factories close together so as to release the tension. Replication factories may in turn fuse together and chromosomes compact. Middle S-PCCs are seen as well known 'pulverized chromosomes' appearance. (C) Late S-phase. Most of DNA finished replication and conformation was changed. Late S-PCCs are seen as 'tandem band arrayed structured chromosomes' like as mitotic chromosomes. (D) G2 to prophase. After finishing of DNA replication, chromosome conformation changed like as mitotic chromosomes, but still so elastic that packed in nucleus. Before fixation, each chromosome occupies individual chromosome territory (CT) in interphase nucleus, thus observed as compartment regions (colorized). (E) Mitosis. After prophase, chromosomes further shortening in longitudinal axis of chromosomes, consequently a straight rod shaped recognizable chromosome formed as usually seen by cytologists under a microscope. For simplicity, the model is shown as two-dimensional and the scaling is arbitrary. The model intends not to depict actual events of chromosome conformation change but to help imagine how DNA replication is involved in chromosomal conformation. As the real chromosomes condense as three-dimensionally, other elements such as coiling and helical winding should be considered together to construct a stereoscopic hierarchical structure of eukaryote chromosomes (Woodcock and Dimitrov, 2001; Swedlow and Hirano, 2003). (Figure reproduced from Figure 3 of Chromosoma 2007; 116(5):453-462. By Gotoh).



Summary and Conclusion A basic and principle question of cell biology is: how DNA folds to chromosome? Numbers of evidence have suggested the involvement of DNA replication in chromosome structure formation. To visualize the dynamics of chromosome structure formation coupled with DNA replication, Cy3-dUTP direct-labeled active replicating DNA was observed in prematurely condensed chromosomes (PCCs) utilized with drug-induced premature chromosome condensation technique, which facilitates the visualization of interphase chromatin as well as the condensed chromosome form. S-phase PCCs revealed clearly the drastic dynamic transition of chromosome formation during S-phase along with the progress of DNA replication: from a 'cloudy nebula' structure in early S-phase to numerous number of 'beads on a string' in middle S-phase and finally to 'striped arrays of banding structured chromosome' in the late S-phase as usual observed in mitotic chromosomes. The drug-induced PCC is clearly provided a new insight that the eukaryote DNA replication is tightly coupled with the dynamics of chromosome condensation/compaction for the construction of eukaryote higher ordered chromosome structure. Based on these findings, a hypothetical model for chromosome compaction involved the role of DNA replication is proposed. In this model, conformational change is simply illustrated as two-dimensional but the real architecture is a three-dimensionally chromosome constructs, with much more complex fashion. It is mostly unclear how DNA replication/transcription conducts to make up a three-dimensional hierarchical structure of chromosomes coupled with twisting/folding/winding or other factors. It should be a most principle challenge in cell science.

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NK cell receptors: evolution and diversity Gwenoline Borhis, Salim I Khakoo

Department of Hepatology, Division of Medicine, Imperial College London, UK (GB, SIK)


Online updated version : http://AtlasGeneticsOncology.org/Deep/NKCellRecEvoDivID20095.html DOI: 10.4267/2042/46021


Summary Natural Killer cell functions are regulated by combinations of activating and inhibitory receptors, derived from a number of different gene families. This review focuses on receptors for MHC class I, which include the killer cell immunoglobulin-like receptors (KIR) and the CD94:NKG2 family of receptors. In particular the KIR are diverse and rapidly co-evolving with their classical MHC class I ligands. Thus NK cells are part of the innate immune system that are continuing to adapt to the challenges of pathogens.

Introduction NK cells are an important component of the innate immune system, which participate in the early immune defence against intracellular pathogens and tumour transformation. They were originally defined by their ability to spontaneously eliminate rare cells lacking expression of class I Major Histocompatibility Complex (MHC class I) self molecules, a process commonly referred to as "missing self" recognition (Biron et al., 1999; Purdy and Campbell, 2009). Upon activation, NK cells can mediate direct cytotoxicity or secrete cytokines and chemokine that modulate subsequent steps in the adaptive immune response. These functions are regulated by the combination of signals from activating and inhibitory receptors (Lanier, 1998). The MHC class I receptors are particularly important for NK cells to discriminate "self" (healthy cells) from "altered-self" (infected- and transformed-cells) or "missing self". MHC class I receptor:ligand interactions can induce inhibitory signals that counteract activating receptor signals and lead to NK cell inhibition. In contrast down-regulation or loss of MHC class I expression, during viral infection or carcinogenesis, shifts the balance towards NK cell activation and target cell destruction by removing this inhibitory signal. Thus in health NK cells

are tolerant towards host cells, but in disease this tolerance can be readily broken.

MHC class I receptors on NK cells MHC class I receptors on NK cells can be either inhibitory or activating. The inhibitory receptors for MHC class I regulate NK cell function by generating a tonic inhibitory signal as hypothesized in the "missing-self" model (Ljunggren and Karre, 1990). The role of the activating receptors for MHC class I appears less clear, but genetic studies have implicated them in recognition of virally infected cells. Several inhibitory receptors have been identified, but there are two main families involved in NK regulation by MHC class I: the Killer cell Immunoglobulin-like Receptors (KIR) and the C-type lectin-like CD94/NKG2A heterodimers. KIR interact with the classical MHC class Ia (HLA-A, -B and -C) while CD94/NKG2A recognizes the non-classical MHC class Ib, HLA-E. Both synergize and permit NK cells to sense and respond to changes in MHC class I expression. These receptors are expressed in a combinatorial fashion on NK cells to generate an NK cell repertoire. The importance of this is gradually being realised. Expression of an MHC class I inhibitory receptor appears to confer additional reactivity on these NK cells, a phenomenon originally termed "licensing" (Kim et al., 2005). Thus NK cells without inhibitory receptors for MHC class I are thought to be relatively hypofunctional, although in specific scenarios, these cells can become important for viral eradication as shown by studies in murine CMV infection (Orr et al., 2010). Furthermore in disease states in which a specific inhibitory receptor may be beneficial, such as KIR2DL3 in hepatitis C virus infection, then individuals with more beneficial repertoires may be more likely to clear infection (Alter et al., 2010). In addition to these key receptors, members of the Ig-Like Transcripts (ILTs) family, which are genetically related to KIR (Wilson et al., 2000), can also recognize

NK cell receptors: evolution and diversity Borhis G, Khakoo SI


MHC class I. For example, LILRB1 (LIR-1, ILT-2), which binds a broad range of MHC class I molecules including HLA-G (Vitale et al., 1999), is able to inhibit the NK cell line NK92 (Kirwan and Burshtyn, 2005). It is also expressed in a variegated fashion on NK cells (Davidson et al., 2010). However it does not appear to play a part in NK cell education and it has yet to be demonstrated that this gene family play a major role in inhibiting NK cells in vivo (Yawata et al., 2008). CD94/NKG2A and KIR molecules have adopted two different recognition strategies. To a large extent CD94/NKG2A ignore MHC class I diversity by recognizing HLA-E. This non-polymorphic MHC class I molecule binds leader peptide sequences derived from classical MHC-A, -B and -C molecules and also from HLA-G (Llano et al., 1998). In contrast the KIR family embrace the diversity of MHC class I through direct recognition of polymorphic determinants. This strategy leads to a highly variable and polymorphic KIR system with diversity comparable to that of MHC class I (Valiante et al., 1997a).

KIR structure and signalling function The KIR family (assigned the designation of CD158) is a member of the immunoglobulin superfamily that comprises 15 expressed receptors (KIRDL1-5B, KIR3DL1-3, KIR2DS1-5 and KIR3DS1): which can be either inhibitory or activating (Table 1). All KIR are type I transmembrane glycoproteins formed from either two (KIR2D) or three (KIR3D) extracellular Ig-like domains, a stem region, a transmembrane region and a cytoplasmic tail. Depending on the length of the cytoplasmic tail KIR can be subdivided into long-tailed and short-tailed receptors. In general these structural characteristics correlate with their function. Long-tailed KIR are generally inhibitory and short-tailed KIR are activating (Vilches and Parham, 2002). An exception to this rule is the receptor KIR2DL4 which has a long intracytoplasmic tail but stimulates potent cytokine production, although only minimal cytotoxicity (Rajagopalan et al., 2006). Inhibitory KIR contain one or two Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs; V/I/LxYxxL/V), which are require for NK cell inhibition via recruitment of the protein tyrosine phosphatases SHP-1 and SHP-2. SHP-1/2 activation leads to the suppression of activating receptor signals (Long, 2008). Activating KIR possess a positively charged residue (usually arginine) in the transmembrane region, which facilitate the association with accessory molecules, DAP12 or FcεRIγ (KIR2DL4) and NK cell activation (cytotoxicity and/or cytokine production) (MacFarlane and Campbell, 2006). The exception, KIR2DL4 contains both ITIMs and a positively charged residue (lysine), which facilitates the association with FcεRIγ and the induction of the activating signals (Kikuchi-Maki et al., 2005).

The KIR genes can be divided into six lineages based on phylogenetic analysis. This has allowed "tracking" of the KIR across species and given insights into the evolution of the KIR gene families. Lineage I KIR have two extracellular domains in the D0D2 conformation; lineage II KIR are specific for MHC-A and -B; lineage III KIR bind HLA-C; lineage V KIR are related to the human KIR framework gene KIR3DL3; and lineage IV and VI KIR are expansions specific to the rhesus macaque and new world monkeys respectively.

KIR locus and diversity Genotyping of individuals for specific KIR genes demonstrated an unexpected diversity in gene content amongst the population (Uhrberg et al., 1997). The genotype of these individuals correlated with expression of KIR genes thus demonstrating that this genetic diversity would be important for NK cell function. This seminal study started a detailed investigation into KIR genetics. Sequencing of two KIR haplotypes from a single individual showed that the KIR are encoded by a compact family of genes which occupy about 150 kb of the Leukocyte Receptor Complex (LRC) on chromosome 19q13.4 (Wilson et al., 2000; Wende et al., 1999). The locus is flanked by the LILR and the FCAR genes and contains up to 17 KIR genes and pseudogenes (Kelley et al., 2005). Because the KIR genes have high sequence homology to each other (90-95%) and are closely distributed within the LRC, they have been proposed to evolve by non-allelic homologous recombination (Carrington and Cullen, 2004). This mechanism could explain the expansion and contraction of the KIR locus and provide a basis for the substantial diversity observed (Hsu et al., 2002a; Hsu et al., 2002b; Shilling et al., 2002; Uhrberg et al., 2002; Whang et al., 2005; Martin et al., 2003). Haplotypic diversity The number of putatively expressed KIR genes usually ranges from 7 to 12, depending primarily on the presence or absence of activating KIR loci (Wilson et al., 2000; Uhrberg et al, 2002; Witt et al., 1999). This variation in gene content is one component of KIR diversity. Despite this extreme variability some systematic features are conserved in the organisation of the KIR locus. Four KIR genes, KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2, are found in all individuals and have been named framework loci (Bashirova et al., 2006). KIR3DL3 and KIR3DL2 define the ends of the KIR-gene region and KIR3DP1-KIR2DL4, the middle. Regions of genetic variability are located between KIR3DL3 and KIR3DP1, and between KIR2DL4 and KIR3DL2 (Wilson et al, 2000; Martin et al., 2000). Two distinct forms of haplotype, termed A and B, can be distinguished on the basis of gene content. Haplotype A has a fixed gene content (KIR2DL1, KIR2DL3, KIR2DL4, KIR2DS4, KIR3DL1, KIR3DL2, KIR3DP1 and KIR3DL3) (Uhrberg et al., 1997) and fewer genes than B haplotype but the most functionally relevant distinction between these two haplotypes is the



number of activating receptors. Haplotype A contains only a single activating KIR gene, KIR2DS4, whereas haplotype B contains various combinations of KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5, KIR3DS1 and KIR2DS4. Furthermore, the KIR2DS4 gene has a null allele with a population frequency of about 84% (Maxwell et al., 2002), thus some homozygous A haplotype individuals don't express any activating KIR (Hsu et al., 2002b). Although A haplotypes are fixed in term of the number and type of genes present, they show extensive allelic variation at several of the genes. In contrast to A haplotype, B haplotype displays a much greater variety of gene contents. Based on segregation analysis, more than 20 different B haplotypes have been described (Hsu et al., 2002a; Yawata et al., 2002a). These haplotypes contain various combinations of KIR genes, including several activating KIR but there is a very high linkage disequilibrium (LD) between many pairs of genes, as KIR2DL1/KIR2DL3 or KIR3DL1/KIR3DL2 alleles (Uhrberg et al., 1997; Shilling et al., 2002; Witt et al., 1999; Norman et al., 2002; Norman et al., 2001; Toneva et al., 2001; Crum et al., 2000). However despite the broad categorizations, there are several exceptions to these simple rules. For instance despite the fact that KIR3DL1 (inhibitory) and KIR3DS1 (activating) segregate as alleles of a single locus in the vast majority of individuals, haplotypes have been described in which they occur on the same chromosome (Martin et al., 2008; Norman et al., 2009). Similarly some KIR haplotypes have fewer than expected KIR. For instance a recently described haplotype contains only 3 KIR genes KIR3DL3, KIR2DS1 and KIR3DL2 (Traherne et al., 2010). Thus the rules for the KIR locus appear unusually flexible, perhaps due to the combination of a high sequence homology between the genes, and an overlap in function of these receptors with the well conserved CD94:NKG2 family of receptors. Furthermore some genes (KIR2DS3 and KIR2DS5) appear to occur in two different chromosomal locations. This has led to the splitting of the locus into two separate sections a centromeric section (KIR3DL3-KIR3DP1) and a telomeric section (KIR2DL4-KIR3DL2) which appear to have diversified independently (Pyo et al., 2010). Sequence analysis of a number of KIR haplotypes shows that allelic diversity of the centromeric section is predominant in the A haplotypes, but it is in the

telomeric section of the B haplotypes that allelic diversity is most noticeable. The distribution of A and B haplotypes varies widely between distinct ethnic groups. The A and B haplotype frequencies are relatively even in Caucasian populations (Uhrberg et al., 1997; Hsu et al., 2002b). However the A haplotype dominates in the Korean, Japanese and Han Chinese populations with an approximate 75% frequency (Whang et al., 2005; Yawata et al., 2002b; Jiang et al., 2005) as compared to the Australian Aborigines, who have a very low frequency of the A haplotypes of about 13% (Toneva et al., 2001). These differences may reflect both founder effects and selection by pathogens and may account for some variation in worldwide disease susceptibility. Allelic polymorphism Point mutation and homologous recombination generate allelic polymorphism (Table 1) (Norman et al., 2009; Shilling et al., 1998). This allelic polymorphism gives an additional dimension to KIR diversity in that unrelated individuals are unlikely to have identical KIR alleles, similar to the situation for MHC diversity (Gardiner et al., 2001). Allelic polymorphism has been described for all the inhibitory KIR genes and names for alleles at several of the most polymorphic loci have been specified based on nomenclature used for HLA loci (Shilling et al., 2002). This polymorphism significantly influences their ligand affinities and levels of cell surface expression. For example, distinct alleles of KIR3DL1, one of the most polymorphic KIR genes encode molecules that appear to be expressed at different levels on the surface of NK cells (Gardiner et al., 2001; Yawata et al., 2006; Pando et al., 2003). Moreover this allelic variability can occur at positions encoding residues that affect interaction with HLA class I (Boyington et al., 2000; Fan et al., 2001) and influences both the binding affinity and the inhibitory capacity. Similarly the genes KIR2DL2 and KIR2DL3 also segregate as alleles of a single locus and although they have broadly similar MHC class I specificity, bind their ligands with substantially different avidities (Moesta et al., 2008). The synergy of haplotype variability and, allelic polymorphism has generated substantial diversity across both individual populations, but also across different ethnic groups (Rajalingam et al., 2001). This diversity is likely driven by both encounters with pathogens, but also by reproductive fitness.

Table 1: KIR nomenclature, lineages and ligands(IPD - KIR Database; Cadavid and Lun, 2009)

Gene name CD nomenclature No. of alleles

No. of protein

Lineage Ligand(s) Function

KIR2DL1 CD158a 43 24 III HLA-C2 inhibitory

KIR2DL2 CD158b1 29 12 III HLA-C1 (weakly HLA-C2) inhibitory

KIR2DL3 CD158b2 33 17 III HLA-C1 (weakly HLA-C2) inhibitory

KIR2DL5A* CD158f 45 18

I Unknown inhibitory

KIR2DL5B* I Unknown inhibitory



KIR3DL1 CD158e1 74 58 II HLA-BBw4 and HLA-ABw4 inhibitory

KIR3DL2 CD158k 84 62 II Certain HLA-A3 and HLA-A*11

inhibitory

KIR3DL3 CD158z 107 56 V Unknown inhibitory

KIR2DL4 CD158d 47 22 I HLA-G activating

KIR2DS1 CD158h 15 7 III HLA-C2A activating

KIR2DS2 CD158j 22 8 III Potentially HLA-C1 (binding not detectable)

activating

KIR2DS3 14 5 III Potentially HLA-C1 activating

KIR2DS4 CD158i 30 13 III HLA-Cw4 and HLA-11 activating

KIR2DS5 CD158g 15 10 III Unknown activating

KIR3DS1 CD158e2 16 12 II Potentially HLA-BBw4 (binding not detectable)

activating

KIR2DP1 22 0 III / pseudogene

KIR3DP1 CD158c 23 0 V / pseudogene

*KIR2DL5 gene is duplicated and encoded by two separate loci within the LRC gene cluster.

KIR recognition and peptide selectivity Individual KIR recognize distinct subsets of the classical human MHC class I allotypes. This binding specificity is determined both by residues of the MHC class I and those of the peptide bound by the MHC class I molecule. Inhibitory KIR are able to recognize all the known HLA-C allotypes (C1 and C2 subgroup) and some subsets of HLA-A and HLA-B allotypes. KIR2DL1 binds HLA-C2 allotypes, which all have a lysine at position 80 (Colonna et al., 1993). KIR2DL2 and KIR2DL3, which segregate as alleles of the same locus, bind mainly HLA-C1 allotypes (with an asparagine at position 80), some HLA-C2 allotypes and a few HLA-B allotypes which have an asparagine at position 80 and also a valine at position 76 (Moesta et al., 2008; Wagtmann et al., 1995; Pende et al., 2009). KIR3DL1 recognize the "Bw4" motif present in 40% of the known HLA-B allotypes and in some HLA-A allotypes, with a higher affinity for the Bw4 motifs containing an isoleucine at position 80 (Cella et al., 1994; Gumperz et al., 1995). KIR3DL2 is only known to bind HLA-A3 and HLA-A11 allotypes whilst ligands for KIR2DL5 and KIR3DL3 have not yet been identified. Given the high sequence homology between the extracellular domains of some activating and inhibitory KIR (~99%), several activating KIR have been reported to bind the same HLA molecules as their inhibitory counterparts, although with significantly weaker affinity (Biassoni et al., 1997; Valés-Gómez et al., 1998; Stewart et al., 2005). Due to their low affinities, the activating KIR-HLA binding specificity is quite uncertain. Moreover the KIR-HLA affinities can be enhanced by specific peptides presented in the HLA molecules, as has been shown for KIR2DS1

interactions with Epstein-Barr virus-infected cells (Stewart et al., 2005). One potential model is that these receptors may bind specific viral peptides that have yet to be determined. In addition to the MHC class I heavy chain, all inhibitory KIR tested to date have some degree of peptide selectivity (Boyington et al., 2000; Malnati et al., 1995; Rajagopalan and Long, 1997; Hansasuta et al., 2004). This appears to have a functional relevance in that NK cells expressing KIR2DL3 are exquisitely sensitive to the peptide bound by MHC class I. This is because peptides that stabilise MHC class I, but bind KIR weakly can antagonize the inhibition due to MHC class I:peptide complexes that bind KIR strongly (Fadda et al., 2010). This process appears to be more efficient than MHC class I downregulation in activating NK cells, and may be important for recognition of infected targets (Rajagopalan and Long, 2010).

KIR evolution A comparison of the KIR genes in human and chimpanzees revealed unexpectedly rapid evolution of the KIR locus, in many ways exceeding the pace of their MHC class I ligand (Khakoo et al., 2000). This contrasts with the high conservation of the CD94:NKG2A system (Shum et al., 2002). Work in the higher primates has revealed that in these species the KIR genes have expanded substantially. Mice, which are the most frequently used immunological model for the immune system of man and his response to disease, do not have KIR as regulators of NK cell activity (Figure 1). Instead they have an expansion of the C-type lectin-like receptors, the Ly49 genes which also bind classical MHC class I molecules. These genes are related to the NKG2A family of receptors, and both these gene families in addition to the CD94 gene are found on murine chromosome 6 in a region designated



the natural killer cell complex (NKC) (Vance et al., 1998). In mice CD94:NKG2A binds the non-classical MHC class I molecule Qa-1, which also binds MHC class I leader sequences. Thus comparison of humans and rodents has revealed two distinct evolutionary pathways for NK cell receptors: one leading to diversification of KIR and the other to diversification of Ly49. Both species have inhibitory NK cell receptors for classical class I molecules and both for a non-classical MHC class I molecule, although NKG2A in mouse and human are not strictly orthologous. Remnants of non-functional genes can be found in the alternate species: the KIR are represented by a gene on murine chromosome X and Ly49 is a pseudogene in humans (Kelley et al., 2005) (Figure 1). Studies in other species have revealed the uniqueness of KIR in the simian primates. KIR genes have been found in species as diverse as cattle, horses, dogs and pinnipeds (Parham et al., 2010). These are thought to have derived from duplication of an ancestral KIR3D gene over 100 million years ago. This resulted in two genes: KIR3DL and KIR3DX (Guethlein et al., 2007). The KIR3DL gene is thought to have spawned the KIR genes of the primates, and the KIR3DX gene given rise to the multigene KIR family in cattle (Sambrook et al., 2006). KIR3DX is retained in humans, however it is a

non-functional pseudogene in the LRC amongst the LILR gene. The adoption of different solutions to the issue of NK cell receptor variability is further illustrated by the expansion of the NKG2 family of genes in the prosimians (Averdam et al., 2009) and the observation that the pinnipeds (seals and sea lions) seem to cope with having only one functional KIR and one functional Ly49 gene (Hammond et al., 2009). Genetic studies implicating specific combinations of KIR in infectious diseases imply that pathogens are a major driving force in KIR evolution. This follows naturally from the observation that natural killer cells are important in clearing viral infections. Pathogens can drive KIR selection both by a direct effect on specific KIR genes and also via an indirect selective pressure through driving the evolution of MHC Class I. This is well illustrated by the co-evolution of KIR and the MHC-C locus in the great apes. The KIR can be divided into lineages based on sequence homologies. The lineage III KIR have MHC-C ligands. The most divergent species from man with an MHC-C allele is the orangutan (Adams et al., 1999). In this species this locus is present in only about half the individuals. Nevertheless, in the orangutan and man's more closely related ancestors

Figure 1

(the gorilla, the common chimpanzee and the pygmy chimpanzee) lineage III KIR have expanded, implying that MHC and the KIR are co-evolving (Abi-Rached et al., 2010b). Similarly in the old world monkeys the expansion of MHC-A/B locus has led to expansion of lineage II KIR, and in the hoolock gibbon loss of MHC-G corresponds to loss of the lineage I gene KIR2DL4, which has been shown to be relatively conserved amongst higher primates (Abi-Rached et al., 2010a; Parham et al., 2010). Whilst humans and chimpanzees share MHC-A, -B and -C loci, they share relatively few KIR genes. These include the framework genes KIR3DL3, KIR2DL4 and

KIR3DL2 (which in the chimpanzee is a chimera of human KIR3DL1 and KIR3DL2 called Pt-KIR3DL1/2), and the genes KIR2DL5 and KIR2DS4 (Khakoo et al., 2000). Humans have retained lineage III inhibitory KIR which bind strongly to HLA-C, however the activating KIR for HLA-C are low avidity. Conversely the common chimpanzee has retained high avidity activating and inhibitory for both group 1 and group 2 HLA-C allotypes. Furthermore, although the lineage II human KIR bind both HLA-A and -B allotypes, the most relevant interaction appears to be that of KIR3DL1 with HLA-B allotypes with the Bw4 serological motif. Although this KIR does bind HLA-A



allotypes with the same serological motif. The binding of other KIR to HLA-A allotypes is less well documented. KIR3DL2 binds HLA-A3 and HLA-A11, although the avidity of this interaction is not well studied (Dohring et al., 1996; Pende et al., 1996; Valiante et al., 1997b). It has also been shown to bind tetramers of HLA-B27 homodimers (Kollnberger et al., 2007). KIR2DS4 also binds HLA-A11 (Graef et al., 2009). However the functional relevance of these interactions with HLA-A is not as well documented as for HLA-B: and KIR. Conversely Pt-KIR3DL2, binds both MHC-A and -B allotypes, and can demonstrably inhibit chimpanzee NK cells in a manner not restricted by the Bw4 serological motif, even though this motif is present on a number of chimpanzee MHC-B allotypes (Khakoo et al., 2002). Thus even where the most simple motifs for KIR binding on MHC appear relatively conserved between species there is substantial evidence for a more rapid evolution at the KIR locus. Within different human populations there is a great diversity in the frequencies of individual KIR genes (Gonzalez-Galarza et al., 2011). The essential role for unequal crossing over in generating this diversity is illustrated by in depth study of the KIR3DL1/KIR3DS1 locus in which it has been demonstrated that this process can account for both duplication and deletion within the KIR gene complex (Norman et al., 2009). Further selection may occur on the basis of the interaction of KIR with its MHC class I ligands, to maintain a functional relationship and which may be fine tuned by the affinity of the KIR for its MHC class I ligand (Single et al., 2007). Thus in the Yucpa population there is a relatively high frequency of the "strong educating" HLA-C*07 allele, and correspondingly higher frequencies of KIR2DL3 alleles with low avidities for HLA-C (Gendzekhadze et al., 2009). This implies that evolutionary pressures have combined to ensure that inhibitory signals to NK cells can be easily overcome and so NK cells can be readily activated in response to pathogens.

Impact of KIR diversity on Human health and disease The impact of KIR diversity on human health is well illustrated by disease association studies. Whilst infection is thought to be the major driving force for the evolution of KIR, there is substantial evidence that KIR diversity impacts a number of pregnancy associated disorders, including pre-eclampsia and recurrent spontaneous abortion (Moffett-King, 2002). During placentation the trophoblast burrows into the placenta, and natural killer cells appear to be important for this process. Analysis of maternal and foetal KIR and MHC class I genotypes demonstrate that if these result in greater foetal NK cell activation then pregnancy is more likely to be successful, due to improved placentation. Thus in cases where the KIR haplotype of the fetus has a preponderance of activating receptors,

such as a type B KIR haplotype, then there I a lower probability of pre-eclampsia (Hiby et al., 2004). Conversely if the fetus has only one activating KIR, as is found in a group A KIR haplotype and the mother has a strong inhibitory MHC class I type for example two group 2 HLA-C alleles then there is a greater risk of pre-eclampsia, foetal growth retardation and recurrent spontaneous abortion (Hiby et al., 2010). This likely drives the evolution of the KIR locus towards a preponderance of activating receptors. Early studies in infectious disease would concur with this evolutionary direction. In HIV infection the activating receptor KIR3DS1 and its HLA-B ligands Bw4 with isoleucine at position 80 (Bw480I) are associated with a slower progression to AIDS (Martin et al., 2002). This begs the question as to the persistence of the "inhibitory" A haplotypes in the human population. Further studies of HIV infection have revealed a second model in which a hierarchy of inhibitory interactions between the inhibitory receptor KIR3DL1 and its HLA-BBw4 ligands influences the progression to AIDS (Martin et al., 2007). Interestingly in this genetic analysis the most protective allele KIR3DL1*004 is one which is not expressed on the cell surface (Pando et al., 2003). This is a feature of other KIR alleles, including KIR2DL2*004 and a number of alleles of KIR2DS3 (VandenBussche et al., 2006; VandenBussche et al., 2009). This implies that care must be taken with the interpretation of these genetic analyses as the presence of the receptor and its ligand does not necessarily mean that there is a simple functional relationship. The importance of inhibitory receptor:ligand interactions is probably best illustrated by consideration of hepatitis C virus (HCV) infection. This is a positive stranded RNA virus that has relatively little specific effects on MHC class I expression, and so may act as a more general template for understanding the role of NK cells in viral infection. Genetic analysis has shown that weaker inhibitory interactions are associated with a more beneficial outcome of HCV infection. It was originally shown that KIR2DL3 and its group 1 HLA-C ligands were associated with spontaneous resolution of HCV infection, which in the vast majority of individuals leads to chronic infection (Khakoo et al., 2004). Binding analysis reveals that KIR2DL3 is a weaker binder to HLA-C than its allele KIR2DL2, which has a similar MHC class I specificity (Winter et al., 1998). It is therefore thought that a weaker inhibitory receptor:ligand interaction can be more easily perturbed than a strong one and hence is more likely to lead to NK cell activation. This protection has been observed in other HCV exposed cohorts and disease settings, including in the clinically important treatment setting (Romero et al., 2008; Knapp et al. 2010; Vidal-Castiñeira et al., 2010). Furthermore, similar to HIV, it can be mapped to the allelic level, but in this case at the HLA-C locus. Thus the common group 1 HLA-C*07 alleles are not protective whereas



most other group 1 HLA-C alleles are protective in combination with KIR2DL3 (Knapp et al., 2010). Thus it appears that there is a balancing selection on the KIR "A" and "B" haplotypes in humans which has permitted maintenance of both in the extant human populations (Gendzekhadze et al., 2009).

Conclusion By virtue of the expression of KIR, natural killer cells are a branch of the innate immune system that are undergoing constant evolution in response to both pathogens and the challenge of successful reproduction. The KIR have diversified in response to MHC driven selective pressures, following exposure to pathogens. Due to the independent segregation of KIR and their MHC class I ligands, in any given individual some KIR may be redundant. Nevertheless, on a population level, by fine tuning natural killer activity these receptors are key regulators of the innate immune response to pathogens.

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http://www.ebi.ac.uk/ipd/kir/stats.html


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Somatostatin (SS), SS receptors and SS analog treatment in tumorigenesis

Liliana Steffani, Luca Passafaro, Diego Ferone, Paolo Magni, Massimiliano Ruscica

Department of Endocrinology, Pathophysiology and Applied Biology, Universita degli Studi di Milano, Milan, Italy (LS, LP, PM, MR), Department of Internal Medicine, Universita degli Studi di Genova, Genoa, Italy (DF)


Online updated version : http://AtlasGeneticsOncology.org/Deep/SomatostatinID20094.html DOI: 10.4267/2042/46022


Abstract Somatostatin (SS) is an inhibitory tetradecapeptide hormone with exocrine, endocrine, paracrine, and autocrine activities, which plays an important regulatory role in several cell functions, including inhibition of endocrine secretion and cell proliferation. Most of the effects of SS and of its currently available analogs are mediated via five different G protein-coupled receptor (GPCRs), codenamed sst1-5. SS receptors (ssts) are expressed in a tissue- and subtype-selective manner in both normal and neoplastic cells, and the majority of SS target tissues express multiple ssts. Recent data suggest that when ssts are coexpressed, they may interact forming homo- and hetero-dimers also with other GPCRs, thus altering their original pharmacological and functional profiles. The formation of dimers can be not only constitutive, but also ligand-promoted: hence, compounds with high affinity for the different receptor subtypes can be used to achieve effects elicited by specific dimers. A feature common to most GPCRs is the cyclic process of signaling, desensitization, internalization, resensitization, and recycling to the plasma membrane. These events prevent cells from undergoing excessive receptor stimulation or periods of prolonged inactivity. SS receptors differently internalize after agonist binding and, specifically, sst2, sst3 and sst5 are internalized to a greater extent than sst1 or sst4. ssts are linked to several second messenger systems which are involved in their downstream intracellular response (i.e., adenylyl cyclase, calcium and potassium ion channels, Na+/H+ antiporter, phospholipase C, phospholipase A2, mitogen activated protein kinase, NO/cGMP, and

serine-, threonine, and phosphotyrosyl- protein phosphatase). Interestingly, SS and SS analogs can control tumor development and progression/metastatization by direct actions, mediated by the ssts, and indirect actions, independent of receptor involvement. The direct antiproliferative effects include inhibition of autocrine/paracrine growth-promoting hormone/growth factor synthesis, arrest of cell division (by blockade of growth factor-mediated mitogenic signals), suppression of cell invasion and induction of apoptosis (programmed cell death). Indirect antitumor effects of SS include suppression of synthesis or/and release of growth factors and growth-promoting hormones, such as insulin, prolactin, insulin like-growth factor 1, epidermal growth factor, transforming growth factor-, gastrin, cholecystokinin and growth hormone. A specific pattern of ssts activation thus seems to elicit relevant antitumoral actions and deserves further exploitation with the aim of validating novel therapeutic approaches to cancer.

1. Somatostatin Somatostatin (SS) was first identified in the ovine hypothalamus as a tetradecapeptide that inhibited the release of growth hormone (Brazeau et al., 1973). SS-producing cells are present at high densities throughout the central and peripheral nervous systems. In the periphery, SS is also secreted by pancreas and gut and in a lesser extent by thyroid, adrenals and submandibular glands, kidneys, prostate, and placenta (Polak et al., 1975). SS mediates a variety of biological effects, the most

Somatostatin (SS), SS receptors and SS analog treatment in tumorigenesis Steffani L, et al.


Figure 1. Amino acid sequences of the human prosomatostatin.

important occurring at the pituitary (inhibition of growth hormone (GH) and tireotropic stimulating hormone (TSH) secretion) and gastroenteropancreatic (GEP) levels (inhibition of insulin, glucagon, and secretin secretion; inhibition of hydrochloric acid production and intestinal fluid absorption) (Konturek et al., 1976). In addition to inhibition of hormone secretion, SS also shows antiproliferative and anti-angiogenetic properties, that have been largely investigated both in cell lines (i.e., human prostate cancer cells, human non small lung cell carcinomas and pituitary adenomas) and GH-secreting tumors. The SS form originally identified in the hypothalamus was SS-14, while SS-28, a congener of SS-14 extended at the N-terminus, was discovered subsequently (Shen et al., 1982). The single human SS gene is located on chromosome 3q28 and the correlate SS mRNA codes for a 116-amino acids (aa) prepro-SS protein (MW 12,727 Da). Prepro-SS has a sequence of hydrophobic aa at the N-terminus which is cleaved at the gly-ala junction at position -78 (from the N-terminus to the C-terminus). Pro-SS undergoes both monobasic (Arg-15) and dibasic (Arg-2Lys-1) cleavages to release the two biofunctional hormones SS-28 and SS-14 (Funckes et al., 1983; Brakch et al., 2002) (Figure 1).

2. Somatostatin receptors In mammals, the biological actions of SS are mediated by at least six G protein-coupled SS receptors (sst) encoded by five different genes, named sst1-sst5. Sst2 exists in two splice variants, sst2A (a long form) and sst2B (a short form), which differ only in the length of the cytoplasmic tail. Sst2 displays a cryptic intron at the 3' end of the coding region, which gives rise to the two spliced variants (Baumeister and Meyerhof, 2000; Olias et al., 2004). In the human gene, the spliced exon encodes for 25 aa residues compared to 38 residues in the unspliced form. The encoded receptor proteins range in size from 356 to 391 aa residues, showing the greatest sequence similarity in the putative transmembrane region, and diverge at their N- and C-terminal segments (Patel, 1999). Human ssts genes are localized to chromosome 14q13 (sst1), 17q24 (sst2), 22q13.1 (sst3), 20p11.2 (sst4), and 16p13.3 (sst5) (Yamada et al., 1993) encoding for proteins of 391 aa,

369 aa, 418 aa, 388 aa, and 363 aa, respectively (Yamada et al., 1992; Corness et al., 1993; Rohrer et al., 1993; Panetta et al., 1994). Structurally, those receptors belong to the so-called "superfamily" of G protein-coupled receptors (GPCRs). All sst isoforms possess a highly conserved sequence motif, YANSCANPI/VLY, in the seventh transmembrane region, which serves as a signature sequence for this receptor family (Kreienkamp et al., 2002). On the other hand, genes for sst1, 3, 4, and sst5 lack classical introns. Interestingly, the estimated sequence identity between sst1 and sst2 receptors is 46%. The deduced aa sequence of human sst3 receptors displays the following degrees of similarity with other members of the sst family: 62% (sst1), 64% (sst2), and 58% (sst4). Moreover, four ssts have been identified in fish and variant forms of several ssts also exist: sst3a, sst3b, sst5a, sst5b, and sst5c in goldfish (Canosa et al., 2004), and sst1a and sst1b in trout (Slagter and Sheridan, 2004). As was the case with SS genes, phylogenetic analysis suggests that sst genes appear to have arisen from a series of gene duplication events. 2.1 Homo- and hetero-dimerization of somatostatin receptor subtypes When ssts in the cell membrane are coexpressed, they may interact forming homo- and hetero-dimers also with other GPCRs, thus altering their original pharmacological and functional profiles. A series of studies, carried out on transfected cell lines, have shown that dimers can consist of two identical sst subtypes (homodimers) or two different subtypes (heterodimers), with a range of possible combinations depending on the specific subtype and, probably, on the specific sst-expressing population (Baragli et al., 2007). The five SS receptor isoforms can be involved in the formation of different dimers, namely, sst1 and sst5 bind efficiently together, while stable sst4-sst5 dimers have not been observed. These interactions are capable to provide greater signalling diversity, affecting the downstream intracellular effects mediated by receptor activation, such as ligand binding affinity, agonist-induced regulation and trafficking. In fact, sst1 endocytosis is enhanced when sst1 and sst5 are co-expressed in the same cell and sst5 is activated;



conversely, the internalization of sst2 is delayed by sst5 and sst2 co-expression. Moreover, ssts can form also heterodimers with other GPCRs: sst2 interacts with the µ-opioid receptor, and sst5 binds to the D2 dopamine receptor (D2R). Interestingly, the sst5-D2R dimer enhances the effects of both receptors, leading to a more potent inhibition of adenylyl cyclase (AC) (Møller et al., 2003). The dimer formation can be not only constitutive, but also ligand-promoted: hence, compounds with high affinity for the different receptor subtypes can be used to achieve effects elicited by specific dimers. In the last years, a variety of mono-, bi- and pan-specific SS analogs has been synthesized, allowing the characterization of the intracellular effectors involved in the downstream signalling of the different ssts (Saveanu et al., 2001). The new receptor specific compounds showed to be useful under many aspects; among them, the understanding of the synergistic effect caused by the simultaneous activation of different receptors. In cultured pituitary cells, a sst2-D2R chimeric compound (BIM-23A387) showed a more potent action in inhibiting prolactin (PRL) and GH secretion compared to the related mono-specific analogs, either alone or in combination (Ferone et al., 2007). A similar pattern of action has been observed for the anti-secretory and anti-proliferative activity in prostate and lung in vitro models, where the treatment with the chimeric sst2-sst5 and sst2-sst5-D2R compounds were more effective than the respective mono-specific SS and D2R analogs (Arvigo et al., 2010). This evidence suggests that the concurrent activation of different GPCRs triggers their dimerization, leading to an enhanced effect. 2.2 Trafficking of somatostatin receptor subtypes A feature common to most GPCRs is the cyclic process of signaling, desensitization, internalization, resensitization, and recycling to the plasma membrane. These events prevent cells from undergoing excessive receptor stimulation or periods of prolonged inactivity (van Koppen et al., 2004). SS receptors differently internalize after agonist binding and, specifically, sst2, sst3 and sst5 are internalized to a higher extent than sst1 or sst4. Among all subtypes, the agonist-mediated trafficking of both sst2 splicing isoforms are the mostly described (Jacobs and Schulz, 2008). Investigations in neuroendocrine tumors showed that both sst2A and sst2B isoforms are rapidly desensitized and internalized after agonist-mediated phosphorylation. Receptor phosphorylation, which involves sites located in the third intracellular loop and in the C-terminal tail, is followed by recruitment of β-arrestin to the receptor forming a stable complex, which is internalized into the same

endocytotic vesicles. Interestingly, the binding affinity of the agonist plays an important role in the degree of receptor internalization. A high binding affinity of the agonist is a prerequisite for triggering sst2 internalization. In fact, the bi-specific sst2/sst5 analog BIM23244, which has a greater sst2 affinity compared to L-817/818 analog is able to induce a greater internalization (Jacobs and Schulz, 2008). Sst5 differs from sst2A in its cellular localization and appears to be predominantly located in intracellular components even without agonist treatment, whereas after stimulation, a large amount of intracellular receptors is recruited to the cell surface. The sst5 third intracellular (i3) loop and the C-terminal tail have been found to regulate receptor internalization, which occurs via clathrin-dependent mechanisms. In cultured pituitary cell lines, where sst5 underwent different kinds of point mutations within the i3 loop, there is a reduction of receptor internalization upon SS-28 treatment. Moreover, by using different C-terminus truncated forms of the receptor, an enhanced sst5 internalization has been observed, thus showing that the sst5 C-terminal tail, or at least a part of it, has an inhibitory role in receptor internalization (Peverelli et al., 2008). Sst3, which shows high affinity for SS-14, internalizes efficiently after agonist stimulation through a clathrin-dependent mediated pathway. Without stimulation, sst3 is almost exclusively located at the plasma membrane, whereas after agonist withdrawal only a small amount of sst3 is recycled to the cell surface (Peverelli et al., 2008). Hence, due to the differential expression of SS receptors in tumors, the comparison of their ability to undergo agonist-induced desensitization and internalization may provide important clues for the clinical use of SS analogs. In this context, an in vitro study demonstrated that short-term administration of the multiligand (sst1/sst2/sst3/sst5) pasireotide (SOM230) modulates SS receptor trafficking in a manner clearly distinct from octreotide (sst2/sst5) (Tulipano and Schulz, 2007). SOM230 was less potent than octreotide in inducing signaling and internalization of the sst2 receptor. Whereas octreotide-activated sst2 receptors cointernalized with β-arrestin-2 into the same endocytic vesicles, SOM230-mediated sst2 activation led to the formation of unstable complexes that dissociated at or near the plasma membrane. Sst2 receptors recycled faster to the plasma membrane in SOM230- than in octreotide-treated cells. The accelerated recycling of SOM230-activated receptors may counteract homologous desensitization in sst2-expressing cells and, hence, result in longer lasting functional responses of SOM230 (Lesche et al., 2009).



Figure 2. Schematic representation of a ligand-driven somatostatin receptor internalization. GRK: GPCR kinase; CCV: clathrin-coated vesicle (modified from van Koppen et al., 2004). 2.3 Somatostatin receptor signalling pathways All five SS isoforms (ssts) bind/interact to G proteins to activate their signalling pathways. They couple to all three Gi subunits (Gi1, Gi2, and Gi3) leading to a potent inhibition of AC activation, and then of cyclic AMP (cAMP) synthesis. Specifically, sst1 is coupled to AC via Gi3; sst2A is able to associate with Gi1, Gi2, Gi3, and Gao2; sst3 interacts with Gi1, Gi2, Gi4, and Gi6 (Reisine and Bell, 1995). Several second messenger systems are involved in their downstream intracellular response: AC, calcium (Ca2+) and potassium (K+) ion channels, sodium (Na+)/H+ antiporter, phospholipase C (PLC), phospholipase A2 (PLA2), mitogen activated protein kinase (MAPK), NO/cGMP, and serine-, threonine-, and phosphotyrosyl-protein phosphatase (PTP) (Patel, 1999). Sst2 and sst4 are the main receptors that activate voltage-gated K+ current (Yang and Chen, 2007). As a result of their activation, membrane hyperpolarization occurs, hindering any subsequent spontaneous membrane potential and leading to a reduction in intracellular Ca2+. Ssts can differently modify Ca2+ currents; in AtT-20 murine cell line, both sst2 and sst5 can couple negatively to an L-type Ca2+ channel reducing Ca2+ influx (Tallent et al., 1996), whereas,

conversely, in the GH3 rat pituitary tumor cell line, only sst2 blocks voltage-gated Ca2+ current (Yang and Chen, 2007). The human ssts also stimulate PTP through a pertussis toxin-sensitive pathway involving Gi2, but differencies among the various species have been found, since sst5 in rat does not regulate PTP. The first evidence of ssts-mediated activation of PTP was given by the counteraction driven by ssts on tyrosine kinase receptors-mediated proliferative effect (Florio, 2008a). One of the main downstream effects of ssts-mediated PTP activation is the inhibition of MAPK ERK1/2 activity. Several data exist about the inhibition of the MAPK signalling cascade by three of the five sst subtypes: sst2, sst3 and sst5. In AtT-20 and in transfected CHO-K1 cells, sst5 constitutively restrains ERK1/2 phosphorylation (Ben-Shlomo et al., 2007), and sst2 and sst3 mediated the same inhibitory signal in SHSY-5Y neuroblastoma cells and in NIH3T3 cells, respectively. Conversely, sst1 and sst4 stimulate the MAPK pathway (Patel, 1999). Glutamate receptor ion channels are also involved in ssts signalling: sst2 inhibits AMPA/kainate receptor-mediated glutamate currents, while sst1 stimulates AMPA/kainate receptor activity in cultured mouse hypothalamic neurons.



Inositol 1,4,5-trisphosphate (IP3) represents another intracellular signalling pathway linked to sst2. In CHO-DG44 cells, it takes place via sst2-mediated activation of phosphatidylinositol 3-kinases (PI3K), whereas in astrocytes and in intestinal smooth muscle cells it is driven by PLC (Florio, 2008a). Experimental data in rat pituitary F4C1 cells indicate that the activation of sst2, but not sst1, stimulates PLC activity and increases cytosolic Ca2+level, due to Ca2+ release from intracellular stores (Rosskopf et al., 2003). In hippocampal neurons, SS effect on PLA-2-dependent stimulation of arachidonate production has been associated with sst4, which is able to elicit arachidonate synthesis through phospholipase A2 (PLA-2) activation (Patel, 1999). In colon carcinoma, enteric endocrine and hepatic cells, the Na+/H+ exchangers can be also activated by sst1, sst3 and sst4, but not by sst2 and sst5 (Florio, 2008a). Interestingly, in human sst5 there are two regions, the BBXXB domain and the DRY motif, located in the third intracellular (i3) and second intracellular (i2) loops, respectively, which are needed to activate the signalling pathways mediated by this receptor subtype. Namely, the BBXXB domain, although being required in the subtype 5 downstream effectors generation, is not directly involved in interactions with Gi protein, since a mutation in the first BBXXB residue does not affect the receptor ability of inhibiting cAMP accumulation. Conversely, the DRY motif was found to be crucial in coupling with Gi protein, since mutations in the DRY sequence do not impair sst5-driven inhibition of cAMP production. However, both regions are necessary to mediate the other sst5 intracellular responses, such as cytoplasmic Ca2+ reduction and inhibition of ERK1/2 phosphorylation (Peverelli et al., 2009).

3. Tumorigenesis Tumorigenesis is a collection of complex genetic diseases characterized by multiple defects in the homeostatic mechanisms that regulate cell growth, proliferation and differentiation. In humans, several lines of evidence indicate that tumorigenesis is a multistep process which reflects genetic alterations that drive the progressive transformation of normal cells into highly malignant derivatives. Tumorigenesis is thought to require four to six stochastic rate-limiting mutation events to occur in the lineage of one cell. Hanahan and Weinberg (Hanahan and Weinberg, 2000) suggest that six cellular alterations, or hallmarks, collectively drive a population of normal cells to become a cancer. The six hallmarks are (i) self-sufficiency in growth signals (SG), (ii) insensitivity to antigrowth signals (IA), (iii) evasion of apoptosis (EA), (iv) limitless replicative potential (LR), (v) sustained angiogenesis (SA), and (vi) tissue invasion and metastasis. Genetic instability (GI) is defined as an "enabling characteristic" that facilitates the acquisition of other mutations due to defects in DNA repair. These

hallmarks form a candidate set of rules that underlie the transformation of a normal tissue to a cancerous one and are shared in common by most and perhaps all types of human tumors (Spencer et al., 2006). 3.1 Antitumor actions of somatostatin and somatostatin analogs SS has been shown to display several biological actions which include inhibition of exocrine and endocrine secretions, gut motility, cell proliferation, cell survival and angiogenesis. SS analogs show antineoplastic and antiproliferative activity in many experimental in vivo and in vitro models and this activity is principally attributed to activation of sst2 and sst5. SS analogs treatment can be effective in the control of tumor growth in humans and in 37-82% of patients receiving SS analogs, as primary medical therapy, tumor shrinkage has been observed. The antiproliferative and antitumoral effects of SS analogs occur independently of their antisecretory and antihormonal effects. From these results we can infer that antisecretory and antitumor effects of SS and SS analogs are mediated by different receptors/signalling pathways and that the antiproliferative effect of these synthetic compounds may depend on tumor ssts profile, but also on the specific target cell intracellular signalling (Pyronnet et al., 2008). SS and SS analogs can control tumor development and progression/metastatization by two separate mechanisms: direct actions, mediated by the ssts, and indirect actions, independent of the receptors. 3.1.1 Direct somatostatin antitumor actions Direct effects of SS and its analogs on tumor cell growth and spread derive from interaction with specific tumor cell membrane receptors. The direct antiproliferative actions include inhibition of autocrine/paracrine growth-promoting hormone/growth factor synthesis, arrest of cell division (by blockade of growth factor-mediated mitogenic signals), suppression of cell invasion and induction of apoptosis (programmed cell death) (Pyronnet et al., 2008). The exact antitumoral mechanism initiated by SS analogs depends on the tumor cell type and the ssts to which it binds. In this way, each receptor subtype is able to mediate different biological actions (Susini and Buscail, 2006). Cell cycle arrest is mediated by interaction of SS with its five receptors and the consequent initiation of several intracellular signalling pathways, which are either activated or inhibited according to the sst subtype, the downstream recruited enzyme and cell environment. These pathways include activation of tyrosine kinases (JAK, c-src) and tyrosine phosphatases (SHP1, SHP2, PTP), activation/inhibition of nitric oxide synthase/cGMP-dependent protein kinase, Ras/ERK pathway and inhibition of PI3 kinase/Akt pathway, which in turn lead to induction of the cyclin-dependent kinase inhibitor p27kip1 or p21Cip1 and cell cycle arrest (Pyronnet et al., 2008). SS also induces cell growth inhibition through restoration of functional gap



junctions. These structures are composed of connexins and play a pivotal role in maintaining the differentiated state and cell-contact inhibition. Actually, in most cancer cells, it has been observed an impaired expression of connexins (Lahlou et al., 2005). It has been demonstrated that SS is also a potent anti-migratory and anti-invasive agent for various tumor cells. Inhibition of cell invasion occurs through molecular mechanisms which are cell type specific and depend on sst expression pattern, on sst effector coupling as well as on the signalling cascade involved in target cells (Pola et al., 2003). SS analogs are also thought to inhibit cell proliferation by inducing apoptosis. The receptor subtypes primarily involved in SS-induced apoptosis are sst3 and sst2. Apoptotic effect is achieved by regulation of the two main signalling pathways, the cell-extrinsic pathway (triggered by death receptors) and the cell-intrinsic pathway (also called the mitochondrial pathway) (Pyronnet et al., 2008; Florio, 2008b). SS and its chemically designed analogs are potential therapeutic agents, in particular for the treatment of endocrine diseases that cause hormone hypersecretory syndromes. SS and its commercially available analogs exert antisecretory and antiproliferative effects by interacting with one or more of the five ssts, which then trigger various intracellular signalling pathways according to the tissue, thus possibly leading to different actions. The tissue expression patterns of ssts, the binding profile of agonists and ssts effector coupling confer functional and therapeutic specificity to ligand activity (Zatelli and degli Uberti, 2009). The two SS analogs currently used in the clinics are Octreotide and Lanreotide. They have demonstrated efficacy in reducing GH and IGF-1 levels in up to 60% of patients with acromegaly and therefore have been widely used in the treatment of GH hypersecretion (Shimon et al., 1997). The main pharmacological target of these compounds is sst2, the receptor subtype which is the most frequently expressed in human GH-secreting pituitary adenomas, but they also bind, with a lesser affinity, to sst5. However, a significant proportion of patients with acromegaly is resistant to the treatment with ocreotide. Pasireotide (SOM-230), a compound that interacts with multiple ssts (sst1-2-3-5) is able to inhibit GH secretion in octreotide-resistant pituitary adenomas, representing a potential therapy for octreotide-resistant acromegaly patients (Petersenn et al., 2010). The efficacy of pasireotide in overcoming octreotide resistance has been attributed to its ability of binding to all ssts and, in particular, to its greater affinity for sst5, which is up-regulated in such tumors. This ssts multiligand compound showed in vitro a significant reduction of cell viability in many non-functioning pituitary adenomas (NFPAs) probably through the inhibition of VEGF secretion. Several results suggested that pasireotide could be a potential therapeutic agent for conditions characterized by an excess of ACTH. In

patients with Cushing's disease, the administration of pasireotide decreased urinary free cortisol levels and significantly improved symptoms associated with the disease. Moreover, in ACTH-secreting pituitary tumor cells, pasireotide reduced ACTH secretion and cell proliferation (Bode et al., 2010). NFPAs represent a possible therapeutic target also for selective sst1 agonists as these tumors have been demonstrated to express sst1. The sst1 agonist BIM-23926 has exhibited antisecretory and antiproliferative effects in a group of NFPAs in vitro. Moreover, several findings support the hypothesis that chimeric ssts/DR agonists can be effective in suppressing in vitro cell proliferation in the majority of NFPAs. Indeed, BIM-23A387, a chimeric sst2/DR2 selective agonist inhibits cell viability in most NFPA primary cultures, as well as BIM-23A760, a compound with high affinity for DR2, sst2 and sst5 significantly suppresses DNA synthesis in the 60% of the NFPA cultures tested (Florio et al., 2008). It has been observed that SS and its analogs can decrease plasma calcitonin levels and improve symptoms in patients with medullary thyroid carcinoma (MTC), but their antiproliferative effects remain controversial. As a matter of fact, in TT cells, a human MTC cell line expressing all ssts, sst2 activation leads to inhibition of DNA synthesis and cell proliferation, whereas sst5 activation has an opposite effect. Thus, we can infer that sst2 and sst5 agonists can antagonize the activity of one another in contrast to what happens in pituitary adenomas. Potent sst1-selective ligands (BIM-23296 and BIM-23745) could have a therapeutic role in MTC because they are effective in reducing DNA synthesis, the viability of TT cells, calcitonin secretion and gene expression (Zatelli et al., 2006). Ssts are also highly expressed in most neuroendocrine tumors with a variable expression patterns. Treatment with Octreotide and Lanreotide is ineffective in inhibiting hormone secretion in some patients with neuroendocrine tumors because they develop tachyphylaxis. Conversely, pasireotide has shown a considerable reduction of symptoms in the majority of patients with metastatic gastroenteropancreatic endocrine tumors (Desai et al., 2009). Experimental data on prostate cancer showed that four (sst1-2-3-5) out of five ssts receptors were found to be expressed in the LNCaP cell line, an in vitro model of human androgen-dependent PCa. Their activation by selective SS agonists resulted in a significant anti-proliferative effect with a peculiar pattern according to receptor subtype, ligand affinity and, possibly, receptor dimerization. Moreover, such treatments were also able to modulate the profile of the IGF system, known to be involved in PCa progression. Interestingly, these data provide strong evidence for an inhibitory role of sst1 activation on PCa cell proliferation, suggesting that SS agonists with enhanced sst1 affinity and selectivity may have great potentiality as pharmacological tools for at least androgen-dependent PCa treatment. In addition, the antiproliferative effect of sst1 and sst5 mono-



specific agonists may be due, at least in part, to the inhibition of IGF-I secretion (Ruscica et al., 2010). 3.1.2 Indirect somatostatin antitumor mechanisms Indirect antitumor effects of SS include suppression of synthesis or/and release of growth factors and growth-promoting hormones such as insulin, prolactin, IGF-1, epidermal growth factor (EGF), transforming growth factor- (TGF-), gastrin, cholecystokinin (CCK) and GH. Several experimental in vitro and in vivo results indicate that another indirect action of SS and SS analogs on tumor growth may be the inhibition of angiogenesis. Angiogenesis, the formation of new blood vessels from an existing capillary network, is necessary for tumor neovascularization, which is essential for tumor growth, invasion and for dissemination of metastasis. By limiting the blood supply, tumor growth can be effectively controlled (Kvols and Woltering, 2006). SS and SS analogs exert antiangiogenic actions through different mechanisms like suppression of endothelial cell proliferation and arrest of monocyte and endothelial cell migration. Normal endothelial cells lack sst2 receptors and the expression of this receptor subtype on endothelial cells uniquely appears as they proliferate to form new blood vessels (Kvols and Woltering, 2006). So, the inhibition of angiogenesis may result from the up-regulation of sst2 during the angiogenic switch from resting to proliferating endothelium. However, other ssts such as sst3 and sst5 may also play a role. At the molecular level, this effect results from SS-mediated inhibition of MAP kinase activity and endothelial NO synthase (eNOS) activity. Another mechanism by which SS analogs suppress angiogenesis is through a broad inhibition of both the release and the effect of growth factors, some of which are angiogenic, including vascular endothelial growth factor (VEGF), platelet-derived growth factor, IGF-1 and basic fibroblast growth factor. These growth factors, secreted by tumor cells and infiltrating inflammatory cells, stimulate endothelial and smooth muscle cell proliferation and migration, important processes in angiogenesis (Zatelli et al., 2007).

4. Somatostatin receptor activation and tumorigenesis: future directions According to the evidence reported in the present paper, a specific pattern of ssts activation seems to elicit important antitumoral actions with potential relevance to some solid tumors expressing these receptor isoforms. In addition to the well-established anti-secretory effects, which may affect the cancer-associated paraneoplastic syndrome as well as the possible autotrophic actions of tumor-produced secretory proteins, a consistent body of data indicates that stimulation of tumor-expressed ssts results in a

multi-step restrain of tumorigenesis. These mechanisms thus deserve further exploitation with the aim of validating novel therapeutic approaches to cancer.

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Case Report Section Paper co-edited with the European LeukemiaNet

A new case of translocation t(14;14)(q11;q32) in B lineage ALL Elvira D Rodrigues Pereira Velloso, Priscila Pereira dos Santos Teixeira, Karina Prandi Melillo, Luciana J Rodrigues da Silva, Cristina Alonso Ratis, Daniela Borri, Cristóvão LP Mangueira

Clinical Laboratory, Hospital Israelita Albert Einstein, Sao Paulo, Brazil (EDRPV, PPdST, LJRdS, CAR, DB, CLPM); CASE Intermedica, Sao Paulo, Brazil (KPM)


Online updated version : http://AtlasGeneticsOncology.org/Reports/t1414BALLVellosoID100048.html DOI: 10.4267/2042/46023


Clinics Age and sex 43 years old male patient.

Previous history no preleukemia ; no previous malignancy ; no inborn condition of note.

Organomegaly hepatomegaly , splenomegaly , no enlarged lymph nodes , central nervous system involvement.

Blood WBC : 227X 109/l HB : 5,9g/dl Platelets : 51X 109/l Blasts : 12% Bone marrow : aspirate: 90% lymphoblast%

Cyto-Pathology Classification Cytology LLA-L2

Immunophenotype HLA-DR+, TdT+, CD79a+, CD19+, cyCD22+, CD20+, CD10+

Rearranged Ig Tcr rearranged IGH (FISH)

Pathology not done

Electron microscopy not done

Diagnosis common B-ALL

Survival Date of diagnosis: 02-2009

Treatment: Cancer and Leukemia Group B (CALGB) protocol Complete remission : not evaluated Treatment related death : Neutropenia and lung infection Relapse : no

Status: Death. Last follow up: 03-2009

Survival: 20 days

Karyotype Sample: Bone marrow

Culture time: 24 and 48 hours without stimulating agents

Banding: G

Results: 46,XY,t(14;14)(q11;q32.1)[20]

Karyotype at Relapse: not done

Other molecular cytogenetics technics FISH using IGH Break Apart Rearrangement Probe, Vysis

Other molecular cytogenetics results nuc ish(IGHx2)(5'IGH sep 3'IGHx1)[154/200]/ (5'IGHx2,3'IGHx1)(5'IGH con 3'IGHx1)[33/200]

A new case of translocation t(14;14)(q11;q32) in B lineage ALL Rodrigues Pereira Velloso ED, et al.


G- banded partial karyotypes showing the t(14;14).

Interphase FISH shows IGH gene rearrangements (IGH Dual Color, Break Apart Rearrangement probe).

Comments Translocation t(14;14)(q11;q32) in B lineage acute lymphoblastic leukemia was described in few cases, some of them associated with other recurrent

rearrangements such as t(4;11) and t(8;14). Lui et al, in 2004 showed IGH rearrangement in two cases, although the partner was unknown. Akasaka et al in 2007, described CEBPE involvement in a patient with B-ALL and t(14;14)(q11;q32). In 2008, Han et al showed through FISH analysis the presence of trisomy 4 as a simultaneous involvement of IGH and CEPBE genes. The t(14;14)(q11;q32) CEBPE/IGH may be associated with good prognosis in B-ALL. In 4 cases with clinical follow-up, complete remission was achieved and those were alive at the time of report. In the case described herein, the t(14;14) was the sole anomaly, IGH rearrangement was detected but CEBPE involvement was not studied. This patient has well known bad prognostic features as high WBC count and CNS involvement and died few days after diagnosis.

References Liu S, Bo L, Liu X, Li C, Qin S, Wang J. IGH gene involvement in two cases of acute lymphoblastic leukemia with t(14;14)(q11;q32) identified by sequential R-banding and fluorescence in situ hybridization. Cancer Genet Cytogenet. 2004 Jul 15;152(2):141-5

Akasaka T, Balasas T, Russell LJ, Sugimoto KJ, Majid A, Walewska R, Karran EL, Brown DG, Cain K, Harder L, Gesk S, Martin-Subero JI, Atherton MG, Brüggemann M, Calasanz MJ, Davies T, Haas OA, Hagemeijer A, Kempski H, Lessard M, Lillington DM, Moore S, Nguyen-Khac F, Radford-Weiss I, Schoch C, Struski S, Talley P, Welham MJ, Worley H, Strefford JC, Harrison CJ, Siebert R, Dyer MJ. Five members of the CEBP transcription factor family are targeted by recurrent IGH translocations in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Blood. 2007 Apr 15;109(8):3451-61

Han Y, Xue Y, Zhang J, Wu Y, Pan J, Wang Y, Shen J, Dai H, Bai S. Translocation (14;14)(q11;q32) with simultaneous involvement of the IGH and CEBPE genes in B-lineage acute lymphoblastic leukemia. Cancer Genet Cytogenet. 2008 Dec;187(2):125-9


Rodrigues Pereira Velloso ED, Pereira dos Santos Teixeira P, Prandi Melillo K, Rodrigues da Silva LJ, Alonso Ratis C, Borri D, Mangueira CLP. A new case of translocation t(14;14)(q11;q32) in B lineage ALL. Atlas Genet Cytogenet Oncol Haematol. 2011; 15(9):806-807.



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A new case of translocation t(14;14)(q11;q32) in B lineage ALL Rodrigues Pereira Velloso ED, et al.


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