(CANCER RESEARCH57. 2276-2280. June 1, 1997)

ABSTRACT

Cancer arises from aberrations in the genetic mechanisms that controlgrowth and differentiation. HMGI-C and HMGI(Y) are members of the

HMGI family of architectural factors expressed in embryonic or undifferentiated cells and highly associated with transformation. Translocations of 12q13-15 in lipomas (fat cell tumors) disrupt HMGI-C and fuse its

DNA-binding domains to novel transcriptional regulatory domains. Thisstudy shows that in a rare, karyotypically distinct group of human lipomas, rearrangements of 6p2l—23produce internal deletions withinHMGI(Y). Activation of the rearranged alleles leads to expression ofaberrant HMGI(Y) transcripts in differentiated adipocytes. A molecularanalysis of these transcripts demonstrates that fusion of HMGI DNAbinding domains to putative transcriptional regulatory domains was notnecessary for lipoma formation. However, such fusions may facilitatetumor development because activation of the wild-type HMGI allele,normally required for tumorigenesis, is bypassed in lipomas which expresschimenc HMGI proteins. We hypothesize that HMGI misexpression in adifferentiated cell Is a pivotal event in benign tumorigenesis, and themolecular pathway of tumor development depends upon the precise anCure of 11MG! disruption.

INTRODUCTION

Elucidation of the genetic mechanisms that control growth anddifferentiation (1) have led to a greater understanding of mammaliandevelopment and its various abnormalities, including, most prominently, cancer (2, 3). A number of genes that function both indevelopment and transcriptional regulation (4) are known to be mutated in tumor cells. In addition to the well-documented role of c-mycin B-cell ontogeny and leukemias (5), other examples include the c-rdprotein, expressed in the hematopoietic organs of the mouse embryo(6), as well as the oncoproteins ets-l and ets-2, which act duringdevelopment of lymphoid and neuronal tissues (7).

Recently, the embryonically expressed accessory transcription factor HMGI-C was shown to be disrupted in human fat cell tumors,lipomas. HMGI-C is a member of the HMGI family of architecturalfactors (8), which at present consists of two members, HMGI-C andHMGI(Y). The important structural feature shared by both HMGI

proteins is the presence of three DNA-binding domains, termed AThooks because of their ability to bind to AT-rich DNA in the minorgroove (9). A series of elegant experiments with HMGI(Y) at thehuman IFN-!3 promoter have demonstrated its involvement in transcriptional regulation (10). HMGI(Y) was shown to be an essentialcomponent of the enhanceosome (1 1), a higher order transcriptionenhancer complex that forms when several distinct transcription factors assemble on DNA in a stereospecific manner (12). As an architectural component of the enhanceosome, HMGI(Y) promotes activation of gene expression by subtly modulating DNA conformation (12).

Received 12/31/96; accepted 417/97.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

@ This work was supported by NIH Grant GM38731 (to K.K.C.).2 To whom requests for reprints should be addressed, at Department of Biochemistry,

UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854-5635. Phone:(908) 235-4768; Fax: (908) 235-4783.

A number of studies, in particular with HMGI-C, have provided

insight into the role of the HMGI family in growth and development.Hmgi-c is expressed exclusively during murine embryogenesis (13)and is undetectable in newborn and adult tissues (13, 14). Similarly,Hmgi(y) is predominantly expressed until 16.5 days post coitum in themouse embryo (I 3). Inactivation of Hmgi-c produced a dramaticdisruption of both pre- and postnatal growth, resulting in the pygmyphenotype (15). Pygmy mice exhibit significant growth retardation,which results in a 60% weight reduction compared to their wild-typelittermates (16). Interestingly, Hmgi-c inactivation does not affect thegrowth hormone/insulin-like growth factor endocrine pathway (13),suggesting that Hmgi-c functions in a previously unknown growthregulatory mechanism.

During development, the processes of proliferation and differentiation are often interdependent (17), and studies with the HMGIgenes have implied that HMGI expression may be also associatedwith the biological, rather than only the proliferative, state of thecell ( 18). Expression of Hmgi-c and Hmgi(y) in a thyroid cell lineis dramatically increased after transformation, although the rate ofproliferation of the transformed cells does not increase (19, 20).Interestingly, several cell lines described in these reports differedmarkedly in their degree of transformation. A partially transformedcell line that retained its differentiated phenotype revealed lowlevels of Hmgi expression, whereas Hmgi expression was high infully transformed cell lines that lost their differentiation markers(20). Finally, preliminary studies from our laboratory have demonstrated that in the mouse embryo, Hmgi-c is localized to lessdifferentiated mesenchymal cells and is no longer present in theirterminally differentiated counterparts (Ref. 13 and data notshown). In combination, these results indicate that the function ofthe HMGI proteins may be to maintain the undifferentiated cellularstate (21).

A direct role for HMGI-C in tumorigenesis has been demonstrated

by the analysis of several distinct types of solid human tumors (22,23). Disruptions ofHMGI-C were linked to the pathogenesis of a widevariety of benign mesenchymal neoplasms that have consistent rearrangements at chromosomal band l2ql3—15 (24). Translocations ofHMGJ-C generate novel chimeric transcripts that consist of theHMGI-C DNA-binding domains fused to ectopic sequences providedby the translocation partner (22, 23). These novel sequences werepredicted to encode for transcriptional regulatory domains, suggestingthat aberrant transcription of the HMGI-C target genes contributes totumor formation (22).

The diverse set of mesenchymal neoplasms in which HMGJ-C isfrequently disrupted by translocations of l2ql3—15 includes lipomas,uterine leiomyomas, pulmonary hamartomas, and pleomorphic adenomas of salivary gland (22, 23). Another cytogenetic subgroup thatcan be identified in this set of tumors is characterized by rearrangements at 6p2l—23 (24, 25). Intriguingly, HMGJ(Y) has previouslybeen localized to this chromosomal area (26). Conservation of theHMGI DNA-binding domains (9) and the cognate expression patterns

of the HMGI proteins (13, 14) suggested HMGI(Y) as a likely candidate for involvement in mesenchymal neoplasia.

2276

Misexpression of Disrupted HMGI Architectural Factors Activates AlternativePathways of Tumorigenesis'

Alexei Tkachenko, Hena R. Ashar, Aurelia M. Meloni, Avery A. Sandberg, and Kiran K. Chada2

Department of Biochemistry, UMDNJ-Robert Wood Johnson Medical School, Piscazaway, New Jersey 08854-5635 (A. T., H. R. A., K. K. CI, and The Cancer Center of SouthwestBiomedical Research, Institute of Genetrix, Scottsdale, Arizona 85251 (A. M. M., A. A. S.)

on June 14, 2020. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

HMGI(Y) REARRANGEMENTSIN LIPOMAS

08203 t(6;1 1) with translocations involving 6p21—23(24). RNA wasisolated directly from frozen tumor samples (27), and 3'RACE (28)was performed with an HMGI(Y)-specific exon five primer to amplifyHMGI(Y) transcripts to give an expected wild-type product of approximately 1.5 kb. Surprisingly, aberrant HMGI(Y) products of 592 and591 bp were observed in tumors ST92-24269 and ST88-08203, respectively (Fig. 1A). At the same time, HMGI-C expression was notdetected in these tumors, and both HMGI(Y) and HMGI-C genes werenot expressed in normal, adult fat (Fig. 1B). Sequencing of theaberrant HMGI(Y) cDNAs revealed heterologous sequences that fol

HMGI(Y) lowedthe5' endof HMGI(Y)in bothlipomas.The presenceofaberrant transcripts in ST92-24269 and ST88-08203 was confirmedby an independent RT-PCR in which a novel sequence-specific reverse primer, rather than oligo-(dT), was used for reverse transcriptionand subsequent PCR (Fig. 2).

The anomalous HMGJ(Y) cDNAs were further characterized by adetailed molecular analysis. In lipoma ST92-24269, comparison of thenovel sequence to the GenBank database revealed that it was derivedfrom the 3'UTR of wild-type HMGJ(Y) (Fig. 3). PCR analysis of thegenomic DNA from tumor 5T92-24269 determined that the transcriptwas produced by an internal deletion of both exonic and intronicsequences (data not shown) that removed 922 bp from the wild-typeHMGJ(Y) cDNA (Fig. 3). Sequencing of the aberrant transcript inlipoma ST88-08203 revealed a fully intact HMGJ(Y) open readingframe. Further analysis demonstrated that this transcript removed 923bp of the wild-type sequence from exon eight (Fig. 3). Interestingly,the rearrangement was limited to only the 3'UTR of the gene, leavingthe coding sequenceintact. Therefore, the aberrant transcripts isolatedfrom the lipomas with rearrangements of 6p2l—23 are produced byinternal deletions within the HMGI(Y) gene. Although the translocations do not directly disrupt the HMGI(Y) gene, the existence ofsubmicroscopic molecular aberrations in the close proximity to achromosomal translocation site is often observed (30).

A

B

1.6kb

1.0kb

0.5kb

0.3kb

0.2kb —

Fig. 1. HMGI expression in human lipomas with chromosomal rearrangements of6p2I—23. In A, 3'RACE with HMGJ(Y)-specific primers on RNA from lipomas ST92-24269 and ST88-08203 yields aberrant 592- and 591-bp products, respectively; thepredicted wild-type 1.5-kb fragment is amplified only in ST88-08203. No detectableexpression of HMGJ(Y)was observed in normal s.c. fat (Fat), which is mainly composedof differentiated adipocytes (24). OLD-i is a colorectal adenocarcinoma cell line thatexpresses wild-type HMGI(Y) and HMGI-C (29). Previously, aberrant transcripts involving cryptic splice sites have been observed with HMGI-C(23) but not in our present studyor other studies with HMGI(Y) (26). Left, molecular weight markers. In B, HMGI-Cexpression is not detected by specific RT-PCR in lipomas with rearrangements of6p2l—23and in normal fat.

1.6kb —

1.0kb —

0.5kb —

0.4kb —

0.3kb —

—

Fig. 2. Sequence-specific RT-PCR confirms the presence of HMGI(Y) aberrant products in RNA isolated from lipomas with rearrangements of 6p2l—23. RT-PCR productswere amplified using primers located on either side of the deletion within HMGJ(Y).Expected product sizes are: 1314 bp from wild-type transcript; 392 bp from the aberranttranscript in ST92-24269 cDNA; and 391 bp from the aberrant transcript in ST88-08203cDNA. In ST92-24269 and STh8-08203, 359- and 358-bp bands, respectively, areproduced by the altemative splicing of HMGJ(Y)(26).

2277

a@ c@,(0 0

I- I..@Cl) Cl) IL1 0

MATERIALS AND METhODS

Identification and Characterization of Aberrant Transcripts in Lipomm. Lipomaspecimenswereobtainedfrompatientsat thetimeof surgeryand processed as described (24). Karyotypes of lipomas were: ST88-08203

[46, XX, t(6;l l)(p23;ql3), t(l l;l2)(q13;q22)] and ST92-24269 [46, XX, t(4;6)(q27;p2l)]. Isolation of RNA from frozen tumors, reverse transcription,3'RACE3 and specific RT-PCR were performed essentially as described (22).3'RACE on RNA isolated from lipomas ST88-08203 and ST92-24269 wasperformed using an HMGI(Y) exon 6 primer 5'-GTGCCAACACCTAAGAGAC and nested with an exon 6 primer 5'-CCTCGGGGCCGACCAAAGOGG. HMGI-C-specific RT-PCR in lipomas ST88-08203 and ST92-24269 was performed using oligo(dT)-primed cDNA and HMGI-C primers5'-ACTfCAGCCCAGGGACAACC (exon 1) and 5'-CATTFCCTAGGTCTGCCTCTFOG (exon 3).

Aberrant products obtained in 3'RACE were isolated from the gel, cloned,and sequenced. For lipoma ST92-24269, the genomic fragment was PCRamplified using exon 8 sense primer 5'-GAGGA000CATCTCGAGG-3' andantisense primer 5'-CACCACAGGAGACTCCAGTC-3' complementary tothe sequence that is located downstream of the poly(A) signal and is notincluded in the HMGJ(Y)transcript. In lipomas ST88-08203 and ST92-24269,the aberrant HMGI(Y)transeripts were specifically reverse transcribed using anHMGJ(Y) exon 8 antisense primer 5'-CAGAACAGGAGGCAATGAGG andPCR amplified using the same antisense primer and exon 5 sense primer5'-GGCAGGCCGCGCAAGCAGCCT.

RESULTS

Isolation and Analysis of Aberrant HMGI Transcripts. Tumorsobtained from patients at the time of surgery were processed forculturing and karyotyped as described previously (24). A molecularanalysis was performed on lipomas ST92-24269 t(4;6) and 5T88-

3 The abbreviations used are: 3'RACE, 3' rapid amplification ofcDNA ends; RT-PCR,

reverse transcription-PCR; UTR, untranslated region; ORF, open reading frame.

1 HMGI-C

0) C@)(0 0CbiI C@4

c410I

@- @- @1

Cl) Cl) 0

on June 14, 2020. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

HMGI(Y)REARRANGEMENTSIN LIPOMAS

0OHMGI(Y)

exon 5 6 7 8Fig. 3. Rearrangements of fip2l—23in human lipomas

result in deletions within the HMGJ(Y) gene. Hatchedboxes, exons; open boxes, introns; dashed line, deletedsequences. Sequences that code for DNA binding domains(DBI. DBJI. and DBIII) are represented by the black boxeswithin exons. Stop codon (TGA) and 3'UTR are indicated.The nucleotide sequences flanking the deletions are shown,and the breakpoints are indicated by arrows. The untranslated first four exons of the gene are not shown. In lipomaST92-24269, the deletion includes 10bp of exon 6, 5 1bp ofexon 7, and 861 bp of exon 8. Of this sequence. I Il bpencodes for the COOH terminus of HMGI(Y). In lipomaST88-08203. the 923-bp deletion is limited to exon 8 anddoes not disturb the HMGI(Y) ORF.

Distinct Features of the Proteins Encoded by the AberrantHMGI Transcripts. In lipoma ST92-24269, the predicted HMGI(Y)fusion protein consists of the first two DNA-binding domains ofHMGI(Y) fused in frame to an uninterrupted ORF encoding for 108

amino acid residues (Fig. 4). A detailed examination of the ORFrevealed an unusually high content of proline (17%), which is indicative of a potential transcriptional regulatory domain (31). Therefore,the overall structure of this HMGI(Y) fusion protein is remarkablysimilar to proteins produced by disruptions at l2q13—l5 that juxtaposed the DNA-binding domains of HMGI-C to putative transcriptional regulatory domains (22).

In the tumor ST88-08203, translation of the HMGJ(Y) aberranttranscript predicted a normal protein. This is in contrast to the lipomasdescribed previously (22, 23), where chimeric HMGJ-C transcriptsencoded for novel fusion proteins, the formation of which was proposed to be necessary for lipoma development (22). Furthermore,disruption of HMGJ-C by t( 12; 13) in lipoma ST91-l98 (22) resultedin a truncated protein that consists mainly of the HMGI-C AT-hooks(data not shown). We conclude that fusion of transcription regulatorydomains to the DNA-binding domains of disrupted HMGI proteins isnot required for lipoma formation.

Lipomas Can Bypass Expression of the Wild-Type HMGJ Allele. Expressionof the wild-type HMGIproteinsis highly associatedwith transformation (20, 32) and can be detected in a wide variety oftumors. Moreover, inhibition of HMGJ-C synthesis was shown torender several distinct cell types intransigent to retroviral transformation, suggesting that HMGI expression is required for tumorigenesis(33). In agreement with this hypothesis, appreciable levels of wildtype HMGI(Y) expression were observed in tumor ST88-08203 (Figs.

ST92-24269

MSESSSKSSQ PLASKQEKDG TEKI@@ KQPPVSPGTA LVGSQ

KEPSEVPTPK I@RGR*GSK NKGAA@LTLL @TRHSAIS@S SDGA@

IRss@c@L@ T@LLLLP@1@QGSGSITP@IFT NYLWTVVLJT VQCSI

LRH@SLLLL@A@.NvH@.HcLLT@PRS@RY SLWGRG&GAWQ@*

Fig. 4. Novel sequence in lipoma ST92-24269 encodes for a potential transcriptionalregulatory domain. HMGI(Y) DNA-binding domains are boxed. Novel sequence is inboldface, and proline residues are underlined.

1 and 2). Surprisingly, the nonrearranged allele was not expressed inlipoma ST92-24269 (Figs. 1 and 2), where an HMGI(Y) fusionprotein was identified.

Next, we examined expression of wild-type HMGI-C in lipomaswith rearrangements of l2ql3—l5 that fuse HMGI-C DNA-bindingdomains to heterologous sequences (22). Again, the nonrearrangedallele was expressed in tumor ST9I-198 (data not shown), wheret(l2;13) produced a truncated HMGI-C but not in lipoma 5T93-724(data not shown), in which HMGI-C DNA-binding domains werefused to LIM transcriptional regulatory domains (22, 34).

DISCUSSION

Rearrangements within HMGI Genes Are Required for LipomaDevelopment. The studies on HMGJ-C and HMGI(Y) now provideinsights into the molecular mechanism of tumor formation in lipomasand, by extrapolation, in related solid mesenchymal neoplasms. In all12 analyzed lipomas (22, 23), chromosomal rearrangements haveproduced disruptions in translocated HMGI alleles. An aberrationcommon to these tumors is a deletion of, or within, the highlyconserved and unusually large 3'UTR that is a distinguishing featurein HMGJ genes (35). The best example is lipoma ST88-08203, wherethe aberrant transcript codes for the wild-type HMGI(Y) protein, and

the deletion is restricted to the 3'UTR. Rearrangementsof the 3'UTRof HMGI-C have also been observed in leiomyoma and pleomorphicadenoma of salivary gland (23). This suggests that 3'UTRs of theHMGI genes may contain sequences required for the regulation ofHMGJ expression and that disruptions of the 3'UTRs are necessaryfor the activation ofthe disrupted allele. We conclude that the minimalrequirement for tumor formation is a disruption within an HMGJ genethat involves a deletion within the 3'UTR.

The aberrant transcripts isolated from lipomas with rearrangementsof 6p2l-23 were generated by internal deletions within the translocated HMGI(Y) allele. This observation suggests that in lipomas andrelated benign mesenchymal tumors, 11MG! genes may contain internal deletions and other submicroscopic rearrangements that would beundetectable by cytogenetic techniques. Therefore, the contribution ofthe HMGJ genes to tumorigenesis is likely to be underestimated andmore significant than thought previously.

2278

TGAI UfliD@ 001

@“““@@

...ctgc caa... ...tgc tccc...

ST92-24269

ST8B-08203

on June 14, 2020. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

HMGI(Y)REARRANGEMENTSIN LIPOMAS

Tumor

WtI@ (+)

WtM@ (+)

Gain of atranscrl@m@ dom@n

Tumor formation

wtbM@(-)

Fig. 5. Model oflipoma formation. Black boxes, AT-hooks; stippled box, exons coding for the COOH-terminal domain of an HMGI protein that has no transcriptional activity (10);hatched bo@ transcriptional regulatory domain(s). Broken arrow, transcription of the allele. wtHMGI(—)and wtHMGJ(+) refers to the expression of nonrearranged allele. The cell inwhich lipoma development is initiated can theoretically be either wtHMGJ(—)or wtHMGI(+).

Misexpression of HMGI Genes in a Differentiated Cell Resultsin Twnorigenesis. Lipomas are composed of mature adipocytes (24)that, similar to other terminally differentiated cells, normally do notexpress 1-1MG! proteins (13, 14). However, transcriptionally activeHMGJ alleles are consistently found in solid mesenchymal tumorswith karyotypic aberrations of 12q13—15(22, 23) and 6p2l—23 (thisreport). Therefore, rearrangements of 12q13—15or 6p2l—23 activatean HMGI allele normally silent in adult cells, and the resultingmisexpression of the HMGI protein in the context of a differentiatedmesenchymal cell is a crucial step in tumor development. In thismodel, tumorigenesis results from the temporally inappropriate expression in an adult cell of a gene that is normally expressed duringprenatal development in an embryonic cell of the same lineage.

These observations are somewhat similar to the studies in B-cellleukemias where activation of c-myc expression in a precursor of theB-cell lineage results in neoplasia (5). Unlike the HMGI familymembers, however, the endogenous expression of c-myc is not restricted to embryogenesis, and its inappropriate expression takes placeat the same time in the life of the organism when it is normallyexpressed (36). In greater contrast to the misexpression of HMGIproteins in lipomas is the ectopic expression of HOX1 1 in a subset ofT-cell acute lymphoid leukemias, which result from activation ofHOX11 in cells of the inappropriate lineage (37).

Distinct Molecular Pathways of Tumorigenesis Exist in Lipo

man. The molecular analysis of the lipomas described above yieldsvaluable information about the expression state of the nonrearrangedHMGI alleles. Wild-type HMGJ expression, normally associated withtumorigenesis (20, 32), was readily detectable in lipomas 5T88-08203and ST91-198, where chromosomal rearrangements resulted in normal HMGI(Y) and truncated HMGI-C proteins, respectively. In contrast, the nonrearranged HMGI allele was not expressed in tumorsST92-24269 and ST93-724, where the aberrant HMGI transcripts

encoded for proteins consisting of the HMGI DNA-binding domainsfused to putative transcriptional regulatory domains.

Lack of expression of the nonrearranged HMGI allele in lipomas inwhich chimeric transcripts encode for transcriptional regulatory domains can now be combined with results reported previously (22, 23)into a mechanistically coherent model oflipoma development (Fig. 5).Although speculative at this stage, tumor development appears to beinitiated when the chromosomal rearrangement disrupts an HMGIallele, resulting in HMGI misexpression in a differentiated mesenchymal cell. A deletion within the 3'UTR is probably the minimalrearrangement necessary for tumor formation. Subsequently, one ofthe alternative tumorigenic pathways is selected based on the precisenature of the HMGJ disruption. When a chromosomal rearrangementproduces an HMGI protein with no intrinsic transcriptional activity,tumor development is dependent upon subsequent activation of thenonrearranged allele. However, the requirement for HMGI expressionin tumorigenesis (33) is circumvented when, as a result of a translocation, HMGI DNA-binding domains are fused with a transcriptionalregulatory domain (Fig. 5). The reduced number of events involved intumor formation would readily explain the most frequently observedtranslocation in lipomas, t(3;12)(q29;q15) (25, 38), because it juxtaposes DNA-binding domains of HMGI-C with LIM domains that areinvolved in transcriptional regulation (34). The alternative explanations for activation of the non.rearranged allele by an HMGI autoregulatory mechanism is unlikely because it would postulate that thepresence of transcriptional regulatory domains in the fusion proteinsfound in lipomas ST92-24269 and 5T93-724 interferes with a putativeautoregulatory function. In conclusion, distinct rearrangements of asingle gene can activate alternative molecular pathways of tumorpathogenesis.

HMGJ(Y) and HMGJ-C, two distinct members of the embryonicallyexpressed HMGI family of architectural factors (39), have now been

2279

wtM@(-)

wtIM@(-)

on June 14, 2020. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

HMGI(Y) REARRANGEMENTSIN LIPOMAS

shown to be disrupted in identical tumors. Rearrangements ofHMGI-C, first reported in lipomas (22), were later described in othermesenchymally derived neoplasms with translocations of 12q13—15(23). We predict that, similar to HMGI-C, disruptions of HMGI(Y)will be also responsible for uterine leiomyoma, pulmonary hamartoma, pleomorphic adenomas of salivary gland, and other mesenchymal tumors with recurrent aberrations at 6p2l—23 (24—26). Previously, aberrations of l2q13—15 were reported as the most frequentkaryotypic abnormality in lipomas, whereas rearrangements of6p2l—23occur rarely in these neoplasms (24, 25). Notably, HMGI-Cencompasses a much larger genomic region (>100 kb; Refs. 22 and23) than HMGJ(Y) (10.1 kb; Ref. 26), which may explain the differentfrequency of the involvement of these two genes in lipomas.

Benign tumors, unlike their malignant counterparts, are characterized by one or only a few highly specific genetic alterations (40).Therefore, it has been proposed that the molecular analysis of theseneoplasms would identify genes of major importance for growth andproliferation (41). The above studies with HMGJ(Y) and HMGI-C inlipomas (22, 23) demonstrate the significance of the HMGI family inadipogenesis and tumor progression (42, 43).

ACKNOWLEDGMENTS

We thank Dr. Susan Fried for providing us with human fat samples and Dr.Danny Reinberg for critical reading of the manuscript.

REFERENCES

1. Aaronson, S. A. Growth factors and cancer. Science (Washington DC), 254: 1146—1152,1991.

2. Hay, E. 0., and Zuk, A. Transformations between epithelium and mesenchyme:normal, pathological and experimentally induced. Am. J. Kidney Dis., 26: 678—690,1995.

3. van Leuwen, F., and Nusse, R. Oncogene activation and oncogene cooperation inMMTV-induced mouse mammary cancer. Scm. Cancer Biol., 6: 127—133,1995.

4. Rabbitis, T. H. Chromosomal translocations in human cancer. Nature (Lond.), 372:143—149,1994.

5. Rabbitts, T. H., and Boehm, T. Structural and functional chimerism results fromchromosomal translocations in lymphoid tumors. Adv. Immunol., 50: 119—146,1991.

6. Carrasco, D., Weih, F., and Bravo, R. Developmental expression of the mouse c-relproto-oncogene in hematopoietic organs. Development (Camb.), 120: 2991—3004,1994.

7. Maroulakou, I. G., Papas, T. S., and Green, J. E. Differential expression of ets-1 andets-2 proto-oncogenes during murine embryogenesis. Oncogene, 9: 1551—1565,1994.

8. Wolffe, A. Architectural factors. Science (Washington DC), 264: 1100—1101, 1994.9. Reeves, R., and Nissen, M. S. The AT DNA binding domain of mammalian high

mobility group I chromosomal protein: a novel peptide motif for recognizing DNAstructure. J. Biol. Chem., 265: 8576—8582, 1990.

10. Thanos, 0., and Maniatis, T. The high mobility group protein HMGI(Y) is requiredfor NF-scB-dependent virus induction of the human IFN-@3gene. Cell, 71: 777—789,1992.

11. Bazett-Jones, 0. P., Leblanc, B., Herfert, M., and Moss, T. Short range DNA loopingby the Xenopus HMG-box transcription factor xUBF. Science (Washington DC), 264:1134—1137,1994.

12. Falvo, J. V., Thanos, D., and Maniatis, T. Reversal of intrinsic DNA bends in theIFN-f3 gene by transcription factors and the architectural protein HMGI(Y). Cell, 83:1101—1111,1995.

13. Thou, X., Benson, K. F., Ashar, H. R., and Chada, K. Mutation responsible for themouse pygmy phenotype in the developmentally regulated factor HMGI-C. Nature(Lond.), 376: 771—774,1995.

14. Manfioletti, G., Giancotti, V., Bandiera, A., Buratti, E., Sautiere, P., Cary, P.,Crane-Robinson, C., Coles, B., and Goodwin, G. H. cDNA cloning of the HMGI-Cphosphoprotein, a nuclear protein associated with neoplastic and undifferentiatedphenotypes. Nucleic Acids Rca., 19: 6793—6797, 1991.

15. Xiang, X., Benson K. F., and Chada, K. Mini-mouse: disruption of the pygmy locusin a transgenic insertional mutant. Science (Washington DC), 247: 967—969,1990.

16. Benson, K., and Chada, K. Mini-mouse: phenotypic characterization of a transgenicinsertional mutant allelic to pygmy. Genet. Res., 64: 27—33,1994.

17. Skapek, S. X., Rhee, J., Spicer, 0. B., and Lassar, A. B. Inhibition of myogenicdifferentiation in proliferating myoblasts by cyclin 01-dependent kinase. Science(Washington DC), 267: 1022—1025,1995.

18. Vartainen, E., Palvimo, J., Mahonen, A., Linnala- Kankkunen, A., and Maenpaa P. H.Selective decrease in low-Mr HMG proteins HMG I and HMG Y during differentiation of mouse teratocarcinoma cells. FEBS Leu., 228: 45—48,1988.

19. Giancotti, V., Berlingieri, M. T., DiFiore, P. P., Fusco, A., Vecchio, G., and CraneRobinson, C. Changes in nuclear proteins on transformation of rat epithelial thyroidcells by a murine sarcoma retrovirus. Cancer Rca., 45: 6051—6057, 1985.

20. Giancoui, V., Pani, B., Andrea, P. D., Berlingieri, M. T., DiFiore, P. P., Fusco, A.,Vecchio, G., Philp, R., Crane-Robinson, C., Nicolas, R. H., Wright, C. A., andGoodwin, G. H. Elevated levels of a specific class of nuclear phosphoroproteins incells transformed with ras and v-mos oncogenes and by co-transfection with c-mycand polyoma middle T genes. EMBO J., 6: 1981—1987,1987.

21. Bustin, M., Lehn, 0. A., and Landsman, 0. Structural features ofthe HMG chromosomal proteins and their genes. Biochim. Biophys. Acts, 1049: 231—243,1990.

22. Ashar,H. R.,Fejzo,M. S.,Thachenko,A., Thou,X., Fletcher,J.A., Weremowicz,S.,Morton, C. C., and Chada, K. K. Disruption of the architectural factor HMGI-C:DNA-binding A-T hook motifs fused in lipomas to distinct transcriptional regulatorydomains. Cell, 82: 57—65,1995.

23. Schoenmakers, E. F. P. M., Wansehura, S., Mols, R., Bullerdiek, J., Van der Berghe,H., and Van de Ven, W. J. M. Recurrent rearrangements in the high mobility groupprotein gene, HMGI-C, in benign mesenchymal tumours. Nat. Genet., 10: 436—444,1995.

24. Sreekantaiah, C., Leong, S. P. L., Karakousis, C. P., McGee, D. L., Rappaport, W. 0.,Villar, H. V., Neal, D., Fleming, S., Wankel, A., Herrington, P. N., Carmona, R., andSandberg, A. Cytogenetic profile of 109 lipomas. Cancer Res., 51: 422—433,1991.

25. Mark, J., Havel, G., Grepp, C., Dahienfors, R., and Wedell, B. Chromosomal panernin human benign uterine leiomyomas. Cancer Genet. Cytogenet., 44: 1—13,1990.

26. Friedmann,M., Holth, L. T., Zoghbi,H. Y., andReeves,R. Organization,inducibleexpression and chromosome localization of the human HMGI(Y) non-histone proteingene. Nucleic Acids Res., 21: 4259—4267, 1993.

27. Chirgwin, J., Przybyla. A., MacDonald, R., and Rutter, W. J. Isolation of biologicallyactive ribonucleic acid from sources enriched in ribonuclease. Biochemistry, 18:5294—5299,1979.

28. Frohman, M. A., Dush, M. K., and Martin, G. R. Rapid production of full-lengthcDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Nail. Acad. Sci. USA, 85: 8998—9002,1988.

29. Ashar, H. R., Cherath, L., Przybysz, K. M., and Chada, K. Genomic characterizationof human HMGI-C, a member of the accessory transcription factor family found attranslocation breakpoints in lipomas. Genomics, 31: 207—214,1996.

30. Bornkman, G. W., Polack, A., and Eick, D. c-myc deregulation by chromosomaltranslocation in Burkitt's lymphoma. In: G. Klein (ed), Cellular Oncogene Activalion, pp. 223—273.New York: Marcel Dekker, 1988.

31. Mitchell, P. J., and Tjian, R. Transcriptional regulation in mammalian cells bysequence-specific DNA binding proteins. Science (Washington DC), 245: 371—378,1989.

32. Gianconi, V., Buratti, E., Perissin, L., Zorzet, S., Balmain, A., Portella, G., Fusco, A.,and Goodwin, G. H. Analysis of the HMGI nuclear proteins in mouse neoplastic cellsinduced by different procedures. Exp. Cell Res., 184: 538—545, 1989.

33. Berlingieri, M. T., Manfioletti, G., Santoro, M., Bandiera, A., Visconti, R., Giancotti,V., and Fusco, A. Inhibition of HMGI-C protein synthesis suppresses retrovirallyinduced neoplastic transformation of rat thyroid cells. Mol. Cell. Biol., 15: 1545—1553,1995.

34. Sanchez-GarcIa, I., Osada, H., Forster, A., and Rabbitts, T. H. The cysteine-rich LIMdomains inhibit DNA binding by the associated homeodomain in Isl-l. EMBO J., 12:4243—4250, 1993.

35. Eckner, R., and Birnsteil, M. L. Cloning of cDNAs coding for human HMG I andHMG Y proteins: both are capable of binding to the octamer sequence motif. NucleicAcids Res., 17: 5947—5959, 1989.

36. Marcu, K. B., Bossone, S. A., and Patel, A. J. myc function and regulation. Armu.Rev. Biochem., 61: 809—860, 1992.

37. Hatano, M., Roberts, C. W. M., Minden, M., Crist, W. M., and Korsmeyer, S. J.Deregulation of a homeobox gene. HOXIJ, by the t(l0;14) in T cell leukemia.Science (Washington DC), 253: 79—82, 1991.

38. Mandahl, N., Heim, S., Arheden, K., Rydholm, A., Willen, H., and Mitelman, F.Three major cytogenetic groups can be identified among chromosomally abnormallipomas. Hum. Genet., 79: 203—208,1988.

39. Grosschedl, R., Giese, K., and Pagel, J. HMG domain proteins: architectural elementsin the assembly of nucleoprotein structures. Trends Genet., 10: 94—100, 1994.

40. Sandros, J., Stenman, G., and Mark, J. Cytogenetic and molecular observations inhuman and experimental salivary gland tumours. Cancer Genet. Cytogenet., 44:153—167,1990.

41. Sreekantaiah, C., and Sandberg, A. A. Clustering of aberrations to specific chromosome regions in benign neoplasms. tnt. J. Cancer, 48: 194—198,1991.

42. Bussemakers, M. J. G., Van de Ven, W. J. M., Debruyne, F. M. J., and Schalken, J. A.Identification of high mobility group protein 1(Y) as a potential progression markerfor prostate cancer by differential hybridization analysis. Cancer Res., 51: 606—611,1991.

43. Ram, T. G., Reeves, R., and Hosiek, H. L. Elevated high mobility group 1(Y) geneexpression is associated with progressive transformation of mouse mammary epithehal cells. Cancer Rca., 53: 2655—2660,1993.

2280

on June 14, 2020. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1997;57:2276-2280. Cancer Res   Alexei Tkachenko, Hena R. Ashar, Aurelia M. Meloni, et al.   Activates Alternative Pathways of TumorigenesisMisexpression of Disrupted HMGI Architectural Factors

  Updated version

  http://cancerres.aacrjournals.org/content/57/11/2276

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/57/11/2276To request permission to re-use all or part of this article, use this link

on June 14, 2020. © 1997 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from