Tu1681

Pathological Significance of MSI-L/EMAST and 9p24.2 LOH in ColorectalCancersMelissa Garcia, Chan Choi, Hyeong-Rok Kim, Junichi Koike, Hiromichi Hemmi, TakeshiNagasaka, Yuji Toiyama, C. Richard Boland, Minoru Koi

(Background) We have demonstrated that down-regulation of hMSH3 results in low levelsof microsatellite instability (MSI-L) and elevated microsatellite alterations at selected tetranu-cleotide repeats (EMAST) in tissue cultured cell lines. Primary tumor tissues exhibiting MSI-L and/or EMAST from stage II/III CRC show intra-tumor depression of hMSH3 and up-regulation of GLUT1 that is a marker for hypoxia. In vitro, hypoxia induces down-regulationof hMSH3 and the development of EMAST in CRC cell lines. Finally, Stage II/III primaryCRC exhibiting MSI-L/EMAST has a shorter recurrence-free survival than that of microsatellitestable counterparts not exhibiting MSI-L/EMAST. These results support the concept thatintra-tumor hypoxia may induce MSI-L/EMAST through down-regulation of hMSH3 andenhance the aggressiveness of primary CRC, leading to recurrence and/or distant metastasis.Thus, MSI-L/EMAST could be a marker for identifying genetic and/or epigenetic changesthat control recurrence and/or distant metastasis from primary CRC. We previously reportedthat LOH at the SMARCA2 locus on 9p24.3 is significantly high in liver metastasis (LM)exhibiting MSI-L/EMAST (termed moderate MSI: MSI-M) from primary CRC. (Aim) Thepurpose of this study is to determine the pathobiological significance of MSI-L/EMAST andLOH at SMARCA2 region in CRC. (Methods) Eighty three LMs and 186 primary CRC withknown clinicopathological factors and disease outcome were analyzed for MSI/EMAST andLOH at the 9p24.2 region. Significant associations between factors were determined byvarious statistical analyses. (Results) We proposed that a locus affected by 9p24.2 LOHmight promote metastasis from primary CRC; such a locus was mapped to an 80 kb regionat the 3' boundary of the SMARCA2 gene. Stage II and III primary CRCs exhibiting MSI-M and 9p24.2 LOH were significantly more likely to give rise to a solitary LM suitablefor hepatectomy, whereas MSI-M-positive counterparts without 9p24.2 LOH, or highlymicrosatellite stable (HMSS) tissues were more likely to give rise to simultaneous metastasesto multiple organs, and therefore, not suitable for surgical management. Multivariate analysisshowed that MSI-M with 9p24.2 LOH in the primary CRC is an independent positiveprognostic factor following recurrence (HR = .1981, P=.0007). (Conclusions) The presence orabsence of 9p24.2 LOH in Stage II/III primary CRC with MSI-M is an important determinant ofthe aggressiveness of the recurrent metastasis.

Tu1682

Defective DNA Mismatch Repair Activity Is Common in Sporadic SebaceousNeoplasms, and an Ineffective Approach to Screen for Lynch SyndromeAnu R. Lamba, Angela Y. Moore, Jennifer Rhees, Mildred (Millie) A. Arnold, C. RichardBoland

Background: Lynch syndrome (LS) is the form of hereditary colorectal cancer caused by agermline mutation in a DNA mismatch repair (MMR) gene. Small subsets of individualswith LS have a variant called Muir-Torre syndrome (MTS). MTS patients develop multiplesebaceous neoplasms and keratoacanthomas. When a person develops these skin lesions, itsuggests possible LS. Tumors caused in the setting of LS have microsatellite instability (MSI)and neoplasms caused by inactivation of the DNA MMR system are typically associated withloss of expression of the cognate protein in the tumor tissue at immunohistochemistry (IHC).Absence of gene expression and MSI have been well-documented in LS; however it is unclearwhether the presence of these abnormalities in sebaceous neoplasms would indicate thelikely presence of otherwise unsuspected LS. Aim: To determine whether the presence ofMSI or abnormal IHC in sebaceous neoplasms would alert the clinician to the unsuspectedpresence of LS. Methods: De-identified, paraffin-embedded pathological specimens of cuta-neous sebaceous neoplasms were obtained. No patient had a prior diagnosis of LS and nopatient had more than 2 lesions. IHC was performed for the DNA MMR genes MSH2 andMLH1. A 5 marker mononucleotide repeat panel was analyzed to detect MSI, and 2 or moremutated markers were determined to be MSI-high (MSI-H). Results: 164 sebaceous neoplasmswere obtained from 162 patients, including sebaceous adenomas (95/164, 58%), sebaceousepitheliomas (46/164, 28%), sebaceous carcinomas (15/164, 9%), hamartomas (5/164, 3%),keratoacanthoma (1/162) and 2/164 unknown samples. IHC data was obtained from 162samples and MSI data was obtained from 138 samples. 50/162 (31%) had abnormal IHCdetermined by loss of staining for either MSH2 (41/162, 25%), MLH1 (13/162, 8%) or both(4/162, 2%). When analyzed by pathology, 27/91 (30%) of adenomas had abnormal IHC,1/15 (7%) of carcinomas had abnormal IHC, and 21/43 (49%) of epitheliomas had abnormalIHC. 37% (51/138) of the tumors were MSI-H. When analyzed by neoplasm, 26/79 (33%)of adenomas, 16/38 (42%) of epitheliomas, 7/14 (50%) of carcinomas and 1/4 (25%) ofhamartomas were MSI-H. 82% (111/136) of those with both IHC and MSI results correlatedas expected, whereas 18% (25/136) showed discordance between IHC and MSI. A total of68/162 (42%) had either abnormal IHC or MSI, indicating deficient DNA MMR activity.Conclusion: Given the substantial proportion of DNA MMR deficiency in these sebaceousneoplasms, it would appear that inactivation of this DNA repair system is a typical mechanismfor tumorigenesis in sporadic sebaceous neoplasia. Screening for DNA MMR defects insebaceous neoplasms is not an effective way to distinguish patients with LS or MTS fromthose with sporadic skin lesions, and at ordinary risk for LS-associated cancers.

S-817 AGA Abstracts

Tu1683

MicroRNA-18a Induces Apoptosis in Colon Cancer Cells via theAutophagosomal Degradation of hnRNP A1Hiroaki Konishi, Mikihiro Fujiya, Katsuyoshi Ando, Kazuyuki Tanaka, Aki Sakatani,Tatsuya Dokoshi, Yuhei Inaba, Shugo Fujibayashi, Takuma Goto, Shin Kashima,Nobuhiro Ueno, Junpei Sasajima, Takahiro Ito, Hiroki Tanaka, Katsuya Ikuta, YutakaKohgo

Background and Aims: Colon cancer is the most common cause of cancer-related death inboth Western and Eastern countries. The etiology of colon cancer is associated with geneticand epigenetic abnormalities. Conversely, the abnormal expression of microRNAs (miRs) invarious cancers, including colon cancer, has been explored. While many mRNAs targetedby miRs have been identified, whether or not miRs directly target oncogenic proteins stillremains unknown. The present study showed that miR-18a directly binds to heterogeneousnuclear ribonucleoprotein A1 (hnRNP A1) in the cytoplasm of colon cancer cells and inhibitsthe oncogenic properties of the protein via autophagosomal degradation. Methods: Cellgrowth was evaluated using an MTT assay and in vivo xenograft model. The expression ofgenes was assessed with Western blotting, RT-PCR and immunocytochemistry. Protein-protein and RNA-protein binding was evaluated with immunoprecipitation and a bindingassay, respectively. The degree of protein degradation was assessed using isotope-labeledamino acids. Results: 1. The MTT assay and xenograft model showed that the overexpressionof miR-18a suppressed the progression of the colon cancer cell line SW620. TUNEL stainingand a Western blotting analysis of cleaved-caspases and -PARP showed that the transfectionof miR-18a induced the apoptosis of SW620 cells; however, treatment with miR-18 did notchange the translation of mRNA of cell growth- and apoptosis-associated molecules. 2. Thebinding assay revealed that miR-18a binds to hnRNP A1 in the cytoplasm of SW620 cells,while Western blotting showed that miR-18a inhibits the hnRNP A1 function, including theupregulation of cyclin D1 and CTGF. 3. A competitor RNA that included the complementarysequence of the region of the miR-18a-hnRNP A1 binding site repressed the effects of miR-18a on the induction of cancer cell apoptosis. 4. miR-18a formed a complex with hnRNPA1, which subsequently induced the ubiquitination of hnRNP A1 and the degradation ofthe protein. 5. 3-MA, an inhibitor of autophagy and siRNAs of autophagosomal degradationrelated-molecules (ATG7, p62 and BAG3), repressed the degradation of hnRNP A1 by miR-18a. Conclusions: miR-18a induces the apoptosis of colon cancer cells via the autophagolyso-mal degradation of oncogenic hnRNP A1. The findings of this study therefore suggest thatmiRs directly targets and degrades oncogenic proteins, thereby suppressing cancer progres-sion.

Tu1683a

Regulation of Gastric Cancer by Metastasis by Mir-206-Pax3-Met SignalingLin Zhang, Yanglin Pan, Xuegang Guo, Daiming Fan

Using Transcription Factor/DNA Array, we identified that paired box gene 3 (PAX3) maybe involved in human gastric cancer (GC) metastasis. PAX3 is a member of the PAX familyof transcription factors which play a crucial role in embryogenesis but are also implicatedin tumorigenesis. However, the expression and function of PAX3 in gastric cancer remainlargely unclear. In this study, we found that PAX3 expression was up-regulated in GC cellsespecially in high metastatic potential cells. Immunohistochemical (IHC) assays also showedthat PAX3 expression was markedly higher in GC tissues than in adjacent noncanceroustissues. GC patients with positive PAX3 expression had shorter overall survival times.Transwell assays and in vivo metastasis assays demonstrated that up-regulation of PAX3 inlow metastatic potential GC cells which expressed low PAX3 protein level significantlypromoted the invasiveness and pulmonary metastasis of the cells. On the other hand,knockdown of PAX3 in high metastatic potential GC cells markedly reduced cell metastaticpotential. Mechanistically, PAX3 overexpression relies on loss of the tumor suppressor miR-206, which was reported to be down-regulated in GC and to inhibit GC cells proliferationthrough directly targeting the CyclinD2 (Cancer Lett. 2013 May 10; 332(1):94-101). Wedemonstrate here that miR-206 suppressed PAX3 expression by targeting its 3'UTR. Further-more, PAX3 and miR-206 expression exhibited a significant negative correlation in GCtissues. MiR-206 significantly represses PAX3 expression at both mRNA and protein levelsin GC cells. Further investigation showed that showed that miR-206 inhibit GC metastasisthrough down-regulating PAX3. In addition, the pro-metastasis function of PAX3 was revealedto be mediated by up-regulating downstream target MET. Luciferase reporter assays demon-strated that PAX3 directly bound to the promoters of MET. Real-time PCR and western blotassays also demonstrated that up-regulation of PAX3 in GC cells increased MET expression.Down-regulation of PAX3 exhibited the opposite effect. Moreover, the protein levels of PAX3and MET were positively correlated in matched human GC specimens, and their co-expressionwas associated with poor prognoses. In conclusion, our results reveal that miR-206-PAX3-MET signaling is critical to GC metastasis. Targeting the pathway described here may opennew therapeutic prospects to restrict the metastatic potential of gastric cancer.

Tu1684

In Vitro, 3D Culture of Human Colon Cancer (CC) Cell Lines Appears MoreAppropriate Than 2D Culture to Study Resistance to 5-FU/Oxaliplatin(FOLFOX) Chemotherapy. First Result Suggesting a Role for ABCG2Frédérique Prévost, Céline Quesnelle, Jean-Marie Reimund, Mathilde Lechevrel

Anticancer drugs mode of action and mechanisms of resistance to these agents are usuallyfirst studied in vitro using 2D (monolayer) cultures of human cancer cell lines. However,2D cultures have been suspected to be less appropriate than 3D cultures (colonospheres)suggested to better represent the pathophysiology of human tumors (cell-cell interactions,3D architecture, etc.). In addition, recent work suggested that the ABC drug transporterABCG2 is associated to resistance to FOLFOX both in vitro and in vivo. Our aim was tocompare viability (and resistance) of human CC cell lines cultures in 2D versus 3D and tostudy the potential role of ABCG2 in in vitro resistance to FOLFOX. Viability of HCT116and HT29 culture in 2D and in 3D, as well as colony forming efficiency (CFE) was assessed

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sin presence or not of FOLFOX (50 μM of 5-FU and 1.25, 2.5 or 5 μM of oxaliplatin; 50μM of 5-FU/2.5 μM of oxaliplatin being considered as the circulating blood concentrationof patients treated by FOLFOX) (luminescence viability assay). ABCG2 mRNA expressionin 2D vs. 3D HT29 cultures (HT29 being more resistant than HCT116; see results) wasalso studied (Q-PCR). FOLFOX significantly decreased HT29 and HCT116 viability withoutany dose-dependency. HCT116 cells were more affected than HT29 cells (e.g. 20±2% vs43±2% for FOLFOX 50/2.5; p<0.05). Using 3D cultures, FOLFOX decreased dose-depen-dently HCT116 viability but at a lesser extent than in 2D (72±1% for FOLFOX 50/2.5;p<0.01). By contrast, viability of HT29 colonospheres was not affected, these cells culturedin 3D therefore appearing as completly resistant to FOLFOX. CFE was significantly decreasedboth in HCT116 and HT29 cells. In HT29 cells, basal expression of ABCG2 mRNA wassignificantly higher in 2D vs. 3D cultures (p=0.001). However, as FOLFOX increased two-times ABCG2 mRNA expression in 2D HT29 cultures, it increases by 6-fold ABCG2 mRNAexpression in HT29 colonospheres (p<0.01 compared to untreated colonospheres). In conclu-sion, HCT116 and at a much greater level HT29 cells cultured in 3D are significantly moreresistant to FOLFOX than in 2D cultures, suggesting that 3D culture is more appropriateto study in vitro resistance to anticancer drugs in CC. This chemoresistance seems to be atleast partly explained by increased expression of ABCG2 drug transporter. Acknowlegments:F.P was supported by an academic grant from the French Research Ministery ("AnnéeRecherche"). This work was also supported by grants from "Alsace contre le Cancer" andfrom "Association Coeur-Cancer" from the Département de la Manche, France.

Tu1685

Loss of Cohesin Is a Common Early Event in Colorectal Cancer: PotentialMediator of Chromosomal InstabilityRamesh K. Wali, Richard Kalman, Mart DeLaCruz, Jaclyn Weinstein, Navneet Momi,Ehsan Chitsaz, Lisa I. Jepeal, Ari B. Davis, Audrey H. Calderwood, Hemant K. Roy

Background: Chromosomal instability (CIN) with concomitant loss of heterozygosity fortumor suppressor genes is a hallmark of the major subset of colorectal cancers (CRCs) asreported recently in Cancer Genome Atlas (Nature 2012). Several candidate genes such asPIGN, MEX3C and ZNF516 have been implicated in CIN (Burrell et al., nature 2013).However, these are located on chromosome 18 whose loss is generally a late event in CRC.Other molecular drivers of CIN include the spindle assembly proteins Bub1 and Bub3 butthese encompass only a small proportion of CRCs. However, a recent study implicatesgermline alterations in Bub1 and 3 as CRC risk factors, implicating CIN as a relatively earlyevent (De Veor et al. Gastro 2013). Thus elucidating the molecular determinants of CIN iscritical in early carcinogenesis. It has recently been established that cohesin family of proteinsare critical in chromosomal segregation process which act as a multimeric complex comprisingof structural maintenance proteins (SMC3), sister chromatid cohesion proteins (SA-1 andSA-2) and the chromatin loader (NIPBL). Previous reports have shown that these are mutatedin a small proportion (~3%) of all CRCs (Barber et al., PNAS 2008) but the expressionlevels have not been assessed in early and late colon carcinogenesis. Aims: To study alterationsin the cohesion complex markers during early-late cancer progression. Methods: Immunohis-tochemistry (IHC) was performed on 72 tumors (different grades), 10 tumor-adjacent unin-volved and 10 control samples for cohesions (SMC3, and SA-1) and NIPBL. The data wasdichotomized as positive or negative from an observer graded 4 point intensity score.Cohesion expression from human CRC cell lines HT29 (CIN+) and HCT116 (CIN-) wasmeasured by Western blot. Results: While 100% of normal control samples presented positiveimmunoreactivity for SA-1, a large proportion of adenocarcinoma samples (65%) werenegative. In addition, 20% of adjacent uninvolved (field carcinogenesis) and 60% of adenomasalso showed loss of reactivity signifying early loss in carcinogenesis (Fig 1). The averageintensity score also revealed significant loss in SA-1 (p<0.0001) expression in cancer com-pared to normal (Fig 2). NIBPL showed a similar trend with a 40% decrease in adenocarcino-mas (p<0.009). SMC3 showed a statistically insignificant decrease (19%; p<0.09) in adenocar-cinomas. Furthermore CIN+ HT-29 expressed 1.4 fold increases in SA-1 compared to CIN-HCT 116 cells (p<0.003). Conclusions: We demonstrate herein, for the first time, that lossof SA-1 and NIPBL are common and early events in colon carcinogenesis. This dramaticdownregulation occurs at the polyp stage and is more expected in the CIN positive ratherthan CIN negative. This suggests that cohesion mediated CIN via epigenetic silencing maybe a common pathway in early colonic carcinogenesis.

S-818AGA Abstracts

Tu1686

Attenuation of Pancreatic Cancer Cell Migration and Invasion Through aTargeted Inhibition of the Rac GEF Vav1Gina L. Razidlo, Christopher Magnine, Arthur C. Sletten, Rachel M. Hurley, Mark A.McNiven

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of human cancer,due in large part to a high incidence of metastasis and resistance to conventional chemothera-pies. As such, new therapies are needed to halt the metastatic process. The proto-oncogeneVav1 is ectopically expressed in over half of human pancreatic cancers, and its expressioncorrelates with a poor prognosis in humans and promotes survival and transformation inisolated tumor cells. We have found that Vav1, through its GEF activity towards Rac1, alsopotently induces migration by pancreatic tumor cells. In addition, Vav1 also signals throughCdc42 to drive the formation of invadopodia and matrix degradation, which also promotestumor cell invasion. Importantly, the activation of Vav1 circumvents the requirement foractive Src in this process. Because Vav1 is ectopically expressed in pancreatic cancers andis a potent regulator of tumor cell survival, proliferation, and invasive migration, it couldprovide a specific therapeutic target for these tumors. Recently, the anti-inflammatory agentazathioprine was described as an inhibitor of Vav1/Rac1 activity in hematopoietic cells.Therefore, we hypothesized that azathioprine could also inhibit the Vav1-driven processesof proliferation and invasion in pancreatic tumor cells. Indeed, we have determined thatazathioprine treatment of cultured human PDAC cells reduces viability and proliferation.Further, similar to siRNA-mediated depletion of Vav1, azathioprine treatment reduces cellmigration, as well as the matrix degradation required for invasion. Vav1-expressing tumorcells were more sensitive to azathioprine than Vav1-negative cells, supporting the premisethat the inhibitory effects of azathioprine are mediated through Vav1. Finally, azathioprinealso reduces metastasis in a xenograft model of pancreatic cancer. Taken together, thesedata uncover a role for aberrantly expressed Vav1 in regulation of the cytoskeletal machineryrequired for migration and invasion, and in the promotion and metastasis of pancreaticcancer. Even with the expression of multiple Rho family GEFs, tumor cells that upregulate