activity of canonical WNT signaling nor any kind of interaction between β-catenin andtelomerase components. Summary: The interaction of β-catenin and telomerase and theTERT dependent reduction in transcriptional activity of canonical WNT signaling seems tobe cell type specific during esophageal carcinogenesis and might shed some light on themolecular mechanisms of the telomere elongating independent function of telomerase.

S1993

Tumor Suppressor At Motif Binding Factor 1 (ATBF1) Negatively RegulatesCell Growth Through the Nuclear Translocation with Runt DomainTranscription Factor 3 (RUNx3) in Cooperation with TGF-β SignalTransductionMotoshi Mabuchi, Hiromi Kataoka, Tsutomu Mizoshita, Eiji Kubota, Tsuneya Wada,Satoshi Tanida, Makoto Sasaki, Takeshi Kamiya, Takashi Joh

Background and aims: AT motif binding factor 1 (ATBF1), a homeotic transcription factor,was identified as a tumor suppressor (Nat. Genet., 2005) and frequent loss of heterozygosityat ATBF1 locus in gastric cancer was recently reported (Clin Cancer Res., 2007). Wepreviously showed that ATBF1 expression inversely correlated with the malignant characterof gastric cancer (Oncogene, 2001) and ATBF1 enhanced the promoter activity of the p21(Cancer Res., 2003). We also found that ATBF1 translocates between cytoplasm and nucleus(Int. J. Cancer, 2007), but its precise mechanism has not been fully elucidated. In thisstudy, we investigated the mechanism of nuclear translocation of ATBF1 with runt domaintranscription factor 3 (RUNX3) in cooperation with TGF-β signal transduction. Materialsand Methods: 1. To analyze the expression of ATBF1 in gastric cancer cells, we performedimmunohistochemistry of 123 resected gastric cancer tissues. 2. ATBF1 and RUNX3 wereover expressed by transfecting myc-tagged ATBF1 expression vector and flag-tagged RUNX3expression vector. Immunoprecipitation (IP) between ATBF1 and RUNX3 was performed.3. To investigate the nuclear translocation of endogenous ATBF1 and RUNX3, we examinedthe subcellular localization of ATBF1 and RUNX3 in SNU-16 gastric cancer cells treatedwith from 0.2 to 2.0 ng/ml of recombinant TGF-β1 by confocal mirosope. 4. We confirmedATBF1 and RUNX3 protein expression in cytoplasm and nucleus extracted fraction bywestern blot. 5. Cell cycle analyses by using FACScan were performed. 6. Dual-luciferaseassays were performed by transfecting ATBF1 and RUNX3 with p21 reporter vector. Results:1. The nuclear localization of ATBF1 was observed in 33 (26.8%) gastric cancers and thecytoplasm localization of ATBF1 was observed in 61 (49.6%) gastric cancers. ATBF1 expres-sion was not observed in 29 (23.6%) gastric cancers. 2. IP revealed that the physicalinteraction of ATBF1-RUNX3. 3. In SNU-16 gastric cancer cells, ATBF1 and RUNX3 existedin the nucleus before recombinant TGF-β1 stimulation, but after 24 hours of TGF-β1stimulation, endogenous ATBF1 and RUNX3 translocated to the nucleus by confocal micro-scopic observation. 4. Western blot also revealed ATBF1 and RUNX3 protein in the nucleuswas increased after TGF-β1 stimulation. 5.ATBF1 induced the accumulation in the G0/G1phase. 6. ATBF1 and RUNX3 synergistically up-regulated p21 promoter activity. Conclusion:ATBF1 associates with RUNX3 and translocates to the nucleus with RUNX3 by TGF-β signaltransduction and works as tumor suppressor and transcription regulator in the nucleus.

S1994

The Intracellular Tyrosine Kinase PTK6 Plays Distinct Roles in Colon andBreast Cancer Cell LinesAnsu O. Perekatt, Jessica Gierut, Yu Zheng, Angela L. Tyner

PTK6 is expressed at high levels in differentiated epithelial cells of the normal colon. However,PTK6 is also expressed in two-thirds of breast cancers where it has been shown to enhanceproliferative signaling by ErbB receptors. To determine if PTK6 has different functions incolon and mammary gland epithelial cells, we compared the role of PTK6 in ErbB receptorsignaling in the colon cancer cell line SW480 and the breast cancer cell line T47D. Thesetwo cell lines respond to Heregulin (HRG), the ligand for ErbB3 and ErbB4, and expresscomparable levels of PTK6. In T47D cells, HRG stimulation activates the Mitogen ActivatedProtein Kinases (MAPKs) Erk5 and p38, and knockdown studies have shown that PTK6 isrequired for this activation. In contrast, Erk5 and p38 kinases were not activated in theSW480 colon cancer cell line. Using In Vitro kinase assays, we found that although PTK6is activated in T47D cells, it is not activated in SW480 cells in response to HRG stimulation,and this may account for the lack Erk5 and p38 activation. We then analyzed HRG regulatedsignaling in additional intestinal cell lines, including the non-transformed rat intestinalepithelial cell line IEC-18, and the HCT116 colon cancer cell line, both of which expressPTK6. Unlike our findings with SW480 cells, Erk5 and p38 were activated in the IEC-18and HCT-116 cell lines. However, shRNA-mediated knockdown of PTK6 in HCT116 cellsenhanced Erk5 and p38 activation, indicating that PTK6 plays an inhibitory role in HRGmediated Erk5 and p38 activation in this colon cancer cell line. These data indicate thatPTK6 has distinct functions in colon and mammary epithelial cells. Currently, we are studyingthe effect of HRG stimulation on PTK6 and ErbB receptor activation in HCT116 and IEC-18 cells. Our studies will help to clarify if PTK6 is a viable therapeutic target in the treatmentof colon and/or breast cancer.

S1995

The Beta Spectrin ELF Interacts with SMAD3/SMAD4 to Repress TelomeraseReverse Transcriptase (TERT) in Hepatocellular Cancer ProgressionZhongxian Jiao, Zhixing Yao, Anish Shah, Kyi Soe, Wenguo Yao, Wilma S. Jogunoori,Lopa Mishra, Bibhuti Mishra

Βackground and Aims: Telomerase is a specialized reverse transcriptase that synthesizesrepeat DNA sequences at telomeres- the specialized ends of chromosomes regulated bymultiple proteins that include TGF-β, c-Myc and Smad3. Absence of telomerase activity inmost normal human cells results in the progressive shortening of telomeres with each celldivision, ultimately leading to chromosomal instability and cellular replicative senescenceor growth arrest. Emerging evidence indicates that ELF, a Smad3/Smad4 adaptor protein

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required for TGF-β signaling, is a powerful tumor suppressor. Elf+/-, elf+/-/Smad3+/- and elf+/-/Smad4+/- mice dramatically develop foregut cancers, Because TERT is markedly activated inelf +/- hepatocellular cancer tissues compared to Smad3-/- tissues, we hypothesized that ELFinteraction with Smad3 could be an important factor in regulating TERT expression. Results:We tested human TERT (hTERT) expression levels in several HCC cell lines that havedifferent levels of ELF. hTERT expression levels negatively correlate with ELF by WesternBlot analysis. Over-expression of ELF and/or Smad3 decreases the RNA level of telomerase(hTERT) in PLC/PRF/5 and SNU-398 (hepatocarcinoma) cell lines. Co-transfection of anElf expression vector and a 1.9kb hTERT promoter-luciferase construct significantly inhibitedthe hTERT promoter in PLC/PRF/5 and SNU-398 cells. In Vitro RNAi and Deletion studiesof Elf and Smad3 binding elements in the human TERT promoter obtained consistentresults which show increased promoter activities by luciferase assay. Furthermore, chromatinimmunoprecipitation assay (CHIP) suggests binding of Smad3/ELF protein to hTERT pro-moter. Conclusion: Taken together our data suggest that divergent pathways converge onELF and Smad3 that then regulate TERT. Inactivation of the TGF-β signaling pathway withTERT activation provides a strong strategy for generating targeted therapeutics at TERT tothese lethal human cancers.

S1996

A New Preclinical Mouse Model for Sporadic Primary and Metastatic ColonCancerKenneth Hung, Larissa Georgeon Richard, Umar Mahmood, Raju Kucherlapati

Highly accurate mouse cancer models are critical for translational research. Most modelsfor primary colon cancer are based on germline or tissue-wide gene modification. Whereasthese may serve as models for inherited cancer predisposition syndromes, they are poorsurrogates for sporadic colon cancer. Metastatic colon cancer models utilize injection oftumor cells into the bloodstream or liver, making these poor surrogates for the metastaticprocess. Adenovirus expressing cre recombinase (a-cre) has been used to modify floxedgenes involved in colon carcinogenesis, but the rate of tumor formation has been quite low.Instead, we have employed a surgical approach to increase the penetrance. We reasonedthat a low tumor burden would allow adequate time for spontaneous macroscopic metastasesto form. A-cre was administered to floxed Apc mutant mice (Apc CKO) and Apc CKO micewith a latent mutant Kras allele (Apc CKO/LSL-Kras). The same mice, but injected withempty adenovirus (a-wt), were used as controls. After a-cre treatment, the penetrance oftumor formation was 92% (n=59), whereas that for a-wt was 0%. The mean number oftumors in the Apc CKO and Apc CKO/LSL-Kras treated animals were 1.3 and 3.6, respectively(p=0.00161). The mean distance from the anus in the Apc CKO and Apc CKO/LSL-Krastreated animals were 22.5 and 19.6 mm, respectively. Tumors presented along the entirespectrum of human disease - adenomas, carcinoma in situ, invasive carcinoma, and macro-scopic liver metastasis. There was a loss of Apc in tumors, but not in the surroundingepithelium. Cytoplasmic and nuclear b-catenin was seen in tumors. Endoscopy was usedto generate growth curves for each individual primary tumor. Interestingly, the growth ratesof Apc CKO and Apc CKO/LSL-Kras were not significantly different (p=0.406). Microarrayanalyses demonstrated a higher Pearson correlation coefficient between sporadic Apc CKOand Apc CKO/LSL-Kras and human tumors (0.255 and 0.249, respectively) than did ApcMin(0.049), demonstrating these tumors to be more akin to those found in human disease. Thefindings of preclinical trials with combination drug therapy will be presented. In summary,we have devised a method to model colon cancer in mice with very high penetrance (>90%)along the entire adenoma-carcinoma-metastasis axis with primary tumors in the distal colonand metastatic tumors in the liver. Based on transcriptomic signatures, these tumors moreclosely mimic human disease than traditional models. The reproducible location of theprimary tumors in the distal-most colon permits longitudinal surveillance by endoscopy.Taken together these are ideal models for translational research.

S1997

Overexpression of Proto-Oncogene RBM3 Induces Potent Tumor Formation ByConverting Normal Cells to Cancer Stem CellsSatish Ramalingam, Gopalan Natarajan, Randal May, Stan A. Lightfoot, VibhuduttaAwasthi, Courtney W. Houchen, Shrikant Anant

Introduction: The cancer stem cell hypothesis is an evolving concept of oncogenesis thathas recently gained wide acceptance. Here, we propose a model suggesting that a smallfraction of tumor cells acquire stem cell properties due to oncogene-induced changes ingene expression. RBM3 is a protooncogene whose expression increases in GI cancers. It isa translation modulator that regulates the expression of genes involved in proliferationand/or apoptosis thereby protecting cells during mitosis. Recently, we demonstrated thatdoublecortin and CaM kinase-like-1 (DCAMKL-1), a microtubule-associated kinase, is aintestinal stem cell marker. Aim: To determine whether overexpression of RBM3 inducespotent tumorigenesis and increases cancer stem stem cells. Method: RBM3 overexpressingSW480 colon cancer and NIH3T3 normal mouse fibroblast cells were plated in soft agarto isolate transformed cells that grew as spheroids. The spheroids were injected into flanksof athymic nude mice. Total RNA and protein were isolated for Real Time RT-PCR and westernblot analyses, respectively. Tumors were formalin fixed and used for immunohistochemistry.Results: We have determined that both SW480 and NIH-3T3 cells overexpressing RBM3induce tumors in nude mice and contain malignant DCAMKL-1 positive cells. Furthermore,~5% SW480-RBM3 cells were DCAMKL-1+. In contrast only 0.5% of control SW480 cellswere DCAMKL-1+. NIH-3T3-RBM3 DCAMKL-1+ cells were sorted out and grown in tissueculture dishes. These cells demonstrated outgrowth of neurites and high levels of notchprotein expression. Notch signaling has been implicated in the regulation of cell-fate decisionssuch as self-renewal of adult stem cells and differentiation of progenitor cells along a particularlineage. Notch signaling is also known to promote self-renewal of mammary stem cells. Todemonstrate stemness in the DCAMKL-1+ cells, only 5000 cells were injected into the flanksof athymic nude mice. After 4 weeks, tumors were observed not only in the flanks, but alsoin the lungs, liver and spleen. This was observed in both cell types NIH-3T3-RBM3 andSW480-RBM3. These data, taken together imply that overexpression of RBM3 results in

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