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stowards reducing co-carcinogenic effects of PG peptides. These studies were supported byNIH grants # CA097959 and CA 114264 to PS.

S1988

The Absence of the Proto-Oncogene Fer Increases Colon Cancer DevelopmentMaitham A. Khajah, Stefan J. Urbanski, Donna-Marie McCafferty

Background: The group IV non-receptor protein tyrosine kinase Fer kinase has been describedas a proto-oncogene Fer and is implicated in signal transduction pathways regulated bygrowth factors. Fer is highly expressed in different malignant cell lines and In Vitro down-regulation of Fer reduces the proliferation of prostate carcinoma cells and attenuates thedevelopment of prostate cancer. The Aim of this study was to determine the role of Ferkinase in an In Vivo model of colitis-associated adenocarcinoma: the interleukin 10 (IL-10-\-) model. Methods: WT (129SvEv), IL-10-\-, and IL-10-\-FerDR/DR double knockout micewere used at 3 months of age. Macroscopic and histological scoring of inflammation anddysplasia were performed on the colon (n= 13-25 mice). Myeloperoxidase (MPO) activitywas determined as an indication of neutrophil recruitment to the colonic tissues. Bonemarrow-derived neutrophils chemotaxis toward different chemoattractants (WKYMVm; fMLPpeptide, keratinocyte derived cytokine; KC, macrophage inflammatory protein; MIP-2, andgranulocyte macrophage colony stimulating factor; GM-CSF) was performed using the underagarose assay In Vitro. Results: A significant increase in inflammatory parameters (macroscopicand histological score, and MPO activity) was noted in IL-10-\- compared to WT mice at 3months of age. IL-10-\- mice had a 34 % incidence of mucosal hyperplasia (polyps) withno evidence of neoplastic changes, with the exception of 1 (of 14) mouse with low gradedysplasia. A moderate but significant increase in inflammation was observed in IL-10-\-FerDR/DR double knockout compared to IL-10-\- mice (p<0.05). Surprisingly, in the absence of Ferkinase, IL-10-\- mice had a 52% incidence of polyps with a 61% incidence of adenocarcinoma.Multiple adenocarcinomas were noted in some mice (2 mice). Using bone marrow derivedneutrophils we demonstrated a significant increase in chemotaxis of neutrophils from IL-10-\- mice compared with wild type (116±12 vs. 48.8±8.3 toward WKYMVm, 45.3±6.1 vs.21.6±5.4 toward KC, 55.8±8 vs. 23.6±5.3 toward MIP-2, and 101±4.2 vs. 42±4.1 towardGM-CSF respectively). Neutrophil chemotaxis was further enhanced in the absence of Ferkinase (166±8.9) toward WKYMVm only. Conclusions: These data demonstrate for the firsttime an important role for Fer kinase in regulating the development adenocarcinoma in achronic model inflammation In Vivo. This may be associated with enhanced leukocyterecruitment to the colon in the absence of Fer kinase contributing to the inflammation-dysplasia-adenocarcinoma sequence.

S1989

Identification of a Functional p53 Responsive Element Within the Promoter ofXAF1 Gene in Gastrointestinal Cancer CellsQing Gu, Wenjing Zhang, Jide Wang, Juan Ma, Zesong Li, Hui Y. Lan, Benjamin C.Y.Wong

BACKGROUND: XAF1 was first identified as a XIAP-interacting protein and functioned asa tumor suppressor to sensitize cells to apoptosis. XAF1 expression in gastric cancer tissueswas negative correlated with p53 status with cells harboring wildtype p53 expressed lowlevel of XAF1. The purpose of this study was to clarify the regulatory mechanism ofp53 on XAF1 expression. METHODS: XAF1 expressions and promoter activities in severalgastrointestinal (GI) cancer cell lines with different p53 status were detected by westernblot and dual luciferase assay. The effects of ectopic over-expressed wildtype and mutantp53 on XAF1 expression were evaluated after transient transfection. A 107bp of XAF1 corepromoter were separated into five probes to examine their binding capacity to the recombinantp53 protein. Site-directed mutation of the putative p53 binding sequence and p53 knock-down by siRNA were performed to confirm the interaction between p53 and XAF1 promotersegments. RESULTS: The protein expression and the promoter activities of XAF1 in celllines with null p53 were higher than that in cell lines with wildtype and mutant p53. Ectopicover-expression of wildtype p53 suppressed XAF1 protein expression and transcriptionalactivities. A halfsite (-95nt~-86nt, translation starting codon was defined as +1) and aquartersite of p53 responsive element (-4nt~+1nt) were found within XAF1 promoter. Bothsequences bound to recombinant p53 effectively determined by electrophoresis mobilityshift assay (EMSA) and the binding could be blocked specifically by non-labelled consensusp53 oligonucleotide. Site-mutation of the putative p53 responsive sequences abrogated thebinding between probes and p53 protein. However, only the mutation of halfsite but notthe quartersite sequence increased XAF1 promoter activities. Suppression of p53 by siRNAcould not only decrease the binding capacity of p53 responsive halfsite but also increaseXAF1 expression and its promoter activity. CONCLUSIONS: p53 could suppress the tran-scription of XAF1 through interaction with a high affinity responsive element (-95nt~-86nt)within XAF1 promoter. The significance of this exclusive interaction between these twotumor suppressors remains to be elucidated.

S1990

Opposing P21waf1 Regulation By TGFβ/SMAD4 and Activin/SMAD4 in ColonCancer CellsBarbara H. Jung, Jennifer Cabral, Przemyslaw K. Slowik, Jessica Gomez, Stayce E. Beck,John M. Carethers

BACKGROUND: Activin and transforming growth factor β (TGFβ) are growth suppressiveTGFβ family ligands that signal through a primary receptor type 2 receptor (activin receptor2, ACVR2, and TGFβ receptor 2, TGFBR2, respectively) to activate specific type 1 receptors,ACVR1 and TGFBR1. This is followed by phosphorylation of cytosolic SMAD2/3 that pairswith SMAD4 to translocate to the nucleus as a transcription factor. Despite the utilizationof identical SMADs, microsatellite unstable colon cancers inactivate both activin and TGFβsignaling simultaneously. A key downstream target of TGFβ signaling is the cdk2 inhibitorp21waf1, which has growth suppressive properties in the nucleus, but may act as a tumor

A-308AGA Abstracts

promoter in the cytosol. Here, we dissected ligand-specific effects on p21waf1 expression,modulation and its functional consequences. METHODS: ACVR2/TGFBR2 (WT)/SMAD4(WT) FET and ACVR2/TGFBR2 (WT)/SMAD4 (null) SW480 colon cancer cells were assessedfor p21waf1 expression after ligand treatment and subcellular localization using Westernblots. p21waf1 and SMAD4-specific siRNA were used to establish functional consequencesof p21waf1 loss and SMAD4 dependency. Cellular viability, apoptosis, and migration weredetermined using MTT assay, ADP/ATP apoptosis assay, and Boyden chambers. p21-specifictransactivation was determined using p21-luciferase, and p21waf1 transcription using quant-itative PCR. Expression of activin/TGFβ pathway members was correlated to p21waf1 subcel-lular localization in primary colon cancers via immunohistochemistry. RESULTS: Activinwas a more potent inducer of apoptosis, while TGFβ was more growth suppressive, andboth functions were SMAD4-dependent. TGFβ-induced growth suppression coincided withincreased nuclear p21waf1 protein through increased p21waf1 transactivation, transcriptionand protein stabilization. However, activin decreased nuclear p21waf1 protein with a nuclearto cytosolic shift. Induction of p21waf1 by TGFβ was SMAD4-dependent, but decrease ofnuclear p21waf1 by activin was SMAD4-independent. Knock down of p21waf1 reversedgrowth suppressive effects, enhanced survival, and increased the migratory capacity of bothligands. Increased cytosolic p21waf1 in primary colon cancers correlated with intact ACVR2.CONCLUSIONS: Despite identical downstream SMAD signaling, activin and TGFβ haveopposing effects on the cdk2 inhibitor p21waf1, resulting in differing effects on growthsuppression, migration, and survival in colon cancer. Activin-specific modulation of p21waf1functions through non-canonical, SMAD-independent pathways and may represent a poten-tial target for therapy of metastatic disease.

S1991

The PI3K-AKT Pathway Is a Downstream Effector of CD24Inna Naumov, Sarah Kraus, Diana Kazanov, Shiran Shapira, Eyal Sagiv, Nadir Arber

Background: CD24 is a cell-surface molecule, thought to be involved in adhesion andsignaling processes. However, a signaling cascade for CD24 has not been identified. CD24is a potential oncogene reported to be overexpressed in a large variety of human malignancies.We have shown that CD24 is overexpressed in 90% of gastrointestinal tumors at a fairlyearly stage in the multistep process of carcinogenesis (Gastroenterology, Vol 131(2) 630-639, 2006). Anti-CD24 mAb induced a significant growth inhibition in CD24-expressingcolorectal and pancreatic cancer cells (Clin Can Res, Vol 13(22), p. 6807-6815, 2007).Xenograft models showed that tumor growth was significantly reduced in mAb-treated mice.Similarly, growth inhibition of cancer cell lines was achieved by down-regulation of CD24expression using short hairpin RNA (shRNA) (Can Res, Vol 68(8), 2803-2812, 2008). ThePI3K-AKT pathway has been implicated in the molecular pathogenesis of a variety of cancersand its overexpression was recently demonstrated to be an early event in colorectal carcino-genesis. Aim: To examine the role of PI3K-AKT in the signaling cascade leading to growthinhibition induced by anti-CD24 mAb treatment. Methods: Pancreatic cancer cells, Colo357,were treated with anti-CD24 mAb for various time-points (24, 48, 72hrs) and AKT activationwas examined by Western blotting with anti-phospho-AKT (Ser473). Cell proliferation wasdetermined by methylene blue assay following exposure to mAb alone or combined withwortmannin (PI3K inhibitor). Cell cycle and apoptosis was evaluated by FACS analysisfollowing PI staining. Results: Cell growth was significantly inhibited in a time- and dose-dependent manner. The level of apoptosis was 25% and 49% after 48 and 72hrs respectively,without a change in cell cycle. Combined mAb treatment with wortmannin increasedapoptosis by 11%. Surprisingly, mAb treatment resulted in the down-regulation of AKTactivation with a significant AKT degradation. Conclusions: Here we report, for the firsttime, that AKT is a downstream effector of CD24. CD24 inhibits AKT activity. The combinedtargeting of CD24 and AKT might serve as a potent anticancer treatment. Understandingthe functional aspects of CD24-induced signaling will pave the way for successful cancertherapy using anti-CD24 antibodies and selective promotion of apoptosis.

S1992

The Interaction of β-Catenin and Telomerase and Its Role DuringCarcinogenesisFalk Mancke, Angela Queisser, Heike Kunert, Steffen Heeg, Nina Hirt, Frank Götschel,Andreas Hecht, Steve Artandi, Oliver G. Opitz

Introduction: Maintenance of telomere length is one major determinant of immortalizationand thus malignant transformation. Most human tumor cells maintain their telomere lengththrough reactivation of telomerase. Recently, several lines of evidence demonstrated thattelomerase possesses additional functions during carcinogenesis and hair follicle stem cellbiology, independent of its ability to elongate telomeres. Studies investigating the molecularmechanisms of this telomere elongating independent function of telomerase analyzed thegenomewide transcriptional response to acute changes of TERT levels, the protein componentof telomerase, in mouse skin. Further statistical comparison to other micro array gene setsrevealed that TERT exerts its function on hair follicle stem cells through transcriptionalcontrol of WNT related genes. Taking these findings in consideration our aim was to furtherelucidate the telomere elongating independent functions of telomerase during carcinogenesis,investigating the potential interaction between telomerase and WNT signaling components.Methods: We analyzed distinct cell types representing different steps in the malignanttransformation of squamous cell carcinoma and adenocarcinoma of the esophagus. Weperformed transient transfection assays determining the transcriptional activity of the canon-ical WNT pathway, co-immunoprecipitation experiments looking for protein-protein interac-tions and immunoprecipitation (IP) experiments with telomerase repeat amplification proto-col (TRAP). This allowed us to study interactions of the catalytically active enzyme. Toknock down TERT expression we carried out siRNA transfection experiments. Results: IP-Immunoblot experiments demonstrate a interaction of β-catenin and TERT in 293T cells.IP-TRAP experiments identified β-catenin and catalytically active telomerase as interactionpartners in 293T cells and fully malignant adenocarcinoma cells. Using siRNA mediatedknock down of TERT we observed a TERT dependent reduction in transcriptional activityof canonical WNT signaling, suggesting a functional relevance of the described interaction.Despite robust telomerase activity squamous cell carcinoma cells show neither transcriptional