AGA Abstracts Sa1787 Regulation of Chemotherapy Resistance Through Epithelial-Mesenchymal Transition by Zinc Finger E-Box-Binding Proteins Shinya Ohashi, Seiji Naganuma, Mitsuteru Natsuizaka, Shingo Kagawa, Hideaki Kinugasa, Harry Subramanian, Naoko Taoka, Steffen Heeg, Shugo Ueda, Andres J. Klein-Szanto, Oliver G. Opitz, Shujiro Yazumi, Hiroshi Itoh, Hiroshi Nakagawa Introduction: Zinc finger E-box-binding (ZEB) proteins ZEB1 and ZEB2 are transcription factors essential in transforming growth factor (TGF)-β-mediated epithelial-to-mesenchymal transition (EMT) and cancer stem cell functions. We have demonstrated recently that ZEBs are upregulated in esophageal squamous cell carcinoma (ESCC) cells with mesenchymal characteristics and increased malignant potential such as anchorage independent growth, invasiveness and tumorigenicity upon xenograft transplantation (Cancer Res. 2011;71:6836- 47). However, the functional roles of ZEBs in chemotherapeutic resistance remain to be elucidated. Methods: Primary tumor tissues from patients who underwent surgery with or without neoadjuvant chemotherapy were analyzed. ESCC cell lines and In Vitro transformed human esophageal cells EPC2T were treated with either 5-fluorouracil or cisplatin, and subjected to WST-1 colorimetric cell proliferation assays, phase contrast imaging, flow cytometry, quantitative RT-PCR and Western blotting analyses. Short hairpin RNA was stably transduced by lentivirus to knockdown ZEB1 and ZEB2 in a regulatable manner (Tet-On system). Results: 70% of primary tumor tissues from patients who received neoadjuvant chemotherapy (n=10) contained ZEB1 positive ESCC cells, reminiscent of EMT. By contrast, ZEB1 positive ESCC cells were found in 33% of patients without neoadjuvant chemotherapy (n=51). EMT was present in a subset of cultured ESCC cells. The percentage of mesenchymal cells correlated with downregulation of E-cadherin and upregulation of N-cadherin, Vimentin and ZEBs. Moreover, flow cytometry revealed that the mesenchymal ESCC cells express a high level of CD44, a cancer stem cell marker, implicated in invasion, metastasis and chemoresistance. Interestingly, chemotherapeutic drug sensitivity was reduced as a function of the frequency of mesenchymal subset of the cells when four different cell lines were compared. In two cell lines (TE1 and TE5) with moderate levels of mesenchymal cells, the cells surviving 5-fluorouracil treatment were predominantly mesenchymal. In HCE7 cells displaying fully mesenchymal characteristics, knockdown of either ZEB1 or ZEB2 increased 5-fluorouracil sensitivity. Moreover, TGF-β induced EMT in EPC2T cells, resulted in a significantly increased cell viability rate upon cisplatin treatment and that was antagonized by knockdown of either ZEB1 or ZEB2. Conclusions: These data demonstrate that ZEB1 and ZEB2 confer chemotherapy resistance through EMT-mediated enrichment of a unique subset of ESCC cells defined by a CD44 upregulation. Neoadjuvant therapy may induce ZEB1 positive cells, thus having broader implications upon EMT and cancer therapy. Sa1788 Resistance to TRAIL is Mediated by DARPP-32 Through Regulation of NF-kB/ FLIP(S) Signaling in Gastric Cancer Abbes Belkhiri, Shoum Zhu, Zheng Chen, Jun Hong, Mohammed Soutto, Wael El-Rifai Background: Various chemotherapeutic drugs have been used in gastric adenocarcinomas, but drug resistance has hampered the effectiveness of chemotherapy leading to unfavorable clinical outcome. We have previously reported that Dopamine and cAMP-regulated phospho- protein, Mr 32000 (DARPP-32), promoted cell survival and drug resistance in upper gastroin- testinal carcinomas. Death receptor-mediated cell death is one of the major apoptosis path- ways. Death receptors transduce a death signal upon binding TNF-α-related apoptosis inducing ligand (TRAIL) leading to apoptosis in several types of human cancer cells. Activation of the TRAIL/death receptors pathway could provide a therapeutic advantage to eradicate chemoresistant tumors. However, TRAIL resistance is common in cancer. In this study, we uncovered a novel mechanism by which DARPP-32 promotes TRAIL resistance in gastric cancer. Methods and Results: To investigate the role of DARPP-32 in regulating TRAIL- induced apoptosis, we reconstituted DARPP-32 expression in MKN-28 cells and treated them with TRAIL (200 ng/ml) for 24h. The clonogenic survival assay data indicated that cell survival was 3-fold higher in DARPP-32-expressing cells than control cells (p<0.01) in response to TRAIL. Conversely, shRNA-mediated knockdown of endogenous DARPP-32 sensitized the resistant MKN-45 cells to TRAIL. We investigated if DARPP-32-mediated increase in survival was dependent on regulation of apoptosis in MKN-28 cells after treatment with TRAIL. The immunofluorescence and Western blot results showed a 5-fold less cyto- chrome c release (p<0.001) and a significantly less activation of caspases 8, 9, and 3 in DARPP-32-expressing cells than control cells. In addition, Quantitative RT-PCR and Western blot results indicated a 3-fold increase in mRNA (p<0.01) and a significantly higher level of BCL-xL protein in DARPP-32-expressing cells than control cells. This suggested a possible role for BCL-xL in blocking TRAIL-induced activation of the intrinsic apoptosis pathway. S-326 AGA Abstracts Western blot analysis indicated that TRAIL can induce caspase-dependent cleavage of NF- kBp65 protein as a mechanism to inhibit NF-kB survival pathway. This cleavage was abrogated by DARPP-32. The luciferase reporter and immunofluorescence assays confirmed that DARPP-32 can significantly suppress TRAIL-induced inhibition of NF-kB, hence maintaining NF-kB activity and the expression of its anti-apoptotic target FLIP(S) protein. This suggests that the NF-kB/FLIP(S) axis may be a negative feedback loop that blocks TRAIL-induced activation of caspase 8, thereby inhibiting the extrinsic apoptosis pathway. Conclusions: Our findings uncover a novel mechanism of TRAIL resistance mediated by DARPP-32, whereby it inhibits the intrinsic apoptosis pathway through up-regulation of BCL-xL, and the intrinsic pathway through the NF-kB/FLIP(S) axis in gastric cancer. Sa1789 Cisplatin and Ultra-Violet-C Synergistically Down-Regulate Receptor Tyrosine Kinases in Human Colon Cancer Cells Junji Kawaguchi, Seiji Adachi, Ichiro Yasuda, Takahiro Yamauchi, Masanori Nakashima, Tomohiko Ohno, Masahito Shimizu, Hisataka Moriwaki Platinum-containing anti-cancer drugs such as cisplatin are widely used for patients with various types of cancers. We have recently reported that high dose ultra-violet-C (UV-C) (200 J) suppresses colon cancer cell proliferation by desensitization of epidermal growth factor receptor (EGFR), which leads oncogenic signaling in these cells. In this study, we investigated the combination effect of low dose cisplatin (10 μM) and low dose UV-C (10 J) on cell growth and apoptosis in several human colon cancer cells (SW480, DLD-1, HT29 and HCT116). First, we demonstrated that the combination inhibited cell cycle and colony formation indicating the suppressive effect on cell proliferation, while either cisplatin or UV-C alone had little effect. We also observed that the combination induced apoptosis in these cells. To clarify its mechanism, we focused on receptor tyrosine kinases (RTK) and found that the combination caused the downregulation of EGFR as well as HER2. In fluorescence microscopy study and quantification analysis for cell surface EGFR, UV-C caused transient internalization of the EGFR but with time EGFR recycled back to the cell surface, while cisplatin did not affect its localization. Moreover, the combination use caused persistent internalization of the EGFR, indicating that cisplatin inhibits the recycling of the internalized EGFR induced by UV-C, which results in the lasting ubiquitination of the EGFR. Taken together, these results suggest that the combination use of cisplatin and UV- C synergistically inhibit cell growth by down-regulating RTK. Our present findings could provide evidence of a possible combination therapy against human colon cancer. Sa1790 MiR-124 Systemic Delivery as a Novel Therapeutic Approach for Hepatocellular Cancer Maria Hatziapostolou, Christos Polytarchou, Alexandra Drakaki, George A. Poultsides, Dimitrios Iliopoulos Background & Aims: Although there is knowledge about the etiologic agents responsible for hepatocellular carcinoma (HCC) pathogenesis, the molecular mechanisms leading to HCC have not been well characterized. Delivery of microRNAs in the liver is more efficient in comparison to other tissues and recent studies revealed that microRNA administration is able to suppress hepatitis C leading to the initiation of clinical trials in HCV-infected patients. The focus of this study was to identify microRNAs deregulated during HCC formation and investigate their applicability as therapeutic tools by suppressing tumor formation in liver cancer mouse models with no side effects. Methods: Real-time PCR was performed in 12 normal and 45 HCC samples to assess miR-124 and IL6R expression. The levels of STAT3 phosphorylation were assessed by ELISA and Western blot. Liver paraffin- embedded sections from 25 normal and 31 HCC patients were used for miR-124 in situ hybridization and phospho-STAT3 immunohistological staining. MicroRNA negative control (miR-NC) or miR-124 (5mg/kg) was systemically administered 32 weeks post in N,N- diethylnitrosamine (DEN) treatment in mice, in a weekly basis, for 4 cycles. On week 36, mice were sacrificed and tumor burden was assessed. Cleaved caspase-3 and cleaved PARP were assessed by ELISA and Western blot. Results: MiR-124 was significantly down-regulated in HCC relative to normal tissues. Bioinformatics and molecular analyses revealed that miR- 124 directly targets the 3'UTR of IL6R. In accord, the levels of IL6R were significantly up-regulated in HCC relative to normal tissues. IL6R de-repression resulted in increased phosphorylation of STAT3 in SNU-449 liver cancer cells. Immunohistochemical analysis for phosphorylated STAT3 and in situ hybridization for miR-124 revealed that in 13/30 (43.3%) of HCC tumors the pathway is active, suggesting its relevance to human cancer. We tested whether systemic administration of miR-124 is able to suppress HCC tumor growth in DEN- treated mice. Initially, we examined if systemic delivery of miR-124 had any cytotoxic effects in normal mice. Our data demonstrated that miR-124 administration did not have any cytotoxic effects in the function of essential organs (liver, kidneys, spleen, lungs, heart). Furthermore, systemic administration of miR-124 suppressed >80% HCC tumor growth and size through induction of apoptosis. Interestingly, miR-124 administration resulted in restoration of physiological miR-124 expression, IL6R suppression and inhibition of STAT3 phosphorylation. Conclusions: We show for the first time that systemic administration of a microRNA has therapeutic effects on liver cancer with no signs of toxicity, suggesting that miR-124 administration could be a viable anti-cancer approach for liver cancer patients that could be tested in phase I clinical trials.