Abstract B214: Antagonizing CBP/beta-catenin signaling-enhanced radiation sensitivity and altered epithelial-mesenchymal transition markers in head and neck squamous cell carcinoma.

Introduction: Survivin is often overexpressed in many types of cancers. An elevated survivin expression level in cancers is correlated with enhanced proliferative index, reduced apoptosis levels, and resistant to chemotherapy and radiation therapy. In addition, cancers often have elevated levels of S100A4 expression, which is known to be involved in metastasis. Metastasis has been linked with the ability of cancer cells to undergo epithelial-mesenchymal transition (EMT). Both survivin and S100A4 are known to be highly expressed in the head and neck cancers(HNC), which is the 6th most common cancer in the world and its 5-year survival rate is often less than 50%. ICG-001, developed by Michael Kahn, is a novel small molecule inhibitor of Wnt signaling. Previous studies have shown that ICG-001 inhibits CBP/beta-catenin interaction and its down-stream target genes, including survivin and S100A4. The purpose of this study is to examine the effect of this small molecule inhibitor in head and neck squamous cell carcinoma (HNSCC), which constitutes more than 90% of all HNC, and radiation resistance and metastasis are the main cause of a poor prognosis. Experimental Procedures: Three HNSCC cell lines (USC-001, SCC-71, and SCC-15) were used in this study. Expression of survivin, cyclin-D1, S100A4, and E-cadherin were analyzed by realtime qRT-PCR. Survivin expression is further analyzed by western blotting and immunohistochemistry. Survivin and Wnt/b-catenin promoter activities were analyzed by survivin-luciferase and TOPFLASH assays, respectively. Viable cells were determined by trypan blue exclusion assay and WST-1 assay. Dead cells were determined by Sytox-green/Hoechst staining. Caspase activity was determined by caspase-6 assay. Summary of results: Our in-vitro data showed that ICG-001 treatment (5, 10, and 25uM) reduced viable HNSCC cells and Ki67positive cells as much as 50%, in a dose-dependent manner. Western blotting, RT-PCR, immnohistochemistry and survivin-promotor analysis showed that survivin expression level was significantly reduced by ICG-001 treatment. Moreover, Wnt/b-catenin promoter activity was reduced by ICG-001. In addition, ICG-001 treatment significantly downregulated S100A4(mesenchymal marker) expression and upregulated E-cadherin (epithelial cell marker) expression level in all of the cell lines we tested. Pre-treatment with ICG-001 enhanced radiation induced cell death in vitro, determined by WST-1 and Sytox-Green/Hoechst assays. In order to delineate the molecular mechanisms of ICG-001 coupled radiation-mediated cell death, caspase activity was measured. Our preliminary results showed that Caspase-6 activity was enhanced in a dose dependent manner in ICG-001 pre-treated, radiation-treated cells. Conclusion: Antagonizing CBP/b-catenin signaling by ICG-001 induced anti-proliferative effect in HNSCC and could enhance radiation sensitivity in vitro, possibly by inducing apoptosis due to a reduced survivin expression. Moreover, the small molecule inhibitor may reduce EMT process by reducing S100A4 expression levels. Modulating downstream target genes of CBP/b-catenin signaling by the small molecule inhibitor ICG-001 may be a powerful tool to enhance radiation sensitivity and to reduce metastatic potential in HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B214.