Abstract 825: Wnt/beta-catenin signaling regulates the expression of O6-methylguanine DNA-methyltransferase in cancer cells: a new strategy to overcome resistance for DNA alkylators in cancer therapy
Authors: Dyberg Cecilia H. , Baryawno Ninib , Milosevic Jelena , Wickström Malin , Sveinbjörnsson Baldur , Northcott Paul A. , Taylor Michael D. , Kool Marcel , Kogner Per , Johnsen John Inge 
Published: August 2012

Abstract
Background: A number of DNA-damaging agents attack the O6 position on guanine and thereby form the most potent cytotoxic DNA adducts known. The DNA-repair protein O6-alkylguanine (O6-AG) DNA alkyltransferase (AGT) encoded by the gene O6-Methylguanine (O6-MG)-DNA-methyltransferase (MGMT) repair DNA adducts caused by alkylating agents. MGMT has important implications in cancer treatment since its expression correlates inversely with sensitivity to agents that form O6-alkylguanine adducts, such as temozolamide. It is therefore of great interest to find agents that induce MGMT deficiency, increase the sensitivity and possible overcoming resistance to alkylating chemotherapeutic agents. Methods: Cell lines from medulloblastomas, gliomas, colon cancer and neuroblastomas were examined for Wnt/beta-catenin activity and MGMT expression. We used Western blot, Real-Time quantitative PCR (Q-PCR), siRNA knockdown and cDNA overexpression of beta-catenin, MGMT promotor reporter plasmids and cells with inducible siRNAs targeting beta-catenin to study beta-catenin mediated regulation of MGMT. The correlation between Wnt activity and MGMT expression was also investigated in primary medulloblastomas, gliomas and colon cancer using gene expression cohorts. Cell cytotoxicity and clonogenicity of chemotherapeutic drugs in combination with celecoxib were examined in cell lines using fluorometric microculture cytotoxicity assay and clonogenic assay, respectively. Compounds targeting Wnt signaling was investigated in combination with temolzolamide in vitro and in vivo. Results: MGMT expression level was shown be correlated to Wnt signaling activation both in primary tumors and cell lines of different origins. Proinflammatory prostaglandin E2 activates the Wnt/beta-catenin signaling and increase MGMT expression. Transfection experiments and cells with inducible siRNAs revealed that beta-catenin directly regulates MGMT expression via Tcf/LEF binding. Wnt inhibiting drugs and compounds potentiates the cytotoxic effect of the DNA alkylating drug, temozolomide in cells with elevated MGMT expression in vitro and in vivo. Conclusions: Our data demonstrate that MGMT is a direct Wnt/beta-catenin target, and agents that inhibit Wnt signalling reduces the transcription of MGMT through a prostaglandin E2-Wnt/beta-catenin route and thus increases the sensitivity to temozolomide. This provides a rational approach for improved efficacy of chemotherapeutic drugs inducing DNA alkylation in cancer treatment. The data also suggest that Wnt/beta-catenin is an important target for therapeutic interventions.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 825. doi:1538-7445.AM2012-825