Abstract 4035: Inactivation of the putative tumor suppressor gene AGTR1 by promoter hypermethylation in primary human cancer

Abstract

Abstract Introduction: Primary human cancers are thought to arise from genetic and epigenetic alterations of tumor suppressor genes and oncogenes. Transcriptional and DNA copy-number studies have improved our understanding and classification of solid tumors. Recent studies have established that like mutation, methylation-mediated gene silencing often leads to tumorigenesis. The aim of this study was to comprehensively explore the “ovarian cancer methylome” and to validate the novel methylation marker AGTR1 in a large independent series of ovarian cancer samples. Patients and methods: Fifteen ovarian cancer samples and 10 normal ovarian surface epithelium brushings were hybridized to Affymetrix U133 plus 2.0 arrays. Furthermore, 3 normal ovarian and 3 isogenic (resistant and sensitive to chemotherapy) ovarian cancer cell lines, with or without treatment with a demethylating agent were hybridized to the same array. Selection of cancer specific methylated genes was based on differential expression between normal and cancer samples, with cancer samples having lower expression than normal samples, and upregulation in cancer cell lines after treatment with a demethylating agent. A number of selected genes that showed low or no expression in primary cancer tissues and re-expressed after treatment with a demethylating agent were then tested for promoter methylation by bisulfite sequencing. Quantitative methylation specific PCR (QMSP) was developed for the AGRT1 gene and tested in a total of 353 ovarian cancer, 17 ovarian cystadenoma and 16 ovarian borderline tumor samples. Results: Fourteen primary ovarian tumor DNA samples were available from the samples that were hybridized for expression array analysis. Twelve out of 14 samples were methylated for AGRT1 which is inversely correlated with expression from our array analysis. We then tested 13 normal ovarian epithelium samples and no methylation was detected in any of the samples. Validation in a large independent set of samples confirmed that ovarian cancer samples (207/353, 53%) were more frequently methylated than ovarian cystadenoma (3/17, 18%; P=0.004). Conclusion: We have identified a set of potential tumor suppressor genes by a comprehensive discovery approach to uncover the cancer methylome. Among these genes, AGTR1 methylation was identified as a potential cancer specific methylated gene that may hold promise for further studies to establish it as a biomarker. Functional studies are warranted to evaluate the biologic role of AGTR1 methylation in the carcinogenic process of solid tumors. 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 4035. doi:1538-7445.AM2012-4035