Epigenomic analysis of DNMT3B-mediated cervical cancer invasion by methylated DNA immunoprecipitation identifies PTPRR as a metastasis suppressor gene

AACR Annual Meeting-- Apr 18-22, 2009; Denver, COIntroduction Epigenetic changes are important events in cancer development. The understanding of epigenetic regulation in the cancer invasion is limited. The present study is to interrogate the role of de novo DNA methylation in cervical cancer invasions. Methods and Results: A subclone of HeLa with invasive phenotype, HeLa3rd, was selected. The analysis of differential mRNA expression of DNMTs (1, 3a, and 3b) reveals significant over-expression of DNMT3b in HeLa3rd. Interference of DNMT3b using RNAi with the efficiency of 60% reduced 62% of transwell invasion ability in vitro and completely inhibited lung metastasis in mouse models through tail vein injection. To discover genes mediated by DNMT3B in cancer metastasis, we carried out a genome-wide DNA methylation analysis using methylated DNA immunoprecipitation coupled with promoter microarray,mDIP-on- chip, comparing HeLa3rd and HeLa3rd with DNMT3B knockdown. We uncovered a protein tyrosine phosphatase, receptor type, R (PTPRR) differentially methylated in cancer cells with different invasion phenotype. Demethylation treatment using 5-AzadC in HeLa3rd cells confirmed the re-expression of mRNA and demethylation of the promoter of PTPRR. Chromatin immunoprecipitation coupled with PCR validated the targeting of DNMT3b on PTPRR promoter. Re-expression of PTPRR in an inducible system inhibited transwell invasion ability by 25%, but not interfering in cell proliferation. The methylation of PTPRR in clinical samples was also analyzed using quantitative methylation specific PCR. The methylation rate of PTPRR was significantly higher in squamous cell carcinoma (76%, n=30) and adenocarcinoma (55%, n=20) than that in normal cervix (5%, n=30) (P< 0.001). Conclusion: The present study demonstrates for the first time that DNMT3b plays an important role in the cervical cancer invasion and metastasis. The methylation - silencing of PTPRR through DNMT3b may serve as part of the mechanism of DNMT3b-related cancer invasion. The high methylation rate of PTPRR in invasive cancer but not in normal cervix may be used as a useful biomarker for cervical cancer screening in the future.Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5192.