Frequent DNA Hypermethylation at the RASSF1A and APC Gene Loci in Prostate Cancer Patients of Pakistani Origin

DNA methylation has emerged as a potentially robust biomarker for prostate cancer (PCa). Since DNA methylomes appear to be disease as well as population specific, we have assessed the DNA methylation status of RASSF1A, APC, and p16 (potential biomarkers of PCa) in Pakistani population. Primary prostate cancer tissues were obtained from 27 formalin-fixed paraffin-embedded blocks (FFPE) of cancer patients who underwent radical prostatectomy and transurethral resection of prostate (TURP) during 2003–2008. As controls, twenty-four benign prostatic FFPE tissues were obtained from patients who underwent TURP for benign prostatic hyperplasia during 2008. DNA was extracted, and methylation-specific PCR was used to assess the methylation status for RASSF1A, APC, and p16 gene promoters. Our results revealed that the RASSF1A promoter was hypermethylated in all the tested cancer samples but was also hypermethylated in 3 out of 24 control tissues. The APC promoter was hypermethylated in 15 out of 27 cancer samples and in none of the control samples. Strikingly, none of the samples showed methylation at the p16 promoter. Our findings suggest that RASSF1A and APC gene promoters are frequently hypermethylated in the Pakistani population and therefore have the potential to develop into universally dependable biomarkers for detecting PCa. 1. Introduction A well-characterized epigenetic mechanism is DNA methylation. DNA methylation typically occurs at CpG islands that are located in the promoter regions of about 50% of human genes. In general, hypermethylation of gene regulating regions (promoters) turns off gene expression, whereas hypomethylation has the opposite effect. Unprogrammed changes in the DNA methylome can culminate in the establishment of a disease state by activating or repressing genes related to cell cycle, growth, and apoptosis [1–3]. Aberrant DNA methylation profile (either hyper or hypo) has been linked to variety of malignancies and emerged as a potentially useful biomarker for monitoring neoplasia [4–6]. Current methods of detecting PCa including PSA and transrectal biopsy fall far short to be ideal methods for diagnosing clinically significant PCa [7, 8]. Epigenomic alterations appear to contribute significantly to PCa onset. A number of gene promoters including GSTP1, APC, RASSSF1A, COX2, MDR1, ER , hMLH1, and p14/INK have also been found to be frequently hypermethylated in PCa [9–12]. Accumulating data on the methylation status of various genes indicates that biomarkers based on specific methylomes may serve to differentiate between cancerous and