Influence of DNA methylation in determining cellular phenotype and its usefulness as a diagnostic biomarker for detecting prostate cancer
Date of Award
Doctor of Philosophy in Health Science (PhD)
Biological and Biomedical Sciences
Alterations in DNA methylation patterns are associated with many types of cancers. DNA methylation affects the expression profile of a lexicon of genes in cancer cells and therefore plays a causal role in the development and progression of neoplasia. Several genes which are abnormally hypermethylated in prostate cancer (PCa) have been identified, but how DNA methylases preferentially target these gene promoters remains elusive. / In somatic cancer cells, DNMT1 is the main maintenance methylase while DNMT3a and DNMT3b are capable of methylating DNA at the CpG dinucleotides de novo. Given the supposedly well-defined roles of DNMT1 and DNMT3b one would expect that depletion of DNMT1 in cancer cells impacts gene expression patterns much more dramatically than removal of either DNMT3a or DNMT3b. Previous studies in which the respective roles of DNMT1 and DNMT3b were evaluated in colon and gastric cancer cell lines have shown contrasting results. To study the roles of these enzymes in prostate cancer cells we employed small interfering RNA (siRNA) to silence DNMT1 and DNMT3b expression in prostate cancer derived PC3 cells and subsequently studied the resulting phenotypic and gene expression changes in the engineered cell lines. Strikingly, our results reveal that loss of DNMT1 markedly reduced growth but significantly increased migratory and invasive potential of silenced cells compared to the mock transfected control cells. DNMT3b silenced cells proliferated poorly but showed decreased migratory and invasive potential. Interestingly, among the l6 gene promoters whose methylation status was interrogated by methylation specific PCR (MSP), loss of either DNMT1 or DNMT3b in the engineered cells culminated in demethylation of Rb1 and MR-beta gene promoters. Silencing of DNMT3b resulted in specific demethylation of the APC gene promoter. Furthermore, gene expression profile of 268 prostate cancer specific genes in the silenced cell lines was investigated using a DNA microarray. Our results revealed significantly increased expression of seven genes in DNMTI silenced cells (>2-fold increase in expression), from which CDKN3 and claudin-3 genes were the most notable. Loss of DNMT3b in PC3 cells culminated in increased expression of four genes including caspase 7, cytochrome b5, CDKN3 and protein kinase C. These findings suggest that loss of either DNMTI or DNMT3b results in locus specific changes in methylation status and gene expression profile. Gene silencing in response to DNA methylation is directed by methyl binding proteins (MBPs) which bind to methylated CpGs and recruit repressor complexes containing HDAC activities. A number of MBDs have been identified to date but it is unclear whether they have unique or overlapping functions. To determine whether the two well characterized CpG methylated DNA binding proteins, MeCP2 and MBD1, have distinct or redundant functions, we employed RNAi to silence their expression in the prostate cancer derived PC3 cells, and subsequently, compared cell growth, invasion and migration properties of these cell lines in addition to their respective mRNA expression profiles. Cells devoid of MeCP2 proliferated more poorly as compared to MBDI deficient cells and the parental PC3 cells. Boyden chamber invasion and wound-healing migration assays showed that MBD1 silenced cells were both more invasive and migratory compared to MeCP2 silenced cells. Genechip microarray analyses showed striking differences in the mRNA expression profiles obtained from MeCPZ and MBD1 depleted cells relative to each other as well as to control cells. Our results suggest that MeCP2 and MBD1 appear to have non-overlapping biochemical functions in vivo and that discrete sets of genes involved in cellular proliferation, apoptosis, invasion and migration are targeted by each protein. Since DNA methylation is strongly associated with PCa, the DNA methylation status of four bona fide biomarkers of disease RASSFIA, APC, p16 and Rb1 in 27 primary prostate cancer and 24 benign prostatic tissues was studied using methyl-sensitive PCR (MSP) to determine whether their DNA methylation status could serve as a robust marker of disease. Strikingly, our results revealed that RASSF,/A promoter was hypermethylated in all tested cancer samples (sensitivity L007o); unexpectedly, RASSF/A was also found to be hypermethylated in 3 out of 24 control tissues (specificity (87Vo). APC was found to be hypermethylated in 15127 cancer samples (sensitivity 557o) while p16 and Rb1 promoters did not appear to be modified. Hypermethylation at APC gene promoter was also found to be significantly associated with early stage cancer with odds ratio of 5.162 (p-value 0.049). Overall, our findings suggest that hypermethylation of RASSF1A and APC gene promoters have a potential to develop into a sensitive and specific diagnostic tools for detection of PCa.
Yaqinuddin, A. (2008). Influence of DNA methylation in determining cellular phenotype and its usefulness as a diagnostic biomarker for detecting prostate cancer (Unpublished doctoral thesis). Aga Khan University, Karachi, Pakistan.