Epigenetic Modification Suppresses PKCα Expression in Epithelial Cancers

Abstract

Members of the protein kinase C (PKC) family of serine/threonine kinases are key mediators of signaling pathways that are altered in cancer. The conventional PKC, PKCα, has anti-proliferative and tumor-suppressive roles in many epithelial systems. Consistent with these roles, PKCα expression is reduced in about 60% of colorectal and endometrial cancers. The importance of PKCα loss in these tumors is highlighted by findings that (a) low levels of PKCα expression correlate with poor survival in patients, and b) PKCα deficiency enhances tumor aggressiveness in mouse models of intestinal and uterine cancer. The objective of this study was to understand the mechanisms involved in the loss of PKCα in tumors and to explore strategies for restoration of PKCα expression for cancer treatment. Our analysis of cell lines and patient samples has determined that loss of PKCα is the result of reduced PRKCA transcription. However, no differences in the activity of individual promoter elements were observed between PKCαhigh and PKCαlow cell lines, pointing to an epigenetic effect. Thus, we hypothesized that PKCα loss in colon and endometrial cancers is caused by changes in DNA methylation of the PRKCA promoter region and that clinically relevant demethylation agents offer a strategy for therapeutic re-expression of this tumor suppressive kinase. MBDCap-seq analysis of endometrial tumors and normal controls revealed tumor-associated de novo hypermethylation in the right CpG shore of PRKCA, with no consistent changes in the PRKCA promoter or left shore. Bisulfite treatment and pyrosequencing analysis of 27 colon and endometrial cancer cell lines revealed different methylation levels of the PRKCA right CpG shore that inversely correlated with PKCα mRNA and protein expression. The methylation pattern in the PRKCA promoter region as well as the inverse correlation between right shore methylation and PKCα mRNA expression were confirmed in colon cancer samples. Treatment of PKCαlow endometrial and colon cancer cells with the FDA approved de-methylating agent, 5-aza-2′-deoxycytidine (decitabine, Dacogen®), significantly reduced the methylation of the PRKCA right CpG shore and increased PKCα expression. Ongoing studies in PtenΔ4-5/+ mice, which develop endometrial lesions lacking PKCα expression, will determine if decitabine can restore PKCα expression in endometrial tumors in vivo. In conclusion, the tumor suppressive protein kinase, PKCα, is transcriptionally repressed by hypermethylation of the right CpG shore of the PRKCA promoter region in colon and endometrial cancers, and the expression of PKCα can be restored by treatment with a clinically relevant, FDA approved de-methylation agent.