Calcium handling genes are regulated by promoter DNA methylation in colorectal cancer cells

Calcium signaling regulates various cellular processes, including proliferation and cell death. DNA methylation of gene promoters is an epigenetic modification that facilitates transcriptional suppression. Disruption of calcium homeostasis and DNA methylation in cancer are each linked to tumor development and progression. However, the possible connection between these two processes has not been thoroughly studied. Therefore, we measured the expression of six gene families involved in calcium regulation (ATP2A, ITPR, ORAI, RyR, STIM, and TRPC) in a colorectal cancer cell model, HCT116, with either genetic (Double Knock-out/DKO) or pharmacological (5-aza-2'-deoxycytidine/DAC) inhibition of DNA methyltransferases. Fourteen of the 20 examined calcium handling genes were expressed at higher levels in DKO cells as compared to HCT116. Expression of five genes was increased in HCT116 cells treated with DAC, three matching DKO. Due to a unique expression pattern of the three ATP2A genes in our model, encoding the Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase (SERCA) pumps, we chose to evaluate the methylation status of these genes, protein expression, and potential associated physiological effects, using the SERCA inhibitor thapsigarin (TG). We observed an expected pattern of promoter methylation coinciding with reduced expression and vice versa. This differential mRNA expression was associated with altered SERCA3 protein expression and cytosolic calcium levels with TG exposure. As a result, DKO cells displayed less TG-induced cytotoxicity, as compared to HCT116 cells. Overall, it is likely that at least several calcium regulatory genes are transcriptionally regulated by DNA methylation, and this may play a role in tumorigenesis through altering apoptosis in cancer.