Predictive efficacy of proapoptotic epigenetic pharmacotherapies: An ovarian cancer paradigm

B21

Objectives: Epigenetic aberrancies such as altered methylation patterns that occur throughout the cell's genome are a classic molecular indication of most cancers including ovarian malignancies. Towards a better understanding of molecular and cellular mechanisms that could be targets for ovarian cancer therapeutics, we sought to investigate the co-selection of hypermethylated genes responsible for cisplatin resistance. Our hypothesis is that loss of function of additional genes and pathways may be co-selected with the mismatch repair gene MLH1 to give rise to cisplatin-refractory ovarian cancers.Experimental Design and Methods: The transcriptional activities of the genes VDR, CYP27A1, CYP27B1, CYP24A1, MLH1, and BRCA1, which putatively are all subject to epigenetic controls, were examined in ovarian cancer cell line and clinical specimens. The cell lines include a set of cisplatin-sensitive parental cells and their daughter sublines isogenic for a cisplatin-selected drug resistant phenotype. To assess whether gene downregulations were due to aberrant hypermethylation, cancer cells were treated with Decitabine (5'-aza-deoxycytine), a compound which inhibits the DNA methyltransferase (DMNTs) isoenzymes, and the abundance of gene transcripts were measured and normalized to uninduced levels. Sequencing analysis for methylation gains across gene promoter regions was conducted of bisulfite-converted DNAs and the COBRA method was applied to interrogate the methylation status of specific CpG dinucleotides of genomic DNAs isolated from normal ovarian tissues, primary carcinomas, and recurrent cancers.Results: Altered levels of the VDR, CYP27A1, CYP27B1, and MLH1 transcripts were detected for ovarian cancer cells treated with Decitabine. By contrast the BRCA1 gene was constitutively transcribed and not further epigenetically-induced. Epigenetic mechanisms alone were responsible for the down-regulation the VDR, CYP27B1, and MHL1 genes in the cells investigated. However, for CYP27A1 both genetic and epigenetic mechanisms appeared to be responsible for this gene's activity loss. Dense methylation of CpG dinucleotides across the CYP27B1 gene promoter was found for the ovarian cancer cells, consistent with the finding that gene silencing was due to an epigenetic event. COBRA assays of the VDR and CYP27B1 genes of clinical specimens of normal ovarian tissues, primary carcinomas, and recurrent cancers detected methylation in only two cancer cases. Interestingly, one patient had responded poorly to initial cisplatin chemotherapy while the second case was a recurrent disease distinctive of the neuroectodermal ovarian cancer type.Conclusions: An accurate understanding of chemotherapy failures and disease relapse is imperative to the individualization of cancer pharmacotherapeutics and patient care. We present here a developing utility of cancer molecular profiles for the pre-assessment of effective therapies of three cytotoxic compounds - platinums, secosteroids, and epigenetic inhibitors - in a panel of cisplatin-sensitive and resistant ovarian cancer cells. Co-selection of additional genes with the mismatch repair gene MHL1 was not observed within the parent/daughter cell lines examined here.