TGFbeta/SMAD4 Signaling and Altered Epigenetics Contribute to Increased Ovarian Cancer Severity

Ovarian cancer is the eighth most common cancer and is the fifth most common cause of cancer related death among women. Early stage ovarian cancer is very responsive to treatments and more than 93% of patients diagnosed with early stage disease achieve a five year survival rate. By contrast less than 30% of patients who are diagnosed with late stage disease achieve a five year survival rate, yet more than 60% of all cases present as late stage. Treatment is typically surgery followed by a chemotherapy regiment of a platinum-based chemotherapeutic and a taxane derivative. While this treatment plan works well for early stage disease, recurrent and late stage cancers are less responsive. Here we present work investigating the altered epigenetics and TGFbeta/SMAD4 signaling pathway in ovarian cancer in an effort to better understand the difference in disease severity at a molecular level.;We have identified a microRNA hsa-mir-9-3 which is epigenetically repressed by DNA methylation in primary ovarian cancer patients. Quantitative analysis of DNA methylation in the CpG island which contains the hsa-mir-9-3 microRNA revealed significant hypermethylation in both patient samples and cell lines compared to normal tissue. Functional studies reveal that the repression of this microRNA leads to increased proliferation rates and a decrease in apoptosis. We believe that the hypermethylation of the hsa-mir-9-3 locus serves as a novel biomarker for ovarian cancer.;TGFbeta/SMAD4 signaling is commonly dysregulated in ovarian cancers while being a key growth inhibition signal for the ovarian surface epithelium during menstruation. Here we present a genome-wide profile of SMAD4 binding by ChIP-sequencing following TGFbeta stimulation in the ovarian cancer cell line A2780. We believe this to be the first truly genome-wide profiling of SMAD4 binding using next generation sequencing approaches in an ovarian cancer model. Comparison of ChIP-seq results with previously reported ChIP-chip studies show dramatic biological and technical differences including more than 70% of all SMAD4 binding loci being more than 10kb away from the nearest transcription start site. Gene expression analysis following TGFbeta stimulation revealed a group of 318 genes whose expression changed following SMAD4 binding to the distal promoter of the gene. Of those 318 genes, a subset of them was used to predict patient survival in two independent patient cohorts. These results suggest that the loss of long distance SMAD4 gene regulation following TGFbeta stimulation may play a key role in ovarian carcinogenesis.;Additionally, we identified a novel biomarker, CLDN11, whose epigenetic repression is associated increased cisplatin resistance in a tissue culture model system. Examination of CLDN11 expression levels in a previously reported patient cohort revealed lower expression levels associated with increased tumor grade. Finally, loss of CLDN11 expression is associated with increased cellular motility.;In conclusion we have investigated and correlated several different epigenetic and signaling abnormalities associated with an increase in the severity of ovarian cancer while demonstrating the importance of recent technological advances in genome-wide methodologies. Together these results are likely to aid in both future discovery methods and patient prognosis and treatment.