Differential Response of MED12 Downregulated Breast Cancer to Targeted Therapy

Abstract

MED12 is a subunit of a large multi‐subunit protein complex called Mediator. Mediator plays critical roles in gene regulation by repressing or activating transcription through modulation of RNA polymerase II. The specific role of MED12 within Mediator is to regulate the activity of CDK8 kinase. Many studies in the last several years have uncovered that mutations within MED12 or downregulation of MED12 leads to dysregulation of CDK8 kinase activity which has many physiological implications, including promotion of tumorigenesis in a variety of different cancers. Specifically, MED12 mutations have been found in up to 67% of breast cancer patients. Although many strides have been made towards finding superior treatment strategies for breast cancer, there is still a critical need for uncovering targeted therapies for specific genetic subsets of breast cancer. Therefore, we are aiming to find a superior treatment strategy for breast cancer patients who carry MED12 mutations. Natural compounds are a promising treatment option since more than 50% of drugs currently available in the pharmaceutical industry are derived from natural sources. Consequently, we have employed a screen of a natural compound library to discover novel therapeutics for MED12 mutant breast cancer. Specifically, we generated a stable MED12 knockdown MCF‐7 cell line through the use lentiviral‐mediated shRNA. Since MED12 mutations abolish MED12 functions, the MED12 knockdown cell line should mimic a MED12 mutant setting. We first confirmed that the MED12 knockdown cell line exhibits enhanced proliferation through the employment of proliferation assays. Next, we performed a natural compound screen as assayed by MTT cell viability assays and found that MED12 knockdown cells are differentially affected by the compounds. Our results indicated that MED12 knockdown cells appear to be more resistant to treatment when compared to MED12 wildtype cells, although we did also uncover compounds that differentially targeted MED12 knockdown cells. We selected out the compounds that differentially targeted MED12 knockdown cells and subjected them to quantitative PCR analysis. MED12 has previously been shown to place a constraint on GLI3‐mediated SHH signaling, a finding that is of interest since SHH signaling is often hyperactivated in breast cancer. We therefore examined the expression of GLI3 target genes in our MED12 knockdown cell line that were either treated with control vehicle or selected natural compounds. Interestingly, we found that GLI3 signaling is upregulated in MED12 knockdown cells, and more importantly, the upregulation is drastically reversed in the compound‐treated cells. Overall, our results demonstrate that natural compounds could prove to be an effective targeted therapy for MED12 mutant breast cancer by targeting the GLI3‐mediated SHH signaling pathway.