Abstract TMIM-066: EZH2 REGULATES THE EPIGENETIC REPROGRAMMING OF OVARIAN CANCER PROMOTING CARCINOMA-ASSOCIATED MESENCHYMAL STEM CELLS
Authors: Frisbie Lenard G. , Shen Hui , Coffman Lan 
Published: June 2021

Abstract
Ovarian tumor cells reside within a complex tumor microenvironment (TME) critical to the formation and function of ovarian cancer. Carcinoma-associated mesenchymal stem cells (CA-MSCs) are stromal progenitor cells within the TME which significantly enhance ovarian tumor cell growth and chemotherapy resistance. We demonstrated that CA-MSCs arise from ovarian tumor cell mediated reprograming of normal tissue MSCs. The resulting CA-MSC phenotype has a unique and durable expression profile without acquisition of genetic mutations, suggesting epigenetic mechanisms underpin the induction of a CA-MSC. In support of this, EPIC methylation array and ATACseq data demonstrate CA-MSCs have significant global hypermethylation and overall “closed” chromatin structure compared to normal MSCs. Further, promoter methylation alterations correlate with the gene expression profile which characterize CA-MSCs. We thus sought to investigate the mechanism underlying the cancer-mediated epigenetic reprograming of CA-MSCs.
The histone-lysine N-methyltransferase Enhancer of Zeste homolog 2 (EZH2) is induced early in the conversion of a CA-MSC through tumor secreted factors. EZH2 promotes transcriptional repression through histone H3 lysine 27 trimethylation (H3K27me3) leading to a closed chromatin state. EZH2 also acts as a scaffold for DNA-methyltransferases (DNMTs) thus enhancing DNA methylation. Western blotting confirmed increases in EZH2 and the EZH2 target repressive marks H3K27me3 and H2AK119ub in CA-MSCs versus MSCs. We therefore hypothesize that ovarian cancer mediated induction of EZH2 in normal tissue MSCs is critical to the formation of a CA-MSC through epigenetic reprogramming.
Normal tissue MSCs derived from benign human ovary or omentum and CA-MSCs derived from high grade serous ovarian cancer patient samples were isolated as previously described. To model the tumor-mediated induction of CA-MSCs, direct co-culture of normal tissue MSCs with high grade serous cell lines CAOV3, PEO1 or OVCAR3 were used. MSC/tumor cell co-cultures were treated with the EZH2 inhibitor Tazemetostat and/or the DNMT inhibitor 5-Azacitidine. A mathematical model, referred to as the CA-MSC classifier which uses the expression of 6 genes to accurately distinguish normal MSCs from CA-MSCs, was utilized to assess the impact of EZH2 and DNMT inhibition on the formation of a CA-MSC. In this model, values closest to 1 indicate a high likelihood of being a CA-MSC (a value of &gt;0.8 meets the threshold for a CA-MSC and &lt;0.2 meets the threshold for a normal MSC).
Pharmacologic EZH2 inhibition significantly decreased the induction of a CA-MSC phenotype in tumor cell co-culture without impacting tumor cell or CA-MSC viability. DNMT inhibition alone yielded a modest decrease in CA-MSC induction. However, the combination of EZH2 inhibition and DNMT inhibition blocked the formation of a CA-MSC with a classifier score going from 0.9 (in untreated co-cultures) to 0.1 in dual-inhibition co-cultures.
Collectively, our data suggests that ovarian tumor cell mediated EZH2 induction plays a critical role in the conversion of a MSC into a CA-MSC through epigenetic reprogramming. This reprogramming can be interrupted through targeting EZH2 and DNMT. This presents a novel target to block the formation of the tumor enhancing stromal niche thus offering a potentially powerful new approach to the treatment of ovarian cancer.
Citation Format: Lenard G. Frisbie, Hui Shen, and Lan Coffman. EZH2 REGULATES THE EPIGENETIC REPROGRAMMING OF OVARIAN CANCER PROMOTING CARCINOMA-ASSOCIATED MESENCHYMAL STEM CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-066.