Abstract A097: Targeting ovarian cancer metastasis initiating cells induced by the microenvironment through ERK/EZH2/DNMT1 mediated miR-193b downregulation
Authors: Dasari Subramanyam , Wang Ji , Cheng Frank , Halprin Melissa , Pepin David , Mitra Anirban K. 
Published: March 2024

Abstract
Extensive metastasis and frequent relapse are major contributors to the high mortality rate of ovarian cancer (OC) patients. However, the regulation of metastasis and disease recurrence is poorly understood and there is no development of specific treatment strategies to target them. Using an organotypic 3D culture model of the human omentum, we have studied the productive crosstalk between metastasizing OC cells and its microenvironment that is critical for establishment of metastasis. To identify the clinically relevant microRNAs that can regulate both early and advanced metastasis, we combined our 3D omentum culture approach with the end point analysis of microRNA expression profiles of matched primary and metastatic tumors from OC patients. miR-193b was a key microRNA thus identified to be downregulated in early and advanced metastasis. The decrease in miR-193b promoted metastatic colonization by enhancing the ability of the OC cells to invade through the outer layers of the omentum and increased cancer stem cell-like phenotype, helping metastasis initiation. Overexpressing miR-193b resulted in a significant decrease in metastases in OC xenografts while inhibition had the opposite effect. Moreover, treating a chemoresistant OC patient derived xenograft (PDX) model of metastasis with miR-193b significantly reduced metastasis. We have identified the microenvironmental signals and the resulting mechanism of miR-193b downregulation via the ERK/EZH2/DNMT1 axis, using heterotypic coculture models, conditioned medium experiments, secretome analysis, inhibition, and rescue experiments. Basic FGF2 (bFGF2) and IGF binding protein 6 (IGFBP6) were the key mesothelial factors that were responsible for the downregulation of miR-193b in cancer cells. These factors were found to activate ERK in the cancer cells that induced EZH2 and DNMT1 expression. Using ChIP and MeDIP, we discovered that EZH2 induces H3K27me3 in the miR-193b promoter, which helps recruit DMNT1 that catalyzes DNA hypermethylation at the miR-193b promoter. ScRNA-seq analysis of patient metastases confirmed that mesothelial cells are the source of FGF2 and IGFBP6 in the TME. We next proceeded to study the mechanism by which miR-193b decrease promotes metastasis. By performing RNA-seq in OC cells overexpressing miR-193b, we identified cyclin D1 (CCND1) as a key target, which was validated at RNA and protein levels. Knockdown of CCND1 mimicked the decreased expression of stem cells markers (ALDH1A1, OCT4, SOX2 and Nanog), and spheroid formation, caused by miR-193b overexpression. The induction of OC stem cells upon miR-193b inhibition could be rescued by simultaneous overexpression of CCND1. In conclusion, we have identified the mechanism of microenvironment induced downregulation of miR-193b in OC cells that helps establish metastatic tumors by inducing cancer stem cells via its target CCND1. Treating a chemo resistant OC PDX with miR-193b significantly reduced metastases, indicating that miR-193b replacement therapy could be a promising approach to treat OC patients.
Citation Format: Subramanyam Dasari, Ji Wang, Frank Cheng, Melissa Halprin, David Pepin, Anirban K. Mitra. Targeting ovarian cancer metastasis initiating cells induced by the microenvironment through ERK/EZH2/DNMT1 mediated miR-193b downregulation [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A097.