Metastasis and frequent relapse contribute to the high mortality rate of ovarian cancer (OC) patients. However, the mechanisms of regulation of critical steps during metastasis is poorly understood and treatment strategies have not been developed to specifically target them. Using an organotypic 3D culture model of the human omentum, we have studied the productive cross-talk between metastasizing OC cells and the metastatic microenvironment that is essential 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 42 matched primary and metastatic tumors from OC patients. miR-193b and miR-4454 was thus identified as an important metastasis suppressor, downregulated by signals from the metastatic microenvironment.
Both miR-193b and miR-4454 were downregulated by signals from the site of metastasis. Decreased microRNA expression promoted metastatic colonization by enhancing the ability of the OC cells to attach and invade through the outer layers of the omentum. These metastasis initiating cells have cancer stem cell like characteristics. The induction of cancer stem cell-like phenotype by the decreased expression of miR-193b was important for establishment of metastatic tumors, and could potentially regulate chemoresistance and recurrent disease in OC. Stably overexpressing miR-193b resulted in a significant decrease in metastases in OC xenografts while stable inhibition had the opposite effect. Moreover, treating a chemoresistant OC patient derived xenograft (PDX) model of metastasis with miR-193b significantly reduced metastasis. Using heterotypic coculture models, conditioned medium experiments, secretome analysis, inhibition, and rescue experiments, we have identified the microenvironmental signals and the mechanism of miR-193b downregulation via the ERK/EZH2/DNMT1 axis. By performing RNA-seq in OC cells overexpressing miR-193b, we identified cyclin D1 (CCND1) as a key target. Knockdown and functional rescue experiments confirmed CCND1 as the functional effector of miR-193b responsible for the metastasis initiation phenotype.
miR-4454 was downregulated in the metastasizing ovarian cancer cells through paracrine signals from microenvironmental fibroblasts, which promoted migration, invasion, proliferation, and clonogenic growth in ovarian cancer cells as well as their ability to penetrate through the outer layers of the omentum. Stable overexpression of miR-4454 decreased metastasis in ovarian cancer xenografts. Its mechanism of action was through the upregulation of its targets, secreted protein acidic and cysteine rich (SPARC) and BCL2 associated athanogene 5 (BAG5), which activated focal adhesion kinase (FAK) signaling, promoted mutant p53 gain of function by its stabilization, and inhibited apoptosis. Our studies suggest the possible application of miR-193b and miR-4454 replacement therapy as a novel approach to treat OC metastasis.
Audience Take Away:
- Cross-talk between cancer cells and its tumor microenvironment (mesothelial cells and fibroblasts)
- Epigenetic regulation by microenvironmental signals
- Potential clinical application of microRNAs to treat OC metastasis