Silencing ROR2 inhibits metastatic behavior of ovarian cancer cells

Abstract

Objectives: Adhesion of migrating ovarian tumor cells to peritoneal mesothelial cells and their subsequent clearance of the mesothelial monolayer is a key early event in metastatic seeding of ovarian cancer. We sought to identify mediators of ovarian cancer adhesion and invasion contributing to this tumor-microenvironment interplay. Methods: To identify potential drivers of ovarian cancer adhesion and invasion, a multicellular assay incorporating tumor (epithelial ovarian cancer cells) and microenvironment (patient derived omental fibroblasts) was adapted to carry out a high throughput genetic screen using a human kinome siRNA library. Based on screen candidates, ROR2, a noncanonical Wnt receptor that binds Wnt5a, was identified. ROR2 expression was evaluated in primary patient derived ovarian cancer cells and in high grade ovarian cancer cell lines (ES2, A2780, OVCAR3, OVCAR5, OVCAR8, ID8Trp53-/-BRCA2-/-). Biologic roles of ROR2 as well as the therapeutic effect of ROR2 silencing were examined in vitro using a validated mesothelial cell clearance assay. We further examined the effect of ROR2 silencing on force generation by ovarian cancer cell spheroids using immunofluorescent staining of phospho-myosin light chain (pMLC). Results: ROR2 was found to be expressed at a protein level across established ovarian cancer cell lines as well as primary ovarian cancer cells from patient derived tumors. Analysis in multicellular assays demonstrated that ROR2 silencing with siRNA dramatically decreases mesothelial cell clearance by tumor cell spheroids when compared to control siNEG spheroids. This result was observed across multiple cell lines (ES2, OVCAR8, OVCAR5, and OVCAR3) (P Conclusions: ROR2 plays a critical role in mediating the ability of ovarian cancer cells to clear a mesothelial cell monolayer, a key step leading to metastasis in ovarian cancer. Mesothelial clearance has been demonstrated to be a force dependent process, and our data suggests that ROR2 may play a role in force generation by ovarian cancer spheroids. Understanding the factors driving prometastatic tumor-microenvironment interactions is critical to develop targeted therapeutics to block the spread of ovarian cancer. As ROR2 is an actionable target, further investigation of ROR2 targeting in ovarian cancer is needed as it may represent a novel treatment strategy.