Loss of the obscurin-RhoGEF downregulates RhoA signaling and increases microtentacle formation and attachment of breast epithelial cells

Abstract

Obscurins are RhoGEF-containing proteins whose downregulation has been implicated in the development and progression of breast cancer. Herein, we aim to elucidate the mechanism for increased motility of obscurin-deficient cells. We show that shRNA-mediated obscurin downregulation in MCF10A cells leads to >50% reduction in RhoA activity relative to scramble control (shCtrl), as well as decreased phosphorylation of RhoA effectors, including myosin light chain phosphatase, myosin light chain, lim kinase, and cofilin, in both attached and suspended cells. These alterations result in decreased actomyosin contractility, allowing suspended cells to escape detachment-induced apoptosis. Moreover, ~40% of shObsc-expressing cells, but only ~10% of shCtrl-expressing cells, extend microtentacles, tubulin-based projections that mediate the attachment of circulating tumor cells to endothelium. Indeed, we show that MCF10A cells expressing shObsc attach in vitro more readily than shCtrl cells, an advantage that persists following taxane exposure. Overall, our data suggest that loss of obscurins may represent a substantial selective advantage for breast epithelial cells during metastasis, and that treatment with paclitaxel may exacerbate this advantage by preferentially allowing obscurin-deficient, stem-like cells to attach to the endothelium of distant sites, a first step towards colonizing metastatic tumors.