ROR2-Vangl2 complexes direct ovarian cancer cell motility in response to Wnt5a (265)

Abstract

<b>Objectives:</b> In both developmental and cancer contexts, collective migration has shown to be at least partially dependent upon asymmetric localization of core protein complexes within leader and follower cells. A critical function of non-canonical Wnt signaling in vertebrates is to regulate planar cell polarity (PCP), the polarity axis that organizes the plane of a tissue. Cells at the invasive front of cellular aggregates exhibit elevated expression of genes associated with motility pathways, including Wnt/PCP signaling. Using microfluidic devices, we have previously shown the soluble non-canonical Wnt ligand, <i>WNT5A</i>, influencing the directional motion of ovarian cancer cells via the ROR2 receptor. We sought to determine whether asymmetrical localization of ROR2 could guide <i>WNT5A-</i>directed motility. <b>Methods:</b> A DNA sequence encoding a fusion protein of ROR2 with a C-terminal Emerald (Em) fluorescent protein tag was introduced into ES2 ovarian cancer cells. Aggregated spheroids of ES2 ROR2-Em cells were plated onto a fibronectin-coated surface, and time-lapse fluorescent images were obtained of the radial migration in the presence or absence of <i>WNT5A</i>. Based on the direction of motion, cells were divided into leading or trailing half for analysis of the localization of ROR2 protein. Data were presented as mean +/- SEM. For co-immunoprecipitation experiments, ES2 cells expressing a Myctagged ROR2 DNA construct were utilized, with Myc epitope used for pulldown. <b>Results:</b> In the absence of <i>WNT5A</i>, ROR2 was uniformly distributed among the leading and lagging edge of the migrating cells (46.9% +/-2.452 ROR2 localized on the leading edge). In the presence of <i>WNT5A</i>, ROR2 was preferentially located to the leading edge of migrating cells (64.6% +/- 3.908 ROR2 localized on the leading edge). <i>VANGL2</i>, a core component in planar cell polarity, formed Wnt-induced receptor complexes with ROR2 in developing limb buds and other tumor types. <i>VANGL2</i> had been shown to reside at the protrusive edge of leader cells. To address whether <i>VANGL2</i>-ROR2 complexes contributed to the leading-edge localization of ROR2 in ovarian cancer cells, co-IP experiments were performed. Complex formation of ROR2-<i>VANGL2</i> was present in ovarian cancer cells at baseline (<i>VANGL2</i>:ROR2 IP ratio 1), yet was increased in the presence of <i>WNT5A</i> (<i>VANGL2</i>:ROR2 IP ratio 1.5). <b>Conclusions:</b> We have previously shown that <i>WNT5A</i> promotes the motility of ovarian cancer cells by influencing both directionality of motion as well as the degree of displacement in a ROR2 dependent manner. Asymmetric localization of Wnt/PCP core component complexes within leader and follower cells is crucial for cell motility, an intrinsic metastatic property. Our studies suggest that under the influence of <i>WNT5A</i>, ROR2 localizes to the leading edge of migrating cells. Additionally, ROR2 complexes with <i>VANGL2</i> in ovarian cancer cells, and this complex is enhanced in the presence of <i>WNT5A</i>. As <i>WNT5A</i> is produced at high levels by peritoneal mesothelial cells and omental adipose tissue in the microenvironment of ovarian cancer metastasis, disruption of the ROR2-<i>VANGL2</i> complex represents an attractive target for disrupting metastatic colonization of ovarian tumors.