Abstract P1-01-05: Wnt5a induces ROR1 to associate with cortactin, which undergoes tyrosine phosphorylation, and enhances migration of breast cancer cells

Background: ROR1is an evolutionarily conserved, oncoembryonic surface-antigen expressed in breast cancer. Previously, we found that ROR1 can serve as a receptor for Wnt5a, which can induce non-canonical Wnt signaling that enhances cancer-cell migration. Recently we found that Wnt5a could induce ROR1 to complex with a cytoskeletal protein designated HS1, which recruited ARHGEF1, enhanced activation of RhoA, and promoted leukemia-cell migration. These effects of Wnt5a on CLL cells could be inhibited by cirmtuzumab, a humanized, high-affinity monoclonal antibody (mAb) specific for ROR1. As expression of HS1 is limited to hematopoietic cells we examined breast cancer cell lines and primary breast cancer cells for expression of cortactin. Cortactin (also called EMS1), is a cytoskeletal protein, which is homologous in structure and function to HS1 and is broadly expressed in human cancers, including breast cancer. Methods: We performed immunoprecipitation studies using high-affinity mAb to ROR1 or cortactin and immunoblot analysis to examine for the association of ROR1 with cortactin and tyrosine phosphorylation of cortactin at Y421. We also assessed the expression levels of cortactin, ROR1, and ARHGEF1, in the MCF7 breast cancer cell line and in breast-cancer patient-derived xenografts (PDX). We also generated MCF7-ROR1 cells, which were stably transfected to express ROR1 or various mutant forms of ROR1 generated to study the structure-function requirements for effective ROR1-cortactin interactions. Results: We found that ROR1 associates with cortactin in freshly-isolated breast cancer PDX tumors or in PDX cells cultured with exogenous Wnt5a. We corroborated these results in breast cancer cell line MCF7-ROR1 cells, which were transfected to express ROR1 and found to migrate more effectively than the parental MCF7 lacking expression of ROR1. Wnt5a also induced cortactin tyrosine phosphorylation at Y421, recruitment of ARHGEF1, and activation of RhoA, which we found associated with enhanced breast-cancer cell migation. The capacity of Wnt5a to induce such changes could be blocked by treatment of the cells with cirmtuzumab. We generated truncated forms of ROR1 without a proline-rich domain (PRD) and found PRD was necessary for this association or Wnt5a-induced cortactin phosphorylation and enhanced cancer-cell migration. Accordingly, we introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at positions 784, 808, 826, or 841 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→A mutants, ROR1P(841)A had impaired capacity to recruit cortactin and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate cortactin or activate ARHGEF1, and was unable to enhance the motility of the MCF7 cells transfected with this mutant form of ROR1. Conclusions: Collectively, these studies indicate the capacity of cortactin to complex with ROR1 plays an important role in ROR1-dependent Wnt5a-enhanced breast cancer cell migration. These studies also demonstrate that cirmtuzumab can inhibit the formation of cortactin-ROR1 complexes, cortactin phosphorylation, and cancer-cell migration, and metastasis. Citation Format: Hasan MK, Tripple V, Widhopf II GF, Yu J, Zhang S, Parker BA, Kipps TJ. Wnt5a induces ROR1 to associate with cortactin, which undergoes tyrosine phosphorylation, and enhances migration of breast cancer cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-05.