Abstract 3687: Wnt5a induces DOCK1 to complex with ROR1 and activate Rac1 leading to enhanced growth of breast cancer cells
Authors: Hasan Md Kamrul , Widhopf George F. , Zhang Suping , Lam Sharon M. , Parker Barbara A. , Kipps Thomas J. 
Published: September 2020

ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic surface antigen that is expressed on breast cancer, but not on normal postpartum tissues. We found ROR1 was a receptor for Wnt5a, and high-level expression of ROR1 in tumors of breast cancer patients associated with enhanced cell growth, aggressive disease, and shorter overall survival (Zhang S., et al., PLOS ONE, 7(3): e31127, 2012). More recently, ROR1 could activate RhoGTPases and enhance tumor growth in breast cancer (Zhang S., et al., PNAS, 116(4), 2019). Such effects of Wnt5a on breast cancer patient-derived-xenograft (PDX) tumors could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb, which is undergoing clinical evaluation in patients with cancer. However, the molecular mechanism(s) for how ROR1 contributes such functions remained elusive. We performed mass spectrometry-based proteomic analysis on anti-ROR1 immune precipitates from breast cancer PDX tumors and detected DOCK1 in addition to ROR1, implying that ROR1 associates with DOCK1. DOCK1 (Dedicator of cytokinesis 1, also known as DOCK180) that expressed in breast cancer PDXs and cell lines, is a cytoplasmic protein, and a member of the DOCK-A subfamily of guanine exchange factors (GEFs) specific for Rac1. Moreover, DOCK1 consists SH3 domain, which allows it to bind characteristic motifs (-P-X-X-P-), which often are found in the proline rich domains (PRD) of other proteins. Immunoblot analysis confirmed that ROR1 associated with DOCK1 in breast cancer PDX tumors or MCF7 cells transfected to express ROR1 (MCF7-ROR1) in response to Wnt5a, leading to enhanced activation of Rac1. To explore functional role, we performed a 2-week colony-formation assay using MCF7-ROR1 cells, and found that treatment of Wnt5a could enhance number of colonies of MCF7-ROR1 cells and that such colony formation capability could be inhibited by reducing expression of DOCK1 with specific siRNA, but not by control siRNA, suggesting a dependency on DOCK1 for Wnt5a-enhanced MCF7-ROR1 cells colony formation capability. Moreover, such effects of Wnt5a to enhance colony formation ability of MCF7-ROR1 cells could be inhibit by cirmtuzumab. We also introduced single amino-acid substitutions of proline (P) to alanine (A) in the ROR1 proline-rich-domain (PRD) at positions 784, 808, 826, or 841 in potential SH3-binding sites. In contrast to wild-type ROR1, or other ROR1P⇒A mutants, ROR1P(808)A was unable to recruit DOCK1 to ROR1, or activate Rac1 in response to Wnt5a. Next we examined the tumor-growth potential of MCF7-Ctrl, MCF7-ROR1, or MCF7-P808A cells. The cells (5 × 104 cells/inoculate in RPMI 1640 together with an equal volume of Matrigel) were injected subcutaneously into the second right mammary pad of 4-to 6-week-old Rag2−/−γc−/- mice pretreated with estrogen pellets (17β-estradiol), which were placed subcutaneously in the intrascapular region one week before the cells inoculation. We found that MCF7-ROR1 cells (n=6) had significantly higher tumor growth relative to MCF7 cells (n=8), which had the same tumor growth as that of MCF7-P808A cells (n=6). Moreover, MCF7-ROR1P808A tumors had less activation of Rac1 compared to MCF7-ROR1 cells expressing wild-type ROR1. This study reveals that the recruitment of DOCK1 to ROR1 may be critical for the capacity of Wnt5a to enhance breast cancer tumor growth.

Citation Format: Md Kamrul Hasan, George F. Widhopf II, Suping Zhang, Sharon M. Lam, Barbara A. Parker, Thomas J. Kipps. Wnt5a induces DOCK1 to complex with ROR1 and activate Rac1 leading to enhanced growth of breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3687.