The orphan receptor tyrosine kinase Ror1 mediates canonical Wnt signaling and is required for NSCLC cell survival in vitro and in vivo.

4946 Deregulation of the Wnt mediated self-renewal, proliferation and differentiation mechanisms is associated with malignant transformation and tumor growth. Insights into the key regulators of the pathway may help provide novel therapeutic strategies, specifically targeting the self-renewal pathways in cancer. The receptor tyrosine-kinase like orphan receptor-1 (Ror1) plays a crucial role in developmental morphogenesis. Ror1 over-expression in B-CLL patient samples and a number of cancer cell lines prompted an investigation into its biological role in cancer. Using 293T cells stably expressing the SuperTopFlash (STF) reporter system, we identified Ror1 as a mediator of canonical Wnt3a signaling. Although, Ror1 expression did not influence canonical Wnt pathway activation to the same extent as either LRP6 or Dvl3 over-expression, it was observed to be a positive regulator of the pathway, activating the STF reporter 2.5 -7 fold, in a dose-dependent manner. Mutant Ror-1 constructs, lacking the frizzled-like domain or the entire intracellular domain were unable to enhance STF reporter activation to the same extent as the full-length receptor. In addition, co-expression of Ror1 with Dvl3 resulted in a stronger activation of the STF reporter than by either construct alone, demonstrating synergy between the two proteins and canonical Wnt pathway activation. Using Ror1 targeting shRNA we assessed the role of endogenous Ror1 receptors in Wnt3a canonical signaling. Ror1 shRNA inhibited Wnt3a mediated activation of the STF reporter to the same extent as LRP6 shRNA. STF reporter activation directly correlated with the levels of Ror1/LRP6 surface expression, suggesting that Ror1 is as equally important for canonical Wnt signal transduction as LRP6. Effects of stable Ror1 knockdown was then evaluated in A549 and H1299 NSCLC cell lines, expressing high levels of endogenous Ror1. In both A549 and H1299 cells, Ror1 shRNA decreased cell proliferation and induced cell cycle arrest in vitro. An even stronger effect of Ror1 knockdown was observed in vivo, where infection of A549 cells with Ror1 shRNA completely abrogated tumor growth, as compared to non-targeting control shRNA. These data demonstrate Ror1 to be a positive regulator of canonical Wnt signaling and crucial for NSCLC cell survival in vitro and in vivo.