The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transcriptional target of the lineage-survival oncogene NKX2–1/TTF-1 in lung adenocarcinomas. In addition to its kinase-dependent role, ROR1 functions as a scaffold protein to facilitate interaction between caveolin-1 (CAV1) and CAVIN1, and consequently maintains caveolae formation, which in turn sustains pro-survival signaling toward AKT from multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET (proto-oncogene, receptor tyrosine kinase), and IGF-IR (insulin-like growth factor receptor 1). Therefore, ROR1 is an attractive target for overcoming EGFR-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Here, we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer.
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We thank Yukako Shimada and Naoe Hotta for their technical support, and Kiyoshi Yanagisawa for the valuable discussion regarding this study. We are also grateful for the expert assistance from N Tsurumaki for image analysis. This work was supported in part by grants-in-aid for Scientific Research (A) and (C) from the Japan Society for the Promotion of Science (JSPS), the Project for Cancer Research and Therapeutic Evolution (P-CREATE) program of the Japan Agency for Medical Research and Development (AMED), and a grant-in-aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
TY and TT conceived and designed the research. TY, MH, LI, CL and MS performed the biochemical experiments. TY, MH, LI, CL and HI performed the cell experiments. TY and MH performed the molecular biological experiments. TY, MH, CL and MY performed the immunofluorescence microscopy examinations. JC and TF performed the electron microscopy examinations. TY, TF and TT wrote the manuscript.
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Yamaguchi, T., Hayashi, M., Ida, L. et al. ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma. Oncogene 38, 5142–5157 (2019) doi:10.1038/s41388-019-0785-7