Cross-activating c-Met/β1 integrin complex drives PNAS PLUS metastasis and invasive resistance in cancer Arman Jahangiria, Alan Nguyena, Ankush Chandraa,1, Maxim K. Sidorova,1, Garima Yagnika, Jonathan Ricka, Sung Won Hana, William Chena, Patrick M. Flanigana, Dina Schneidman-Duhovnyb,c, Smita Mascharaka, Michael De Laya, Brandon Imbera, Catherine C. Parkd, Kunio Matsumotoe, Kan Lua, Gabriele Bergersa, Andrej Salib,c,f, William A. Weissg, and Manish K. Aghia,2 aDepartment of Neurosurgery and Brain Tumor Research Center, University of California, San Francisco, CA 94143; bDepartment of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94143; cDepartment of Pharmaceutical Chemistry, University of California, San Francisco , CA 94143; dDepartment of Radiation Oncology and Breast Program, University of California, San Francisco, CA 94143; eCancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-8641, Japan; fCalifornia Institute for Quantitative Biosciences (QB3), University of California, San Francisco, CA 94720; and gDepartment of Neurology, University of California, San Francisco, CA 94143 Edited by Mina J. Bissell, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA, and approved September 1, 2017 (received for review February 3, 2017) The molecular underpinnings of invasion, a hallmark of cancer, have uncertain. Here, we demonstrate that c-Met and β1, not α5, integrin in- been defined in terms of individual mediators but crucial interactions teract physically and functionally during two invasive cancer processes: between these mediators remain undefined. In xenograft models and metastases, the cause of 90% of cancer deaths (3), and glioblastoma patient specimens, we identified a c-Met/β1 integrin complex that (GBM) resistance to VEGF neutralizing antibody bevacizumab, a com- formed during significant invasive oncologic processes: breast cancer mon occurrence after short-lived response to this agent (4–9). metastases and glioblastoma invasive resistance to antiangiogenic VEGF neutralizing antibody, bevacizumab. Inducing c-Met/β1complex Results formation through an engineered inducible heterodimerization system Physical Interaction Between c-Met and β1 Integrin Increases During promoted features crucial to overcoming stressors during metastases Metastases. To determine which integrins interacted with c-Met in or antiangiogenic therapy: migration in the primary site, survival under breast cancer cells, we performed a Western blot in cultured HCC1143, hypoxia, and extravasation out of circulation. c-Met/β1 complex forma- HCC3153, and MDA-MB-231 human breast adenocarcinoma cells, tion was up-regulated by hypoxia, while VEGF binding VEGFR2 seques- which revealed that c-Met was expressed in all three lines, while, of a MEDICAL SCIENCES tered c-Met and β1 integrin, preventing their binding. Complex panel of screened integrins, αV, α3, α5, β1, β3, and β5 integrins were formation promoted ligand-independent receptor activation, with expressed in all three lines (SI Appendix,Figs.S1andS2). Immuno- integrin-linked kinase phosphorylating c-Met and crystallography re- precipitation then revealed that, of these six integrins, β1 integrin was β β vealing the c-Met/ 1 complex to maintain the high-affinity 1integrin the most oncologically pertinent one (5, 10) that bound c-Met in all conformation. Site-directed mutagenesis verified the necessity for three lines (Fig. 1A and SI Appendix,Fig.S3). To determine if this β c-Met/ 1 binding of amino acids predicted by crystallography to medi- c-Met/β1 integrin interaction increased during metastases, we implan- ate their extracellular interaction. Far-Western blotting and sequential ted MDA-MB-231 cells into the mammary fat pads of immunodeficient immunoprecipitation revealed that c-Met displaced α5 integrin from n = β mice ( 5), after which spontaneous metastases arose in one mouse. 1 integrin, creating a complex with much greater affinity for fibronec- Using proximity ligation assays (PLAs), we detected c-Met/β1integrin tin (FN) than α5β1. Thus, tumor cells adapt to microenvironmental complex formation at the invasive front in all four primary tumors, stressors induced by metastases or bevacizumab by coopting receptors, which normally promote both cell migration modes: chemotaxis, move- ment toward concentrations of environmental chemoattractants, and Significance haptotaxis, movement controlled by the relative strengths of periph- eral adhesions. Tumor cells then redirect these receptors away from Invasion is a major cause of cancer mortality, as exemplified by their conventional binding partners, forming a powerful structural metastatic spread of peripheral malignancies or local intracranial c-Met/β1 complex whose ligand-independent cross-activation and ro- invasion of glioblastoma. While individual mediators of invasion bust affinity for FN drive invasive oncologic processes. are identified, functional or structural interactions between these mediators remain undefined. We identified a structural cross- resistance | glioblastoma | angiogenesis | hypoxia | invasion activating c-Met/β1 integrin complex that promotes breast can- cer metastases and invasive resistance of glioblastoma to the antiangiogenic therapy bevacizumab. We show that tumor cells ooperation between integrins and receptor tyrosine kinases adapt to their microenvironmental stressors by usurping c-Met and (RTKs) contributes to migration in normal and cancer cells. C β1 integrin, with c-Met displacing α5 integrin from β1integrinto Previous studies have demonstrated colocalization of RTK c-Met β α β form a c-Met/ 1 complex with far greater fibronectin affinity than with 5or 1 integrin (1, 2) but the mechanisms and biologic α5β1 integrin. These findings challenge conventional thinking consequences of these interactions remain undetermined. Invasion about integrin–ligand interactions and define a molecular target of tumor cells is directed by haptotaxis, cellular locomotion in re- for disrupting metastases or invasive oncologic resistance. sponse to concentration gradients of adhesive molecules in the extracellular matrix (ECM), and chemotaxis, cellular locomotion in Author contributions: A.J. and M.K.A. designed research; A.J., A.N., A.C., M.K.S., G.Y., J.R., response to concentration gradients of diffusible factors. Given the S.W.H., W.C., P.M.F., D.S.-D., S.M., M.D.L., and B.I. performed research; A.J., D.S.-D., C.C.P., K.M., A.S., and M.K.A. contributed new reagents/analytic tools; A.J., A.N., A.C., G.Y., ability of integrins and c-Met to mediate haptotaxis and chemo- D.S.-D., K.M., K.L., G.B., W.A.W., and M.K.A. analyzed data; and M.K.A. wrote the paper. taxis, respectively, these complementary functions suggest that in- Conflict of interest statement: M.K.A. is a member of the Scientific Advisory Board for teraction between c-Met and integrins could have a significant Oncosynergy, Inc. biologic impact on tumor cell migration. This article is a PNAS Direct Submission. α While chemotactic c-Met and haptotactic integrins like 5or 1A.C. and M.K.S. contributed equally to this work. β 1 each contribute individually to tumor cell migration, the mecha- 2To whom correspondence should be addressed. Email: [email protected]. nism through which they interact to regulate migration and the spe- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cific aspects of cancer cell biology that rely on their interaction remain 1073/pnas.1701821114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1701821114 PNAS | Published online September 26, 2017 | E8685–E8694 Downloaded by guest on September 28, 2021 particularly in the primary tumor with gross metastases (Fig. 1B and SI revealed increased human vimentin staining when injecting cells Appendix,Fig.S4). PLA revealed more signal (red dots indicative of pretreated with AP21967 versus those pretreated with vehicle (P = c-Met/β1 integrin complex) in gastric (Fig. 1C and SI Appendix,Fig.S5) 0.02; Fig. 2F and SI Appendix, Fig. S10) and PCR for the FKBP-c- and brain (Fig. 1C and SI Appendix,Fig.S6) metastases relative to the Met fusion sequences revealed no detectable signal in all mice re- primary tumor that metastasized, with quantification revealing more ceiving cells pretreated with vehicle, compared with consistent signal than double the amount of PLA signal in the gastric metastasis (P = in all mice receiving cells pretreated with AP21967 (Fig. 2F). Seven 0.0006) and nearly fivefold more PLA signal in the brain metastasis days after tail vein injection of cells, there was increased lung human (P = 0.01) relative to the primary tumor (Fig. 1D). Similarly, immu- vimentin staining relative to 2 h when injecting cells pretreated with noprecipitation (IP) of lysates from MDA-MB-231-BR, an MDA-MB- AP21967 compared with no detectable staining when injecting cells 231 derived cell line selected through serial propagation in vivo for its pretreated with vehicle (Fig. 2G and SI Appendix, Fig. S11). PCR at propensity for brain metastases (11), exhibited greater c-Met/β1integrin this 7-d time point revealed dramatic increase in the FKBP-c-Met complex in culture than MDA-MB-231 cells (Fig. 1E), suggesting du- fusion sequence transcript in all mice 7 d after receiving cells pre- rability of the complex within the metastatic phenotype. treated with AP21967 while only two of five mice receiving cells pretreated with vehicle had detectable transcript at this time point The c-Met/β1 Complex Promotes Breast