Cross-activating c-Met/β1 integrin complex drives metastasis and invasive resistance in cancer

Abstract

The molecular underpinnings of invasion, a hallmark of cancer, have been defined in terms of individual mediators but crucial interactions between these mediators remain undefined. In xenograft models and patient specimens, we identified a c-Met/β1 integrin complex that formed during significant invasive oncologic processes: breast cancer metastases and glioblastoma invasive resistance to antiangiogenic VEGF neutralizing antibody, bevacizumab. Inducing c-Met/β1 complex formation through an engineered inducible heterodimerization system promoted features crucial to overcoming stressors during metastases or antiangiogenic therapy: migration in the primary site, survival under hypoxia, and extravasation out of circulation. c-Met/β1 complex formation was up-regulated by hypoxia, while VEGF binding VEGFR2 sequestered c-Met and β1 integrin, preventing their binding. Complex formation promoted ligand-independent receptor activation, with integrin-linked kinase phosphorylating c-Met and crystallography revealing the c-Met/β1 complex to maintain the high-affinity β1 integrin conformation. Site-directed mutagenesis verified the necessity for c-Met/β1 binding of amino acids predicted by crystallography to mediate their extracellular interaction. Far-Western blotting and sequential immunoprecipitation revealed that c-Met displaced α5 integrin from β1 integrin, creating a complex with much greater affinity for fibronectin (FN) than α5β1. Thus, tumor cells adapt to microenvironmental stressors induced by metastases or bevacizumab by coopting receptors, which normally promote both cell migration modes: chemotaxis, movement toward concentrations of environmental chemoattractants, and haptotaxis, movement controlled by the relative strengths of peripheral adhesions. Tumor cells then redirect these receptors away from their conventional binding partners, forming a powerful structural c-Met/β1 complex whose ligand-independent cross-activation and robust affinity for FN drive invasive oncologic processes.

Role of c-Met/β1 integrin complex in the metastatic cascade

“…While c-Met and b1 integrin are each known to individually contribute to metastases (23,24), the mechanisms through which these drive metastases or invasive resistance remain uncertain, as their high baseline expression levels do not change tremendously during acquisition of metastases (23,24). We previously addressed this knowledge gap by identifying a structural complex between c-Met and b1 integrin formed at significantly higher levels in metastatic tumors relative to their primary tumors (7). Here, we build upon that observation by determining which steps of the metastatic cascade the c-Met/b1 integrin complex drives and whether the complex promotes organ-specific metastases.…”

“…This finding is consistent with a demonstrated role of b1 integrin in tumor cell adhesion to endothelial cells (25) via binding of tumoral a4b1 integrin to endothelial VLA-4. Because the c-Met/b1 integrin complex promotes ligandindependent conformational changes in b1 integrin that structurally resemble activation (7) it is likely that the c-Met/b1 complex promotes b1 integrin functionality in general, including VLA-4 binding. Interestingly, our demonstration that the c-Met/b1 integrin complex does not promote extravasation suggests distinct mediators of tumor cell trafficking into versus out of circulation.…”

“…Because of the findings of activated stem cell pathways from c-Met/β1 complex formation ( Figure 1E) and because breast cancer stem cells have been shown to be a subset of breast cancer cells with enriched metastatic capacity (12), we then asked whether the ability of the c-Met/β1 complex to drive metastases (7) reflected an ability of the complex to enrich the breast cancer stem cell population. Indeed, AP21967 increased the expression of stem cell genes c-Myc (13), Klf4 (14), Oct4 (13), Sox2 (13), and Nanog (13) in MDA-MB-231-iDimerize-c-Met-β1 cells (P<0.001; Figure 1F; Supplemental Figure 5).…”

“…One potential mechanism of this c-Met/β1 complex-induced intravasation was noted when conditioned media from the breast cancer stem cells enriched by c-Met/β1 complex induction increased intravasation of breast cancer cells in our cell culture assay (P=0.0098; Figure 2D; Supplemental Figure 7). Because we previously showed that VEGF neutralizing antibody bevacizumab treatment increased c-Met/β1 complex formation in glioblastoma cells (7), we then analyzed the effects of bevacizumab on MDA-MB-231 cells. As with glioblastoma cells, bevacizumab increased c-Met/β1 complex formation in MDA-MB-231 cells (Supplemental Figure 8).…”

“…MDA-MB-231 human breast adenocarcinoma cells (ATCC HTB-26) were passaged fewer than six months and verified by providing sources using short tandem repeat (STR) profiling and confirmed to be Mycoplasma free. MDA-MB-231-iDimerize-c-Met-β1 cells containing the Lenti-X iDimerize inducible heterodimer system were created as described previously (7). Complex formation was induced in these cells by treating with AP21967…”