Introduction: Invasive resistance limits anti-angiogenic therapy for glioblastoma. We previously reported upregulated c-Met and ß1 integrin in bevacizumab-resistant glioblastomas. We hypothesized that chemotactic c-Met and haptotactic ß1integrin interact in resistant tumors.
Methods: We utilized immunoprecipitation, Proximity ligation assays (PLAs), cell-culture biologic assays, general cloning and immunohistochemistry.
Results: Immunoprecipitated lysates of intracranial U87-BevR and U87-BevS xenografts, isogeneic models of bevacizumab resistance and responsiveness, revealed robust physical c-Met/ß1integrin interactions in bevacizumab-treated U87-BevR, and minimal c-Met/ß1integrin complex in IgG-treated U87-BevR or IgG/bevacizumab-treated U87-BevS. PLAs mirrored immunoprecipitation results. In culture, complex formation increased dose-dependently by bevacizumab, c-Met ligand HGF, and hypoxia. VEGF165 and endothelial cell conditioned medium inhibited complex formation. Using far-western blotting, we found that only the extracellular c-Met domain bound ß1integrin. This c-Met/ß1integrin interaction led to cross-activation, with increasing concentrations of ß1integrin ligand fibronectin increasing ligand-independent c-Met phosphorylation. This activation was inhibited with integrin-linked kinase knockdown. Inducing the complex through the iDimerize system, in which we tagged ß1integrin and c-Met with complementary proteins binding upon heterodimer addition, increased migration (P<0.05). Site-directed biopsy in a bevacizumab-resistant glioblastoma patient revealed increased complex formation progressing from core to enhancing edge to invasive cells outside enhancement. PLAs revealed 20-fold more c-Met/ß1 complexes after bevacizumab resistance in patient specimens compared to a two-fold increase in bevacizumab-naïve glioblastomas at recurrence (P<0.05). Furthermore, the percentage of ß1integrin bound to c-Met in 20 patient glioblastomas at diagnosis correlated inversely with survival (P<0.05). Treating xenografts with 3 mg/kg bevacizumab was as effective as 10mg/kg; however, only the latter contained c-Met/ß1 complexes. Similarly, a patient receiving low dose bevacizumab demonstrated negligible complex by PLA.
Conclusions: ß1integrin forms a complex with c-Met in bevacizumab-resistant glioblastoma leading to allosteric cross-activation driving invasive resistance. Complex formation at high but not low anti-angiogenic therapy doses despite equal efficacy suggests that, for anti-angiogenic therapy, “less is more.”
Patient Care: The described complex formation of c-Met/β1 integrin in our study not only serves as an important biologic marker of resistance to anti-angiogenic therapy, it also serves as a potential target in order to prevent resistance from taking place. These findings can have a substantial impact in improving the efficacy of a therapy that is initially very active in glioblastoma patients.
Learning Objectives: By the end of this presentation, attendees should be able to have a general understanding of: 1) therapeutic resistance in glioblastoma following anti-angiogenic therapy 2) upregulation of c-Met/ß1 complex 3) The mechanism by which this complex drives invasion and proliferation 4) the importance of lower dosing of bevacizumab to prevent the development of resistance