Abstract
Invasive resistance limits anti-angiogenic therapy's efficacy. We previously reported upregulated c-Met and b1 integrin in bevacizumab-resistant glioblastomas. We hypothesized chemotactic c-Met/HGF and haptotactic b1 integrin physically interact, forming an alliance driving invasive growth. Immunoprecipitated lysates of intracranial xenografts of U87-BevR and U87-BevS, models of bevacizumab-resistance and responsiveness, revealed robust c-Met/b1 integrin interactions in bevacizumab-treated U87-BevRs, and minimal complex in bevacizumab-treated U87-BevS, IgG-treated U87-BevR, or U87-BevS. Proximity ligation assays (PLAs) mirrored results of our immunopreicipitation. In culture, complex formation increased dose-dependently by bevacizumab-induced VEGF depletion, exposure to c-Met ligand HGF, and hypoxia. Complex formation was inhibited by VEGF165, the predominant VEGF isoform, and endothelial cell conditioned medium. Far-western blotting demonstrated only extracellular c-Met domain to bind b1 integrin. The c-Met/b1 integrin interaction led to cross-activation, with increasing concentrations of b1 integrin ligand fibronectin which increased c-Met phosphorylation. This was inhibited with integrin-linked kinase (ILK) knockdown. Complex induction via iDimerize, where b1 integrin/c-Met were tagged with complementary proteins, forming a complex upon heterodimer addition, increased migration substantially (P < 0.05). Site-directed biopsy of 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/b1 complexes after bevacizumab resistance in patient-specimens compared to bevacizumab-naive glioblastomas at recurrence (P < 0.05). Furthermore, the percentage of cMet/b1 integrin from 20 patients taken at diagnosis correlated inversely with survival (P < 0.05). Anti-angiogenic therapy dose on complex formation in vivo by treating xenografts with 3 mg/kg bevacizumab was as effective as 10 mg/kg; however, only the latter contained c-Met/b1 complexes. A patient receiving 5mg/kg of bevacizumab, had no increased complex under PLA. In summary, we have demonstrated a mechanism where b1 integrin forms a complex with c-Met in bevacizumab-resistant glioblastoma, leading to allosteric cross-activation, driving the invasive resistant phenotype.
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