LAUSR

Introduction Overexpression of epidermal growth factor (EGFR) drives glioblastoma (GBM) cell invasion and tumour progression. EGFR has been a major therapeutic target in GBM. However, clinical trials were disappointing, and we are still missing molecular basis to explain these poor results. In other solid tumours, extracellular matrix proteins found in the tumour microenvironment and their heterodimeric integrin receptors trigger resistance to EGFR targeted therapy. We recently established that the fibronectin receptor, α5β1 integrin is a pertinent therapeutic target in GBM. Therefore, we seek to evaluate whether α5β1 integrin is involved in therapy targeting EGFR in GBM. Material and methods We used GBM cell line (U87) overexpressing or down expressing α5 integrin subunit. EGFR was inhibited by clinically approved tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, lapatinib). For cell motility assays, we performed spheroid dissemination assays, in 2D and 3D environment and chemotaxis assays through Boyden chambers. We also evaluated the impact of integrin expression on cell clonogenicity and cell grow th (2D, spheroids) in presence of EGFR inhibitors. Confocal microscopy confirmed integrin, EGFR and endosomes markers localization. Protein colocalization in early-endosomes was confirmed by confocal image analysis and dSTORM super-resolution. Results and discussions We showed that loss of integrin α5 expression increased U87 cell sensitivity to TKIs in spheroid cell evasion assays but did not affect TKIs efficiency on chemotaxis, cell growth (2D or 3D) or cell clonogenicity. EGFR is thus critical for cell dissemination from GBM tumour spheroïds and overexpression of α5 integrin can circumvent EGFR inhibition by TKIs. Using confocal imaging, we showed that TKIs provoked a massive relocalization of integrin and EGFR in Rab5, EEA1 or LRP1 positive endosomes. Dynamic studies confirmed that TKIs increased EGFR receptor endocytosis. By colocalization studies and super-resolution dSTORM imaging, we clearly showed that in intracellular vesicules integrin and EGFR are in close proximity, suggesting a potential interaction. Conclusion We showed in GBM cells that α5β1 integrin expression triggers resistance to EGFR-targeting TKIs and that TKIs alter EGFR and integrin membrane trafficking. These data point out the potential importance of integrin and EGFR endocytosis and trafficking in GBM resistance to TKIs.