Hypoxia modulates A431 cellular pathways association to tumor radioresistance and enhanced migration revealed by comprehensive proteomic and functional studies

Abstract

Tumor hypoxia induces cancer cell angiogenesis, invasiveness, treatment resistance, and contributes to poor clinical outcome. However, the molecular mechanism by which tumor hypoxia exerts a coordinated effect on different molecular pathways to enhance tumor growth and survival and lead to poor clinical outcome is not fully understood. In this study, we attempt to elucidate the global protein expression and functional changes in A431 epithelial carcinoma cells induced by hypoxia and reoxygenation using iTRAQ quantitative proteomics and biochemical functional assays. Quantitative proteomics results showed that 4316 proteins were quantified with FDR<1%, in which over 1200 proteins were modulated >1.2 fold, and DNA repair, glycolysis, integrin, glycoprotein turnover, and STAT1 pathways were perturbed by hypoxia and reoxygenation-induced oxidative stress. For the first time, hypoxia was shown to up-regulate the nonhomologous end-joining pathway, which plays a central role in DNA repair of irradiated cells, thereby potentially contributing to the radioresistance of hypoxic A431 cells. The up-regulation of Ku70/Ku80 dimer, a key molecular complex in the nonhomologous end-joining pathway, was confirmed by Western blot and liquid chromatography/tandem mass spectrometry-MRM methods. Functional studies confirmed that up-regulation of glycolysis, integrin, glycoprotein synthesis, and down-regulation of STAT1 pathways during hypoxia enhanced metastastic activity of A431 cells. Migration of A431 cells was dramatically repressed by glycolysis inhibitor (2-Deoxy-d-glucose), glycoprotein synthesis inhibitor (1-Deoxynojirimycin Hydrochloride), and STAT1α overexpression that enhanced the integrin-mediated cell adhesion. These results revealed that hypoxia induced several biological processes involved in tumor migration and radioresistance and provided potential new targets for tumor therapy.