Hypoxia-Induced Autophagy Promotes EGFR Loss in Specific Cell Contexts, Which Leads to Cell Death and Enhanced Radiosensitivity

Abstract

Treatment failure through radio resistance of tumors is associated with activation of the epidermal growth factor receptor (EGFR). Tumor cell proliferation, DNA-repair, hypoxia and metastases-formation are four mechanisms in which EGFR signalling has an important role. However, the effect of hypoxia on EGFR expression is still controversial. In this study, we evaluated the in vitro and in vivo effects of hypoxia on EGFR and its mechanism, as well as its relationship with radiosensitivity. Treatment failure through radio resistance of tumors is associated with activation of the epidermal growth factor receptor (EGFR). Tumor cell proliferation, DNA-repair, hypoxia and metastases-formation are four mechanisms in which EGFR signalling has an important role. However, the effect of hypoxia on EGFR expression is still controversial. In this study, we evaluated the in vitro and in vivo effects of hypoxia on EGFR and its mechanism, as well as its relationship with radio sensitivity. In a panel of cancer cells with low or high cell density under hypoxia, viable cell counting assay was used to calculate the percentage of cell death. Western blot was used to detect the levels of proteins involved in EGFR signaling and autophagy. The localization of EGFR and autophagy-related molecules was observed by confocal microscopy. The xenograft model in nude mice was used to elucidate the in vivo correlation of hypoxia with EGFR and p62. Colony formation assay was performed to assess the radiosensitivity. In this study, we demonstrated that hypoxia enhanced EGFR expression and sustained cell survival in SiHa, CAL 27 and A549 cells at both low and high cell densities, while in MCF-7, MDA-MB-231 and HeLa cells, EGFR and cell survival were regulated by hypoxic treatment in a cell-density dependent manner: upregulated at low cell density and down regulated at high cell density. In MCF-7 and HeLa xenografts in nude mice, EGFR expression varied inversely with the pimonidazole level that was used as an indicator of hypoxia, accordant with the effect of hypoxia at high cell density in vitro. Hypoxia induced more remarkable cell autophagy at high cell density than at low cell density. Autophagy inhibitor 3MA, rather than proteasome inhibitor MG132 inhibited hypoxia-mediated EGFR loss and shifted cell death to cell survival in HeLa cells. The MCF7 cells’ sensitivity to ionizing radiation (IR) under hypoxia was also conditional on the cell densities when the hypoxia treatment was introduced, inversely associated with the expression levels of EGFR. Hypoxia can decrease EGFR expression in some cell lines by enhancing autophagy at high cell density, leading to cell death and hypersensitivity to radiotherapy. This study may help understand how hypoxia influences EGFR expression and radiosensitivity.