To elucidate whether the impact of hypoxia
To elucidate whether the impact of hypoxia on EGFR expression is cell context specific or culturing density dependent, we chose the following cancer MK-8776 driven by EGFR and evaluated the EGFR expression after hypoxia treatment at different culturing density: a highly aggressive and poorly differentiated triple-negative breast cancer cell line MDA-MB-231, a well differentiated breast adenocarcinoma cell line MCF-7, a lung adenocarcinoma cell line A549, a squamous cervical carcinoma cell line SiHa, a cervical adenocarcinoma cell line HeLa, an oral adenosquamous carcinoma cell line CAL 27. Upregulation of EGFR was found in all examined cells when they were cultured at low cell density, while high cell density resulted in downregulation of EGFR in some of the cell lines. As an important mechanism that can be triggered by hypoxia for EGFR degradation and signaling limitation (So et al., 2014; Xu et al., 2016; Fang et al., 2015), autophagy was examined and proved to be associated with the cell density dependent EGFR loss under hypoxia. Radioresistance mediated by hypoxia was further assessed at different cell densities. This study may help to understand how hypoxia influences EGFR expression and the development of therapeutic methods.
Materials and methods
Discussion Most studies suggested that EGFR is upregulated in hypoxia (Nijkamp et al., 2013; Keulers et al., 2015; Tan et al., 2016). However, Heinz et al. recently established a novel method named multiplex immunofluorescence and single-cell segmentation, and demonstrated that EGFR expression was significantly downregulated with increasing distance from tumor vessels. Furthermore, they found that the phosphorylated ribosomal protein S6, a well-known downstream target of the EGFR-AKT signaling pathway, showed a preferential expression adjacent to blood vessels (Mayer et al., 2016). Kaanders et al. found that EGFR was present mainly in better oxygenated areas (Hoogsteen et al., 2012). Uhlen et al. demonstrated that EGFR expression was attenuated or missing in the distant cell layers of normal oral mucosa (Uhlén et al., 2015). In this study, we found that at low cell density, hypoxia obviously increased EGFR expression in all examined cells; while at high cell density, hypoxia led to EGFR loss in MCF-7, MDA-MB-231 and HeLa cells, but upregulated EGFR expression in A549, SiHa and CAL 27 cells. Based on accumulating evidences, we speculated that the effect of hypoxia on EGFR was correlated with cell density and histopathological patterns, which remains to be explored. Autophagy is an important mechanism for EGFR degradation and signaling limitation. For example, the protein kinase CK2 inhibitor CX-4945 or the herbal plant derivative celastraol induced autophagic degradation of EGFR and consequently inhibited the survival of cancer cells (So et al., 2014; Xu et al., 2016). In the present study, we showed that hypoxia could induce autophagy in HeLa cells at HD to a high degree that might contribute to EGFR degradation. At the same time, PI3K/AKT and MAPK/ERK, two major downstream signaling pathways of EGFR, were obviously downregulated by hypoxia, which might impair the cell survival. We have confirmed that hypoxia enhanced EGFR expression in SiHa, CAL 27 and A549 cells, while in MCF-7, MDA-MB-231 and HeLa cells, EGFR was downregulated at high cell density. As hypoxia-induced autophagy is associated with the downregulation of EGFR, we speculated that different cell types with high cell density may have a distinct autophagic flux during hypoxia, which determines the level of EGFR in response to hypoxia. Hypoxia-mediated EGFR downregulation is of great clinical significance. EGFR overexpression is generally believed to mediate radioresistance, and antibody-mediated EGFR downregulation could lead to radiosensitization (Bonner et al., 2006; Santiago et al., 2010). In our experimental system, hypoxia-mediated EGFR loss at HD promoted the cancer cells’ death, and the remaining cells became more sensitive to radiotherapy compared with cells undergoing hypoxia at LD, where EGFR was upregulated. Based on our data, we hypothesized that further exacerbated hypoxia mimicked by high culturing density might improve radiosensitivity instead of radioresistance, probably through downregulation of EGFR. We will examine additional cell types and conduct clinical trials to further support our conclusion.