ERO1α is a novel endogenous marker of hypoxia in human cancer cell lines.

BACKGROUND: Hypoxia is an important factor that contributes to tumour aggressiveness and correlates with poor prognosis and resistance to conventional therapy. Therefore, identifying hypoxic environments within tumours is extremely useful for understanding cancer biology and developing novel therapeutic strategies. Several studies have suggested that carbonic anhydrase 9 (CA9) is a reliable biomarker of hypoxia and a potential therapeutic target, while pimonidazole has been identified as an exogenous hypoxia marker. However, other studies have suggested that CA9 expression is not directly induced by hypoxia and it is not expressed in all types of tumours. Thus, in this study, we focused on endoplasmic reticulum disulphide oxidase 1α (ERO1α), a protein that localises in the endoplasmic reticulum and is involved in the formation of disulphide bonds in proteins, to determine whether it could serve as a potential tumour-hypoxia biomarker. METHODS: Using quantitative real-time polymerase chain reaction, we analysed the mRNA expression of ERO1α and CA9 in different normal and cancer cell lines. We also determined the protein expression levels of ERO1α and CA9 in these cell lines by western blotting. We then investigated the hypoxia-inducible ERO1α and CA9 expression and localisation in HCT116 and HeLa cells, which express low (CA9-low) and high (CA9-high) levels of CA9, respectively. A comparative analysis was performed using pimonidazole, an exogenous hypoxic marker, as a positive control. The expression and localisation of ERO1α and CA9 in tumour spheres during hypoxia were analysed by a tumour sphere formation assay. Finally, we used a mouse model to investigate the localisation of ERO1α and CA9 in tumour xenografts using several cell lines. RESULTS: We found that ERO1α expression increased under chronic hypoxia. Our results show that ERO1α was hypoxia-induced in all the tested cancer cell lines. Furthermore, in the comparative analysis using CA9 and pimonidazole, ERO1α had a similar localisation to pimonidazole in both CA9-low and CA9-high cell lines. CONCLUSION: ERO1α can serve as a novel endogenous chronic hypoxia marker that is more reliable than CA9 and can be used as a diagnostic biomarker and therapeutic target for cancer.

Endoplasmic reticulum oxidase 1α is critical for collagen secretion from and membrane type 1-matrix metalloproteinase levels in hepatic stellate cells.

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.

Hypoxia-inducible ERO1α promotes cancer progression through modulation of integrin-ß1 modification and signalling in HCT116 colorectal cancer cells.

Endoplasmic reticulum disulphide oxidase 1α (ERO1α) is an oxidase localized in the endoplasmic reticulum that plays a role in the formation of disulphide bonds of secreted and cell-surface proteins. We previously showed that ERO1α is overexpressed in various types of cancer and we further identified ERO1α expression as a novel factor related to poor prognosis in cancer. However, the biological functions of ERO1α in cancer remain unclear. Here, we investigated the cell biological roles of ERO1α in the human colon-cancer cell line HCT116. ERO1α knockout (KO) by using CRISPR/Cas9 resulted in decreased tumourigenicity in vivo and reduced cell proliferation only under hypoxia in vitro, which suggested that ERO1α promotes cancer progression specifically in a low-oxygen environment. Thus, we evaluated the function of ERO1α in cell proliferation under hypoxia, and found that under hypoxic conditions, ERO1α KO resulted in a contact-inhibited morphology and diminished motility of cells. We further showed that ERO1α KO induced a change in integrin-ß1 glycosylation and thus an attenuation of cell-surface integrin-ß1 expression, which resulted in the aforementioned phenotype. Our study has established a previously unrecognized link between ERO1α expression and integrin activation, and thus provides new evidence for the effectiveness of ERO1α-targeted therapy for colorectal carcinoma.