Abstract
BackgroundHypoxia 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.MethodsUsing 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.ResultsWe 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.ConclusionERO1α 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.
Highlights
Hypoxia is an important factor that contributes to tumour aggressiveness and correlates with poor prognosis and resistance to conventional therapy
We have previously reported that the expression of endoplasmic reticulum disulphide oxidase 1α (ERO1α), an oxidase localized in the endoplasmic reticulum that plays a role in the formation of disulphide bonds, is higher in various types of cancer when compared to normal tissues, and that the expression levels of this molecule are associated with cancer progression and prognosis [18,19,20,21]
ERO1α expression is enhanced in hypoxic conditions We focused on the HCT116 and HeLa cell lines, which have low (CA9-low) and high levels (CA9high) of hypoxia-induced carbonic anhydrase 9 (CA9), respectively, and measured the changes in ERO1α and CA9 protein expression in cells grown under normal and low oxygen concentration by western blotting (WB)
Summary
Hypoxia is an important factor that contributes to tumour aggressiveness and correlates with poor prognosis and resistance to conventional therapy. 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. Carbonic anhydrase 9 (CA9) and glucose transporter 1 (GLUT-1) are upregulated under hypoxic conditions through the upregulation and stabilisation of hypoxiainducible factor 1 (HIF-1) and are considered hypoxia markers [6,7,8]. Indicate that the expression of CA9 and GLUT-1 varies in different cancer cell lines [10, 11] Immunohistochemistry for these markers in relation to pimonidazole staining has yielded conflicting results [12, 13]. It is necessary to identify hypoxia biomarkers that are precise and can be used retrospectively, for example, in pre-existing paraffin sections, without the need for the administration of a substrate prior to tumour removal
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
