Carbonic Anhydrase 9 Confers Resistance to Ferroptosis/Apoptosis in Malignant Mesothelioma Under Hypoxia


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Zan Li1,Li Jiang1,Shinya Toyokuni1
1Nagoya University Graduate School of Medicine

Abstract

Background

Malignant mesothelioma (MM) is an aggressive cancer characterized by iron-addiction. Previous studies showed carbonic anhydrase 9 (CA9) was overexpressed in MM. CA9 is regarded as an endogenous marker for hypoxia and regulates cellular pH in many tumors. However, the function of CA9 in MM is incompletely known. Herein, we aim to elucidate whether CA9 has a role in adaption of MM cells to tumor microenvironment by iron-homeostasis maintenance.

Method

Three human MM and one non-carcinomatous mesothelial cell lines were cultured in normal and hypoxic (1% O2) conditions. The expression of CA9 and iron-associated proteins were assessed by immunoblotting. MTT and wound healing assays were performed to evaluate the anti-tumor effects by CA9 inhibitors (S4 and U104). Intracellular catalytic Fe (II) and lipid peroxidation were detected by corresponding probes (siRhoNox and BODIPY) to validate ferroptosis. AnnexinV and cleaved-caspase3 were examined to detect apoptosis

Results

CA9 overexpression was accompanied by increased catalytic Fe (II) in response to hypoxia. Treatment with CA9 inhibitors decreased viability and migration ability of MM cells while less effect on non-carcinomatous mesothelial cells, Met-5A. We found mitochondrial fission and enhanced autophagy in response to CA9 inhibition in MM cells which was associated with increased catalytic Fe (II) in mitochondria and lysosomes. Consistently, reactive oxygen species and lipid peroxidation were increased. Finally, the cell death resulted from CA9 inhibitors was suppressed by apoptosis inhibitor and iron chelator. Moreover, erastin-induced ferroptosis exhibited decreased expression of CA9 protein in MM cells

Conclusion

Our results indicated CA9 is indispensable to MM cell proliferation and migration. Importantly, we elucidated the biochemical link between CA9 and iron-metabolism in apoptosis and ferroptosis of MM cells. These findings suggest CA9 is a molecular target for MM therapy.