Carbonic Anhydrase Inhibitors Induce Ferroptosis through Inhibition of AKT/FTH1 Signaling in Ewing Sarcoma Tumor Cells

SIMPLE SUMMARY: Ewing sarcoma (ES) is one of the most common kinds of cancer in youngsters, and ES relapse after therapy treatment remains a problem. Carbonic anhydrases (CAs; EC 4.2.1.1) have emerged as interesting molecular targets for the development of anticancer medicines as a fundamental regulator of cellular homeostasis. In this investigation, we used the commercially available acetazolamide and previously found CA inhibitors to target the CAII isoform, which was overexpressed and positively linked with relapse in ES patients. We detected that CAII inhibitors can trigger ferroptosis in ES cells by downregulating FTH1 and increasing cathepsin B expression, inhibit cell growth, limit invasion, and trigger apoptosis or autophagy-related cell death. The findings imply that cytosolic CAII may be a promising target for ES therapy, and CAII inhibitors may be useful as single-agent or combination antitumor therapies in the treatment of ES. ABSTRACT: Ewing sarcoma (ES) is one of the most frequent types of malignant tumors among children. The active metabolic state of ES cells presents a new potential target for therapeutic interventions. As a primary regulator of cellular homeostasis, carbonic anhydrases (CAs; EC 4.2.1.1) have emerged as promising molecular targets for the development of anticancer drugs. Within the present study, we tested the commercial drug acetazolamide and our previously discovered inhibitors to target the CAII isoform, which was overexpressed and positively correlated with ES patient relapse. We employed molecular biology tests to identify effective inhibitors of CAII that can induce ferroptosis by downregulating FTH1 expression in ES cells. In vitro, we have also demonstrated their ability to reduce cell proliferation, decrease invasion, and induce apoptosis- or autophagy-related cell death. Using Western blotting, we confirmed the induction of cathepsin B in cells treated with CA inhibitors. It was found that the suppression of cathepsin B expression during the treatment reduces the anticancer efficacy of selected CAII inhibitors. These experiments highlighted profound antitumor activity of CAII inhibitors attributive to their remarkable ability to trigger ferroptosis in Ewing sarcoma cells without causing substantial host damage. The obtained results suggest that cytosolic CAII may be a prospective target for ES treatment, and CAII inhibitors can be considered as potential single-agent or combination antitumor agents to be used in the treatment of ES.