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Although tetraarsenic hexoxide is known to exert an anti-tumor effect by inducing apoptosis in various cancer cells, its effect on other forms of regulated cell death remains unclear. Here, we show that tetraarsenic hexoxide induces the pyroptotic cell death through activation of mitochondrial reactive oxygen species (ROS)-mediated caspase-3/gasdermin E (GSDME) pathway, thereby suppressing tumor growth and metastasis of triple-negative breast cancer (TNBC) cells. Interestingly, tetraarsenic hexoxide-treated TNBC cells exhibited specific pyroptotic characteristics, including cell swelling, balloon-like bubbling, and LDH releases through pore formation in the plasma membrane, eventually suppressing tumor formation and lung metastasis of TNBC cells. Mechanistically, tetraarsenic hexoxide markedly enhanced the production of mitochondrial ROS by inhibiting phosphorylation of mitochondrial STAT3, subsequently inducing caspase-3-dependent cleavage of GSDME, which consequently promoted pyroptotic cell death in TNBC cells. Collectively, our findings highlight tetraarsenic hexoxide-induced pyroptosis as a new therapeutic strategy that may inhibit cancer progression of TNBC cells.

Purpose: To investigate the anti-cancer effects of tetraarsenic hexoxide (TAO, As4O6) in cervical cancer cell lines and in a series of patient-derived xenograft (PDX) mouse models. Methods: Human cervical cancer cell lines, including HeLa, SiHa and CaSki, and human umbilical vein endothelial cells (HUVECs), were used to evaluate the anti-cancer activity of TAO. Cellular proliferation, apoptosis, and enzyme-linked immunosorbent assay (ELISA) for matrix metallopeptidase 2 (MMP-2) and 9 (MMP-9) were assessed. The tumor weights of the PDXs that were given TAO were measured. The PDXs included primary squamous cell carcinoma, primary adenocarcinoma, recurrent squamous cell carcinoma, and recurrent adenocarcinoma. Results: TAO significantly decreased cellular proliferation and increased apoptosis in cervical cancer cell lines and HUVEC. The functional studies on the cytotoxicity of TAO revealed that it inhibited the activation of Akt and vascular endothelial growth factor receptor 2 (VEGFR2). It also decreased the concentrations of MMP-2 in both cervical cancer cell lines and HUVECs. Active caspase-3 and p62 were both increased by the treatment of TAO, indicating increased rates of apoptosis and decreased rates of autophagy, respectively. In vivo studies with PDXs revealed that TAO significantly decreased tumor weight for both primary squamous cell carcinoma and adenocarcinoma of the cervix. However, this anti-cancer effect was not seen in PDXs with recurrent cancers. Nevertheless, the combination of TAO with cisplatin significantly decreased tumor weight in PDX models for both primary and recurrent cancers. Conclusions: TAO exerted inhibitory effects on angiogenesis, cellular migration, and autophagy, and it showed stimulatory effects on apoptosis. Overall, it demonstrated anti-cancer effects in animal models for human cervical cancer.

Modified arsenic derivative compounds such as arsenic trioxide (As2O3), arsenic trisulfide (As2S3), and tetraarsenic hexoxide (As4O6) have been known to be one of the potent anti-cytotoxic effects in various cancer cells, including leukemia, glioma, colon, breast, and cervix cancer cells. Particularly, tetraarsenic hexoxide was developed as a chemotherapeutic agent for patients with advanced cancers. Although anti-cancer effect of tetraarsenic hexoxide has been extensively studied in various cancer cells, the molecular basis of its tumor inhibitory activity remains poorly understood. In this study, we suggest a mechanism of tetraarsenic hexoxide induces pyroptosis through the increase of mitochondrial ROS by targeting phosphorylation of mitochondrial STAT3, subsequently activating caspase-3/GSDME that eventually leads to suppression of cancer progression of TNBC cells. Interestingly, we first found that tetraarsenic hexoxide markedly induced pyroptotic cell death through caspase-3 in releading N-terminal fragments of GSDME in TNBC cells. Indeed, tetraarsenic hexoxide-treated TNBC cells exhibited pyroptotic cell death and the release of LDH. Supporting this observation, inhibition of active caspase-3 attenuated tetraarsenic hexoxide-induced pyroptotic features by blocking the cleavage of GSDME. In conclusion, our results suggest a mechanism of tetraarsenic hexoxide by inducing pyroptosis through the increase of mitochondrial ROS by targeting phosphorylation of mitochondrial STAT3, subsequently activating caspase-3/GSDME that eventually leads to suppression of cancer progression of TNBC cells. Also, given that traditional chemotherapeutic agents, tetraarsenic hexoxide may be useful for aggressive TNBC cells showing resistance to apoptosis as a new GSDME-targeted therapeutic strategy. Citation Format: Haein An, Jin Sun Heo, Akira Ooshima, Zhaoyan Wu, Seong-Jin Kim, ILLJU BAE, Kyung-Min Yang. Tetraarsenic hexoxide enhances generation of mitochondrial ROS to promote pyroptosis by inducing the activation of caspase-3/GSDME in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 989.