Abstract 5505: Combination nitazoxanide and auranofin treatment has synergistic anticancer activity in anaplastic thyroid cancer and act by enhanced activation of multiple cell death pathways
Authors: Ghosh Chandrayee , Gunda Viswanath , Hu Jiangnan , Zhang Lisa , Zhang Ya-Qin , Shen Min , Kebebew Electron 
Published: June 2023

Abstract
Purpose: Anaplastic thyroid cancer (ATC) is one of the most aggressive human cancers, with a median survival time of 6 months and with no current curative treatment. We have used quantitative high-throughput screening (qHTS) of clinically approved or investigational agents to identify candidate compounds for ATC therapy. As monotherapy for most cancers is not effective, we used combination drug matrix screening of highly active compounds from our single agent qHTS to identify compound combinations that may show synergistic effect. In this study, we evaluated the anticancer activity of nitazoxanide and auranofin combination treatment and its mechanism of action and biomarkers of treatment response in in vitro and in vivo models of ATC.
Experimental Design: We used four (8505C, C643, SW1736, THJ16T) ATC cell lines in in vitro and in vivo ATC models to evaluate the safety and efficacy of this combination treatment compared to single agent and vehicle control.
Results: Combination nitazoxanide and auranofin treatment synergistically inhibited cellular proliferation (p&lt;0.001) and inhibited colony formation and migration (p&lt;0.001) in ATC cell lines. We observed cellular oxidative stress with significant increase in ROS levels in the combination treatment group (p&lt;0.001). Nitazoxanide treatment alone and in combination with auranofin caused G1/G0 arrest in cell cycle, induced autophagy (increased LC3BII expression) and later apoptosis. Auranofin treatment alone and in combination with nitazoxanide induced ferroptosis (decreased GPX4 expression and intracellular GSH/GSSG ratios). RNA-Seq analysis, to identify biomarkers of response, showed &gt;7-fold increase in HMOX-1 (heme oxygenase 1) with combination treatment which was validated by western blot. Combination treatment significantly inhibited tumor growth as compared to single agents (p&lt;0.01) and had no significant treatment-related toxicity in vivo. Analysis of tumor samples showed significantly increased LC3BII expression by immunohistochemistry and reduced GPX4 mRNA expression (p&lt;0.0001) with combination treatment compared to control in vivo.
Conclusions: Combination nitazoxanide and auranofin treatment has synergistic anticancer activity in both in vitro and in vivo ATC models. The synergistic activity of the combination is due to enhanced G1/G0 arrest, greater activation of autophagy, apoptosis and ferroptosis than single drug treatment. LC3BII, HMOX-1 and GPX4 levels could be useful biomarkers of treatment response to combination nitazoxanide and auranofin.
Citation Format: Chandrayee Ghosh, Viswanath Gunda, Jiangnan Hu, Lisa Zhang, Ya-Qin Zhang, Min Shen, Electron Kebebew. Combination nitazoxanide and auranofin treatment has synergistic anticancer activity in anaplastic thyroid cancer and act by enhanced activation of multiple cell death pathways. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5505.