Disulfiram/Cu Kills and Sensitizes BRAF-Mutant Thyroid Cancer Cells to BRAF Kinase Inhibitor by ROS-Dependently Relieving Feedback Activation of MAPK/ERK and PI3K/AKT Pathways

Abstract

BRAFV600E, the most common genetic alteration, has become a major therapeutic target in thyroid cancer. Vemurafenib (PLX4032), a specific inhibitor of BRAFV600E kinase, exhibits antitumor activity in patients with BRAFV600E-mutated thyroid cancer. However, the clinical benefit of PLX4032 is often limited by short-term response and acquired resistance via heterogeneous feedback mechanisms. Disulfiram (DSF), an alcohol-aversion drug, shows potent antitumor efficacy in a copper (Cu)-dependent way. However, its antitumor activity in thyroid cancer and its effect on cellular response to BRAF kinase inhibitors remain unclear. Antitumor effects of DSF/Cu on BRAFV600E-mutated thyroid cancer cells and its effect on the response of these cells to BRAF kinase inhibitor PLX4032 were systematically assessed by a series of in vitro and in vivo functional experiments. The molecular mechanism underlying the sensitizing effect of DSF/Cu on PLX4032 was explored by Western blot and flow cytometry assays. DSF/Cu exhibited stronger inhibitory effects on the proliferation and colony formation of BRAFV600E-mutated thyroid cancer cells than DSF treatment alone. Further studies revealed that DSF/Cu killed thyroid cancer cells by ROS-dependent suppression of MAPK/ERK and PI3K/AKT signaling pathways. Our data also showed that DSF/Cu strikingly increased the response of BRAFV600E-mutated thyroid cancer cells to PLX4032. Mechanistically, DSF/Cu sensitizes BRAF-mutant thyroid cancer cells to PLX4032 by inhibiting HER3 and AKT in an ROS-dependent way and subsequently relieving feedback activation of MAPK/ERK and PI3K/AKT pathways. This study not only implies potential clinical use of DSF/Cu in cancer therapy but also provides a new therapeutic strategy for BRAFV600E-mutated thyroid cancers.