ChaC1-based drug screenings identify a synergistic lethal effect of auranofin and proteasome inhibitors in hepatocellular carcinoma cells.

Abstract

The gamma-glutamylcyclotransferase, ChaC1, is an enzyme catalyzing glutathione (GSH) degradation. In this study, we performed ChaC1 activity-based drug screening from FDA-approved drug library to identify GSH-detoxifying drugs, and found that ChaC1 overexpression mediated glutathione depletion largely enhanced the anticancer efficacy of auranofin (AUR) in hepatocellular carcinoma (HCC) cells. AUR treatment in ChaC1 overexpressed cells led to constitutive activation of oxidative stress and induction of endoplasmic reticulum (ER) stress response genes, such as ATF4 and ATF3. Proteomic study showed that co-treatment of AUR and ChaC1 overexpression upregulated a series of cell death promoting genes, such as DEDD2 and DDIT4. To mimic ChaC1 overexpression by inducing endogenous ChaC1 high expression, we performed ChaC1 expression-based drug screening for ChaC1 inducing drugs from the same drug library, and found that proteasome inhibitors (PIs), including Bortezomib (BTZ), Ixazomib (IXZ) and Delanzomib (DLZ), dramatically induced endogenous ChaC1 expression in an ATF4 dependent manner in HCC cells. Furthermore, combinational treatment of AUR and PIs synergistically led to cell death, which could be inhibited by N-Acetyl-L-cysteine (NAC) and cycloheximide (CHX), but not Z-VAD-FMK (zVAD), necrostatin-1 (Nec-1), ferrostatin-1 (Fer-1) or chloroquine (CQ). Induction of DEDD2 and DDIT4 was also observed in PIs and AUR co-treated cells. Finally, blockade of the ATF4-ChaC1 pathway suppressed cell death and upregulation of DEDD2 and DITT4 in response to PIs and AUR co-treatment in HCC cells. Together, our study identified a synergistic lethal effect of PIs and AUR in HCC cells through ChaC1-based drug screenings, suggesting that combination of PIs and AUR might be repurposed for HCC treatment.