BRCA1 is a breast and ovarian cancer tumor suppressor protein that associates with BARD1 to form a RING͞RING heterodimer. The BRCA1͞BARD1 RING complex functions as an ubiquitin (Ub) ligase with activity substantially greater than individual BRCA1 or BARD1 subunits. By using NMR spectroscopy and site-directed mutagenesis, we have mapped the binding site on the BRCA1͞BARD1 heterodimer for the Ub-conjugating enzyme UbcH5c. The results demonstrate that UbcH5c binds only to the BRCA1 RING domain and not the BARD1 RING. The binding interface is formed by the first and second Zn 2؉ -loops and central ␣-helix of the BRCA1 RING domain, a region disrupted by cancer-predisposing mutations. Unexpectedly, a second Ub-conjugating enzyme, UbcH7, also interacts with the BRCA1͞BARD1 complex with similar affinity, although it is not active in Ub-ligase activity assays. Thus, binding alone is not sufficient for BRCA1-dependent Ub-ligase activity.

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As histone deacetylase inhibitors (HDACIs) have limited efficacy against solid tumors, we investigated whether and how oxidative stress is involved in sensitivity to HDACIs to develop a novel therapeutic option of HDACIs treatment. We first tested whether a reduction of the antioxidant glutathione (GSH) by glutamine deprivation affects sensitivity to a commercially available HDACI vorinostat and reactive oxygen species (ROS) accumulation. Next we investigated the relationship between a glutamate-cystine transporter xCT and the efficacy of vorinostat using siRNA of xCT and bioinformatic analyses. Finally, we verified the combinatory effects of vorinostat and the xCT inhibitor salazosulfapyridine (SASP) on ROS accumulation, cell death induction, and colony formation. Glutamine deprivation increased vorinostat-mediated cell death with ROS accumulation. Genetic ablation of xCT improved the efficacy of vorinostat, consistent with the results of public data analyses demonstrating that xCT expressions positively correlate with insensitivity to HDACIs in many types of cancer cell lines. Vorinostat caused ROS accumulation when combined with SASP, possibly resulting in synergistic ferroptosis. Our study provides a novel mechanistic insight into the mechanism underlying sensitivity to HDACIs involving xCT, suggesting xCT to be a promising predictive marker of HDACIs and rationalizing combinatory therapy of HDACIs with xCT inhibitors to induce ferroptosis.