Rad6 inhibition sensitizes ovarian cancer cells to platinum drugs by attenuating activation of multiple DNA repair networks

Abstract

Objectives: Ovarian cancer (OC) platinum resistance is often attributable to an increase in DNA repair. Rad6 is an E2 ubiquitin–conjugating enzyme that plays a central role in the activation of several DNA repair pathways. Rad6, in conjunction with its E3 partner Rad18, is vital for the activation of trans-lesion synthesis (TLS) DNA damage tolerance by monoubiquitinating PCNA; and the activation of the Fanconi anemia (FA) DNA repair pathway critical for DNA crosslink repair. We aimed to determine if inhibition of Rad6 could attenuate the DNA damage repair signaling induced by platinum drugs, and thereby overcome OC platinum resistance. Methods: OC cell lines were used to evaluate Rad6-mediated ubiquitin signaling. Both genetic and pharmacologic inhibitors of Rad6 were used and compared with controls in cancer growth and clonogenic assays. Responsiveness to platinum chemotherapy was tested in combination with Rad6 inhibitor. Standard Western blotting techniques were used to measure protein levels with or without Rad6 inhibition. Immunostaining was used to visualize DNA damage foci formation. Results: Rad6B was overexpressed in OC with increasing intensity by immunohistochemistry with increasing stage. Inhibition of Rad6 was effective in blocking DNA repair pathway mediators such as FANCD2, PCNA, yH2AX. Treatment with siRNAs or a small molecule inhibitor led to growth inhibition and attenuated clonogenic potential of OC cells. In addition, OC cells were sensitized to carboplatin by both genetic and pharmacological inhibition of Rad6. Genetic knockdown of Rad6 resulted in a threefold reduction in the IC50 value of carboplatin, and the IC50 value for carboplatin was reduced fivefold after treatment with a small molecule inhibitor. Moreover, Rad6 inhibition potentiated DNA damage induced by platinum drugs and sensitized platinum-resistant OC cells to carboplatin. Immunostaining confirmed that Rad6 inhibition attenuated FANCD2 foci formation, indicating reduced DNA repair. Conclusions: Rad6 inhibition reduced OC cell growth, but also consistently blocked carboplatin-induced PCNA monoubiquitination and FA pathway activation. This resulted in a sustained increase in DNA damage and an increase in cancer cell death. Together, our data indicate that inhibition of Rad6 holds therapeutic potential for the treatment of OC alone or in combination therapy.