Combining Mitomycin C with inhibition of BAD phosphorylation enhances apoptotic cell death in advanced cervical cancer

Abstract

Objective: Mitomycin C (MMC), a DNA-damaging chemotherapeutic, is commonly used clinically for recurrent cervical carcinoma (CC), either alone or in combination. MMC generates DNA damage resulting in CC cell death yet also induces increased AKT-BAD phosphorylation associated with drug resistance and reduced clinical benefit. The present study evaluates the efficacy of combined MMC and a BAD phosphorylation inhibitor in CC. Methods: The association and function of phosphorylation of BAD on serine 99 (pBADS99) for cell survival of both MMC-resistant or sensitive-CC cells was explored. BAD was mutated to BADS99A to examine the requirement of BADS99 for CC cell survival and a novel small-molecule inhibitor of pBADS99 was utilized. Cell proliferation, survival, foci formation, and patient-derived organoids (PDOs) assays were utilized to determine efficacy, synergy and related mechanisms. Results: MMC IC50 was positively correlated to the cell line pBADS99/BAD ratio. Increased BADS99 phosphorylation was observed in both MMC-sensitive or -resistant CC cells after MMC treatment. Inhibition of pBADS99 in CC cell lines produced synergistic apoptosis through BAD-mediated apoptotic pathways and enhanced DNA damage in response to MMC. The concurrent use of pharmacological inhibition of pBADS99 and MMC was synergistic, resulting in diminished cell viability and inducing apoptotic cell death in MMC-sensitive and -resistant CC cell lines or patient-derived organoids. Conclusion: A combination of MMC with inhibition of BAD phosphorylation potentiated efficacy compared to single agent treatment. The potential further development of such strategies may provide outcome benefits to patients with CC.

Keywords

• BAD phosphorylation
• Mitomycin C
• Apoptosis
• cervical cancer
• N-cyclopentyl-3-((4-(2,3-dichlorophenyl) piperazin-1-yl) (2-hydroxyphenyl) methyl) benzamide (NPB)