The disruption of the CCDC6 – PP4 axis induces a BRCAness like phenotype and sensitivity to PARP inhibitors in high-grade serous ovarian carcinoma

Abstract

Abstract Background Treatment with PARP inhibitors (PARPi) is primarily effective against high-grade serous ovarian cancers (HGSOC) with BRCA1/2 mutations or other deficiencies in homologous recombination (HR) repair mechanisms. However, resistance to PARPi frequently develops, mostly as a result of BRCA1/2 reversion mutations. The tumour suppressor CCDC6 is involved in HR repair by regulating the PP4c phosphatase activity on γH2AX. In this work, we reported that in ovarian cancer cells, a physical or functional loss of CCDC6 results synthetic lethal with the PARP-inhibitors drugs, by affecting the HR repair. We also unravelled a role for CCDC6 as predictive marker of PARPi sensitivity in ovarian cancer, and the impact of CCDC6 downregulation in overcoming PARPi resistance in these tumours. Methods A panel of HGSOC cell lines (either BRCA-wild type or mutant) were treated with PARPi after CCDC6 was attenuated by silencing or by inhibiting USP7, a CCDC6-deubiquitinating enzyme, and the effects on cell survival were assessed. At the cellular and molecular levels, the processes underlying the CCDC6-dependent modification of drugs’ sensitivity were examined. Patient-derived xenografts (PDXs) were immunostained for CCDC6, and the expression of the protein was analysed statistically after digital or visual means. Results HGSOC cells acquired PARPi sensitivity after CCDC6 depletion. Notably, CCDC6 downregulation restored the PARPi sensitivity in newly generated or spontaneously resistant cells containing either wild type- or mutant-BRCA2. When in an un-phosphorylated state, the CCDC6 residue threonine 427 is crucial for effective CCDC6-PP4 complex formation and PP4 sequestration, which maintains high γH2AX levels and effective HR. Remarkably, the PP4-dependent control of HR repair is influenced by the CCDC6 constitutively phosphorylated mutant T427D or by the CCDC6 loss, favouring PARPi sensitivity. As a result, the PP4 regulatory component PP4R3α showed to be essential for both the activity of the PP4 complex and the CCDC6 dependent PARPi sensitivity. It's interesting to note that immunohistochemistry revealed an intense CCDC6 protein staining in olaparib-resistant HGSOC cells and PDXs. Conclusions Our findings suggest that the physical loss or the functional impairment of CCDC6 enhances the PP4c complex activity, which causes BRCAness and PARPi sensitivity in HGSOC cells. Moreover, CCDC6 downregulation might overcome PARPi resistance in HGSOCs, thus supporting the potential of targeting CCDC6 by USP7 inhibitors to tackle PARPi resistance.

Keywords

• Biomarker
• PARP inhibitors
• Cisplatin
• Drugs resistance
• PDX
• PP4