Investigation the role of PARP inhibitors in ovarian cancer using in vitro models and clinical samples

Abstract

Ovarian cancer (OC) is the fifth most common cancer in women worldwide with 14.1 million incidences. Approximately 5 out of 10 women with ovarian cancer have BRCA mutations (BRCA-m) or other defects in DNA repair. The current management of BRCAm-associated ovarian cancer is not different from the treatment of the non-BRCA stage-matched cases. However, recent data suggest that these BRCA-m cancers should be treated as a distinct disease entity, since BRCA mutation status has a significant influence on OC patient outcomes. Poly (ADP-ribose) polymerase (PARP) inhibitors effectively can destroy tumour cells with defective BRCA1 or BRCA2 genes through the concept of synthetic lethality. Early results of phase  and  of clinical studies have shown that rucaparib -a new PARP inhibitor-, has an efficacy in BRCA-m OC cells with homologous recombination deficiency (HRD) loss of heterozygosity (LOH), although it might also exert a beneficial effect on non-BRCA-m ovarian cancer. In this work, I evaluate the effects of BRCA2 mutation on homologous recombination (HR) efficiency by using immunofluorescence technique (γ-H2AX assay). Moreover, I report significantly higher levels of γ-H2AX staining in BRCA2-m OC cell lines, compared to control (BRCA2-wt) cell lines which shows that HR pathway can be affected by BRCA2 deficiency. My results indicate the lack of ability to fully repair of DNA damage in cancer cells with BRCA2-mutation. In addition, I show a good connection between the phosphorylation level of γ-H2AX foci and the H2AX gene expression in vitro. I present a complete overview of the alterations at gene and protein level of H2AX in OC and propose its potential as a predictive biomarker. The TCGA databases revealed that H2AX is overexpressed in OC in comparison to controls. In terms of prognostic value, it has been showed that higher expression of H2AX is related to better overall survival (OS), whereas there is no apparent difference on disease free interval (DFI) in the ovarian cancer cohort. Moreover, I prove high level of H2AX expression in different type of OC while there is no correlation between H2AX overexpression and FIGO stage. I also study the effects of Rucaparib in vitro. The BRCA2 mutant ovarian cancer cell line PEO1 exhibited higher PARP1 activity when treated with H2O2 compared to BRCA2 wild type cell lines (PEO4 and SKOV3). The migratory and proliferative capacity of PEO1 cells was compromised following treatment with rucaparib 10 μM compared to BRCA2 wild-type cell lines. These results suggest that ovarian cancer patients with BRCA mutation have the higher likelihood of benefiting from treatment with PARP inhibitors. Finally, I demonstrate that liquid biopsies offer a promising alternative to tissue samples, providing a non-invasive diagnostic approach or aid in serial monitoring of disease evolution. I assess the levels/staining patterns of γ-H2AX and WT1 in a clinical study (CICATRIx) of patients (BRCA-wt and BRCA-m); and analyse these data to identify the clinical validity of this staining with patient outcome. I determine the practicality of γ-H2AX quantification as a predictive biomarker of response in CTCs in OC patients. My results show that overall, the level of CTCs with positive γ-H2AX staining is highest prior to chemotherapy treatment and fell during treatment to the lowest at the end of treatment. Collectively my data suggest that liquid biopsies can be used for monitoring of disease progression in OC patients treated with chemotherapeutic agents and/or PARP inhibitors. Preclinical models developed in this study can also be used in the future to identify effects of novel drugs.