Role of the Fanconi anaemia pathway in mediating response to chemotherapy of high grade serous ovarian cancer

Abstract

High grade serous ovarian cancer (HGSOC) is the most common type of ovarian cancer and carries a poor prognosis for patients, with a 5 year survival rate of just 40%. This is largely due to the development of resistance to platinum based chemotherapy. While HGSOCs are initially highly sensitive to platinum chemotherapy, most tumours will recur with increasing levels of resistance, until they stop responding altogether. Platinum causes cell death by inducing interstrand crosslinks (ICLs) in DNA, which are repaired by the Fanconi anaemia (FA) pathway. This project aims to investigate the significance of alterations in the FA pathway in platinum resistance and identify further functions of FA pathway proteins in HGSOC.

Excluding BRCA1 and BRCA2, which are mutated in 20% of cases, mutations in FA genes are rare in HGSOC. However, bioinformatic analysis showed that expression of FA genes was highly varied, with genes showing different profiles of high and low expression. A panel of proteins was selected for further investigation in HGSOC cell lines, based on expression data and a literature review. Analysis of paired PEO1 and PEO4 cell lines generated before and after the development of chemoresistance in a HGSOC patient showed that FANCD2 expression was increased in the resistant PEO4 line. Expression of RFWD3 was highly varied in a panel of cell lines, and protein appeared deficient in a highly platinum sensitive cell line. Further assessment of these candidates was therefore carried out to determine whether they may mediate chemoresistance in HGSOC.

Knockdown of RFWD3 was shown to enhance platinum sensitivity of HGSOC cell lines. Using CRISPR-cas9 editing, cell line models with reduced expression of RFWD3 were produced. These were shown to be hypersensitive to carboplatin and mitomycin C, but no change in sensitivity to FA pathway inhibitors or drugs associated with synthetic lethality in FA deficient cells was observed. RFWD3 deficiency did however lead to alterations in cell cycle dynamics, and reduced cell migration and proliferation, indicating that RFWD3 may have alternative functions outside its canonical role in ICL repair which promote a malignant tumour phenotype. Low expression of RFWD3 was also associated with higher mutational burden and thus genomic instability. Analysis of tumours from HGSOC patients showed that, consistent with cell line observations, RFWD3 expression between tumours was highly variable. Low expression of RFWD3 was associated with complete response to platinum chemotherapy. Unexpectedly, high RFWD3 expression was associated with longer progression free and overall survival. This may be a result of the conflicting roles of RFWD3 in chemoresistance, malignancy and regulating genomic stability.

FANCD2 knockdown was shown to increase the sensitivity of PEO4 cells to carboplatin and mitomycin C, but the PEO1 cell line was unaffected. The increased FANCD2 expression in the resistant cell line was found to occur as a result of regulation by mTOR, with DNA methylation also playing an undefined role. Despite identifying numerous changes in the response of PEO1 and PEO4 to FANCD2 inhibitors and drugs associated with synthetic lethality in FA deficient cell lines, none of these were mediated by differences in FANCD2 expression. This may imply that FANCD2 expression had less impact on drug response than other chemoresistance mechanisms in these cell lines. Further to its role in ICL repair, loss of FANCD2 expression was also associated with enhanced cell migration. While FANCD2 expression was observed to vary in HGSOC patient tumours, this was not associated with chemotherapy response or survival.

Expression of both RFWD3 and FANCD2 have been demonstrated to play roles in mediating platinum resistance and malignancy of HGSOC. While the FA pathway is often considered as a single entity, it is important to consider the different functions and impacts of individual proteins. Due to the variation and complexity of the roles that the FA pathway fulfils in HGSOC cells, changes in chemosensitivity do not necessarily translate to changes in survival, and further work should be carried out to characterise functions of the FA pathway members. With further study, FANCD2, RFWD3 and other members of the FA pathway may have utility as biomarkers of platinum resistance.