Nuclear PAK4 impairs homologous recombination via RAD51C to promote genome instability in high-grade serous ovarian cancer.

Abstract

p21-activating kinase 4 (PAK4) is a nucleo-cytoplasmic shuttling protein frequently overexpressed in cancer. In particular, nuclear PAK4 (nPAK4) expression correlates with clinical aggressiveness and survival in ovarian cancer, although the mechanism still remains unclear. By analyzing the nPAK4 transcriptome and nPAK4-bound enomic landscape, we previously found that nPAK4 affects transcription of genes involved in DNA damage repair in high-grade serous ovarian cancer (HGSC) cells, and identified RAD51C as a potential direct transcriptional repression target of nPAK4. In this study, we further validated the mode of regulation of RAD51C imposed by nPAK4, and interrogated whether nPAK4 functionally affects DNA repair through RAD51C to disrupt genome stability in HGSC. We demonstrated by ChIP-PCR that nPAK4 occupies the RAD51C promoter region at its consensus DNA binding site in HGSC cells. Overexpression and knockdown studies confirmed nPAK4 negatively regulates RAD51C expression in HGSC cells, whereas nPAK4 expression but not that of cytoplasmic PAK4 or total PAK4, correlates with RAD51C expression in a panel of serous ovarian cancer cell lines. Analysis of 316 ovarian serous cystadenocarcinoma samples from TCGA showed that gene alterations in PAK4 and RAD51C were mutually exclusive, suggesting a potential coregulatory role of PAK4/RAD51C in HGSC. Further investigation on nPAK4-mediated DNA repair activity revealed nPAK4 significantly reduced homologous recombination competence in a DR-GFP reporter assay, he effect of which is in part contributed by RAD51C. Concurrently, we found a significant elevation in the number of micronuclei formed in PAK4 overexpressing SKOV3 compared to control SKOV3 cells. In addition, PAK4 overexpression exacerbates the formation of aberrant chromosomal structures upon exposure to the chemotherapy drug cisplatin. Correlation studies of TCGA data from ovarian serous cystadenocarcinoma patients revealed that genome-wide copy number aberrations are positively correlated with PAK4 expression. Taken together, we found that HGSC nPAK4 is a novel negative regulator of RAD51C. The impact of nPAK4-mediated RAD51C downregulation contributes to genome instability in HGSC by functionally disrupting homologous recombination.