Molecular studies on EGFR and PAK4 in ovarian carcinogenesis

Abstract

Ovarian cancer causes significant morbidity and mortality worldwide yet targeted therapy options are limited. p21-activated kinase 4 (PAK4) and epidermal growth factor receptor (EGFR) are frequently overexpressed in ovarian cancer. PAK4 has been demonstrated to transactivate EGFR and co-regulate ovarian cancer cell proliferation. However, their reciprocal interaction and regulation in other tumorigenic events are still unclear. Delineating the functional dynamics between EGFR and PAK4 may provide novel therapeutic options in ovarian cancer. Herein, we analysed the expression profiles of EGFR and PAK4 in ovarian tumour tissues. Co-expression of high nuclear EGFR and nuclear PAK4 protein was associated with worse survival outcome, advanced disease and serous histology. At the molecular level, we demonstrated PAK4 as a downstream effector of EGFR signalling to control cell migration, chemotaxis and generation of reactive oxidative species. EGF stimulation promoted PAK4 phosphorylation and nuclear trafficking through JNK and PI3K pathways. PAK4 physically interacted with EGFR in ovarian carcinoma cells but not normal ovarian surface epithelial cells. We mapped the PAK4 kinase domain as the necessary region for such interaction and uncovered a novel tyrosine phosphorylation event on PAK4 through a functional EGFR kinase domain. We further interrogated the functional significance of nuclear PAK4, as EGFR activation promoted nuclear PAK4 accumulation specifically in ovarian cancer cells but not in normal ovarian surface epithelial cells. Chromatin immunoprecipitation followed by deep sequencing delineated the PAK4 genomic landscape in OVCAR3 and validated PAK4 to bind DNA specifically at the ‘GCTGGGAYTACAGG’ consensus motif. We further revealed a novel dimerization configuration of PAK4 through its leucine zipper domain in ovarian cancer cells. The existence of PAK4 dimer implies that PAK4 may bind DNA in a similar fashion as other leucine zipper-containing transcription factors. The PAK4 transcriptome in ovarian cancer was characterized by microarray. Gene ontology and pathway enrichment analysis identified significant mRNA deregulation of genes with known functions in DNA damage repair processes, hinting a mechanism by which PAK4 mediates chemoresistance commonly found in ovarian cancer. Functional testing by in vitro experiments demonstrated PAK4 protected ovarian cancer cells from forming cisplatin-DNA adducts and DNA double-strand breaks, confirming a novel PAK4 role in DNA damage repair. Among the many PAK4 deregulated DNA-repair related genes, we detected PAK4 enrichment in the promoter region of RAD51C. Repetitive occurrence of the validated PAK4 motif ‘GCTGGGAYTACAGG’ was found in RAD51C promoter, implying PAK4 might impose direct transcriptional regulation on RAD51C. Co-targeting EGFR and PAK4 with gefitinib and PF-3758309 suppressed ovarian tumour growth synergistically in vitro, while similar additive effect of the two drugs on tumour growth inhibition was observed in a tumour xenograft model with minimal side effect. This study revealed a novel, intimate reciprocal interaction between EGFR and PAK4 crucial for promoting multiple tumourigenic hallmarks in ovarian cancer. This is the first study characterizing the DNA binding ability of nuclear PAK4 and unveiling a novel PAK4 function on DNA damage repair. Our findings suggested that combinatorial targeting of EGFR-PAK4 might be a promising therapeutic strategy for ovarian cancer.