PAK4 mediated mechanisms contributing to the chemoresistance of ovarian cancer

Abstract

Ovarian cancer is one of the most lethal cancers in women worldwide. Surgery and chemotherapy are the major regimens for ovarian cancer patients. In fact, a significant portion of ovarian cancer patients is diagnosed with extra-ovarian dissemination. Relapse of the tumor with development of chemoresistant phenotypes is common. Chemoresistance thus becomes the major obstacle in curing ovarian cancer but the underlying mechanisms have not yet been fully understood. The p21-activated kinases (PAKs) are serine/threonine kinases involved in various cellular functions including cell motility, survival and angiogenesis. PAK4, a member of the PAK family, was previously reported to play a role in chemoresistance and clinical outcome of patients with ovarian cancers. In this study, I aim to delineate the crucial domains in PAK4 that may contribute to the development of chemoresistance in ovarian cancer cells by analysis of various PAK4 mutants. Here, I manipulated the PAK4 constructs to generate different mutants with alteration on different functional domains. MTT assays were conducted to detect the cisplatin resistance among different constructs-expressed ovarian cancer cells. It was found that PAK4 upregulation renders cells more resistant to cisplatin independent of its kinase domain. Moreover, the subcellular localization of PAK4 was investigated to delineate the effect of nuclear PAK4 localization on the chemoresistance of ovarian cancer towards cisplatin. Two PAK4 nuclear localization signals: NLS1 and NLS3 were identified from previous studies and mutations in these regions were performed. Abolishment of nuclear PAK4 in ovarian cancer cells was achieved in NLS1 mutant and the cell chemosensitivity towards cisplatin was assessed. It was found that abolishment of nuclear PAK4 lowered the level of chemoresistance, suggesting that PAK4 nuclear accumulation has a vital function with regard to conferring mechanisms for drug resistance. This study has also tested whether PAK4 upregulation was due to gene amplification or PAK4 gene mutation. To evaluate the difference in gene copy number, quantitative PCR assay was adopted to evaluate the genomic DNA of available ovarian cancer cell lines. Exons 5, 6, 9, 10 of PAK4, which encoded functional domains including the kinase domain, were also sequenced to detect mutations. My results showed that no functional mutations could be detected but amplification was found in 50% of the cell lines. In summary, my study has shown that overexpression of PAK4 is a contributor to cisplatin resistance of ovarian cancer cell line SKOV3. PAK4 gene amplification is a possible mechanism governing PAK4 overexpression. Gene mutation, including that of PAK4 kinase domain, may not be a crucial factor. In contrast, nuclear localization of PAK4 protein is likely to play a significant role in chemoresistance. Further study may shed light on the potential of PAK4 as a novel therapeutic target of ovarian cancer.