Abstract
Abstract Background: Ovarian cancer is one of the most lethal cancers among women worldwide. Despite of an initial response to chemotherapy, relapse may happen due to the chemoresistant phenotypes. However, underlying mechanism conferring chemoresistance is not well defined. p21-activated serine/threonine kinases (PAKs) are serine/threonine kinases involved in various cellular functions including cell motility, survival and angiogenesis.PAK4 is the prototypic member of Group II PAKs. In this study, we 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. Materials and Methods: Ovarian cancer cell line SKOV-3 stably expressing various PAK4 mutants was used as an in vitro model. SKOV-3 was transfected with Flag-tagged wild type PAK4, constitutively active PAK4 (445N/474E), kinase-dead PAK4 (M350), or the control vector, p3XFLAG-CMV-10. To illustrate the functional impact of nuclear PAK4, PAK4 constructs with various nucleus localization signals (NLS) mutation were generated. PAK4 NLS mutants were then stably transfected into SKOV-3 and the expression and nuclear exclusion were confirmed by western blotting and immunofluorescence microscope respectively.They were also treated with cisplatin and the discrepancies in response to cisplatin were accessed by MTT assay and through the evaluation of apoptotic activity using flow cytometry and caspase-3 activation. In order to unveil the significance of PAK4 amplification and mutation status, genomic DNA of two immortalized ovarian epithelial cell lines HOSE6-3 and HOSE-11-12, and eleven ovarian cancer cell lines were extracted. Exon 3, which encodes the Cdc42-Rac interaction/binding (CRIB) domain and exons 5 - 10, which encode the kinase domain were sequenced to detect possible mutation in comparison to the wild-type sequence. Results: By MTT assay, overexpression of wild type PAK4 was demonstrated to enhance the survival of SKOV3 cells when compared with vector-transfected control cells when challenged with cisplatin. Flow Cytometry Analysis confirmed the above survival pattern. No significant differences were found between the two PAK4 kinase domain mutants and the wild type one. At genomic level, no amplification or mutation in PAK4 gene could be found in the ovarian cancer cell lines studied. Wild type PAK4 expression was found in both nucleus and cytoplasm. Mutation of NLS3, but not NLS1, could abolish the nuclear-localization of PAK4. Conclusion: In summary, aberrant expression of PAK4 contributes to resistance of ovarian cancer cells towards cisplatin.Amplification or mutation in PAK4 kinase domain cannot be detected and therefore may not be the crucial factor underlying overexpression in ovarian cancers. Further study on PAK4, including its subcellular localization, may further shed light on its potential as a novel therapeutic target of ovarian cancer. Citation Format: Ya Nan Du, Oscar GW Wong, Ka Kui Chan, Esther SY Wong, Annie NY Cheung. PAK4 mediated mechanisms contributing to chemoresistance of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2621. doi:10.1158/1538-7445.AM2014-2621
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