Abstract 2324: RhoGTPase-based regulation of cell motility by Plk4.

Abstract

Background. Polo-like kinase 4 (Plk4) is a serine-threonine kinase that localizes to centrioles and is essential for centriole duplication. Plk4 expression is increased in colorectal, pancreas and breast cancers, and predicts resistance to therapy and poor survival. While centriolar overduplication and multipolar spindle formation is one mechanism by which dysregulated Plk4 can facilitate oncogenesis, our laboratory has found that Plk4 promotes migration and invasion of fibroblasts and of cancer cells by mechanisms currently under investigation. Haploid levels of Plk4 are associated with an increased incidence of improper cleavage furrow positioning during mitotic division, and we previously showed that RhoA is not adequately activated to effect proper actomyosin ring placement and contraction in Plk4 heterozygous murine embryonic fibroblasts (MEFs). We showed that the effect of Plk4 on RhoA activation is mediated by phosphorylation of the GEF Ect2 by Plk4 (Rosario et al., PNAS 2010). The small Rho GTPases RhoA, Rac1 and Cdc42 are known to regulate cell motility. We hypothesize that Plk4 promotes cancer cell motility by altering the activation of one or more of these RhoGTPases. Methods and Results: Pulldown experiments have shown a two-fold reduction in activated Rac1 in Plk4+/− as compared to Plk4+/+ MEFs. Depletion of Plk4 from wildtype MEFs using siPlk4 (confirmed by real time RT-PCR) reduced levels of activated Rac1, as compared to siLuciferase controls. Cdc42 activation was unaffected by Plk4. To determine the mechanism of the effect of Plk4 on Rho GTPase activation we investigated the upstream regulators that may be affected by Plk4, aside from Ect2. We scanned a library of 149 GEFs and GAPs cloned into the Creator system and identified 8 potential interactors (all GEFs) that contain the Plk4 consensus phosphorylation motif. We are evaluating these 8 candidates for physical interaction with Plk4 by co-transfection and co-immunoprecipitation from HeLa cells. By this method, we find that Plk4 physically interacts with the RhoA GEF ARHGEF1. To establish functional validation of this interaction, we are examining the phenotype of individually- and co- transfected HeLa cells. Transient transfection with Plk4 for 24h results in an arborized phenotype, while transfection with ARHGEF1 results in a rounded morphology; co-transfected cells display an intermediate phenotype. Immunofluorescence imaging shows co-localization of Flag-Plk4 and GFP-ARHGEF1 in lamellipodia, in keeping with a potential functional interaction. Whether the effect of ARHGEF1 on cell morphology is mediated through Rac1, RhoA, or both requires further investigation, as does the precise role of Plk4. Conclusions: Plk4 regulates the activation of Rac1 GTPase, and we demonstrate a physical and functional interaction of Plk4 with the Rac1 GEF ARHGEF1. Regulation of cancer cell motility via this pathway may contribute to promotion of invasion and metastasis by Plk4. Citation Format: Volha (Olga) Brashavitskaya, Karineh Kazazian, Rick Bagshaw, Carla O. Rosario, Francis S W Zih, Yosr Haffani, James W. Dennis, Tony J. Pawson, Carol J. Swallow. RhoGTPase-based regulation of cell motility by Plk4. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2324. doi:10.1158/1538-7445.AM2013-2324

Plk4 Promotes Cancer Invasion and Metastasis through Arp2/3 Complex Regulation of the Actin Cytoskeleton

“…In a search for evidence of a causative role for CA in cancer development and progression, others have shown that Plk4induced CA is associated with acquisition of an invasive phenotype by benign cells (9,14). We and others (14,40) have demonstrated that Plk4 promotes Rac1 activation; however, this was insufficient for Plk4-induced motility in our system. Regulation of actin dynamics and cell motility occurs at multiple levels.…”

Nek2 and Plk4: prognostic markers, drivers of breast tumorigenesis and drug resistance

“…Retrospective analyses of public dataset confirmed that overexpression of Nek2 conferred a poor survival outcome in breast tumors (19, 23). AACR communications mentioned Plk4 overexpression and its predictive relevance to therapy outcome in breast cancer (44, 48, 49). …”

“…Thus, in liver cancer models, haploid levels of Plk4 can disrupt Rho-GTPase signaling during cytokinesis, resulting in aneuploidy and tumorigenesis (50). In addition, studies performed in Plk4 +/− mouse embryonic fibroblasts suggest that this enzyme promotes activation of Rac1 to induce migration (48). Findings from lung cancer cells support a pro-survival role for Plk4.…”

Seize the engine: Emerging cell cycle targets in breast cancer

“…The identification of PLK4 as a promising therapeutic target resulted from a systematic approach combining kinome‐wide RNAi screening and gene expression analysis in cell lines and human BC 97 . PLK4 has been found overexpressed in BC of all subtypes and its increased activity has been consistently linked to disease aggressiveness and epithelial–mesenchymal transition in vitro and in vivo 97–101 . This is likely explained by centrosome amplification, multipolarity and resulting aneuploidy and genomic instability.…”

“… 97 PLK4 has been found overexpressed in BC of all subtypes and its increased activity has been consistently linked to disease aggressiveness and epithelial–mesenchymal transition in vitro and in vivo. 97 , 98 , 99 , 100 , 101 This is likely explained by centrosome amplification, multipolarity and resulting aneuploidy and genomic instability. PLK4 upregulation is associated with a higher incidence of lymph node metastasis, distant metastasis, shorter survival and worse response to neoadjuvant taxane‐based chemotherapy and adjuvant tamoxifen.…”