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The polo family serine threonine kinase Plk4 has been proposed as a therapeutic target in advanced cancers based on increased expression in primary human cancers, facilitation of tumor growth in murine xenograft models, and centrosomal amplification induced by its overexpression. However, both the causal link between these phenomena and the feasibility of selective Plk4 inhibition remain unclear. Here we characterize Plk4-dependent cancer cell migration and invasion as well as local invasion and metastasis of cancer xenografts. Plk4 depletion suppressed cancer invasion and induced an epithelial phenotype in poorly differentiated breast cancer cells. In an unbiased BioID screen for Plk4 interactors, we identified members of the Arp2/3 complex and confirmed a physical and functional interaction between Plk4 and Arp2 in mediating Plk4-driven cancer cell movement. This interaction is mediated through the Plk4 Polo-box 1-Polo-box 2 domain and results in phosphorylation of Arp2 at the T237/T238 activation site, which is required for Plk4-driven cell movement. Our results validate Plk4 as a therapeutic target in cancer patients and reveal a new role for Plk4 in regulating Arp2/3-mediated actin cytoskeletal rearrangement. Cancer Res; 77(2); 434-47. ©2016 AACR.

Nerve fibres are guided to their targets by the combined actions of chemotactic and haptotactic stimuli; however, translating these stimuli to a scaffold that will promote nerve regeneration is nontrivial. In pursuit of this goal, we synthesized and characterized cell-adhesive, biodegradable chitosan scaffolds. Chitosan amine groups were reacted with methacrylic anhydride resulting in a water soluble methacrylamide chitosan (MC) that was then crosslinked by radical polymerization resulting in a scaffold. Biodegradability by lysozyme and penetrability of the scaffold by rat superior cervical ganglion (SCG) neurons were studied. Maleimide-terminated cell adhesive peptides, mi-GDPGYIGSR and mi-GQASSIKVAV, were coupled to a thiolated form of MC to promote cell adhesion. The MC scaffold was found to be porous, biodegradable, and to allow neurite penetration. Interestingly, all of these properties were found to depend upon the amount of initiator used in crosslinking. Covalent modification of the MC scaffold with cell adhesive peptides significantly improved neuronal adhesion and neurite outgrowth. The MC can be crosslinked to form a novel scaffold, where our results demonstrate its suitability in neural tissue engineering and its potential for other engineered tissues, such as cartilage repair, where chitosan has already demonstrated some utility.

Polo family kinase 4 (Plk4) is required for mitotic progression, and is haploinsufficient for tumor suppression and timely hepatocyte polarization in regenerating liver. At the same time, recent evidence suggests that Plk4 expression may have a role in clinical cancer progression, although the mechanisms are not clear. Here we identify a gene expression pattern predictive of reduced motility in Plk4(+/-) murine embryonic fibroblasts (MEFs) and validate this prediction with functional assays of cell spreading, migration and invasion. Increased Plk4 expression enhances cell spreading in Plk4(+/-) MEFs and migration in human embryonic kidney 293T cells, and increases invasion by DLD-1 colon cancer cells. Plk4 depletion impairs invasion of wild-type MEFs and suppresses invasion by MDA-MB231 breast cancer cells. Cytoskeletal reorganization and development of polarity are impaired in Plk4-deficient cells that have been stimulated to migrate. Endogenous Plk4 phosphorylated at the autophosphorylation site S305 localizes to the protrusions of motile cells, coincident with the RhoA GEF Ect2, GTP-bound RhoA and the RhoA effector mDia. Taken together, our findings reveal an unexpected activity of Plk4 that promotes cell migration and may underlie an association between increased Plk4 expression, cancer progression and death from metastasis in solid tumor patients.

Polo like kinase 4 (Plk4) is a tightly regulated serine threonine kinase that governs centriole duplication. Increased Plk4 expression, which is a feature of many common human cancers, causes centriole overduplication, mitotic irregularities, and chromosomal instability. Plk4 can also promote cancer invasion and metastasis through regulation of the actin cytoskeleton. Herein we demonstrate physical interaction of Plk4 with FAM46C/TENT5C, a conserved protein of unknown function until recently. FAM46C localizes to centrioles, inhibits Plk4 kinase activity, and suppresses Plk4-induced centriole duplication. Interference with Plk4 function by FAM46C was independent of the latter's nucleotidyl transferase activity. In addition, FAM46C restrained cancer cell invasion and suppressed MDA MB-435 cancer growth in a xenograft model, opposing the effect of Plk4. We demonstrate loss of FAM46C in patient-derived colorectal cancer tumor tissue that becomes more profound with advanced clinical stage. These results implicate FAM46C as a tumor suppressor that acts by inhibiting Plk4 activity.

Aim:The main objective of this study was to compare the oncological outcomes of patients undergoing abdominoperineal resection (APR) versus low anterior resection (LAR) through a transanal total mesorectal excision (taTME) approach.Method: A total of 360 adult patients with a diagnosis of rectal cancer were enrolled at participating centres from the Canadian taTME Expert Collaboration. Forty-three patients received taTME-APR and received 317 taTME-LAR. Demographic, operative, pathological and follow-up data were collected and merged into a single database. Results are presented as hazard ratio (HR) and 95% confidence interval. All analyses were performed in the R environment (v.3.6). Results:The proportion of patients with a positive circumferential radial margin status was higher in the taTME-APR group than the taTME-LAR group (21% vs. 9%, p = 0.001).Complete TME was achieved in 91% of those undergoing APR compared with 96% of those undergoing LAR (p = 0.25). APR was associated with a greater rate of local recurrence relative to LAR, although it was not significant [crude HR = 3.53 (95% CI 0.92-13.53)]. Circumferential margin positivity was significantly associated with a higher rate of systemic recurrence [crude HR = 3.59 (95% CI 1.38-9.3)]. Conclusion:Our results demonstrate inferior outcomes in those undergoing taTME-APR compared with taTME-LAR. The use of this technique for this particular indication needs to be carefully considered.

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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