Plk4 is the most divergent member of the family of polo like kinases (Plks). Plk4-/- embryos arrest at approximately day 7.5 p.c. but Plk4+/- mice are viable and fertile. However, 50% of Plk4+/- mice develop spontaneous tumours of the liver, lung and soft tissues by 2 years of age. Here I investigate the mechanisms that underlie Plk4-related tumourigenesis. Plk4+/- murine embryonic fibroblasts (MEFs) spontaneously become immortal in vitro with increasing passage number and are tumourigenic in vivo when injected into NOD SCID mice. Cytogenetic analysis showed that Plk4 deficient cells are chromosomally unstable with a large number of chromosomal aberrations and increased ploidy. These results demonstrate that early loss of a single Plk4 allele is sufficient to drive cell immortalization, chromosomal instability and tumourigenicity in vivo. In two independent expression array analyses, gene expression patterns that would decrease cell migration were overrepresented in Plk4+/- MEFs. A series of spreading and migration assays functionally validated these results, supporting the hypothesis that Plk4 regulates cell motility. Endogenous Plk4 localized to filopodia and lamellipodia in motile cells and to protrusions of spreading cells; the latter localization was transient and it disappeared by 4h after cell seeding, at which point Plk4 was located in the centrosomes, as typically observed in interphase cells. Transient transfection with Flag-Plk4 enhanced spreading and migration, as well as actin remodeling. Taken together, these data demonstrate temporal regulation of Plk4 in relation to the process of membrane remodeling, and a functional role for Plk4 in cell motility. Plk4 is haploinsufficient for tumour suppression in mice. Plk4 is located at human chromosome 4q28, a region often deleted in primary liver cancer specimens. Here I show that loss-of-heterozygosity (LOH) occurs at the Plk4 locus in ≈50% of human hepatocellular carcinomas (HCC) as well as in preneoplastic cirrhotic liver nodules. LOH at Plk4 is associated with reduced Plk4 expression in HCC tumours, but not with mutations in the remaining allele. These results implicate Plk4 as a potential haploinsufficient tumour suppressor in the genesis of human HCC. With continuing high rates of the predisposing conditions Hepatitis B and non-alcoholic steatohepatitis, and delayed diagnosis, HCC is a global health issue and carries a grave prognosis. A better understanding of genetic predisposition will help guide future screening programs.

Plk4 is the most divergent member of the family of polo like kinases (Plks). Plk4-/- embryos arrest at approximately day 7.5 p.c. but Plk4+/- mice are viable and fertile. However, 50% of Plk4+/- mice develop spontaneous tumours of the liver, lung and soft tissues by 2 years of age. Here I investigate the mechanisms that underlie Plk4-related tumourigenesis. Plk4+/- murine embryonic fibroblasts (MEFs) spontaneously become immortal in vitro with increasing passage number and are tumourigenic in vivo when injected into NOD SCID mice. Cytogenetic analysis showed that Plk4 deficient cells are chromosomally unstable with a large number of chromosomal aberrations and increased ploidy. These results demonstrate that early loss of a single Plk4 allele is sufficient to drive cell immortalization, chromosomal instability and tumourigenicity in vivo. In two independent expression array analyses, gene expression patterns that would decrease cell migration were overrepresented in Plk4+/- MEFs. A series of spreading and migration assays functionally validated these results, supporting the hypothesis that Plk4 regulates cell motility. Endogenous Plk4 localized to filopodia and lamellipodia in motile cells and to protrusions of spreading cells; the latter localization was transient and it disappeared by 4h after cell seeding, at which point Plk4 was located in the centrosomes, as typically observed in interphase cells. Transient transfection with Flag-Plk4 enhanced spreading and migration, as well as actin remodeling. Taken together, these data demonstrate temporal regulation of Plk4 in relation to the process of membrane remodeling, and a functional role for Plk4 in cell motility. Plk4 is haploinsufficient for tumour suppression in mice. Plk4 is located at human chromosome 4q28, a region often deleted in primary liver cancer specimens. Here I show that loss-of-heterozygosity (LOH) occurs at the Plk4 locus in ≈50% of human hepatocellular carcinomas (HCC) as well as in preneoplastic cirrhotic liver nodules. LOH at Plk4 is associated with reduced Plk4 expression in HCC tumours, but not with mutations in the remaining allele. These results implicate Plk4 as a potential haploinsufficient tumour suppressor in the genesis of human HCC. With continuing high rates of the predisposing conditions Hepatitis B and non-alcoholic steatohepatitis, and delayed diagnosis, HCC is a global health issue and carries a grave prognosis. A better understanding of genetic predisposition will help guide future screening programs.

Class of 2015 Abstract / Objectives: Two characterized peptide substrates were assayed with human Polo-like kinase 4 to determine phosphorylation activity. A pilot library of Type-II kinase inhibitors designed to fit into the ATP-binding pocket will be screened to determine HsPlk4 inhibition activity, which will help characterize a novel drug compound. Methods: Two peptide substrates of varying concentrations (2 uM, 1 uM, and 0.5 uM) were each combined with serial dilutions of HsPlk4 (1.25 uM, 0.625 uM, 0.313 uM, 0.156 uM, 0.078 uM, and 0.039 uM). EZ Reader detected phosphorylation activity by measuring fluorescence of both substrate and product, which separated at respective time points based on electrophoresis. The subsequent part of the experiment will be to inhibit the kinase activity with molecular inhibitors. Results: The results showed HsPlk4 activity with the modified PLKtide, (5FAM)KKKTPSDSLYDDGLSKK(CONH2). All reactions with the various concentrations of substrate 1 and HsPlk4 showed phosphorylation activity. The reaction started within the first 10 minutes, quickly reaching maximal phosphorylation of substrate. No p-values were calculated due to lack of data. Conclusions: No overall conclusions can be drawn based on the current results. Results showed the reaction reached its saturation point, so methods need to be refined to obtain data within the first 10 minutes. HsPlk4 phosphorylation of PLKtide confirmed the presumption that PLK family is a conserved family of Ser/Thr kinases. There are practical limitations for obtaining good kinetics data depicting enzyme activity, such as having EZ Reader quickly sample the reaction.

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