Functional characterization of polo-like kinase 4 (PLK4) in hepatocellular carcinoma

Abstract

The Polo-like Kinase 4 (PLK4), a centrosomal serine-threonine (S/T) kinase, is the master regulator of centrosome duplication. Recent evidence suggested that PLK4 is frequently overexpressed in cancer and overexpression of PLK4 is linked to chemo-resistance and poor prognosis in many types of cancers. The critical role of PLK4 plays in centriole biogenesis is well established, but its role in carcinogenesis only begun to emerge until recently. In this study, by stable overexpressing or silencing PLK4 in HCC cells, it was showed that PLK4 was critical for their tumorigenicity, migration and invasion. To understand how PLK4 promotes hepatocarcinogenesis, RNA-Seq analysis was performed using the stable PLK4 overexpression or knockdown HCC cells. Interestingly, bioinformatic analysis of RNA-Seq data revealed that the focal adhesion pathway was involved in PLK4 mediated migration. Such result was further validated by immunoblotting showing overexpression of PLK4 activated focal adhesion kinase in HCC cells. Mechanistically, it was confirmed that Tyrosine Kinase Expressed in Hepatocellular Carcinoma (TEC) is an interacting-partner of PLK4. TEC is a cytoplasmic signal transducer which functional role in HCC remains uncharacterized. ShRNA-mediated knockdown of TEC inhibited tumorigenicity and metastatic potential of HCC cells while overexpression showed reversed behaviors. SiRNA-mediated knockdown of PLK4 in TEC-expressing HCC cells reversed the enhanced cell invasiveness mediated by TEC overexpression, implying that PLK4 was essential for TEC function. By immunofluorescent staining, PLK4 was shown to localize at the centrosome, and overexpression of which induced supernumerary centrosomes in HCC cell lines. Unlike PLK4, overexpression of TEC had no effect in centrosome number. Though TEC was found to localize in cytoplasm, no TEC could be detected at the centrosomes, suggesting that TEC might activate PLK4 in the cytoplasm to enhance invasion. It was further demonstrated that TEC stabilized PLK4 in vivo via phosphorylation at the tyrosine-86 residue inside PLK4 kinase domain. In contrast to wildtype PLK4, overexpression of phospho-defective mutant (Y86F) of PLK4 did not change the invasiveness of HCC cells. Another chapter of this study focused to explore whether Aurora A (AurkA), a critical mitotic kinase in centrosome biology, is an upstream regulator of PLK4 and TAX1 binding-protein 2 (TAX1BP2) in HCC. Approach using the in vitro kinase assay coupled with mass spectrometry fingerprinting analysis showed that AurkA phosphorylated six S/T residues in the Cryptic-Polo-Box domain of PLK4. The phosphorylation resulted in an inhibition of PLK4 kinase activity demonstrated by in vitro kinase assay and immunoblotting using phospho-specific antibody against the active site of PLK4. It is still unclear what is the major function of this phosphorylation. However, since AurkA and PLK4 are both implicated in cytokinesis, the possibility that AurkA inhibits PLK4 activity to induce cytokinesis failure remains to be explored. Using co-immunofluorescence staining, it was demonstrated that TAX1BP2 localized in close-proximity with AurkA during interphase. In addition, TAX1BP2 was strongly phosphorylated by AurkA in in vitro kinase assay. By means of an overlapping peptide array and in vitro kinase assay, all the potential AurkA phosphorylation sites on TAX1BP2 was examined. Further analysis on the functional role of these phosphorylation on TAX1BP2 in regulating centrosome disjunction should be examined. Taken together, this study identified two mechanisms in which phosphorylation of PLK4 by TEC and AurkA modulate the stability and activity of PLK4. Functionally, TEC stabilizes PLK4 in HCC to promote cell invasiveness, while the significance of AurkA mediated PLK4 inhibition and TAX1BP2 phosphorylation warrant further investigation.