Functional characterization of the role of NIMA (never in mitosis gene a)-related kinase 2(Nek2) in hepatocellular carcinoma

Abstract

The NIMA-related kinase 2 (Nek2), which regulates centrosome cohesion and cell cycle progression, is a potential player in carcinogenesis. Reports have shown that up-regulation of Nek2 has been observed in various types of cancer. However, the functional roles of Nek2 in hepatocellular carcinoma (HCC) is poorly understood. HCC is a primary liver malignancy that arises from hepatocyte. It is notorious for its chemo-resistance nature and HCC patients generally have poor prognosis. Previously, our laboratory showed that Nek2 mRNA and protein levels are overexpressed in HCC and knockdown of Nek2 inhibited metastasis. Using the same cell line, Nek2 was found to be involved in the regulation of YAP, a transcriptional factor that control cell proliferation and organ size.

To study the effect of Nek2 in HCC, a stable inducible Nek2 overexpression system was established in different HCC cell lines. Cell growth was inhibited when Nek2 is overexpressed. For the study of centrosome, we observed that higher proportion of cells with centrosome splitting occurred in the Nek2 overexpressing cells in both inducible HepG2 and SMMC-7721 cell lines. Increased cell population with centrosome over-duplication was however observed in U2OS cells when Nek2 is up-regulated. Moreover, cell cycle analysis on induced Nek2 cells revealed an increased in DNA content, suggesting that Nek2 promotes aneuploidy in HCC cells.

Using confocal co-immunofluorescence staining, I demonstrated that TAX1BP2, a putative tumor suppressor in HCC, is a potential interacting partner of Nek2 at the centrosome. Interestingly, I provided evidence that overexpression of TAX1BP2 can significantly block the effect of Nek2 mediated centrosome separation, suggesting that TAX1BP2 may act downstream of Nek2 signaling. Furthermore, in terms of the mechanism of regulation, I showed that TAX1BP2 is a substrate of Nek2 using in vitro kinase assay. Although several potential Nek2 phosphorylation sites in TAX1BP2 has been predicted, it remains to be determined which phosphorylation site is critical for the regulation of TAX1BP2 by Nek2.

To dissect how Nek2 promotes HCC formation and delineate its signaling pathways, both Flag antibody and biotin ligase affinity pull-down assay were employed. Some potential interacting partners of Nek2 were identified by these affinity pull-down approaches. Such as the CP110, which is involved in centrosome elongation, has been isolated by Flag antibody pull down. In addition, I also isolated several other potential binding partners in biotin ligase pull down. These novel binding partners suggest that Nek2 may play a role in centromere function, apoptosis and centrosome overall structures.

To conclude, Nek2 induces centrosome splitting and aneuploidy in HCC cells, which provides insights to the oncogenic role of Nek2 in HCC. Dysregulation of centrosome owing to the overexpression of Nek2 may be a key event in inducing hepatocarcinogenesis.