Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27(Kip1) ubiquitination pathway in hepatocellular carcinoma

Abstract

Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27(Kip1) protein level specifically increased after KIF14 knockdown. The increase in p27(Kip1) was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27(Kip1) accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27(Kip1) for degradation by the 26S proteasome, leading to accumulation of p27(Kip1). The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.

Figure 1

Kinesin family member 14 (KIF14) expression in various hepatocellular carcinoma (HCC) cell lines and short interfering RNA (siRNA)-mediated downregulation of KIF14 expression. (a) KIF14 protein expression in six HCC cell lines and a normal liver cell line (THLE-2). Alpha-tubulin was used as an internal control for western blot analysis. The y axis represents the relative KIF14 protein expression ratio (siKIF14/siNEG). (b) Downregulation of KIF14 by transfection of three KIF14 siRNAs into SNU-449 cells. After 72 h, the cells were harvested to validate the knockdown of KIF14 expression using quantitative reverse transcription-PCR analysis. The y axis represents the relative KIF14 expression ratio (siKIF14/siNEG). (c) KIF14 protein expression after siKIF14 transfection in a time series. Alpha-tubulin was used as an internal control for western blot analysis. The error bars indicate the s.d. of the mean of three individual experiments.

Figure 2

Effects of kinesin family member 14-specific short interfering RNA (siKIF14) knockdown and overexpression on tumor cell growth and cell cycle progression. (a) Colony formation assay after siKIF14 knockdown in SNU-449 cells. (b) Proliferation assay after siKIF14 knockdown in SNU-449 cells. All measurements were repeated three times, and the mean optical density values with s.d. were plotted for each case. *P<0.01. (c) Proliferation assay after transfection of SNU-761 cells with the KIF14 overexpression vector. KIF14 overexpression was confirmed by western blot analysis (bottom plot). Beta-actin was used as an internal control. KIF14over, pCMV6-KIF14-transfected cells; pcDNA, KIF14 empty vector (pcDNA)-transfected cells. (d and e) Effects of KIF14 depletion and siKIF14+pcDNA3-myc-Skp2 co-transfection on cytokinesis. (d) The bar chart represents the average frequencies of the binucleated cells in the siKIF14-, siKIF14+pcDNA3-myc-Skp2- and siNEG-transfected cells. *P<0.05. (e) Examples of the binucleated cells in the three different condition: siNEG-, siKIF14- and siKIF14+pcDNA3-myc-Skp2-transfected cells. DAPI, 4′-6-diamidino-2-phenylindole.

Figure 3

Effects of kinesin family member 14 (KIF14) knockdown on expression of cyclins and cyclin-dependent kinase inhibitors. (a) Western blot analysis shows the downregulation of cyclins B1, D1 and E1 and the upregulation of p27^Kip1 in the KIF14-specific short interfering RNA (siKIF14)-treated cells. There was no difference in the levels of p16^Ink4a and p21^Cip1 between the siKIF14- and siNEG-transfected cells. (b) Double immunochemical staining shows a negative correlation between KIF14 (green) and p27^Kip1 (red). DAPI, 4′-6-diamidino-2-phenylindole.

Figure 4

Mechanism of the elevated level of p27^Kip1 in the kinesin family member 14 (KIF14) knockdown cells. (a) RNA expression levels of p27^Kip1 and cyclins. The y axis represents the relative expression ratio of the target genes (KIF14-specific short interfering RNA (siKIF14)- or siNEG-treated SNU-449/original SNU-449). The ratio values were subsequently normalized by setting the mean ratio of the value from the siNEG-treated cells equal to 1. (b) KIF14 knockdown mimics a proteasome inhibitor in SNU-449 cells. Upper plot: p27^Kip1 expression level in MG132-treated cells. Intracellular p27^Kip1 accumulated in a time-dependent manner after treatment with the proteasome inhibitor MG132. Middle plot: p27^Kip1 expression level in cycloheximide (CHX)-treated and MG132+CHX co-treated cells. In the CHX treatment only cells, the p27^Kip1 level gradually decreased in a time-dependent manner. However, MG132+CHX co-treatment blocked the decrease of p27^Kip1. Bottom plot: effect of siKIF14 treatment on the decreased expression of p27^Kip1 caused by treatment with CHX. Seventy-two hours after transfection with siKIF14 or siNEG, the cells were co-treated with CHX for 0, 0.5, 1 or 3 h.

Figure 5

Effects of kinesin family member 14 (KIF14) knockdown on Skp1/Cul1/F-box (SCF) complex components in SNU-449 cells. (a) KIF14 knockdown downregulated the mRNA expression of Skp2 and Cks1 but not the mRNA expression of Cul1, Roc1 and Skp1. *P<0.05. (b) Skp2 and Cks1 protein expression after KIF14 knockdown.

Figure 6

Schematic summary of the potential molecular mechanism of growth repression caused by kinesin family member 14 (KIF14) knockdown. KIF14 knockdown downregulates the expression of Skp2 and Cks1, which inhibits the proteasome-dependent p27^Kip1 ubiquitination pathway, leading to its accumulation. The increase in p27^kip1 inhibits the expression of cyclins, including E1, D1 and B1, which leads to the suppression of cell cycle progression, resulting in the suppression of hepatocellular carcinoma tumorigenicity. Downregulated Skp2 and Cks1 may also influence cytokinesis failure.