Tumor Suppressive Function of p21-activated Kinase 6 in Hepatocellular Carcinoma

Abstract

Our previous studies identified the oncogenic role of p21-activated kinase 1 (PAK1) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). Contrarily, PAK6 was found to predict a favorable prognosis in RCC patients. Nevertheless, the ambiguous tumor suppressive function of PAK6 in hepatocarcinogenesis remains obscure. Herein, decreased PAK6 expression was found to be associated with tumor node metastasis stage progression and unfavorable overall survival in HCC patients. Additionally, overexpression and silence of PAK6 experiments showed that PAK6 inhibited xenografted tumor growth in vivo, and restricted cell proliferation, colony formation, migration, and invasion and promoted cell apoptosis and anoikis in vitro. Moreover, overexpression of kinase dead and nuclear localization signal deletion mutants of PAK6 experiments indicated the tumor suppressive function of PAK6 was partially dependent on its kinase activity and nuclear translocation. Furthermore, gain or loss of function in polycomb repressive complex 2 (PRC2) components, including EZH2, SUZ12, and EED, elucidated epigenetic control of H3K27me3-arbitrated PAK6 down-regulation in hepatoma cells. More importantly, negative correlation between PAK6 and EZH2 expression was observed in hepatoma tissues from HCC patients. These data identified the tumor suppressive role and potential underlying mechanism of PAK6 in hepatocarcinogenesis.

Keywords: cancer biology; enhancer of zeste homolog 2; hepatocellular carcinoma; histone methylation; overall survival; polycomb; prognostic factor; serine/threonine-protein kinase PAK 6 (PAK6).

FIGURE 1.

Decreased PAK6 expression predicts poor survival in HCC patients. A, waterfall plot of the PAK6 mRNA level in HCC tissues of patients compared with adjacent paired non-tumor tissues (n = 28). Red bar represents the patients with a higher PAK6 expression in tumor than paired non-tumor; blue bar represents the patients with a lower PAK6 expression in tumor than paired non-tumor. B, representative IHC images of PAK6 expression in non-tumor tissue and paired tumor tissue (original magnification ×200 and 400). C, scatter plots for IHC staining score in paired non-tumors and tumors (n = 83). D, scatter plots for PAK6 IHC staining score from tumor node metastasis stage I to IV. E, Kaplan-Meier analysis of the overall survival dichotomized by PAK6 expression. p value is determined by log-rank test. The scale bar is 50 μm.

FIGURE 2.

PAK6 inhibits xenografted hepatoma cell growth in vivo. A, Western blot for endogenous PAK6 expression in six mammalian HCC cell lines and one immortalized liver cell line. B, Western blot for efficiencies of Huh7 stably transfected with NS-shRNA or PAK6-shRNA and SK-Hep1 stably transfected with empty or PAK6, respectively. Tumor growth curve (C), tumor weight analysis (D), and overall survival (E) for groups of nude mice xenografted with Huh7 cells stably transfected with NS-shRNA or PAK6-shRNA and SK-Hep1 stably transfected with empty or PAK6, respectively, where 3 mice were used in each group of 6 tumor sites for tumor volume and weight analysis, and 10 mice in each group were used for survival analysis. p value of survival analysis is determined by log-rank test.

FIGURE 3.

Overexpression of PAK6 restricts hepatoma cell tumorigenecity in vitro. A, CCK-8 assay for cell proliferation; B, Annexin V assay for cell apoptosis induced by serum starvation for 48 h. C, colony formation; D, migration assay (original magnification ×100); E, invasion assay (original magnification ×100); and F, Annexin V assay for cell anoikis induced by culturing on low-adhesive tissue culture plates with 10% serum and a no anoikis-induced condition as control, were performed in SK-Hep1 cells stably transfected with control or PAK6. The scale bar is 50 μm.

FIGURE 4.

Silence of PAK6 promotes hepatoma cell tumorigenecity in vitro. A, Western blot for efficiency of SK-Hep1 stably transfected with NS-shRNA and PAK6-shRNA. B, CCK-8 assay for cell proliferation; C, annexin V assay for cell apoptosis induced by serum starvation; D, colony formation; E, migration assay (original magnification ×100); F, invasion assay (original magnification ×100); and G, annexin V assay for cell anoikis induced by culturing on low-adhesive tissue culture plates with 10% serum and a no anoikis-induced condition as control were performed in Huh7 and Sk-Hep1 cells stably transfected with NS-shRNA and PAK6-shRNA, respectively. The scale bar is 50 μm.

FIGURE 5.

The kinase activity and nuclear translocation determine PAK6 function. A, Western blot for PAK6 expression of SK-Hep1 cells stably transfected with empty, wild-type PAK6 (PAK6), dead kinase mutant (PAK6-DN), and NLS deletion mutant (PAK6-Δ1–7). B, Western blot for PAK6 expression in the cytoplasm and nucleus of SK-Hep1 cells stably transfected with wild-type PAK6 (PAK6) and NLS deletion mutant (PAK6-Δ1–7), after treatment with leptomycin B at 10 nm for 6 h or without leptomycin B treatment as control. C, CCK-8 assay for cell proliferation; D, annexin V assay for cell apoptosis induced by serum starvation; E, colony formation; F, migration assay (original magnification ×100); G, invasion assay (original magnification ×100); and H, annexin V assay for cell anoikis induced by culturing on low-adhesive tissue culture plates with 10% serum and a no anoikis-induced condition as control were performed in SK-Hep1 cells stably transfected with empty vector, wild type PAK6 (PAK6), dead kinase mutant (PAK6-DN), and NLS deletion mutant (PAK6-Δ1–7). The scale bar is 50 μm.

FIGURE 6.

EZH2-mediated epigenetic repression dictates PAK6 down-regulation. A, Western blot analysis for EZH2, SUZ12, and EED expression in different hepatoma cell lines and one immortalized liver cell line. B, Western blot and RT-PCR analysis for PAK6 expression of Huh7 cells stably transfected with empty vector and EZH2. C, Western blot and RT-PCR analysis for PAK6 expression of SK-Hep1 cells stably transfected with NS-shRNA and EZH2-shRNA. D, Western blot and RT-PCR analysis for PAK6 expression of SK-Hep1 cells treated with DMSO and 3-deazaneplanocin A (10 μm). E, the diagram for primers set for ChIP analysis. Two pairs of primers were designed located in the CpG enrichment and transcriptional starting site at the PAK6 promoter, and one pair of primers was designed at the GAPDH promoter as a negative control. ChIP-qRCR analysis for enrichment of EZH2 and H3K27me3 in Huh7 cells stably transfected with empty and EZH2 (F), SK-Hep1 cells stably transfected with NS-shRNA and EZH2-shRNA (G), and SK-Hep1 cells treated with DMSO and 3-deazaneplanocin A (DZNep) (H) (10 μm). I, Western blot and RT-PCR analysis for PAK6 expression of SK-Hep1 cells transfected with NS-siRNA and two different SUZ12-siRNAs. J, Western blot and RT-PCR analysis for PAK6 expression of SK-Hep1 cells transfected with NS-siRNA and two different EED-siRNAs. K, Western blot and RT-PCR analysis for PAK6 expression of SK-Hep1 cells treated with DMSO and trichostatin A (100 nm).

FIGURE 7.

Negative correlation between PAK6 and EZH2 in HCC specimens. A, representative IHC images of different PAK6 and EZH2 staining in two HCC patients (original magnification ×200 and 400). Black arrows represent positive nuclear EZH2 staining. B, correlation analysis between PAK6 and EZH2 IHC staining in 83 patient tissues (r = −0.52, p < 0.001). C, Kaplan-Meier analysis for four groups cataloged by different PAK6 and EZH2 expression. D, a schematic model of the EZH2-PAK6 regulatory pathway in HCC. p value of survival analysis is determined by log-rank test. The scale bar is 50 μm.