GCN5 expression is frequently up-regulated in human HCC tissues and cell lines
To evaluate the involvement of GCN5 in HCC, we examined the expression of GCN5 in a set of 31 human HCC specimens and four different human HCC cell lines by Western blot analysis. Our results showed that GCN5 protein levels were significantly up-regulated in 17 specimens (54.8 %), but down-regulated in 8 specimens (25.8 %), in total of 31 HCC specimens versus the surrounding non-tumorous liver tissues (Fig. 1a). Intriguingly, some GCN5 band shifts were observed (Fig. 1a). In addition, we observed that the mRNA levels of GCN5 were significantly increased in HCC specimens compared with non-tumorous tissues (Fig. 1b).
Furthermore, we analyzed GCN5 expression in human HCC cell lines MHCC97H, Sk-Hep-1, HepG2 and Huh-7 and hepatocyte cell line L-O2. We observed a significant increase in GCN5 expression in HCC cell lines when compared to hepatocyte cell line L-O2 (Fig. 1c). Hence the elevated expression of GCN5 in human HCC specimens and cell lines indicates that GCN5 may be an imperative candidate in HCC progression.
GCN5 knockdown reduces HCC cell proliferation and colony formation
To determine the role of GCN5 in cell proliferation, HCC cell lines HepG2 and Huh-7 cells as well as hepatocyte cell line LO2 were transiently transfected with pCMV-Myc-GCN5 expression plasmids to overexpress GCN5. As shown in Fig. 2a, GCN5 overexpression significantly enhanced the cell proliferation rate of HepG2, Huh-7, and LO2. Furthermore, two stable GCN5-knockdown HepG2 cell lines were established to determine the effects of GCN5 knockdown on cell proliferation. Knockdown of GCN5 significantly decreased the cell proliferation (Fig. 2b), and colony formation (Fig. 2c). These results indicate that GCN5 could potentially promote HCC cell proliferation and colony formation.
GCN5 knockdown inhibits xenograft tumor formation
To investigate the role of GCN5 in HCC progression in vivo, we assessed the effects of GCN5 knockdown on the growth of HCC xenograft tumors in nude mice. We subcutaneously injected 1 × 106 HepG2 control cells (shCtrl), shGCN5-1 and shGCN5-2 cells in dorsal flanks of five nude mice, respectively. Five days after injection, the tumors were measured every 2 or 3 days for 4 weeks with a Vernier caliper. GCN5-knockdown tumors grew considerably slower as compared to the control tumors (Fig. 3a). At the end of study day (30th) the mice were sacrificed and tumors were excised. The control tumors and shGCN5-knockdown tumors were aligned for comparison. As shown in Fig. 3b, HepG2-shGCN5-1 and shGCN5-2 tumors were much smaller in size as compared to HepG2 control tumors. The tumor volume of GCN5-knockdown group (38 ± 10 mm3) was only 34.4 % of the control group (110 ± 30 mm3) (Fig. 3c). Consistently, the expression of proliferation marker proliferating cell nuclear antigen (PCNA) was significantly decreased in all representative GCN5-knockdown HepG2 tumors (Fig. 3d). These results suggest that GCN5 plays a key role in HCC tumor growth.
GCN5 knockdown inhibits cell cycle progression
Since GCN5 knockdown decreased cell proliferation in HCC cells and attenuated the formation of HCC xenograft tumors in nude mice, we hypothesized that inhibition of HCC proliferation by GCN5 knockdown is due to the cell cycle arrest. To confirm our hypothesis, we analyzed the cell cycle dynamics by flow cytometric analysis. Comparing to the control cells, the cell population in G1 phase was significantly increased in GCN5-knockdown cells, with a corresponding decrease in S and G2/M phases (Fig. 4a). These data suggest that GCN5 is required for the G1-to-S phase transition and its knockdown inhibits the growth of GCN5-knockdown cells by impeding G1/S phase transition of the cell cycle.
To understand the mechanism by which GCN5 knockdown inhibits cell cycle progression, protein levels of several cell cycle-related genes were compared. In two GCN5-knockdown cell lines, while the protein levels of PCNA were significantly decreased as expected, the protein levels of cell cycle inhibitor p21Cip1/Waf1 were significantly increased (Fig. 4b). It has been reported that inhibition of Akt signaling can lead to up-regulation of p21Cip1/Waf1 [16], and GCN5 can regulate the activation of Akt [17], we therefore detected the protein levels of phosphorylated Akt at Ser473. As shown in Fig. 4b, the expression of phosphorylated Akt at Ser473 was significantly decreased in GCN5-knockdown cells, suggesting that up-regulation of p21Cip1/Waf1 is at least in part due to the down-regulation of phosphorylated Akt in GCN5-knockdown cells. AIB1 has been implicated in several cancers [18, 19], and attenuation of AIB1 frequently inhibits the activation of Akt signaling by suppressing the expression of insulin receptor substrate (IRS)-1 and IRS-2 [20–22]. We therefore wondered whether the expression of AIB1 is down-regulated in GCN5-knockdown cells. Our results showed that GCN5 knockdown significantly decreased the protein levels of AIB1 (Fig. 4b). Consistently, GCN5 knockdown significantly decreased the mRNA levels of AIB1, but increased the mRNA levels of p21Cip1/Waf1 (Fig. 4c). These results imply that GCN5 promotes cell cycle progression at least in part through up-regulating AIB1 to inhibit p21Cip1/Waf1 expression.
To further determine whether GCN5 regulates cell proliferation through AIB1, we performed rescue experiment for proliferation in GCN5-knockdown cells: GCN5-knockdown cells were transfected with AIB1 expression constructs, and then cell proliferation was measured by MTT assay. The results showed that transfection of AIB1 expression constructs could restore cell proliferation potential of GCN5-knockdown cells (Fig. 4d), suggesting that GCN5 promotes HCC cell proliferation at least moderately through regulating AIB1 expression. Western blot analysis of AIB1-restored cells showed a decrease in p21Cip1/Waf1 protein expression (Fig. 4e), which further substantiates our results and validates that GCN5 promotes cell cycle progression through up-regulating AIB1 to inhibit p21Cip1/Waf1 expression.
GCN5 regulates AIB1 expression by enhancing de novo transcription of the AIB1 gene
Since the mRNA levels of AIB1 were down-regulated in GCN5-knockdown HepG2 cells, we wondered whether GCN5 can regulate de novo transcription of the AIB1 gene. We therefore examined the effect of GCN5 overexpression on the AIB1 promoter activity by using AIB1 promoter reporter assay. Our results showed that GCN5 significantly enhanced the AIB1 promoter activity (Fig. 5a). Unlike a transcription factor, GCN5 protein does not contain a DNA binding domain. Rather than binding to DNA directly, GCN5 is recruited by transcription factors to specific regions of DNA to regulate gene transcription [23]. Because GCN5 has been shown to be a crucial component of E2F-1-transactivating complexes for stimulating E2F-dependent transcription [24], and E2F1 can enhance the AIB1 promoter activity directly [25], we contemplated whether GCN5 is required for E2F1-mediated up-regulation of AIB1 promoter activity. As revealed in Fig. 5b, overexpression of E2F1 significantly enhanced the AIB1 promoter activity as expected, but knockdown of GCN5 significantly decreased the AIB1 promoter activity induced by E2F1 transfection. These results suggest that GCN5 cooperates with E2F1 to enhance de novo transcription of the AIB1 gene.
Consistently, ChIP assay revealed that GCN5 was recruited to AIB1 promoter at E2F1 binding sites (Fig. 5c). Analysis of AIB1 promoter suggested that −250/+350 bp region contains two E2F1 binding sites (Fig. 5d). We performed mutation analysis to determine the role of these E2F1 binding sites in E2F1/GCN5-mediated activation of AIB1 promoter. We mutated E2F1 binding sites-1 and sites-2 on AIB1 promoter and measured AIB1 promoter activity induced by GCN5, respectively (Fig. 5d). Mutation of E2F1 binding site-1 significantly decreased the AIB1 promoter activity induced by GCN5 (Fig. 5e), whereas mutation of E2F1 binding site-2 had no effect on AIB1 promoter activity induced by GCN5 (Fig. 5e). These results suggest that GCN5 is recruited to E2F1 binding site-1 on AIB1 promoter to promote AIB1 expression. To explore whether E2F1 is essential for GCN5 recruitment on AIB1 promoter, we knocked down E2F1 using siRNA and then performed ChIP assay. As shown in Fig. 5f, down-regulation of E2F1 reduced GCN5 enrichment on AIB1 promoter, suggesting that GCN5 is recruited to AIB1 promoter through associating with E2F1.
To determine whether GCN5 is needed to acetylate H3K9 around E2F1 binding site 1 on AIB1 promoter, we knocked down GCN5 and performed ChIP assays using anti-histone H3(acetyl K9) antibody. Our results showed that H3K9 acetylation of E2F1 binding site 1 on AIB1 promoter was significantly reduced in GCN5-knockdown cells as compared to control cells (Fig. 5g), indicating that GCN5 regulates AIB1 expression by acetylating H3K9 around E2F1 binding site 1 on AIB1 promoter.
The expression of GCN5 positively correlates with AIB1 in human HCC specimens from two GEO profile datasets
To determine whether the positive correlation between the expression of GCN5 and AIB1 can be verified in a larger cohort of human HCC specimens, we analyzed the expression of GCN5 and AIB1 in human HCC specimens from two GEO profile datasets (GSE41619 and GSE62743). The expression of GCN5 was positively correlated with AIB1 in these two GEO profile datasets (Fig. 6). These results further support our notion that GCN5 regulates AIB1 expression.
Collectively, we draft a possible model in which GCN5 enhances HCC proliferation at least partially by enhancing AIB1 expression: GCN5 associates with E2F1 and binds to AIB1 promoter to enhance AIB1 transcription by promoting the H3K9 acetylation on AIB1 promoter, which leads to hyper proliferation of HCC (Fig. 7).