Aberrant regulation of HDAC2 mediates proliferation of hepatocellular carcinoma cells by deregulating expression of G1/S cell cycle proteins

Abstract

Histone deacetylase 2 (HDAC2) is crucial for embryonic development, affects cytokine signaling relevant for immune responses and is often significantly overexpressed in solid tumors; but little is known about its role in human hepatocellular carcinoma (HCC). In this study, we showed that targeted-disruption of HDAC2 resulted in reduction of both tumor cell growth and de novo DNA synthesis in Hep3B cells. We then demonstrated that HDAC2 regulated cell cycle and that disruption of HDAC2 caused G1/S arrest in cell cycle. In G1/S transition, targeted-disruption of HDAC2 selectively induced the expression of p16(INK4A) and p21(WAF1/Cip1), and simultaneously suppressed the expression of cyclin D1, CDK4 and CDK2. Consequently, HDAC2 inhibition led to the down-regulation of E2F/DP1 target genes through a reduction in phosphorylation status of pRb protein. In addition, sustained suppression of HDAC2 attenuated in vitro colony formation and in vivo tumor growth in a mouse xenograft model. Further, we found that HDAC2 suppresses p21(WAF1/Cip1) transcriptional activity via Sp1-binding site enriched proximal region of p21(WAF1/Cip1) promoter. In conclusion, we suggest that the aberrant regulation of HDAC2 may play a pivotal role in the development of HCC through its regulation of cell cycle components at the transcription level providing HDAC2 as a relevant target in liver cancer therapy.

Figure 1. Overexpression of HDAC2 is not regulated by Wnt signaling in human HCC.

(A) Tissue lysates were prepared from non-tumoral liver tissues (N) or tumors (HCC, HBV-positive, Edmondson grade G3) (T) and immunoblotted with indicated antibodies. (B) Protein expressions of HDAC2, β-catenin and c-Myc were also determined by immunoblotting in human liver cancer cell lines. (C) Hep3B cells were transfected with siRNAs targeting β-catenin, cyclin D1 or c-Myc. The protein level of HDAC1 or 2 was assessed by immunoblotting. A typical result from three performed experiments is shown.

Figure 2. Effects of HDAC2 inhibition on the cell growth and apoptosis.

(A) Endogenous expression of HDACs (HDAC1-8) in Hep3B cells by qRT-PCR. Each HDAC mRNA level was normalized against GAPDH. (B) HDAC2 was depleted by HDAC2 specific siRNA in Hep3B cells. To ascertain the knockdown efficiency and suppression of HDAC activity, protein expressions of HDAC2, acetyl-histone H3 and H4 were determined by immunoblotting. (C) Time course analysis of the effect of HDAC2 inhibition on adherent cell number. The cell counts show the results of four independent experiments, expressed as mean ± SD (* p<0.05 relative to Scr siRNA). (D) After 48 h transfection of HDAC2 siRNA, cells were stained with Annexin V-FITC and propidium iodide, and apoptotic cells were analyzed by flow cytometric analysis (left). Protein expressions of the proapoptotic genes (AIF, Bax, Apaf-1, cleaved-caspase3, cleaved-PARP) were also determined by immunoblotting (right).

Figure 3. Effects of HDAC2 inhibition on the proliferation and the cell cycle progression of HCC cells.

(A) Cell proliferating activity was assessed by thymidine incorporation analysis in Hep3B cells after transfection with HDAC2 or scrambled siRNA. At 48 and 72 post-transfection, [³H]-thymidine incorporation was analyzed by scintillation counting. The results represent the mean ± SD of three experiments (* p<0.01). (B) After 48 and 72 h post-transfection of HDAC2 siRNA, the DNA content of PI-stained cells was analyzed by flow cytometry. (C) After 1 mM Valproic acid treatment to Hep3B cells for 72 h, the DNA content of PI-stained cells was analyzed by flow cytometry. (D) Hep3B cells were transient transfected with HDAC2 or scrambled siRNA (100 nM) for 72 h, and 1 mM of Valproic acid was also treated on Hep3B cells for 72 h. The protein expressions of p21^WAF1/Cip1 and CDK2 were determined by immunoblotting. Three experiments with the same results were performed.

Figure 4. Systemic modulation of regulatory components by HDAC2 in G1/S cell cycle transition.

Hep3B cells were transfected with no siRNA (None), oligofectamine only (Reag), 100 nmol/L scrambled siRNA (Scr) or 100 nmol/L of two different HDAC2 specific siRNAs (#1 and #2), and were harvested at 72 h post-transfection. (A) Immunoblotting of CDKs, cyclins and CDK inhibitors of G1/S transition were performed. (B) Effects of HDAC2 depletion on pRb and E2F/DP1 target gene expressions. (C) Protein expressions of p21^WAF1/Cip1, CDK2 and PCNA in human HCC tissues were analyzed by immunoblotting. All experiments were repeated three times with same results.

Figure 5. Identification of large-scale gene expression changes by HDAC2 inactivation in Hep3B cells.

(A) Heatmap analysis of genes recapitulated for HDAC family and cell cycle regulation, and graphical representation of the expression changes of genes functionally involved in HDAC gene families (left) and cell cycle regulation (right) by HDAC2 inactivation of Hep3B cells. (B) Validation of microarray data by quantitative real-time PCR. The relative expression level of each gene was normalized to GAPDH mRNA in the same sample. The result of four independent experiments was shown as mean ± SD (Student's t-test).

Figure 6. Sustained suppression of HDAC2 attenuates the tumorigenic potential of Hep3B cells in vitro and in vivo.

(A) Confirmation of HDAC2 suppression by its specific regulation of cell cycle components in HDAC2 depleted cell lines. A typical result of three performed experiments is shown. (B) Time course cell counting analyses of Hep3B and two stable cell lines (Hep3B_Mock and Hep3B_HDAC2KD). The cell counts show the results of four independent experiments. (C) HDAC2-deficient (Hep3B_HDAC2KD) or Mock (Hep3B_Mock) stable cell line was blocked in G2/M transition by nocodazole and then released in fresh medium. The DNA content was determined by FACS analysis with PI-stained cells at each time point (0, 2, 6, 12, 24, 48 h after release) (left). The percentage of cells in G1 phase was calculated and represented as bar graph (right). A typical result of three performed experiments is shown. (D) In vitro Colony formation assay showed the effect of HDAC2 inhibition on the growth of cell colonies after three weeks incubation. Quantification of colony numbers shown is mean ± SD of three independent experiments (top). The representative scan of 0.5% crystal violet-stained cells (bottom). (E) Tumor growth of Hep3B cell xenografts. (F) Representative tumors obtained at sacrifice after 52 days of growth (upper left) and mice (upper right). The tumor volume at sacrifice was presented as bar graph. Values shown are mean ± SD, n = 5 for Mock; n = 3 for HDAC2KD. Data in (B, D, E and F) were analyzed by Student's t-test. * p<0.05, Hep3B_HDAC2KD vs Hep3B_Mock.

Figure 7. HDAC2 regulates p21^WAF1/Cip1 transcription via Sp1-binding sites in the p21^WAF1/Cip1 promoter.

(A) HDAC2-deficient (Hep3B_HDAC2KD) or Mock stable cell line (Hep3B_Mock) was transfected with a reporter construct containing the human wild-type p21 promoter (−2326 to +1) linked to a luciferase reporter gene (pWWP-Luc). For the positive control, 1 µM of Apicidin was treated on the mock cell line for 24 h and luciferase activity was measured. The luciferase activity from the mock cells transfected with pGL3 basic vector was arbitrarily defined as 1.0. (B) Ectopic expression of HDAC2 suppressed the p21^WAF1/Cip1 transcriptional activity. HDAC2-deficient stable cell line (Hep3B_HDAC2KD) was transfected with FLAG-epitope tagged HDAC2 expression vector (F-HDAC2) or control vector (Con). After 24 h incubation, pWWP-Luc vector was also transfected. The promoter activity was measured and normalized as mentioned above. (C) A schematic of the p21^WAF1/Cip1 promoter depicting the regions analyzed by ChIP-qPCR (black bars, A-D). (D) The association of HDAC2 in the p21^WAF1/Cip1 promoter was assessed by the amplification of each region immunoprecipitated with HDAC2. The amount of DNA precipitated by either anti-HDAC2 or control IgG was expressed as percentage of the total input genomic DNA. The result of four independent experiments was shown as mean ± SD. (E) Acetylations of histone H3 and H4 associated with the proximal p21^WAF1/Cip1 promoter was increased by inhibiting association of HDAC2. Hep3B cells were transiently transfected with control (Con) or HDAC2 siRNA for 48 h and subjected to ChIP-qPCR analysis using acetyl-histone H3 (anti-Ac-H3) and H4 (anti-Ac-H4) antibody or control IgG. Precipitated genomic DNA was amplified for the proximal promoter of the p21 locus (region D represented in Figure 7C) by real-time PCR. The amount of precipitated DNA was expressed as percentage of the total input genomic DNA. The result of three independent experiments was shown as mean ± SD. (F) The indicated constructs, pWWP, pWPdel-BstXI, pWP101, pWPmt-Sp1-3, pWPmt-Sp1-5,6, pWPdel-SmaI and Sp1-luc were transiently transfected into each Hep3B stable cells. The promoter activity was measured, and fold induction by HDAC2 depletion is calculated. On the left, the scheme of each construct was shown. Data in (A, B and F) were analyzed by Student's t-test. * p<0.05.