SIRT6 Depletion Suppresses Tumor Growth by Promoting Cellular Senescence Induced by DNA Damage in HCC

Abstract

The role of Sirtuin 6 (SIRT6) as a tumor suppressor or oncogene in liver cancer remains controversial. Thus, we identified the specific role of SIRT6 in the progression of hepatocellular carcinoma (HCC). SIRT6 expression was significantly higher in HCC cell lines and HCC tissues from 138 patients than in an immortalized hepatocyte cell line, THLE-2 and non-tumor tissues, respectively. SIRT6 knockdown by shRNA suppressed the growth of HCC cells and inhibited HCC tumor growth in vivo. In addition, SIRT6 silencing significantly prevented the growth of HCC cell lines by inducing cellular senescence in the p16/Rb- and p53/p21-pathway independent manners. Microarray analysis revealed that the expression of genes involved in nucleosome assembly was apparently altered in SIRT6-depleted Hep3B cells. SIRT6 knockdown promoted G2/M phase arrest and downregulation of genes encoding histone variants associated with nucleosome assembly, which could be attributed to DNA damage. Taken together, our findings suggest that SIRT6 acts as a tumor promoter by preventing DNA damage and cellular senescence, indicating that SIRT6 represents a potential therapeutic target for the treatment of HCC.

Fig 1. Effects of SIRT6 depletion on HCC cell growth.

(A) The expression of SIRT6 in HCC cell lines and normal liver lysate was assessed by western blotting using actin as a loading control. (B) Band intensities were measured by densitometry using Image J. SIRT6 band intensities were normalized to those of actin. (C) Upregulation of SIRT6 mRNA was assessed in HCC tumors relative to normal adjacent tissues by RT-PCR. (D) Hep3B, Huh-7, SNU475, and SNU449 cells were incubated with lentiviral particles containing plasmids with either negative control (NC) or SIRT6 shRNA. Five different lentiviral particles (C1–C5) targeting different sequences were used to delete the SIRT6 gene. HCC cells were harvested 5 days after viral transduction, followed by western blotting to compare the knockdown efficiency of SIRT6. (E) HCC cells transduced by lentiviral particles were seeded into 6-well plates at a density of 500 cells per well. Transduced HCC cells were selected in the growth medium supplemented with 2 μg/ml of puromycin. Cells were fixed and stained with crystal violet after 15 days. Representative images of plates were obtained using a digital camera. (F) The dye was extracted from stained colonies by 20% acetic acid to quantify the colony-forming ability; the extracted dye was quantified by measuring the absorbance at 595 nm with the percentage of the absorbance being calculated relative to that of NC (100%). (G) Hep3B and Huh-7 cells, transduced by the respective lentiviruses containing NC and shSIRT6, were cultured in soft agar with DMEM for 16 and 19 days, respectively. The representative micrographs were obtained using the iSolution Lite program. (H) Colonies larger than the indicated bar on the colony image were counted. The number of colonies (%) was calculated relative to that of NC, which was set to 100%. All data are representative of three independent experiments. (**P<0.05; ***P<0.001).

Fig 2

Effect of SIRT6 depletion on tumor growth in a xenograft mouse model (A) Hep3B cells were transduced with a lentiviral vector containing NC- or SIRT6-shRNA sequences. Hep3B cells were harvested 4 days after viral transduction, and 1 × 10⁶ cells were subcutaneously injected into mice. Tumor sizes in 21 mice were measured every 4 days after the injection and are shown as means ± SEM. (B) Photographs of mice injected with Hep3B cells were taken after sacrifice. (C) Tumors were obtained from the sacrificed mice, and their sizes were compared. (D) Tumor weights from sacrificed mice are shown as means ± SEM. (E) The expression of Ki-67 was immunohistochemically examined by confocal microscopy, and Ki-67-positive cells were counted in randomly obtained images. The scale bar indicates 50 μM. (*P<0.01, **P<0.05).

Fig 3

Induction of cellular senescence by SIRT6 depletion in the p16/Rb and p53/p21- independent manners (A) Representative micrographs of Hep3B cells 6 days after infection with lentiviruses containing NC or shSIRT6. The scale bar indicates 20 μM. (B) Representative micrographs of SA-ß-gal staining in NC and shSIRT6-depleted Hep3B and Huh-7 cells. The percentage of SA-ß-gal-positive cells was quantified from 6 different areas in the plate. (C) mRNA levels of the selected cytokine genes in control and SIRT6-silenced Hep3B cells are indicated in the histogram. (D) The expression of p16, Rb, p53, and p21 was analyzed by western blotting. Three independent experiments were performed. (**P<0.01).

Fig 4. Microarray analysis of SIRT6-depleted Hep3B cells.

(A) Microarray data show statistically significant effects of SIRT6 knockdown on gene expression with the indicated gene ontology. (B) The heat map shows the effect of SIRT6 depletion (from two independent Hep3B cell infections designated as “a” and “b”) on genes that function in nucleosome assembly. (C) mRNA levels of the selected nucleosome assembly genes in control and SIRT6-depleted Hep3B cells are indicated in the histogram. All data are representative of two independent experiments.

Fig 5. Effect of SIRT6 knockdown on DNA damage and cell cycle progression.

(A) Expression of γ-H2AX was assessed by immunocytochemistry using a confocal microscope. (B) Cells with over 10 foci were quantified by counting cells from the randomly obtained images. The quantified cells were compared in the histogram. The scale bar indicates 30 μM. (C) The expression of indicated proteins was assessed by western blotting. Cells for western blotting and immunocytochemistry were harvested or fixed 5 days after viral transduction. (D) Propidium iodide staining was performed to determine the cell cycle distribution in Hep3B cells 5 days after infection with lentiviruses containing NC or shSIRT6. The numbers of Hep3B cells at the G1, S, and G2/M phases were quantified by FACS analysis. The Modifit program was used for data analysis. (E) Expression of cell cycle-related proteins and SIRT6 were assessed by western blotting. Cells for microarray, RT-PCR, and western blotting were harvested 5 days after viral transduction. All data are representative of three independent experiments. (F) Representative FACS analysis of BrdU incorporation. (G) Cell proliferation rate of NC and shSIRT6-depleted cells as quantified by FACS analysis of four independently performed experiments. (**P<0.01, *P<0.05).

Fig 6. Schematic diagram of SIRT6 depletion in HCC.

SIRT6 depletion eventually leads to cellular senescence via the induction of DNA damage, which causes G2/M phase arrest and downregulation of genes encoding histone variants.