Inhibitors of histone deacetylases target the Rb-E2F1 pathway for apoptosis induction through activation of proapoptotic protein Bim

Abstract

Inhibitors of histone deacetylases (HDACIs) are a new generation of anticancer agents that selectively kill tumor cells. However, the molecular basis for their tumor selectivity is not well understood. We investigated the effects of HDACIs on the oncogenic Rb-E2F1 pathway, which is frequently deregulated in human cancers. Here, we report that cancer cells with elevated E2F1 activity, caused either by enforced E2F1 expression, or by E1A oncogene expression, are highly susceptible to HDACI-induced cell death. This E2F1-mediated apoptosis is neither p53- nor p73-dependent but proceeds through selective induction of proapoptotic BH3-only protein Bim. We show that Bim is a direct target of E2F1 and that HDAC inhibition promotes the recruitment of E2F1 to the Bim promoter. Moreover, silencing of Bim by specific small interfering RNA (siRNA) effectively abolishes the E2F1-mediated cell death sensitization to HDACIs. These findings suggest that the oncogenic E2F1 pathway participates in HDACIs-induced apoptosis in cancer cells and underscore the importance of Bim as a key mediator of oncogene-induced apoptosis. Our study provides an important insight into the molecular mechanism of tumor selectivity of HDACIs and predicts that, clinically, HDACIs will be more effective in tumors with high E2F1 activity.

Fig. 1.

HDACIs SAHA and TSA promote E2F1-mediated cell death. (a) p53-null HCT116-ER-E2F1-expressing cells were treated with or without 4-OHT. Cyclin E and p73 expressions were evaluated by immunoblot analysis. (b) ER-E2F1-expressing or control ER-expressing cells were treated with 1 μM SAHA (Left) or 100 nM TSA (Right), in the presence or absence of 4-OHT. After 48 h, cells were harvested, and cell death was assessed by PI staining using FACS. Mean results of three independent experiments are shown with standard deviations. (c) Colony formation assay. One thousand ER-E2F1- or ER-expressing cells were plated per well in sixwell plates for 48 h and followed by the indicated treatment for 24 h. Cells were washed, fresh DMEM was added, and colonies were stained with crystal violet 12 days later. Representative plates are shown. (d) Saos-2 and HCT116 cells infected with adenovirus containing E2F1 or LacZ were treated with 100 nM TSA for 24 h, and cell death was analyzed by FACS.

Fig. 2.

HDACIs selectively activate E2F1 target genes. (a) ER-E2F1-expressing cells were treated with 100 nM TSA for the indicated times or 1 μM SAHA for 24 h in the presence or absence of 4-OHT. mRNA levels of E2F1 target genes as well as p21 and GAPDH were detected with RT-PCR. (b) Cells were treated in a. The expressions of E2F1 target gene products were assessed by Western blotting with antibodies to Bim, Puma, caspase-3, and p73. (c) Saos-2 and IMR90 cells were infected with Ad-E2F1 or Ad-LacZ for 24 h before treatment with 100 nM TSA for an additional 24 h. Expressions of Bim, p73, Puma, and E2F-1 were assessed by Western blotting with corresponding antibodies.

Fig. 3.

The effect of Bim-specific siRNA on HDACI-induced apoptosis upon E2F1 activation. (a) ER-E2F1 were transfected with nonspecific control siRNA (NC siRNA) or Bim-specific siRNA for 48 h, and then either left untreated (-) or treated with 4-OHT, SAHA, or both for an additional 24 h. The expression of Bim was analyzed by Western blotting (Left). Cell death was analyzed by flow cytometry after propidium iodide staining (Right). The percentages of cells in sub-G₁ are indicated. (b) ER-E2F1 cells were transfected with nonspecific control siRNA (NC siRNA) or Bim-specific siRNA for 48 h, and then either left untreated (-) or treated with 4-OHT, 100 nM TSA, or both for an additional 48 h. The expression of Bim was analyzed by Western blotting (Left). Cell death was assessed as in a, and the graph shows the mean results of three independent experiments with standard deviations (Right). (c) Saos-2 cells treated with Bim or control siRNA were infected with Ad-E2F1 for 24 h and followed by 100 nM TSA treatment for an additional 24 h. Bim expression was analyzed by Western blotting (Left), and cell death was assessed as in a; the graph shows the mean results of three independent experiments with standard deviations (Right).

Fig. 4.

SAHA promotes E2F1 recruitment to the Bim promoter. (a) Schematic representation of human Bim promoter containing putative E2F-binding sites. The indicated regions were isolated and cloned into pGL3 reporter construct. (b) pGL3-basic, Bim -1415/-205 or -2415/-1333 luciferase construct and Renilla luciferase construct were transfected into HCT116 cells together with increasing amounts of E2F1-expressing vector (0, 20, and 40 ng). Relative luciferase activities were measured 48 h after transfection. Results are depicted as fold induction, after normalization to the Renilla luciferase activity. (c Left) Schematic representation of the human Bim promoter containing the E2F1-RE element. (Right) E2F1 binding to the Bim promoter was analyzed by ChIP. Crosslinked chromatin from ER-E2F1-expressing cells treated as indicated were immunoprecipitated with antibody to E2F1 and nonspecific IgG (mock IP), and the Bim promoter fragment was amplified by PCR by using primers flanking the E2F1-RE-containing region. Positive control (input DNA) amplifications are shown.

Fig. 5.

The role of endogenous E2F1 in Bim and apoptosis induction after HDACI treatment. (a) U2OS cells were infected with Ad-E1A or Ad-LacZ for 24 h and treated with SAHA (1 μM) for an additional 48 h. Levels of Bim and p73 were assessed by immunoblotting by using antibodies against the indicated proteins (Left). Cell death was determined by FACS analysis (Right). (b) Normal IMR90 and E1A-transformed IMR90 cells were treated with SAHA at indicated concentrations, and cell proliferation was evaluated for the indicated times. (c) Expression of E1A results in up-regulation of Bim and apoptosis potentiation in response to SAHA and TSA. IMR90 and IMR90-E1A cells were treated with SAHA (2.5 μM) and TSA (1 μM) for 48 h. Cell death was determined by FACS analysis (Left), and the levels of Bim and p73 were assessed by immunoblotting by using antibodies against the indicated proteins (Right). (d) IMR90-E1A cells transfected with Bim siRNA or control siRNA (NC siRNA) were treated with TSA (1 μM) for 24 h. The expressions of Bim and β-actin were assessed by immunoblotting by using antibodies against the indicated proteins. Apoptosis was evaluated by FACS analysis. (e) Saos-2 cells were transfected with E2F1 siRNA or negative control siRNA (NC siRNA) and treated with TSA (100 nM) or SAHA (1 μM). The expressions of E2F1, Bim, and PARP were assessed by immunoblotting by using antibodies against the indicated proteins. NS, nonspecific band as the loading control. Apoptosis was evaluated by FACS analysis.