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. 2013 May 17;288(20):14510-14521.
doi: 10.1074/jbc.M113.458737. Epub 2013 Mar 29.

Lysine acetyltransferase GCN5 potentiates the growth of non-small cell lung cancer via promotion of E2F1, cyclin D1, and cyclin E1 expression

Long Chen  1 , Tingyi Wei  1 , Xiaoxing Si  1 , Qianqian Wang  2 , Yan Li  1 , Ye Leng  1 , Anmei Deng  2 , Jie Chen  2 , Guiying Wang  1 , Songcheng Zhu  3 , Jiuhong Kang  4
Affiliations

Lysine acetyltransferase GCN5 potentiates the growth of non-small cell lung cancer via promotion of E2F1, cyclin D1, and cyclin E1 expression

Long Chen et al. J Biol Chem. .

Abstract

The lysine acetyltransferases play crucial but complex roles in cancer development. GCN5 is a lysine acetyltransferase that generally regulates gene expression, but its role in cancer development remains largely unknown. In this study, we report that GCN5 is highly expressed in non-small cell lung cancer tissues and that its expression correlates with tumor size. We found that the expression of GCN5 promotes cell growth and the G1/S phase transition in multiple lung cancer cell lines. Further study revealed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1. Our reporter assays indicated that the expression of GCN5 enhances the activities of the E2F1, cyclin D1, and cyclin E1 promoters. ChIP experiments suggested that GCN5 binds directly to these promoters and increases the extent of histone acetylation within these regions. Mechanistic studies suggested that GCN5 interacts with E2F1 and is recruited by E2F1 to the E2F1, cyclin D1, and cyclin E1 promoters. The function of GCN5 in lung cancer cells is abrogated by the knockdown of E2F1. Finally, we confirmed that GCN5 regulates the expression of E2F1, cyclin D1, and cyclin E1 and potentiates lung cancer cell growth in a mouse tumor model. Taken together, our results demonstrate that GCN5 specifically potentiates lung cancer growth by directly promoting the expression of E2F1, cyclin D1, and cyclin E1 in an E2F1-dependent manner. Our study identifies a specific and novel function of GCN5 in lung cancer development and suggests that the GCN5-E2F1 interaction represents a potential target for lung cancer treatment.

Keywords: Cancer Prevention; Cancer biology; Cell Cycle; E2F1; GCN5; Gene Regulation; Histone Acetylase; Lung Cancer; Lysine Acetyltransferase (KAT).

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Figures

FIGURE 1.
FIGURE 1.
Increased expression of GCN5 and its correlation with tumor size in human lung cancers. A–C, representative images of GCN5 expression in lung cancer tissue samples (upper row) and corresponding adjacent normal tissue samples (lower row), showing high (A), medium (B), and low (C) levels of GCN5 expression in cancer tissues. D, plot representation of scores based on the nuclear and cytoplasmic expression of GCN5 in 75 lung cancer patients compared with matched normal tissues, p < 0.001. E, total GCN5 score in adjacent normal and tumor tissues. F, GCN5 score correlates with tumor size. **, p < 0.01; ***, p < 0.0001. Error bars, S.D.
FIGURE 2.
FIGURE 2.
GCN5 affects cell cycle progression and cell proliferation. A, Western blotting verification of the knockdown and overexpression of GCN5 in stable A549 cell lines. GAPDH was used as a loading control. B, morphology of the stable cell lines at 48 h after plating the same number of cells in the plates. C, proliferation of stable cell lines derived from A549 (upper panel) and H460 (lower panel) cells was examined using a CCK8 assay. D and E, representative profiles of the cell cycle distribution of the A549 stable cell lines (left panels) and statistical analysis of three independent assays. F, apoptotic analysis of the A549 stable cell lines by annexin V and PI staining followed by FACS; representative profiles of cell population distribution (left panels) and statistical analysis of three independent assays. *, p < 0.05; **, p < 0.01. Error bars, S.D.
FIGURE 3.
FIGURE 3.
GCN5 increases both mRNA and protein levels of cyclin D1, cyclin E1, and E2F1. A and B, Western blotting analysis of the protein levels of cell cycle regulators in GCN5-knockdown A549 cells, or GCN5-overexpressing A549 cells and control cell lines. GAPDH was included to show that equivalent amounts of protein were loaded in each lane. A, proteins that are regulated by GCN5. B, proteins that are not regulated by GCN5. C, RT-qPCR analysis of cyclin D1, cyclin E1, and E2F1 mRNA in the GCN5-knockdown cells, GCN5-overexpressing A549 cells, and control cell lines. D, RT-qPCR analysis of apoptosis genes in GCN5-knockdown cells, GCN5-overexpressing cells, and control cell lines. *, p < 0.05; **, p < 0.01. Error bars, S.D.
FIGURE 4.
FIGURE 4.
GCN5 directly binds to the cyclin E1, cyclin D1, and E2F1 promoters, increasing histone H3 and H4 acetylation levels within these regions. A, cyclin E1, cyclin D1, and E2F1 luciferase reporter assay performed in HEK293T cells. Empty control vector and E2F1- or GCN5-overexpression plasmids were co-transfected with cyclin E1, cyclin D1, or E2F1 luciferase reporter plasmids and were then assayed for luciferase activity at 48 h post-transfection. Protein bolts were used to confirm the overexpression of GCN5 and E2F1. B, GCN5 binding within the promoter region of cyclin E1, cyclin D1, and E2F1 in A549 cells. ChIP was performed with control IgG and GCN5 antibodies. Shown are the results of three independent experiments. C, comparison of histone H3 and H4 acetylation of the cyclin E1, cyclin D1, or E2F1 promoters between control and GCN5-overexpressing cell lines. Shown are the results of three independent experiments. *, p < 0.05; **, p < 0.01. Error bars, S.D. D, acetylation levels of histones H3 and H4 (acH3 and acH4) in GCN5-overexpressing or knockdown cells were detected with acetyl histone antibodies. Histone H3 and H4 levels (H3 and H4) were detected with histone antibodies.
FIGURE 5.
FIGURE 5.
E2F1 is required for GCN5 to promote the proliferation of A549 cells. A, GCN5 interacts with endogenous E2F1. Immunoprecipitation (IP) was performed using control IgG or GCN5 antibody, followed by Western blot analysis for E2F1 and GCN5. B, intracellular localization of GCN5 and E2F1 in A549 cells. Immunostaining was performed using GCN5 and E2F1 antibodies. C, RT-qPCR and Western blotting analysis of E2F1 knockdown by shRNA in the GCN5-overexpressing stable cell line. Error bars, S.D. (n = 3 biological replicates). **, p < 0.01.D, altered cell cycle distribution after E2F1 knockdown in A549 GCN5-overexpressing cell lines. E and F, comparison of GCN5-binding at cyclin E1, cyclin D1, and E2F1 promoters in the GCN5-overexpressing cell line after E2F1 knockdown. G, effect of E2F1 knockdown on the GCN5-mediated regulation of cyclin E1, cyclin D1, and E2F1 promoter activity in 293T cells. The GCN5 overexpression plasmid was co-transfected with control (siCTRL) or E2F1 siRNA, and the luciferase reporter activity was assayed 48 h post-transfection. *, p < 0.05. Error bars, S.D.
FIGURE 6.
FIGURE 6.
Overexpression of GCN5 accelerates tumorigenesis in vivo. A, upper panel, the overexpression of GCN5 in two stable cell lines was confirmed by Western blotting prior to injection. Lower panel, sustained GCN5 expression in tumors isolated from mice at 6 weeks post-injection. Two tumors isolated from mice injected with control (FUW) and GCN5-overexpressing cells (FUW-GCN5) were analyzed. B, tumor growth of control and GCN5-overexpressing cell lines in mice. The tumor size was monitored every 3 days. Error bars, S.D. C, tumors derived from control and GCN5-overexpressing cell lines (upper panel) and statistical analysis of tumor weight at 6 weeks post-injection (lower panel). D, in vivo imaging of tumors derived from the control and GCN5-overexpressing cell lines. E, immunochemical staining for GCN5, cyclin D1, Cyclin E1, E2F1, and proliferating cell nuclear antigen (PCNA) in tumors derived from the control and GCN5-overexpressing cell lines. **, p < 0.01; ***, p < 0.001. Error bars, S.D.
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