Lysine Acetyltransferase GCN5 Potentiates the Growth of Non-small Cell Lung Cancer via Promotion of E2F1, Cyclin D1, and Cyclin E1 Expression^*

Tissue Microarray and Evaluation of Immunostaining

Lung cancer tissue arrays were purchased from the National Engineering Center for BioChips in Shanghai, China. The expression of GCN5 or E2F1 in the tissues was evaluated by immunohistochemical staining with either a GCN5-specific or E2F1-specific antibody. The staining was scored according to the staining intensity and the percentage of cells stained. The final staining scores were calculated as the product of staining intensity multiplied by the percentage of stained cells.

Cell Culture

The A549, H460, and H358 human lung cancer cell lines were purchased from the Cell Bank at the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences and were maintained as instructed by the provider. Human embryonic kidney (HEK) 293T cells were purchased from the American Type Culture Collection (ATCC). All of the cells were maintained at 37 °C with 5% CO₂.

Plasmids, Transfections, and Virus Production

For the overexpression of the GCN5 and E2F1, we used the lentiviral vector FUGW, in which the GFP-coding region was replaced with the GCN5 or E2F1 coding sequences. The lentiviral vector pLKO.1 was used for gene knockdown. The following two siRNAs were selected for human GCN5: sense 5′-GCUCUACACAACCCUCAAATT-3′ and antisense 5′-UUUGAGGGUUGUGUAGAGCTT-3′; sense 5′-GCAUGCCUAAGGAGUAUAUTT-3′ and antisense 5′-AUAUACUCCUUAGGCAUGCTT-3′. The following two siRNAs were selected for human E2F1: sense 5′-GCGCAUCUAUGACAUCACCTT-3′ and antisense 5′-GGUGAUGUCAUAGAUGCGCTT-3′; sense 5′-CCUCUUCGACUGUGACUUUTT-3′ and antisense 5′-AAAGUCACAGUCGAAGAGGTT-3. DNA oligonucleotides for these siRNAs were synthesized, annealed, and cloned into pLKO.1 according to the protocol recommended by Addgene. HEK293T cells were transfected with the ExcellGene transfection reagent. Lentiviruses were generated from the HEK293T cells as described previously (28), and lung cancer cells were infected with the virus in the presence of 8 μg/ml Polybrene.

Cell Proliferation Assays, Cell Cycle Analysis, and Annexin V-PI Staining

Cell proliferation and apoptosis were evaluated using the Cell Counting Kit-8 (CCK8, DOJINDO, Japan) and the annexin V-PI staining kit (Roche Applied Science) according to the manufacturer's protocols. Cellular proliferation was measured at 12, 24, 48, and 72 h post-seeding.

Western Blotting, Immunoprecipitation, Immunofluorescence Staining, and ChIP Assays

Western blotting, immunoprecipitation, and immunofluorescence were performed as described previously (29, 30). The following antibodies were used: GCN5, sc-36532, and sc-20698; cyclin D1 (M-20), sc-718; cyclin E (M-20), sc-481; PCAF (E-8), sc-13124; E2F1, sc-251; p-Rb, BS4164; Rb, BS1310; GAPDH (0411), sc-47724; p53 (BP53.122), sc-73566; c-Myc (A-14), sc-789; Bcl-2, CST 2876; Bax, CST 2772; p27 (N-20), sc-527; p21, Abcam ab7960; acetyl histone H3 (Millipore 17–615); acetyl histone H4 (Millipore 17–630). ChIP assays were performed with EZ ChIP kit (Millipore). Ten million cells were used per immunoprecipitation reaction. Immunoprecipitated DNA and whole cell-extracted DNA were used as templates for RT-qPCR with primers that _targeted the flanking sequences of the E2F1-binding site in the promoters of cyclin D1, cyclin E1, and E2F1.

Tumor Xenografts in Nude Mice

Our animal experiments were approved by the Institutional Animal Care and Use Committee of Tongji University and were in compliance with all regulatory guidelines. Five-week-old nude mice were purchased from the National Resource Center for Rodent Laboratory Animals of China. Six mice were injected subcutaneously with 5.0 × 10⁶ of GCN5-overexpressing (FUW-GCN5) or vector control (FUW) cells. The tumors were monitored, and the volumes were calculated at 3-day intervals.

GCN5 Expression Correlates Significantly with the Size of Lung Tumors

To examine the physiological relevance of GCN5 in human lung cancer development, we analyzed the GCN5 protein levels and its distribution in an NSCLC tissue array by immunohistochemical staining using a GCN5-specific antibody. The array included the cancer tissue samples and corresponding adjacent normal tissue samples from 75 lung cancer patients. The patients consisted of 41 males and 34 females, with a median age of 61 years (range, 37–84 years). Among the patients, 24 (32%) died of tumor-related causes, and 51 (68%) were still alive as of the last follow-up (Table 1). The GCN5 levels in the tissue array specimens were assessed by measurement of the staining density scores. We found that in the cancer tissues, 42 cases exhibited strong immunopositivity (56%) (Fig. 1A), 30 cases exhibited moderate-to-strong immunopositivity (40%) (Fig. 1B), and three cases exhibited weak immunopositivity (4%) (Fig. 1C). Statistical analysis revealed that nuclear, cytoplasmic (Fig. 1D), and total levels (Fig. 1E) of GCN5 were significantly increased in the cancer tissues compared with the normal tissues. Remarkably, the GCN5 expression levels were significantly correlated with tumor size (Fig. 1F) (p = 0.0024), progressively advanced regional lymph node metastasis (p = 0.016), and TNM stage III (Table 1). Our lung cancer tissue array analysis revealed that GCN5 is overexpressed in NSCLC, and its expression is tightly linked to tumor proliferation and development.

GCN5 Promotes Lung Cancer Cell Growth by Facilitating the G₁/S Transition of the Cell Cycle

The increased expression of GCN5 in the NSCLC tissues suggests a role for GCN5 in the regulation of lung cancer cell growth. To address whether GCN5 affects NSCLC cell growth, we were able to stably knock down GCN5 (shGCN5) as well as to overexpress GCN5 (FUW-GCN5) in the A549 lung cancer cell line. shRNA oligonucleotides for _targeting two different published regions of GCN5 were cloned into the lentiviral vector pLKO.1. The shRNA exhibiting the highest knockdown efficiency was used in the remainder of this study. GCN5 was efficiently knocked down (shCTRL versus shGCN5) or overexpressed (FUW versus FUW-GCN5) in A549 cells (Fig. 2A). We observed apparent changes in growth rate when we passaged these stable cell lines. The GCN5-knockdown cell line exhibited the most significant growth impairment (Fig. 2B, upper row), and the GCN5-overexpressing cell line grew significantly faster than the control cell line (Fig. 2B, lower row). We further quantified the cell growth rate of these cell lines using a CCK8 assay and confirmed that the overexpression of GCN5 promotes cell growth, whereas its knockdown inhibits the cell growth (Fig. 2C, upper panel). Importantly, the knockdown or overexpression of GCN5 in the other two lung cancer cell lines H460 and H358 (Fig. 2C, lower panel and data not shown) resulted in similar phenotypes.

To determine how the growth of A549 cell lines is regulated by GCN5, we analyzed their cell cycle dynamics by PI staining and fluorescence-activated cell sorting (FACS). We found that compared with the control cell line (shCTRL), the G₁ population was significantly increased in the GCN5-knockdown cell line (shGCN5), with a corresponding decrease in S and G₂/M phases of more than 10% (Fig. 2D). Consistently, in the GCN5-overexpressing cell line (FUW-GCN5), the S phase population was significantly increased compared with the control cell line (FUW), with a corresponding decrease in the G₁ population of ∼13% (Fig. 2E). The data suggest that GCN5 is required for the G₁-to-S phase transition, and its overexpression promotes the growth of A549 cells by facilitating the G₁/S phase transition of the cell cycle. In contrast to the significant changes in cell cycle dynamics caused by the overexpression or knockdown of GCN5, we observed no significant effect on apoptosis, as indicated by annexin V and PI co-staining (Fig. 2F). Neither the overexpression nor the knockdown of GCN5 induced other types of cell death, as measured by trypan blue staining (supplemental Fig. S1). Importantly, the knockdown of PCAF, a KAT with 73% homology to GCN5, in A549 cells does not significantly affect cell proliferation (supplemental Fig. S2).

GCN5 Promotes the Expression of Cyclin D1, Cyclin E1, and E2F1

Because the knockdown or overexpression of GCN5 significantly altered cell cycle phase distribution, we further measured the protein levels of the cell cycle and apoptotic regulators (Fig. 3, A and B) to determine the potential _targets that are attributable to the altered cell cycle dynamics. The knockdown or overexpression of GCN5 elicited pleiotropic effects on a variety of genes (Fig. 3A) and marginal effect on E2F2, p53, c-myc, BCL2, BAX, and p27 (Fig. 3B). Particularly, we noticed consistent changes in cyclin D1, cyclin E1, E2F1, and CDK2 in GCN5 knockdown and overexpressing cells. These _targets were down-regulated upon GCN5 knockdown and up-regulated upon GCN5 overexpression.

We further measured the mRNA levels of E2F1, cyclin D1, cyclin E1 (Fig. 3C), Bax, Bcl2, Bcl-xl, and Bak (Fig. 3D) in these cell lines using real time quantitative-PCR (RT-qPCR). The mRNA measurements were consistent with the protein level measurements. cyclin D1, cyclin E1, and E2F1 were regulated by GCN5, whereas the apoptosis-related genes were not. The mRNA measurements not only confirmed the protein level measurements but also suggested that GCN5 transcriptionally regulates key cell cycle-related genes.

GCN5 Directly Binds to and Increases the Histone H3 and H4 Acetylation of the Cyclin E1, Cyclin D1, and E2F1 Promoters

We found that the expression levels of cyclin D1, cyclin E1, and E2F1 were markedly regulated by GCN5 at both the protein and mRNA levels. We hypothesized that GCN5 might directly regulate the expression of these genes. To test this possibility, luciferase reporters driven by the promoter regions from cyclin D1 (Addgene 32726), cyclin E1 (Addgene 8458), or E2F1 (Addgene 20950) (31–33) were used to evaluate the effect of GCN5 on the promoter activity of these genes. These promoter regions contain E2F1-binding sites, and we observed that the overexpression of E2F1 significantly increased the activities of these reporters, reflecting the robust E2F1 responsiveness of these reporters. The overexpression of GCN5 enhanced the activity of these reporters to similar levels as those induced by the overexpression of E2F1 (Fig. 4A). Chromatin immunoprecipitation (ChIP) showed that the promoter regions of these genes were significantly immunoprecipitated using the GCN5 antibody, indicating that endogenous GCN5 binds directly to the cyclin D1, cyclin E1, or E2F1 promoters (Fig. 4B). The control p53 promoter exhibited no enrichment of GCN5 (Fig. 4B, far right panel). Additional ChIP assays using the acetyl histone antibody revealed that the overexpression of GCN5 resulted in increased histone H3 and H4 acetylation at the cyclin D1, cyclin E1, and E2F1 promoters (Fig. 4C). We verified the lysine acetylation levels of histone H3 and H4 in the four cell lines that we created. Both histone H3 and H4 acetylation were increased upon GCN5 overexpression and decreased upon GCN5 knockdown (Fig. 4D). Taken together, these experiments demonstrated that GCN5 binds directly to the cyclin D1, cyclin E1, and E2F1 promoters, increasing the histone acetylation at these promoters.

E2F1 Is Required for the GCN5-mediated Regulation of Lung Cancer Cell Growth and for the G₁/S Transition

Unlike a transcription factor, the GCN5 protein does not contain a DNA-binding domain. Rather than binding to DNA directly, GCN5 is recruited by transcription factors to specific regions to regulate gene transcription. Previous studies have found that E2F1 associates with and recruits GCN5 to sites of DNA damage (34). We also noted that cyclin D1 and cyclin E1 are downstream _targets of E2F1 (35) and, furthermore, that GCN5 is enriched at the E2F1-binding site of the cyclin D1, cyclin E1, or E2F1 promoters in our ChIP assays. We hypothesized that E2F1 might be the transcription factor mediating the transcriptional function of GCN5 in lung cancer cell growth. To examine this hypothesis, we first performed co-immunoprecipitation. We found that endogenous GCN5 co-immunoprecipitated with E2F1 (Fig. 5A). Confocal microscopy analysis revealed that in A549 cells, both GCN5 and E2F1 localize within the nucleus, and both GCN5 and E2F1 signals exhibited significant degrees of overlap (Fig. 5B). The data suggest that GCN5 interacts with E2F1 in cells. We also observed significant cytoplasmic GCN5, consistent with the cytoplasmic GCN5 observed in patient cancer tissue (Fig. 1).

To further evaluate the role of E2F1 in GCN5-mediated lung cancer cell growth, we knocked down E2F1 in the GCN5-overexpressing cell line (Fig. 5C). We found that the knockdown of E2F1 abrogated the growth-promoting effects of GCN5, as evidenced by the significant accumulation of the GCN5-overexpressing E2F1 shRNA (A-G-shE2F1)-treated A549 cells in G₁ phase (Fig. 5D). The binding of GCN5 to the cyclin D1, cyclin E1, or E2F1 promoters in GCN5-overexpressing cells was significantly reduced when E2F1 was knocked down (Fig. 5, E versus F). The effect of GCN5 overexpression on cyclin D1, cyclin E1, and E2F1 promoter-driven luciferase reporters was also impaired by treatment with E2F1 siRNA. Collectively, our results suggest that GCN5 is recruited by E2F1 to the cyclin D1, cyclin E1, and E2F1 promoters to regulate lung cancer cell growth in an E2F1-dependent manner.

Overexpression of GCN5 Accelerates Tumorigenesis in Vivo

We examined whether GCN5 promotes tumor growth in vivo. Two GCN5-overexpressing cell lines (FUW-GCN5) and two control cell lines expressing an empty vector (FUW) were independently generated. The overexpression of GCN5 was assessed prior to injection into mice (Fig. 6A, upper panel). Lentiviruses encoding firefly luciferase were also introduced into these cell lines to allow for the in vivo imaging of tumor size and tumor metastasis. The ectopic overexpression of GCN5 was detectable in the isolated tumors (Fig. 6A, lower panel). We found that the time of tumor onset (defined as the day when the tumors become palpable after injection) was reduced to 7 days when GCN5 was overexpressed. The growth of tumors initiated by GCN5-overexpressing cells was significantly faster than those initiated by the control cells (Fig. 6B). At 6 weeks after injection, the tumors were isolated and weighed. The GCN5-overexpressing cells produced significantly larger tumors than the control cells (Fig. 6C). In vivo imaging also confirmed that the GCN5-overexpressing cells produced larger tumors than control cells (Fig. 6D). We further compared the protein levels of cyclin D1, cyclin E1, and E2F1 in the tumor tissues isolated from GCN5-overexpressing and control groups by immunostaining (Fig. 6E). The protein levels of all three genes in the tumors initiated by the GCN5-overexpressing cell line were increased compared with those of the tumors derived from the control cell line and correlated with the PCAN level, a growth potential marker. Our animal experiment confirmed that GCN5 potentiates lung cancer growth and promotes the expression of cyclin D1, cyclin E1, and E2F1 in vivo.