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. 2016 Jul;107(7):955-62.
doi: 10.1111/cas.12957. Epub 2016 Jun 13.

Combined inhibition of EZH2 and histone deacetylases as a potential epigenetic therapy for non-small-cell lung cancer cells

Taichi Takashina  1 , Ichiro Kinoshita  2 , Junko Kikuchi  1 , Yasushi Shimizu  2 , Jun Sakakibara-Konishi  1 , Satoshi Oizumi  1 , Masaharu Nishimura  1 , Hirotoshi Dosaka-Akita  2
Affiliations

Combined inhibition of EZH2 and histone deacetylases as a potential epigenetic therapy for non-small-cell lung cancer cells

Taichi Takashina et al. Cancer Sci. 2016 Jul.

Abstract

Recent discoveries have revealed that human cancer involves aberrant epigenetic alterations. We and others have previously shown that the histone methyltransferase EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), is frequently overexpressed in non-small-cell lung cancer (NSCLC) and that an EZH2 inhibitor, 3-deazaneplanocin A, inhibits the proliferation of NSCLC cells. Transcriptional silencing by EZH2 was recently shown to be required for the activity of histone deacetylases (HDACs) that interact with another PRC2 protein, EED. To develop a more effective epigenetic therapy for NSCLC, we determined the effects of co-treatment with 3-deazaneplanocin A and the HDAC inhibitor vorinostat (SAHA) in NSCLC cells. The co-treatment synergistically suppressed the proliferation of all tested NSCLC cell lines, regardless of their epidermal growth factor receptor (EGFR) status. The synergistic effect was associated with slightly decreased histone H3 lysine 27 trimethylation, modestly increased histone acetylation, and the depletion of EZH2 and other PRC2 proteins. The co-treatment resulted in an accumulation of p27Kip1, decrease in cyclin A, and increased apoptotic fraction in an additive/synergistic manner. Interestingly, the co-treatment strongly suppressed EGFR signaling, not only in EGFR-wild-type NSCLC cells, but also in EGFR-mutant cells, mainly through dephosphorylation of EGFR. Furthermore, the co-treatment suppressed the in vivo tumor growth of EGFR-mutant, EGFR-tyrosine kinase-resistant H1975 cells more effectively than did each agent alone, without visible toxicity. These results suggest that the combined pharmacological targeting of EZH2 and HDACs may provide more effective epigenetic therapeutics for NSCLC.

Keywords: 3-Deazaneplanocin A; EZH2; lung cancer; polycomb-group protein; vorinostat (suberoylanilide hydroxamic acid).

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Figures

Figure 1
Figure 1
Four human non‐small‐cell lung cancer cell lines (H1299, H1975, A549, and PC‐3) were treated with suberoylanilide hydroxamic acid (SAHA) for 72 h and subjected to an MTT‐based assay. Data are representative of three independent experiments. Data represent mean ± SD of triplicate samples. Similar results were obtained from all three independent experiments.
Figure 2
Figure 2
Combined therapy with 3‐deazaneplanocin A (DZNep) and suberoylanilide hydroxamic acid (SAHA) synergistically inhibited non‐small‐cell lung cancer cell (NSCLC) proliferation. (a) Four human NSCLC cell lines were treated with DZNep (dose range, 0.05–0.8 μM) and SAHA (dose range, 0.5–8 μM) at a fixed ratio of 1:10 for 72 h and subjected to MTT‐based assay. (b) Four human NSCLC cell lines were treated with DZNep (dose range, 0.025–0.4 μM) and SAHA (dose range, 0.5–8 μM) at a fixed ratio of 1:20 for 72 h and subjected to MTT‐based assay. Combination index (CI) values were determined using the commercially available software, Calcusyn. Data are representative of three independent experiments. Similar results were obtained in all three independent experiments.
Figure 3
Figure 3
Combined therapy with 3‐deazaneplanocin A (DZNep) (D) and suberoylanilide hydroxamic acid (SAHA) (S) depleted PRC2 proteins and decreased the histone methylation and acetylation levels more effectively than did individual treatment, in non‐small‐cell lung cancer cells. Cells were treated with 0.2 μM DZNep and/or 2 μM SAHA for 72 h. Total cell lysates were then harvested and subjected to Western blot analysis. (a) Representative Western blots of EZH2, SUZ12, EED, trimethylation of lysine 27 on histone H3 (H3K27me3), cyclin A, p27Kip1, and actin from three independent experiments are shown. Actin levels in the lysates served as the loading control (C). Similar results were obtained in all three independent experiments. (b) Representative Western blots of acetylation of lysine (Lys) 9, 14, 18, 27, and 56 of histone H3 and total histone H3 from three independent experiments are shown. Total histone H3 levels in the lysates served as the loading control. Similar results were obtained in all three independent experiments.
Figure 4
Figure 4
Combined therapy with 3‐deazaneplanocin A (DZNep) (D) and suberoylanilide hydroxamic acid (SAHA) (S) induced more apoptosis than individual treatments, in non‐small‐cell lung cancer cells. (a) Flow cytometric analysis of apoptosis with annexin V–FITC and propidium iodide staining. Cells were treated with 0.2 μM DZNep and/or 2 μM SAHA for 72 h. The percentage of apoptotic cells was measured using a flow cytometer. Data represent mean ± SD of triplicate samples. Similar results were obtained in all three independent experiments. *P < 0.05 and ** P < 0.01 between indicated groups by one‐way anova with Tukey's multiple comparison test. (b) Cells were treated with 0.2 μM DZNep and/or 2 μM SAHA for 72 h. Total cell lysates were then harvested and subjected to Western blot analysis. Representative Western blots of cleaved poly(ADP‐ribose) polymerase (PARP), cleaved caspase‐3, and actin are shown. Actin levels in the lysates served as the loading control (C). Similar results were obtained in all three independent experiments. EGFR, epidermal growth factor receptor; Ex19 del, exon 19 deletion.
Figure 5
Figure 5
Combined therapy with 3‐deazaneplanocin A (DZNep) (D) and suberoylanilide hydroxamic acid (SAHA) (S) suppressed the epidermal growth factor receptor EGFR signaling pathway in both EGFR‐wild‐type and EGFR‐mutant non‐small‐cell lung cancer cells. Cells were incubated with 0.2 μM DZNep and/or 2 μM SAHA for 72 h. The cell lysates were then harvested and subjected to Western blot analysis. Representative Western blots of EGFR, phosphorylated (p‐)EGFR, protein kinase B (AKT), p‐AKT, extracellular signal‐regulated kinase (ERK)1/2, p‐ERK1/2, and actin are shown. Actin levels in the lysates served as the loading control (C). Similar results were obtained in all three independent experiments. Ex19 del, exon 19 deletion.
Figure 6
Figure 6
Effects of combined therapy with 3‐deazaneplanocin A (DZNep) (D) and suberoylanilide hydroxamic acid (SAHA) (S) on β‐catenin, a transcriptional activator of epidermal growth factor receptor (EGFR), NKD1, and PPP2R2B, which are the direct epigenetic targets of EZH2 involved in β‐catenin regulation. Cells were incubated with 0.2 μM DZNep and/or 2 μM SAHA for 72 h. Cell lysates were harvested and subjected to Western blot analysis. (a) Representative Western blots of NKD1, PPP2R2B, and actin are shown. Actin levels in the lysates served as the loading control (C). Similar results were obtained in all three independent experiments. (b) Representative Western blots of β‐catenin, cyclin D1, and actin are shown. Actin levels in the lysates served as the loading control (C). Similar results were obtained in all independent experiments. Ex19 del, exon 19 deletion.
Figure 7
Figure 7
Combined therapy with 3‐deazaneplanocin A (DZNep) (D) and suberoylanilide hydroxamic acid (SAHA) (S) inhibited in vivo tumor growth of H1975 xenografts. (a) H1975 cells (5 × 106 cells/mouse) were s.c. implanted into the flanks of BALB/cAJcl‐nu/nu nude mice. After the tumor volume reached approximately 50–100 mm3, the following treatments were given to cohorts of five mice for each treatment: vehicle alone (5% DMSO) (Control), 4 mg/kg DZNep, 40 mg/kg SAHA, or 4 mg/kg DZNep plus 40 mg/kg SAHA twice per week i.p. for 6 weeks. Tumor volume was calculated using the equation 1/2 (length × width2). Data represent means ± SD of quintuple samples. *P < 0.01 between indicated groups at day 39 by one‐way anova with Tukey's multiple comparison test. (b) Body weights were measured at the indicated times. Data represent mean ± SD of quintuple samples. (c) Western blot analysis of H1975 xenograft. Proteins were extracted from tumor tissues soon after the mice were killed. Representative Western blots of EZH2, trimethylation of lysine 27 on histone H3 (H3K27me3), epidermal growth factor receptor (EGFR), phosphorylated (p‐)EGFR, protein kinase B (AKT), p‐AKT, extracellular signal‐regulated kinase (ERK)1/2, p‐ERK1/2, and actin are shown. Actin levels in the lysates served as the loading control.
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