Selective inhibition of esophageal cancer cells by combination of HDAC inhibitors and Azacytidine

Abstract

Esophageal cancers are highly aggressive tumors with poor prognosis despite some recent advances in surgical and radiochemotherapy treatment options. This study addressed the feasibility of drugs targeting epigenetic modifiers in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) cells. We tested inhibition of histone deacetylases (HDACs) by SAHA, MS-275, and FK228, inhibition of DNA methyltransferases by Azacytidine (AZA) and Decitabine (DAC), and the effect of combination treatment using both types of drugs. The drug targets, HDAC1/2/3 and DNMT1, were expressed in normal esophageal epithelium and tumor cells of ESCC or EAC tissue specimens, as well as in non-neoplastic esophageal epithelial (Het-1A), ESCC (OE21, Kyse-270, Kyse-410), and EAC (OE33, SK-GT-4) cell lines. In vitro, HDAC activity, histone acetylation, and p21 expression were similarly affected in non-neoplastic, ESCC, and EAC cell lines post inhibitor treatment. Combined MS-275/AZA treatment, however, selectively targeted esophageal cancer cell lines by inducing DNA damage, cell viability loss, and apoptosis, and by decreasing cell migration. Non-neoplastic Het-1A cells were protected against HDACi (MS-275)/AZA treatment. RNA transcriptome analyses post MS-275 and/or AZA treatment identified novel regulated candidate genes (up: BCL6, Hes2; down: FAIM, MLKL), which were specifically associated with the treatment responses of esophageal cancer cells. In summary, combined HDACi/AZA treatment is efficient and selective for the targeting of esophageal cancer cells, despite similar target expression of normal and esophageal cancer epithelium, in vitro and in human esophageal carcinomas. The precise mechanisms of action of treatment responses involve novel candidate genes regulated by HDACi/AZA in esophageal cancer cells. Together, targeting of epigenetic modifiers in esophageal cancers may represent a potential future therapeutic approach.

Keywords: 5mC, 5-methylcytidine; AZA, Azacytidine; DAC, Decitabine; DNMT, DNA (cytosine-5)-methyltransferase; EAC, esophageal adenocarcinoma; ESCC, esophageal squamous cell carcinoma; FAIM, Fas apoptotic inhibitory molecule; GEJ, gastro-esophageal junction; H3Ac, histone H3 acetylation; H3K4me3, histone H3 trimethylation at lysine 4; H3K9Ac, histone 3 lysine 9 acetylation; HDAC, histone deacetylases; HDACi, HDAC inhibitor; Hes-2, Hairy and enhancer of split 2; SAHA, suberoylanilide hydroxamic acid; TSA, Trichostatin A; azacytidine/gene pathway regulation; epigenetics/HDAC inhibitor; esophageal cancer.

Figure 1.

HDACs are deregulated in esophageal cancer cells. The panels show representative serial sections, respective the same tissue areas, of matching normal esophageal epithelium and ESCC or EAC tissue specimens stained for the epigenetic modifiers HDAC1, HDAC2, HDAC3, H3K9ac (red staining; via AP/Streptavidin, DAKO Real Detection system AP/RED) as well as DNMT1 and 5mC (brown staining, via DAB, DAKO envision FLEX+ Kit). Note reduced H3K9ac as well as loss of DNMT1 expression and 5mC (see arrows) in tumor cells of ESCCs and EACs as compared to normal epithelial cells (see inserts). Refer to Supplementary Data Table S1 for quantification of all cases. Bar represents 100 μm.

Figure 2.

HDACs are deregulated in esophageal cancer cells. (A) Indirect immunofluorescence staining revealed nuclear localization for DNMT1 and HDAC1, HDAC2, and HDAC3 in all 7 cell lines. (B) Protein levels were analyzed by immunoblotting, shown by one representative blot (left) and quantification by densitometry (right), revealing maintained HDAC1, HDAC2, HDAC3 and DNMT1 expression in most esophageal cancer cell lines, but decreased HDAC1, HDAC2, and HDAC3 expression in OE21 and OE33 compared to Het-1A cells. (C) Measurement of general HDAC activity revealed increased HDAC activity in all esophageal cancer cell lines (except OE19 as GEJ cell line) compared to Het-1A cells (dark gray bars). Trichostatin A (TSA) as positive control shows adequate reduction of HDAC activity in all cell lines, proving specificity of the fluorescence signal. Data is represented as mean ± SEM for 3 independent experiments. All activity measurements were performed in technical duplicates. Significance levels are represented as *: 0.05–0.01, **: ≤ 0.01–0.001 and ***: ≤ 0.001.

Figure 3.

Dose response curves for the effect of HDACi/AZA on cell viability. Cells were cultured with DMSO (=0 on x-axis) and various doses of (A) SAHA, (B) MS-275, (C) FK228 as well as (D, E) AZA or (F) DAC for 72 h before cell viability was analyzed. To rule out known difficulties with AZA drug stability, cells were treated with (D) continuous AZA or (E) daily refreshment of AZA. (G-I) Cells were treated with 50 μM AZA and 2 different concentrations of each HDACi for 72 h before cell viability was determined. Data is represented as percent in comparison to vehicle (DMSO) treated cells (set to 100%). Shown is the mean ± SEM for 3 independent experiments, each performed in technical triplicate. Significance levels are represented as *: 0.05–0.01, **: ≤ 0.01–0.001 and ***: ≤ 0.001.

Figure 4.

Downregulation of HDAC activity by HDACi. HDAC activity was measured 24 h post HDACi/AZA addition, showing similar downregulation of HDAC activity in all 7 cell lines. Inhibitor concentrations: SAHA = 0.1 μM, MS-275 = 0.5 μM and FK228 = 0.1 nM. Shown is the mean ± SEM of 3 independent experiments, performed in technical duplicates. Significance levels are represented as *: 0.05–0.01, **: ≤ 0.01–0.001 and ***: ≤ 0.001.

Figure 5.

HDACi/AZA treatment induces DNA damage, apoptosis and cell cycle arrest selectively in esophageal cancer cells. (A) DNA damage was assessed by COMET assays 24 h after addition of MS-275 and/or AZA, revealing induction of DNA damage in OE21 (AZA, MS-275-AZA) and OE33 (MS-275/AZA) cells. The total number of analyzed cells, respective COMETs, by CASP software V1.2.2 is given in the bars. (B) Measurement of apoptosis 72 h post HDACi and/or AZA treatment showed cancer cell selective effects, especially of combined MS-275 and AZA treatment in OE21 and OE33 cells. Apoptosis data is presented as percent of 10 000 recorded cells. (C) S phase distribution and (D) Ki-67 expression was analyzed 72 h post HDACi/AZA treatment. Abbreviations: C = control/DMSO treatment, A = AZA, S = SAHA, M = MS-275 and F = FK228. Shown is the mean ± SEM for 3 independent experiments. Obtained p-values were represented as *: 0.05–0.01 and **: ≤ 0.01–0.001.

Figure 6.

Azacytidine impairs migration specifically in esophageal cancer cells. (A) Cell viability measurement 24 h post inhibitor treatment. Shown is the mean ± SEM for 3 independent experiments, performed in technical triplicates. (B) Cell migration was analyzed 24 h post inhibitor treatment by cell exclusion assay. Cell migration was quantified 24 h post MS-275/AZA addition. Data is represented as the log2 fold change of the cell free area (mean ± SEM) of 3 independent experiments. Representative images are shown in (C).

Figure 7.

RNA transcriptome analyses reveal differentially regulated genes by MS-275/AZA treatment. (A) The diagram represents the absolute numbers of significantly upregulated (red bars) and downregulated (green bars) genes for MS-275, AZA and their combination (comb.) compared to vehicle (DMSO) treated cells. (B) Number of genes regulated by MS-275/AZA treatment depicted by Venn diagram, showing joint or unique regulation between cell lines. Using the curated gene sets from the Consensus Path DB, all gene sets related to apoptosis, drug metabolism, DNA replication and damage as well as cell cycle were extracted, depicting (C) upregulated and (D) downregulated gene sets from OE21, OE33 and Het-1A cell lines. (E) Regulation of CDKN1A/p21 mRNA in the present RNA transcriptome were confirmed (F) by qRT-PCR on mRNA and (G) by immunoblot on protein level for all 3 HDACi/AZA combinations. (H) ChIP analysis was performed 24 h post MS-275/AZA treatment with H3Ac and H3K4me3 followed by real-time PCR of the CDKN1A promotor. Data are represented as fold change of % input DNA (after normalization to H3) compared to vehicle (DMSO) treated cells (mean ± SEM).

Figure 8.

Validation of novel candidate genes associated with specific response of esophageal cancer cells. RNA transcriptome data of 4 selected TOP40 genes is shown as expression levels post 24 h of HDACi/AZA treatment in the left panels. Results of validation by qRT-PCR for relative mRNA levels is shown in the right panels. Shown is the mean ± SEM for 3 independent experiments (right hand). (A) BCL6 (rank #2) and Hes2 (rank #27) were selected as upregulated genes and (B) FAIM (rank #4) and MLKL (rank #9) were selected as downregulated genes. Refer to Table 1 for ranking of the TOP40 gene list.