ALDH1A1 is a novel EZH2 target gene in epithelial ovarian cancer identified by genome-wide approaches

Abstract

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy in the United States. EZH2 silences gene expression through trimethylating lysine 27 on histone H3 (H3K27Me3). EZH2 is often overexpressed in EOC and has been suggested as a target for EOC intervention. However, EZH2 target genes in EOC remain poorly understood. Here, we mapped the genomic loci occupied by EZH2/H3K27Me3 using chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and globally profiled gene expression in EZH2-knockdown EOC cells. Cross-examination of gene expression and ChIP-seq revealed a list of 60 EZH2 direct target genes whose expression was upregulated more than 1.5-fold upon EZH2 knockdown. For three selected genes (ALDH1A1, SSTR1, and DACT3), we validated their upregulation upon EZH2 knockdown and confirmed the binding of EZH2/H3K27Me3 to their genomic loci. Furthermore, the presence of H3K27Me3 at the genomic loci of these EZH2 target genes was dependent upon EZH2. Interestingly, expression of ALDH1A1, a putative marker for EOC stem cells, was significantly downregulated in high-grade serous EOC (n = 53) compared with ovarian surface epithelial cells (n = 10, P < 0.001). Notably, expression of ALDH1A1 negatively correlated with expression of EZH2 (n = 63, Spearman r = -0.41, P < 0.001). Thus, we identified a list of 60 EZH2 target genes and established that ALDH1A1 is a novel EZH2 target gene in EOC cells. Our results suggest a role for EZH2 in regulating EOC stem cell equilibrium via regulation of ALDH1A1 expression.

Figure 1. Identification of EZH2 target genes in human SKOV3 EOC cells

(A) SKOV3 cells were infected with indicated lentivirus encoding shEZH2 or control. Drug-selected cells were examined for expression of EZH2, H3K27Me3 and GAPDH by immunoblotting analysis using indicated antibodies. (B) Schematic of experimental strategies used to identify EZH2 target genes. Genes whose expression was upregulated >1.5-fold upon EZH2 knockdown by two individual shEZH2 were identified by global gene expression microarray analysis. Genomic loci occupied by EZH2/H3K27Me3 were profiled by ChIP-seq analysis. (C) Cross-examination of gene expression profiling and ChIP-seq analysis as illustrated in (B) revealed a list of 60 putative EZH2 target genes in human SKOV3 EOC cells.

Figure 2. Validation of upregulation of the selected EZH2 silenced target genes in SKOV3 human EOC cells upon EZH2 knockdown by qRT-PCR

(A) SKOV3 cells were infected with lentivirus encoding the indicated shEZH2s or control. After drug selection, mRNA was extracted and examined for expression of ALDH1A1 mRNA by qRT-PCR. Expression of β-2-microglobulin was used to normalize the expression of ALDH1A1 mRNA. * p < 0.05 compared with controls. (B) Same as (A), but for SSTR1 mRNA expression. * p < 0.05 compared with controls. (C) Same as (A), but for DACT3 mRNA expression. * p < 0.05 compared with controls.

Figure 3. Validation of occupancy of the genomic loci of the selected EZH2 target genes by EZH2 and H3K27Me3 in SKOV3 human EOC cells using ChIP analysis

Control and shEZH2 #2 expressing SKOV3 cells were subjected to ChIP analysis using antibodies specific to EZH2 or H3K27Me3, respectively. An isotype matched IgG was used as a negative control, and an antibody specific to core histone H3 was used as a positive control. After ChIP analysis, the genomic loci of the indicated genes were subjected to PCR amplification using primers detailed in materials and methods. Please see Figure 1A for shEZH2 knockdown efficacy. Shown are representative images of 3 independent experiments. Note that for H3K27Me3 ChIP, a low number of PCR cycles were used compared with EZH2 or histone H3 ChIP to avoid over saturation of signals.

Figure 4. EZH2 targets ALDH1A1 in high-grade serous subtype EOC

(A) Relative expression of EZH2 mRNA in 53 cases of laser capture and microdissected (LCM) high-grade serous EOC and 10 individual isolations of normal human ovarian surface epithelial (HOSE) cells. (B) Quantitation of (A), * p < 0.001 compared with high-grade serous EOC. (C) Same as (A), but for relative expression of ALDH1A1 mRNA. (D) Quantitation of (C), * p < 0.001 compared with high-grade serous EOC. (E) Correlation between expression of ALDH1A1 and EZH2 as determined by Spearman’s statistical analysis using GraphPad Prism version 5.0 software.