NF-YA underlies EZH2 upregulation and is essential for proliferation of human epithelial ovarian cancer cells

Abstract

Epithelial ovarian cancer (EOC) accounts for the most gynecologic malignancy-associated deaths in the United States. Enhancer of zeste homolog 2 (EZH2), which silences gene expression through generating trimethylation on lysine 27 residue of histone H3 (H3K27Me3), is often overexpressed in EOCs and has been suggested as a therapeutic target. However, the mechanism underlying EZH2 overexpression in EOCs is unknown. Here, we show that EZH2 is upregulated at the transcription level, and two CCAAT boxes in the proximal regions of the human EZH2 gene promoter are critical for its transcription in EOC cells. Indeed, NF-YA, the regulatory subunit of the CCAAT-binding transcription factor NF-Y, is expressed at higher levels in human EOCs than in primary human ovarian surface epithelial (HOSE) cells. In addition, there is a positive correlation between expression of NF-YA and EZH2 in EOCs. Notably, high NF-YA expression predicts shorter overall survival in patients with EOCs. The association of NF-YA with the promoter of the human EZH2 gene is enhanced in human EOC cells compared with primary HOSE cells. Significantly, knockdown of NF-YA downregulates EZH2, decreases H3K27Me3 levels, and suppresses the growth of human EOC cells both in vitro and in a xenograft mouse model. Notably, NF-YA knockdown induces apoptosis of EOC cells and ectopic EZH2 expression partially rescues apoptosis induced by NF-YA knockdown. Together, these data reveal that NF-Y is a key regulator of EZH2 expression and is required for EOC cell proliferation, thus representing a novel target for developing EOC therapeutics.

Figure 1. EZH2 is upregulated at the transcriptional level and two CCAAT sites within proximal region of the human EZH2 gene promoter are critical for its transcriptional activation in human epithelial ovarian cancer (EOC) cells

(A) EZH2 mRNA level was determined by qRT-PCR in human ovarian surface epithelial (HOSE) cells and indicated EOC cell lines. Mean of three independent experiments with SD. (B) SKOV3 EOC cells were transfected with a luciferase reporter driven by the indicated proximal promoter fragment of the human EZH2 gene. 12 hours post transfection, Luciferase reporter activity was determined. A luminescent β-gal reporter (Clontech) was used to normalize the transfection efficacy. Mean of three independent experiments with SD. (C) Two CCAAT sites in proximal region of the human EZH2 gene promoter were mutated to CCAAC, which is known to be sufficient to inhibit the binding of the CCAAT-binding transcription factor, NF-Y (38). Note that the first CCAAT site is inverted. (D) Luciferase activity of wild type EZH2 (WT) gene promoter compared to CCAAT sites mutated EZH2 (MT) gene promoter as diagramed in (C). Mean of three independent experiments with SD.

Figure 2. NF-YA expression positively correlates with EZH2 expression in human EOC cells and a high level of NF-YA predicts shorter overall survival in EOC patients

(A) Expression of NF-YA, EZH2, H3K27Me3 and histone H3 in indicated human EOC cell lines and normal HOSE cells determined by immunoblotting. Note that the arrow points to the long NF-YA isoform and the arrowhead points to the short NF-YA isoform. (B) Normal HOSE cells were infected with a lentivirus encoding NF-YA or control. Drug-selected cells were examined for expression of NF-YA, EZH2 and β-actin by immunoblotting. (C) NF-YA is expressed at higher levels in human EOCs compared with normal HOSE cells. Relative expression of NF-YA mRNA in laser capture and microdissected high-grade serous EOC (n = 53) and normal HOSE cells (n = 10). (D) Same as (C) but for expression of EZH2 mRNA. (E) NF-YA expression positively correlates with EZH2 expression. Correlation between expression of NF-YA and EZH2 was determined by Spearman statistical analysis. (F) High NF-YA expression predicts a shorter overall survival in EOC patients. Kaplan-Meier survival curve for EOC patients with low or high NF-YA expression. (G) NF-YA is expressed at higher levels in human EOCs compared with primary non-malignant human fallopian tube epithelial (FTE) cells. Relative expression of NF-YA mRNA in high-grade serous EOC (n = 13) and laser capture and microdissected primary non-malignant FTE cells (n = 24).

Figure 3. The association of NF-YA and its co-activator p300 with the human EZH2 gene promoter is enhanced in human EOC cells compared with normal HOSE cells

(A) Diagram of the human EZH2 gene promoter with indicated positions of primers used for proximal (−64/+141) and distal (−875/−568) regions of the human EZH2 gene promoter in ChIP analysis. (B) Normal HOSE cells or indicated EOC cell lines were subjected to ChIP analysis using antibodies specific to NF-YA or p300, respectively. An isotype matched IgG was used as a negative control, and antibody against core histone H3 was used as a positive control. After ChIP analysis, the distal and proximal regions of the human EZH2 gene promoter were subjected to PCR amplification using primers detailed in materials and methods and as diagramed in (A). (C) Same as (B) but using an antibody against acetylated histone H3.

Figure 4. NF-YA knockdown suppresses the expression of EZH2 and inhibits the growth of human EOC cells in vitro and in a xenograft EOC mouse model

(A) SKOV3 EOC cells were infected with lentivirus encoding the indicated shNF-YAs or control. Drug-selected cells were examined for expression of NF-YA, EZH2, H3K27Me3 and H3K9Me3 by immunoblotting. Expression of core histone H3 was used as a loading control. (B) Same as (A), but equal number of drug-selected cells was seeded and counted at indicated time points. * p < 0.05 compared with either control or shNF-YA #3. Mean of three independent experiments with SD and linear regression. (C) Same as (A), but equal number of drug-selected cells were assayed for anchorage-independent growth in soft agar. Mean of three independent experiments with SD. * p < 0.05 compared with either control or shNF-YA #3. (D) Same as (A), but equal number of the indicated cells were subcutaneously injected in immunocompromised nude mice (n = 5). Four weeks post injection, tumors were removed from mice. (E) Quantification of (D), the size of tumors was measured. Error bars represent SD. * p < 0.05 compared with either control or shNF-YA #3.

Figure 5. NF-YA knockdown induces apoptosis of human EOC cells

(A) SKOV3 EOC cells were infected with lentivirus encoding the indicated shNF-YAs or control. Guava Nexin assay was performed at the indicated time points. (B) Quantification of (A). Mean of three independent experiments with SD. * p < 0.05 compared with either control or shNF-YA #3. (C) Same as (A) but examined for expression of apoptosis markers, cleaved caspase3 and cleave PARP p85 by immunoblotting. (D) Same as (A) but examined for HRK gene expression by qRT-PCR. * p < 0.05 compared with either control or shNF-YA #3. (E) SKOV3 EOC cells were engineered to express a Tet-inducible EZH2. These cells were infected with a lentivirus shNF-YA with or without simultaneous induction of EZH2 expression. Drug-selected cells were examined for expression of NF-YA, EZH2 and indicated apoptosis markers by immunoblotting. Expression of core histone H3 was used a loading control.