Regulation of pancreatic tumor cell proliferation and chemoresistance by the histone methyltransferase enhancer of zeste homologue 2

Abstract

Purpose: Enhancer of zeste homologue 2 (EZH2), a histone methyltransferase, plays a key role in transcriptional repression through chromatin remodeling. Our objectives were to determine the expression pattern of EZH2 and to assess the anticancer effect of EZH2 depletion in pancreatic cancer cells.

Experimental design: Immunohistochemistry and cytosolic/nuclear fractionation were done to determine the expression pattern of EZH2 in normal pancreas and human pancreatic tumors. We used RNA interference, Western blotting, reverse transcription-PCR, and chromatin immunoprecipitation to study the effect of EZH2 depletion on pancreatic cancer cell proliferation and survival.

Results: We detected nuclear overexpression of EZH2 in pancreatic cancer cell lines and in 71 of 104 (68%) cases of human pancreatic adenocarcinomas. EZH2 nuclear accumulation was more frequent in poorly differentiated pancreatic adenocarcinomas (31 of 34 cases; P<0.001). We found that genetic depletion of EZH2 results in reexpression of p27(Kip1) and decreased pancreatic cancer cell proliferation. Moreover, we showed that EZH2 depletion sensitized pancreatic cancer cells to doxorubicin and gemcitabine, which leads to a significant induction of apoptosis, suggesting that the combination of EZH2 inhibitors and standard chemotherapy could be a superior potential treatment for pancreatic cancer.

Conclusions: Our results show nuclear accumulation of EZH2 as a hallmark of poorly differentiated pancreatic adenocarcinoma; identify the tumor suppressor p27(Kip1) as a new target gene of EZH2; show that EZH2 nuclear overexpression contributes to pancreatic cancer cell proliferation; and suggest EZH2 as a potential therapeutic target for the treatment of pancreatic cancer.

Figure 1. EZH2 is overexpressed and accumulated in the nucleus of pancreatic cancer cells

(A-F) Immunohistochemical analysis of EZH2 expression and localization in (A) normal human pancreas and (B-F) pancreatic adenocarcinoma specimens. A, normal pancreatic duct (NPD) is indicated. B, Malignant pancreatic duct shows nuclear accumulation of EZH2, whereas adjacent normal pancreatic ductal cells show no nuclear EZH2 staining. Pancreatic cancer cells (PC). C, EZH2 nuclear accumulation in a poorly differentiated pancreatic adenocarcinoma. D, Nuclear accumulation of EZH2 found in cancer cells of moderately differentiated pancreatic adenocarcinoma but not in adjacent normal pancreatic ductal cells. E, Higher magnification of the delineated inset of (D) image. F, Nuclear accumulation of EZH2 in cancer cells of well differentiated pancreatic adenocarcinoma. G, Distribution of EZH2 staining patterns in pancreatic carcinomas. Nuclear accumulation (NA). H, Equivalent amounts (50 μg) of nuclear and cytosolic proteins isolated from the indicated pancreatic cancer cell lines and normal human pancreas tissue were separated by SDS-PAGE and immunoblotted.

Figure 2. Suppression of EZH2 inhibits pancreatic cancer cell proliferation

MIA-PaCa2 and Panc04.03 pancreatic cancer cells were transfected with a control vector (shVector) or the shEZH2 silencing vector (shEZH2). A, 48 hours post-transfection, the cell pellet was collected and protein was obtained. Cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to indicated proteins. B, Relative cell viability was measured using colorimetric MTS assay at indicated times as described (13). C, Numbers of viable cells were counted by the trypan blue exclusion assay as described (30).

Figure 3. Depletion of EZH2 leads to re-expression of p27^Kip1 in pancreatic cancer cells

MIA-PaCa2 and Panc04.03 pancreatic cancer cells were transfected with a control vector (shVector) or the shEZH2 silencing vector (shEZH2). A, 48 hours post-transfection, the cell pellet was collected, mRNA was obtained and RT-PCR was performed as described in Materials and Methods. Increased expression of MYT1, a known EZH2 target gene, was used as a positive control. B, 48 hours post-transfection, the cell pellet was collected, protein was obtained. Cell lysates were separated by SDS-PAGE, transferred to PVDF membrane, and probed with antibodies to indicated proteins. C, 48 hours post-transfection, Panc04.03 cell pellet was collected. Panc04.03 cancer cells were treated with DMSO (Control) or MG132 (10 μmol/L) for 6 hours. Cell lysates were prepared, separated by SDS-PAGE (50μg/well), transferred to PVDF membrane, and immunoblotted as indicated. An increase of IκBα expression was used as a positive marker of MG132 treatment. D, Binding of EZH2 to the promoter or exon 1 of p27^Kip1 gene was assayed in Panc04.03 cancer cells by ChIP. E, Panc04.03 cancer cells were transfected with a control vector (shVector) or the shEZH2 silencing vector (shEZH2). 48 hours post-transfection, genomic chromatin fragments were immunoprecipitated with trimethyl-H3-K27 and acetyl-H3-K14 at the p27^Kip1 promoter or exon 1. PCR analysis on input chromatin (first 2 lanes) confirmed that equal chromatin amounts were used for ChIP. (F, G) Immunohistochemical staining of a pair of serial sections from the same pancreatic tumor demonstrates nuclear localization of EZH2 (F) and loss of p27^Kip1 expression (G) in the same cancer cells, whereas absence of EZH2 nuclear staining, but p27^Kip1 nuclear accumulation has been observed in benign pancreatic lesions (G, F). Pancreatic intraepithelial neoplasia (PanIN).

Figure 4. EZH2 depletion affects pancreatic cancer chemoresistance

A, Panc04.03 and SU86.86 pancreatic cancer cells were treated with DW (control), 1 μmol/L Doxorubicin (DOX) or 100 nmol/L Gemcitabine (GEM) for 24 hours. Nuclear and cytosolic fractions were prepared, separated by SDS-PAGE (50μg/well), transferred to PVDF membrane, and probed with the indicated antibodies. (B-C), Panc04.03 (B) and SU86.86 (C) pancreatic cancer cells were treated with DW (control), Doxorubicin (1 μmol/L) or Gemcitabine (100 nmol/L) for 48 hours. Whole cell lysates were prepared, separated by SDS-PAGE (50μg/well), transferred to PVDF membrane, and immunoblotted as indicated. The percentage of apoptotic cells was determined by Hoechst staining as previously described (31). Columns, mean; bars, SD.