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Enhancer of Zeste Homolog 2 (EZH2) Promotes Progression of Cholangiocarcinoma Cells by Regulating Cell Cycle and Apoptosis

  • Translational Research and Biomarkers
  • Published:
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Abstract

Background

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2). When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3, resulting in epigenetic silencing of gene expression and cancer progression. We investigated the expression and function of EZH2 in intrahepatic and extrahepatic cholangiocarcinoma (ICC and ECC).

Methods

The influence of EZH2 on cell growth and apoptosis was assessed by knockdown experiments. Target gene of EZH2 was searched by quantitative RT-PCR. Clinical significance of EZH2 in 86 cholangiocarcinoma patients (45 ICC and 41 ECC) who underwent curative surgery was examined by immunohistochemistry.

Results

In vitro analysis, knockdown of EZH2 reduced cell growth, induced G1 arrest, and induced apoptosis, as confirmed by Annexin V staining and increased sub-G1 populations in cholangiocarcinoma cell lines. The expression levels of p16 INK4a and p27 KIP1 were remarkably increased by knockdown of EZH2 in these cell lines. In immunohistochemical study, EZH2 upregulation correlated with tumor diameter (p = 0.0103) in ICC, lymph node metastasis (p = 0.0292) in ECC, and Ki67 index in both ICC (p = 0.0364) and ECC (p = 0.0017). In addition, EZH2 expression was correlated with poor prognosis in both ICC (p = 0.0447) and ECC (p = 0.0227).

Conclusions

The current study demonstrated relationships between EZH2 expression and acceleration of the cell cycle and antiapoptosis, and poor prognosis in cholangiocarcinoma. These results suggest that EZH2 may represent a potential therapeutic target in patients with cholangiocarcinoma.

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Authors and Affiliations

  1. Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan

    Shigeki Nakagawa MD, Hirohisa Okabe MD, PhD, Yasuo Sakamoto MD, PhD, Hiromitsu Hayashi MD, PhD, Daisuke Hashimoto MD, PhD, Naomi Yokoyama, Keita Sakamoto MD, Hideyuki Kuroki MD, Kosuke Mima MD, PhD, Hidetoshi Nitta MD, PhD, Katsunori Imai MD, PhD, Akira Chikamoto MD, PhD, Masayuki Watanabe MD, PhD, Toru Beppu MD, PhD & Hideo Baba MD, PhD

  2. Department of Multidisciplinary Treatment for Gastroenterological Cancer, Kumamoto University Hospital, Kumamoto, Japan

    Yasuo Sakamoto MD, PhD & Toru Beppu MD, PhD

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  1. Shigeki Nakagawa MD
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  2. Hirohisa Okabe MD, PhD
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  4. Hiromitsu Hayashi MD, PhD
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Corresponding author

Correspondence to Hideo Baba MD, PhD.

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Supplementary material 1 (DOCX 21 kb)

10434_2013_3135_MOESM2_ESM.pptx

Supplementary Fig. 1. EZH2 depletion leads to growth inhibition and cell cycle arrest and apoptosis in OZ cells. a Knockdown of EZH2 protein expression analyzed by western blotting. Expression levels were compared 48 h after transfection with EZH2-targeting siRNAs or control siRNA in OZ cells. b Cell growth assay following transient transfection with synthetic control siRNA (si-control) or EZH2-targeting siRNAs (si-EZH2) in OZ cells. *p < 0.05. c Cell-cycle analyses were performed by FACS. Representative histograms for cell-cycle distribution are indicated. d Compilation of cell cycle analyses from three independent experiments. Standard deviations are indicated. *p < 0.05. e Early apoptosis was assessed by staining with PI and AnnexinV after treatment with synthetic control siRNA (si-control) or EZH2-targeting siRNAs (si-EZH2) in OZ cells. Apoptosis also was evaluated by DNA content and percentage of sub-G1 population after treatment with EZH2-siRNA by FACS analysis. Experiments were performed in triplicate. Standard deviations are indicated. *p < 0.05 (PPTX 188 kb)

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Nakagawa, S., Okabe, H., Sakamoto, Y. et al. Enhancer of Zeste Homolog 2 (EZH2) Promotes Progression of Cholangiocarcinoma Cells by Regulating Cell Cycle and Apoptosis. Ann Surg Oncol 20 (Suppl 3), 667–675 (2013). https://doi.org/10.1245/s10434-013-3135-y

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  • DOI: https://doi.org/10.1245/s10434-013-3135-y

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