Histone demethylase JMJD2B is required for tumor cell proliferation and survival and is overexpressed in gastric cancer

https://doi.org/10.1016/j.bbrc.2011.11.045Get rights and content

Abstract

Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Jumonji domain containing 2B (JMJD2B) is a newly identified histone demethylase that regulates chromatin structure or gene expression by removing methyl residues from trimethylated lysine 9 on histone H3. Recent observations have shown oncogenic activity of JMJD2B. We explored the functional role of JMJD2B in cancer cell proliferation, survival and tumorigenesis, and determined its expression profile in gastric cancer. Knocking down JMJD2B expression by small interfering RNA (siRNA) in gastric and other cancer cells inhibited cell proliferation and/or induced apoptosis and elevated the expression of p53 and p21CIP1 proteins. The enhanced p53 expression resulted from activation of the DNA damage response pathway. JMJD2B knockdown markedly suppressed xenograft tumor growth in vivo in mice. Moreover, JMJD2B expression was increased in primary gastric-cancer tissues of humans. Thus, JMJD2B is required for sustained proliferation and survival of tumor cells in vitro and in vivo, and its aberrant expression may contribute to the pathogenesis of gastric cancer.

Highlights

► JMJD2B is required for cell proliferation and in vivo tumorigenesis. ► JMJD2B depletion induces apoptosis and/or cell cycle arrest. ► JMJD2B depletion activates DNA damage response and enhances p53 stabilization. ► JMJD2B is overexpressed in human primary gastric cancer.

Introduction

Gastric cancer is one of the most common malignancies and second leading cause of cancer-related death worldwide [1], [2]. Numerous studies have demonstrated the role of Helicobacter pylori infection in gastric malignant transformation. Persistent infection with H. pylori causes chronic gastritis, which initiates the pathogenesis of gastric cancer. However, the progression into invasive cancer is a multistep process with numerous alterations, with the underlying molecular mechanism poorly understood [1], [2], [3], [4]. In addition, because of lack of reliable early diagnostic markers, the prognosis of gastric cancer remains poor, with a low 5-year survival rate [2]. Therefore, better defining the pathogenesis of gastric cancer and exploring novel therapeutic targets is urgent.

Posttranslational modifications of histones regulate chromatin structure and gene expression. Histone demethylases (HDMs) remove methyl groups from the lysine residues of the histone tails, thereby regulating the transcriptional activity of target genes [5], [6]. Jumonji domain containing 2B (JMJD2B) is a newly identified member of the histone demethylase JMJD2 family that is characterized by the catalytic Jumonji C (JmjC) domain. JMJD2B specifically targets the trimethylated lysine 9 of histone H3 (H3K9) for demethylation at pericentric heterochromatin and euchromatin [7], [8], [9]. Abnormalities in the balance in methylation status of histones because of dysregulated histone methyltransferases and demethylases have been linked to cancer [10], [11]. Recent studies have indicated the role of JmjC proteins in tumorigenesis [12], [13], [14], [15]. Members of the JMJD2 family that target H3K9me3/me2 and H3K36me3/me2 are highly expressed in prostate cancer. JMJD2C/GASC1 associates with the androgen receptor and promotes proliferation of prostate cancer cells [16]. The gene encoding JMJD2C is amplified in esophageal cancer cell lines and is required for their proliferation [12], [17]. Recently, JMJD2B was found as a direct target of hypoxia-inducible factor 1 α (HIF1α) and regulated by HIF1α and estrogen receptor α (ERα) in breast cancer [15], [18] and promoted hormonally responsive breast carcinogenesis [9], [19]. However, few data exist on the expression and role of JMJD2B in other human cancers.

In the present study, we explored the potential oncogenic activities of JMJD2B and its expression profile in human gastric cancer.

Section snippets

Cell lines and culture conditions

Human gastric cancer cell lines AGS, HGC-27 and BGC-823, and cervical cancer cell line HeLa were cultured at 37 °C, 95% air, 5% CO2 in RPMI 1640 (Invitrogen) containing 10% fetal calf serum, 100 U/mL penicillin (Sigma) and 2 mmol/L l-glutamine.

Small interfering RNA (siRNA) treatment

Chemical modified Stealth™ siRNA targeting JMJD2B and control siRNA were from Invitrogen. The sequence for JMJD2B siRNA was 5′-UCU CCA UCA CCU GCC UCA AGC ACA A-3′. siRNA for p53 was from Santa Cruz Biotechnology (sc-29435). Cells were transfected with siRNA

Impaired clonogenic potentials of tumor cells treated with JMJD2B siRNA

We knocked down the expression of JMJD2B using its specific siRNA in the gastric cancer cells AGS, BGC-823 and HGC-27 and cervical HeLa cells. Efficient silencing of JMJD2B expression in all these cells was verified by RT-PCR and Western blot analysis (Fig. 1A and C). Knockdown of JMJD2B led to significantly reduced foci numbers and sizes of all cell lines as compared with the control (AGS cells: 74 ± 23 vs. 4 ± 3; HeLa cells: 235 ± 45 vs. 37 ± 10; BGC-823 cells: 122 ± 10 vs. 27 ± 9; HGC-27: 189 ± 4 vs. 120 ± 

Discussion

The JMJD2 family of histone demethylases has four members, JMJD2A, B, C and D, that demethylate trimethylated histone H3 lysine 9 (H3K9) [7], [26]. JMJD2 family proteins promote transcriptional activation, thereby affecting important processes such as the hormone response, stem cell renewal, germ cell development, and cellular proliferation and differentiation [18]. The involvement of JMJD2B in tumorigenesis has been supported by recent findings of high expression of JMJD2B in ER-positive

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 81000868, 81171536 and 30972775), Science Foundation for Outstanding Young Scientists of Shandong Province (No. BS2009SW025), the National Basic Research Program of China (973 Program, No. 2012CB911202), the Science Foundation of the Ministry of Education of China (No. 20090131120057), Shandong Provincial Natural Science Foundation (No. ZR2009CZ001 and ZR2009CM002) and the Innovation Foundation of Shandong Province

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