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The Histone Demethylase RBP2 Is Overexpressed in Gastric Cancer and Its Inhibition Triggers SenesceJIPING ZENG,*,‡,§ ZHENG GE,* LIXIANG WANG,l QIAO LI,*,‡ NA WANG,*,‡ MAGNUS BJÖRKHOLM,* JIHUI JIA,‡ and DAWEI XU* *Department of Medicine, Division of Hematology, Karolinska University Hospital Solna and Karolinska Institutet, Stockholm, Sweden; and ‡Department of Microbiology/Key Laboratory for Experimental Teratology of Chinese Ministry of Education and §Department of Biochemistry and lDepartment of Pharmacology, School of Medicine, Shandong University, Jinan, PR China
BACKGROUND & AIMS: The aberrant expression of histone-modifying enzymes such as histone deacetylases contributes to oncogenesis. It is unclear whether RBP2, a newly identified histone demethylase, is involved in can- cer development/progression. We determined RBP2 ex- pression in gastric cancer and its biologic function in cancer cells. METHODS: Cancerous and matched nor- mal gastric specimens from 42 patients with gastric can- cer were analyzed for RBP2 expression using quantitative real-time polymerase chain reaction and immunohisto- chemistry. Gene expression was assessed using quantita- tive real-time polymerase chain reaction and immuno- blotting and depleted with small interference RNA. Clonogenesis and cellular senescence were examined by foci formation and b-Galactosidase staining. Promoter activity was determined by luciferase reporter assay. Chromatin immunoprecipitation was used to detect RBP2 and methylated histone H3-K4 on promoters. RE- SULTS: RBP2 messenger RNA and protein expression were increased in 71.5% (30/42) and 100% (20/20) of gastric cancer specimens, respectively. Significantly di- minished foci numbers coupled with massive senescence/ growth arrest and elevated expression of cyclin-depen- dent kinase inhibitors (CDKIs) p21CIP1, p27kip1, and/or p16ink4a occurred in RBP2-depleted gastric and cervical cancer cells. RBP2 depletion-mediated senescence and clonogenic defect were attenuated by inhibiting p21CIP1 or p27kip1 expression. The promoter activity of all 3 CDKIs genes was enhanced by RBP2 inhibition. RBP2 occupied these promoters in control cells, and the loss of RBP2 occupancy was accompanied by enhanced H3-K4 trim- ethylation following RBP2 depletion. CONCLUSIONS: RBP2 is overexpressed in gastric cancer, and its inhi- bition triggers senescence of malignant cells at least partially by derepressing its target genes CDKIs. Hi- stone demethylase inhibition by targeting RBP2 may be an anticancer strategy. Keywords: Cellular Senescence; Histone Demethylation.
G astric cancer is one of the most common malignan- cies and second leading cause of cancer-related death worldwide.1,2 The compelling evidence supports a role for Helicobacter pylori infection in gastric neoplastic transformation. H pylori infection triggers an early stage of gastric cancer pathogenesis by causing chronic gastri- tis; however, a progression into the invasive cancer is clearly a multistep process with numerous alterations involved in cell proliferation and/or survival, whereas the underlying molecular mechanism is largely unclear.1–6 Additionally, because of poor responses to current ther- apeutic approaches, the outcomes of gastric cancer re- main unfavorable with a low rate of 5-year survival.2 Therefore, better defining of the pathogenesis of gastric cancer and exploring novel therapeutic targets for treat- ment are urgently demanding tasks. The histone-modifying enzymes—responsible for acet- ylation, phosphorylation, and methylation of histone proteins—play a key role in the regulation of gene tran- scription by mediating chromatin reconfiguration.7,8 Among these enzymes, RBP2 is a newly identified mem- ber ofJARID family proteins with a histone demethylase (HDM) activity, and it specifically targets tri- and dim- ethylated lysine 4 of histone H3 (H3-K4) for demethyl- ation.9–11 Recent genome-wide analyses have revealed that RBP2 controls the expression of multiple genes, thereby regulating cell fate determination.12–15 The disruption of a balance of histone modifications and aberrant chromatin remodeling has long been impli- cated in cancer development and progression. One of the best examples is the role of dysregulated histone acetyl- transferases and deacetylases (HDACs) in tumorigene- sis.16 Histone acetyltransferases and HDACs catalyze the acetylation and deacetylation of histones H3 and H4, respectively. HDACs have been shown to be overex- pressed, aberrantly recruited by oncoproteins, or mutated in malignant cells.16 Enhanced HDAC activity leads to the transcriptional repression of tumor suppressor genes,
Abbreviations used in this paper: b2-M, b2-microglobulin; CDKIs, cyclin-dependent kinase inhibitors; GC, gastric cancer; HDACs, histone deacetylases; HDM, histone demethylase; QRT-PCR, quantitative real- time PCR; SA-b-Gal, senescence-associated b-Galactosidase; siRNA, small interference RNA. © 2010 by the AGA Institute 0016-5085/10/$36.00 doi:10.1053/j.gastro.2009.10.004
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Received February 2, 2009. Accepted October 8, 2009. Reprint requests Address requests for reprints to: Dawei Xu, MD, Haematology Laboratory, Centre for Molecular Medicine (CMM), L8:00, Karolinska University Hospital Solna, SE-171 76 Stockholm, Sweden. e-mail: Dawei.Xu@ki.se; fax: (46) 8517 73054 or Jihui Jia, MD, Department of Microbiology, School of Medicine, Shandong University, Jinan, 250012, PR China. e-mail: jiajihui@sdu.edu.cn; fax: (86) 531 8838 2502. Acknowledgments The authors thank Drs S. H. Juan (Taipei Medical University, Taiwan), Y. Sowa (Kyoto Prefectural University of Medicine, Japan), P. A. Cloos and K. Helin (University of Copenhagen, Denmark), and E. Hara (Christie Hospital NHS Trust, Manchester, UK) for plasmids. J.Z., Z.G., and L.W. contributed equally to this work. Conflicts of interest The authors disclose no conflicts. Funding Supported by The Swedish Cancer Society, the Swedish Research Council, Cancer Society in Stockholm, Swedish Society of Medicine, the Karolinska Institutet Foundation, National Natural Science Foundation of China (No. 30770118, 30772545, and 30972775), National Basic Research Program of China (973 Program 2007CB914801), and National Outstanding Young Scientist Award of NSFC (30725015). |
