Abstract
Aberrant regulation of histone deacetylase 2 (HDAC2) was reported for gastric cancers. However, responsive cancer genes in disease onset and progression are less understood. HDAC2 expression was studied by quantitative RT-PCR and Western blotting. The functional consequences of HDAC2 knockdown on cell-cycle regulation, programmed cell death, and gene target identification was investigated by flow cytometry, Western blotting, electron microscopy, anchorage-independent colony formation, and cell migration assay and by whole-genome microarray. Therapeutic efficacy of HDAC2 knockdown was determined in nude mice with small hairpin expressing human gastric cancer cells. Epigenetic regulation of p16(INK4a) was studied by methylation-specific PCR and chromatin-IP to evidence HDAC2 or acetylated-histone-H4 binding at gene specific promoter sequences. HDAC2 gene and protein expression was significantly upregulated in different histopathologic grades of human gastric cancers and cancer cell lines. HDAC2 inactivation significantly reduced cell motility, cell invasion, clonal expansion, and tumor growth. HDAC2 knockdown-induced G(1)-S cell cycle arrest and restored activity of p16(INK4a) and the proapoptotic factors. This treatment caused PARP cleavage and hypophosphorylation of the Rb-protein, repressed cyclinD1, CDK4, and Bcl-2 expression and induced autophagic phenotype, that is, LC3B-II conversion. Some gastric tumors and cancer cells displayed p16(INK4a) promoter hypermethylation but treatment with 5-aza-deoxycitidine restored activity. With others the methylation status was unchanged. Here, chromatin-IP evidenced HDAC2 binding. Nonetheless, expression of p16(INK4a) was restored by HDAC2 knockdown with notable histone-H4-acetylation, as determined by chromatin-IP. Thus, p16(INK4a) is regulated by HDAC2. HDAC2 is a bona fide target for novel molecular therapies in gastric cancers.
Highlights
Gastric cancer is one of the leading causes of cancer deaths worldwide [1] and is prevalent in Asia
Additional report suggested histone deacetylase 2 (HDAC2) overexpression to be associated with poor prognosis by multivariate analyses [13]
We recapitulated HDAC2 gene expression from the large cohorts of gastric cancer patients that are available from the National Center for Biotechnology Information (NCBI), Gene Expression Omnibus (GEO) database, and data are given as scatter plot
Summary
Gastric cancer is one of the leading causes of cancer deaths worldwide [1] and is prevalent in Asia. Over the last few years, several studies contributed toward an understanding of the molecular causes of gastric cancer, thereby facilitating its early diagnosis and improved curability [2, 3]. While the molecular mechanisms leading to undue cell growth in gastric cancers remain uncertain the study of enzymes that modify the histone code has become the Authors' Affiliations: 1Lab of Oncogenomics, Department of Pathology, College of Medicine, and 2Functional RNomics Research Center, The Catholic University of Korea, Seoul, Republic of Korea; and 3Center of Pharmacology and Toxicology, Hannover Medical School, Hannover, Germany. Most HDAC inhibitors are not isoform specific [8] and the regulation and contribution of individual HDACs in cancer development remains unclear
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