title: Targeted inactivation of HDAC2 restores p16INK4a activity and exerts antitumor effects on human gastric cancer.
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.
authors.affiliation: Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
authors.forename: Jeong Kyu
authors.initials: JK
authors.lastname: Kim
authors.forename: Ji Heon
authors.initials: JH
authors.lastname: Noh
authors.forename: Jung Woo
authors.initials: JW
authors.lastname: Eun
authors.forename: Kwang Hwa
authors.initials: KH
authors.lastname: Jung
authors.forename: Hyun Jin
authors.initials: HJ
authors.lastname: Bae
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Shen
authors.forename: Min Gyu
authors.initials: MG
authors.lastname: Kim
authors.forename: Young Gyoon
authors.initials: YG
authors.lastname: Chang
authors.forename: Seung-Jin
authors.initials: SJ
authors.lastname: Kim
authors.forename: Won Sang
authors.initials: WS
authors.lastname: Park
authors.forename: Jung Young
authors.initials: JY
authors.lastname: Lee
authors.forename: Jürgen
authors.initials: J
authors.lastname: Borlak
authors.forename: Suk Woo
authors.initials: SW
authors.lastname: Nam
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D045744:Cell Line, Tumor; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000378:metabolism*; D044127:Epigenesis, Genetic / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D056464:Histone Deacetylase 2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D041681:NIH 3T3 Cells; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D014162:Transfection
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D056464:Histone Deacetylase 2