HDAC2 promotes cell migration ⁄ invasion abilities through HIF-1a stabilization in human oral squamous cell carcinoma Cheng-Chi Chang 1 , Been-Ren Lin 2 , Szu-Ta Chen 3,4 , Tsung-Han Hsieh 1 , Yue-Ju Li 5 , Mark Y.-P. Kuo 5 1 Graduate Institute of Oral Biology, School of Dentistry, National Taiwan University, Taipei, Taiwan; 2 Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan; 3 Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan; 4 Department of Pediatrics, National Taiwan University Hospital Yun-Lin Branch, Taipei, Taiwan; 5 Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, Taipei, Taiwan BACKGROUND: Histone deacetylase 2 (HDAC2) ex- pressions in oral squamous cell carcinoma (OSCC) had been implicated in advanced stage and poor prognosis. It suggests a possible link between the migration ⁄ invasion potential of oral cancer cells and the prevalent expression of HDAC2. METHODS: Five head and neck cancer (HNC) cell lines, including Ca9-22, Cal-27, HSC-3, SAS, and TW2.6, were used. Cells stably overexpressing HDAC2 and shRNA against HDAC2 were established to investigate migra- tion ⁄ invasion ability in vitro and tumorigenesis and pro- gression in vivo. RESULTS: We found that alterations in the HDAC2 level in OSCC cell lines modulated their invasive ability with a positive correlation. Animal model also showed that knockdown of HDAC2 expression in SAS cells, originally containing high endogenous HDAC2 expression, resulted in decrease in tumor initiation and progression. Using high-throughput transcriptome analysis, numerous genes involved in HIF-1a-associated pathways were found. At the mechanism levels, using agents to block de novo protein synthesis or prevent protein degradation by ubiquitination, we found the stability of hypoxia inducible factor 1a (HIF-1a) protein was maintained in OSCC cells with HDAC2 overexpression. In addition, co-immuno- precipitation assay also revealed that HDAC2-mediated HIF-1a protein stability is because of direct interaction of HIF-1a with von Hippel–Lindau (VHL) protein. CONCLUSIONS: Our work demonstrates that HDAC2 maintains HIF-1a stability, probably at the level of protein modification, which in turn leads to the increase in cell invasion ⁄ migration ability in oral cancer progression. These findings implicate the potential of HDAC inhibitors for oral cancer therapy. J Oral Pathol Med (2011) 40: 567–575 Keywords: histone deacetylase 2; hypoxia inducible factor 1a; invasion; migration; oral cancer Introduction Oral squamous cell carcinoma (OSCC) is one of the most frequent cancers worldwide with more than half a million patients being diagnosed (5% of all cancer) each year (1, 2). Epigenetic modulation of gene expression is an important regulatory process in cancer biology (3). Gene regulation occurs in the context of packaging of DNA into the nucleosome that is composed of a DNA strand wound around a core of eight histone proteins and the N-terminal tails of each histone extend outward through the DNA strand (4). The amino acid residues on the histone tail can be modified by posttranslational acetylation, methylation, and phosphorylation (5), thereby changing the secondary structure of the histone protein tails in relation to the DNA strands, increasing the distance between DNA and histones, and increasing the accessibility of transcription factors to gene pro- moter regions (6). Histone modification also influences many developmental and regulatory processes within the cell. Accumulating data implicate histone modifica- tion in the pathobiology of cancer and other diseases. Among histone modifications, histone acetylation is mediated by histone acetyl transferases (7), and the acetyl groups are removed by histone deacetylases (HDACs) (8). At least 18 human HDACs with varying function, localization, and substrates are known (9, 10). The four classes of HDACs are grouped by their homology to yeast proteins. Classes I (HDAC1, 2, 3, 8), II (HDAC4, 5, 6, 7, 9, 10), and IV (HDAC11) all contain a zinc molecule in their active site and are inhibited by the pan-HDAC inhibitors. The seven different class III HDACs (sirtuins) are homologous to the yeast Sir2. They do not contain zinc in the active site and are not inhibited by any currently used HDAC inhibitors (11). In addition, the function of many non- histone proteins is also controlled by acetylation on Correspondence: Mark Y.-P. Kuo, Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University, National Taiwan University Hospital, No.1, Chung-Te St., Taipei, Taiwan. Tel: 886 2 23123456 ext., Fax: 886 2 23820785, E-mail: oddie@ntu.edu.tw Accepted for publication January 10, 2011 doi: 10.1111/j.1600-0714.2011.01009.x J Oral Pathol Med (2011) 40: 567–575 ª 2011 John Wiley & Sons A/S Æ All rights reserved wileyonlinelibrary.com/journal/jop Journal of Oral Pathology & Medicine