Tumor-specific mutation and downregulation of ING5 detected in oral squamous cell carcinoma Beyhan Cengiz 1,2 , Esra Gunduz 3,4 , Mehmet Gunduz 1,5 , Levent Bekir Beder 5 , Ryo Tamamura 1 , Cahit Bagci 2 , Noboru Yamanaka 5 , Kenji Shimizu 3 and Hitoshi Nagatsuka 1 1 Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Shikatacho 2-5-1, Okayama, Japan 2 Department of Physiology, Faculty of Medicine, University of Gaziantep, Universite Bulvari, Tip Fakultesi Prof. Dr. Sabri Gungor Arastirma Merkezi, Gaziantep, Turkey 3 Department of Molecular Genetics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Shikatacho 2-5-1, Okayama, Japan 4 Department of Medical Genetics, Faculty of Medicine, Fatih University, Alparslan Turkes Cad. No: 57 Emek, Ankara, Turkey 5 Department of Otolaryngology Head and Neck Surgery, Wakayama Medical University, 811-1, Kimiidera, Wakayama, Japan Our previous study showed high frequency of allelic loss at chromosome 2q37 region in oral cancer. This location contains several candidate tumor suppressor genes such as PPP1R7, ILKAP, DTYMK and ING5. We previously showed 3 members of inhibitor of growth (ING) family, ING1, ING3 and ING4 as tumor suppressor gene in head and neck cancer. As ING5 shows high homology with other members of ING genes including highly conserved carboxy-terminal plant homeodomain and nuclear localization signal, we first picked up ING5 and examined it as a possible tumor suppressor in oral cancer. For this aim, mutation and mRNA expression status of ING5 in paired normal and oral squamous cell carcinoma samples were examined by reverse transcription polymerase chain reaction (RT-PCR) and sequencing. Three missense mutations located within leucine zipper like (LZL) finger and novel conserved region (NCR) domains in ING5 protein were detected, probably abrogating its normal function. We also found 5 different alternative splicing variants of ING5. Then, we examined mRNA level of ING5 by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) analysis, which demonstrated decreased expression of ING5 mRNA in 61% of the primary tumors as compared to the matched normal samples. In conclusion, tumor-specific mutation and downregulation of ING5 mRNA suggested it as a tumor suppressor gene in oral squamous cell carcinoma. Oral and oropharyngeal squamous cell carcinoma is the 6th most frequently occurring cancer worldwide, with 400,000 new cases diagnosed each year. 1 Similar to other type of can- cer, oral squamous cell carcinoma (OSCC) is the result of accumulation of multiple genetic events in the cell. Among these events, inactivation of tumor suppressor genes (TSG) is considered to be an important step in carcinogenesis. Thus, localization and identification of TSG are of great interest for a better comprehension of cancer and development of molec- ular diagnostics as well as therapies. TSG are referred as genetic elements with negative influ- ence on cell proliferation and growth. 2 Inactivation of TSG causes cells to display one or more phenotypes of neoplastic growth. Knudson’s definition of a classical TSG requires inac- tivation of both alleles of a candidate gene in tumors. 3 Inacti- vation of these classical TSG usually occurs through deletion of one of its allele and mutation in the rest of the allele. However, a new class of TSG with haploid insufficiency, in which one allele is lost and the remaining allele is haploinsuf- ficient, has been described recently, and these hemizygous TSG show a tumor-prone phenotype when challenged with carcinogens. 4,5 One of the critical steps for the identification of TSG is loss of heterozygosity (LOH) analysis. By using LOH analysis, we recently showed high frequency of allelic loss on chromo- somes 13q34, 7q31 and 12p13 in oral, and head and neck cancer. Our continuous effort for detailed analysis of these regions resulted in identification of various inhibitor of growth (ING) tumor suppressor family members, including Key words: ING5, oral cancer, ING1, tumor suppressor gene, mutation, splicing variant Grant sponsor: Ministry of Education, Culture, Sports, Science and Technology; Grant numbers: 21592326, 18-06262, 20791337; Grant sponsors: Japan Science and Technology Agency (Seed Innovation Research), Sumitomo Trust Haraguchi Memorial Cancer Research Promotion, and Astrazeneca Research Grant DOI: 10.1002/ijc.25224 History: Received 15 Apr 2009; Accepted 7 Jan 2010; Online 3 Feb 2010 Correspondence to: Mehmet Gunduz, Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayamashi 700-8558, Japan, Fax: þ81-86-235-6654, E-mail: mehmet.gunduz@gmail.com Cancer Genetics Int. J. Cancer: 127, 2088–2094 (2010) V C 2010 UICC International Journal of Cancer IJC