ANTICANCER RESEARCH 31: 2683-2692 (2011) Gankyrin, A Biomarker for Epithelial Carcinogenesis, Is Overexpressed in Human Oral Cancer JUNAN LI1,2,*, THOMAS J. KNOBLOCH1,2,*, LAURA A. KRESTY3,4, ZHAOXIA ZHANG1, JAS C. LANG2,5, DAVID E. SCHULLER5 and CHRISTOPHER M. WEGHORST1,2 1Division of Environmental Health Sciences, College of Public Health, 2Comprehensive Cancer Center and Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, and 5Department of Otolaryngology, The Ohio State University, Columbus, OH43210, U.S.A.; 3Sylvester Comprehensive Cancer Center, and 4Department of Epidemiology and Public Health, Miller School of Medicine, The University of Miami, Miami, FL33101, U.S.A. Abstract. Little is known about the potential involvement of the alteration of the p16CDKN2A tumor suppressor gene. More oncoprotein gankyrin in human oral cancer progression. In this than 80% of human oral squamous cell carcinomas (OSCCs) study, the levels of gankyrin mRNA and protein expression were (1, 2) and 50% of severe oral dysplasias (3) have been shown assessed in human oral epithelial cell lines, at-risk normal oral to exhibit CDKN2A alterations. Inactivation of the p53 tumor tissues, premalignant oral lesions, and primary oral squamous suppressor gene (TP53) is another frequent alteration cell carcinomas (OSCCs). Materials and Methods: Biopsies observed in OSCC development and affects nearly 50% of included 6 oral epithelial cell lines, 32 OSCC specimens for all cases (4). In contrast to the strikingly prevalent incidence qRT-PCR analysis, 27 OSCC specimens and 12 premalignant of alterations in these important tumor suppressor genes, oral lesions for immunohistochemical analysis. Results: knowledge related to the activation of specific proto- Gankyrin was overexpressed in all tested oral epithelial cell oncogenes in human oral cancer remains relatively limited. lines and the majority of OSCC specimens (32/32 (100%) and Recently, overexpression of a newly defined oncoprotein, 21/27 (71%) at the mRNA and protein levels, respectively). gankyrin (PSMD10), has been found to be associated with Moreover, 6/12 of premalignant oral lesions overexpressed human cancers through diverse mechanisms (5-9). Gankyrin gankyrin protein. Conclusion: Gankyrin overexpression is a facilitates phosphorylation and degradation of RB1 protein prevalent event in human oral cancer and occurs during the (pRb) in vitro and in vivo (10, 11), and activates the E2F early stages of oral carcinogenesis, thus being a viable family of transcription factors. In parallel, gankyrin competes therapeutic or chemopreventive target in oral cancer. with p16 for binding with cyclin-dependent kinase 4 (CDK4), which precludes the inhibition of p16 in CDK4- Carcinogenesis is a multistep process through which normal mediated phosphorylation of pRb, thus leading to cell cycle cells are sequentially transformed via the activation of proto- progression (8). Furthermore, gankyrin also functions in the oncogenes and inactivation of tumor suppressor genes into degradation of the tumor suppressor protein p53 (12). By their malignant counterparts. During human oral cancer binding the ubiquitin ligase, murine double minute 2 development, one of the most prominent genetic changes is (MDM2), gankyrin promotes p53 ubiquitylation and subsequent proteasomal degradation (13). Hence, gankyrin functions as a dual-negative regulator of the two most prominent tumor suppressor pathways: (i) INK4-CDK-pRb, This article is freely accessible online. and (ii) ARF-MDM2-p53. Moreover, gankyrin overexpression has been found to be a prevailing event in *Both Authors contributed to this work equally. human hepatocellular carcinomas (HCCs) (6) and esophageal SCCs (ESCCs) (9), and emerging evidence shows that it Correspondence to: Dr. Junan Li, Division of Environmental Health could be one of the earliest molecular events occurring Sciences, College of Public Health, The Ohio State University, Columbus, OH 43210, U.S.A. Tel: +1 6142924066, Fax: +1 during epithelial cancer development. In a chemically 6142924053, e-mail: [email protected] induced rodent hepatocarcinogenesis model (6, 14), hypermethylation of CDKN2A and mutation of TP53 appear Key Words: Gankyrin, overexpression, oral squamous cell carcinoma. during the later stages (HCC), whereas gankyrin is 0250-7005/2011 $2.00+.40 2683 ANTICANCER RESEARCH 31: 2683-2692 (2011) overexpressed much earlier, shortly after initial carcinogen Patient sample procurement. All oral cancer and normal tissues exposure (liver fibrosis), preceding the loss of pRb were obtained from the Head and Neck Tumor Bank (J.C.L.) or (cirrhosis) and hepatocellular adenoma formation. In human Biorepository and Biospecimen Shared Resource at The Ohio State University Comprehensive Cancer Center following HCCs, Tan et al. reported that the frequencies of gankyrin Institutional Review Board approved protocols. Two biospecimen overexpression in Edmondson’s grade I/II, III, and IV HCCs sample sets were used for the retrospective studies: (i) Archival were 82%, 63%, and 22%, respectively (15). Consistently, tissue blocks for 27 OSCC cases and 12 preneoplastic oral 81% of HCCs at the low TNM stage and 35% of HCCs at lesions, (ii) Total cellular RNA for 29 primary OSCCs, 2 the high TNM stage overexpressed gankyrin (16). In recurrent OSCCs, 1 verrucous carcinoma, and 10 histologically addition, a recent study on ESCCs (9) showed that gankyrin normal oral tissues obtained from OSCC patients at the time of overexpression was positively correlated with lower survival surgery (Table I). rate, extent of the primary tumor, lymph node metastasis, Quantitative real-time RT-PCR. Total RNA was isolated from cells distant lymph node metastasis and stage. These findings and tissues using TRIzol Reagent (Invitrogen) according to the further indicate that gankyrin may be important during the manufacturer’s instructions. First-strand cDNA synthesis was development of the malignant potential in ESCC, and may performed using PowerScript Reverse Transcriptase (BD Sciences, play an important role in its progression (9). Thus, it would San Jose, CA, USA) as previously described (22). Real-time RT-PCR appear that gankyrin is one of few oncogenic proteins which reactions were carried out using a SmartCycler system (Cepheid, negatively modulate both pRb and p53 pathways (11), and Sunnyvale, CA, USA), and custom TaqMan assays designed using its aberrant overexpression could be an early initiating event Primer Express 3.0 (Applied Biosystems, Carlsbad, CA, USA). Each reaction mixture included 200 μM dNTPs, 3 mM MgSO , 1.25 units in carcinogenesis. Since most oral carcinomas have 4 of Taq polymerase (Agilent Technologies, Santa Clara, CA, USA), inactivated pRb and p53 pathways either directly or 1× Additive (0.2 mg/ml bovine serum albumin (BSA), 150 mM indirectly (1-4), it is of interest to investigate the involvement Trehalose, and 0.2% Tween 20), 0.1 volume of first-strand cDNA, of gankyrin during oral cancer development. 0.2 μM each primer, and 0.1 μM fluorogenic probe. The samples Here we report the first investigation into the incidence of were amplified at 96˚C for 2 minutes, followed by 55 cycles of 30 altered gankyrin expression in oral cancer. Using quantitative seconds at 95˚C, 30 seconds at 60˚C, and 30 seconds at 68˚C. real-time RT-PCR (qRT-PCR) and immunohistochemistry, Human hypoxanthine phosphoribosyltransferase (HPRT1) was used for gene expression normalization. The primers and fluorogenic we evaluated the expression of gankyrin in human oral probes were as follows: PSMD10, 5’-TCCTCTTCATA lesions. TTGCGGCTT-3’ (forward), 5’-CTTGAGCACCTTTTCCCAGAA-3’ (reverse), and 5’-TET-TGG CCGGGATGAGATTGTAAAA GCC- Materials and Methods TAMRA-3’ (probe); CDKN2A, 5’-TGCCCAACGCACCGA-3’ (forward), 5’-GGGCGC TGCCCATCA-3’ (reverse), and 5’-TET- Cell culture and siRNA transfection. Seven oral cell lines were TAGTTACGGTCGG AGGCCG ATCCA-TAMRA-3’ (probe); for used, including a normal epithelial cell line (TE1177), one HPRT1, 5’-CGGCT CCGTTATGGCG-3’ (forward), 5’-GGTCA premalignant epithelial cell line (SCC-83-01-82), and five TAACCTGGTTCAT CATCAC-3’ (reverse), and 5’-FAM- malignant cell lines (SCC-83-01-82CA, SCC9, SCC4, CAL27, and CGCAGCCCTGGCGTCGTGA-TAMRA-3’ (probe). Each gene was UM-SCC-22A) (17-20). SCC9, SCC4, and CAL27 were purchased amplified separately, and all experiments were performed in triplicate. from the American Type Culture Collection (Manassas, VA, USA); The expression levels of PSMD10 and CDKN2A were normalized UM-SCC-22A was obtained from the University of Michigan; to that of HPRT1, as well as appropriate calibrators, and the relative SCC-83-01-82, SCC-83-01-82CA, and TE1177 were kindly gene expression level was determined using a comparative Ct provided by Dr. S.M. D’Ambrosio of The Ohio State University method (23): (19, 20). All cells were cultured in Advanced DMEM/F12 medium ΔΔCt=(CtPSMD10−CtHPRT1)tumor−(CtPSMD10−CtHPRT1)normal, or (Invitrogen, Carlsbad, CA, USA) containing 5% fetal bovine serum ΔΔCt=(CtCDKN2A−CtHPRT1)tumor−(CtCDKN2A−CtHPRT1)normal –ΔΔCt (FBS; Invitrogen) in a 90% relative humidity incubator at 37˚C Relative gene expression=2 . A two-fold increase (≥2) or decrease (≤0.5) was considered significant for mRNA supplied with 5% CO2. Synthetic siRNA targeting human gankyrin (26S proteasome overexpression or down-regulation analysis, respectively (23). non-ATPase regulatory subunit 10, PSMD10) (sc-72186; Santa Cruz Biotechnology, CA, USA) and control siRNA (sc-37007, Santa Cruz Western