Gankyrin oncoprotein overexpression as a critical factor for tumor growth in human esophageal squamous cell carcinoma and its clinical significance Cristian M. Ortiz 1 , Tetsuo Ito 2 , Eiji Tanaka 1 , Shigeru Tsunoda 1 , Satoshi Nagayama 1 , Yoshiharu Sakai 1 , Hiroaki Higashitsuji 3 , Jun Fujita 3 and Yutaka Shimada 4 * 1 Department of Surgery, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan 2 Department of Medicine and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 3 Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan 4 First Department of Surgery, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan To elucidate the possible involvement of gankyrin in ESCC progres- sion and the effect of its down-regulation in ESCC, we investigated the expression of gankyrin in ESCC tissues comparing it with the corresponding normal esophageal epithelia and tested a short-hair- pin RNA (shRNA) expression vector for gankyrin in ESCC cell lines. Gankyrin protein expression in 11 ESCC cell lines (KYSE se- ries) was examined by RT-PCR and western blot. The expression of gankyrin mRNA in 30 ESCC tissues was compared with the corre- sponding normal epithelia by Real-time PCR. Expression of gan- kyrin protein was immunohistochemically analyzed in the ESCC of 103 patients. A gankyrin-shRNA vector was stably transfected into KYSE 170 cells to assess the role of gankyrin in cell motility, inva- sion and proliferation in vitro and tumor formation in vivo. Gan- kyrin expression increased in all 11 ESCC cell lines. Real-time PCR revealed that gankyrin expression was higher in the cancerous tissue for all 30 patients. In immunohistochemistry, gankyrin overexpres- sion was correlated with lower survival rate (p 5 0.0001), extent of the primary tumor, lymph node metastasis, distant lymph node me- tastasis and stage (p 5 0.0072, p 5 0.0004, p 5 0.0172 and p 5 0.0002, respectively). A shRNA vector against gankyrin repressed growth, cell motility, invasiveness in vitro and tumor formation in vivo. Gankyrin overexpression is associated with poor prognosis. It may play an important role in ESCC tumor progression and could be a potentially important therapeutic gene target in ESCC. ' 2007 Wiley-Liss, Inc. Key words: gankyrin; oncogene; esophageal cancer; clinical significance; novel; cell cycle Despite considerable advances in surgical techniques, perioper- ative care and neoadjuvant chemoradiotherapy, esophageal squa- mous cell carcinoma (ESCC) still remains one of the most lethal cancers and the seventh leading cause of cancer death world- wide. 1,2 Gene study is widely performed in ESCC 3–5 and new oncogenes associated with the progression of ESCC have been identified and targeted to improve the survival of patients with this type of refractory cancer. Gankyrin is a novel oncoprotein with 7 ankyrin repeats. 6–8 Overexpression of gankyrin increases the phosphorylation and degradation of retinoblastoma (RB1) by its RB1-binding motif, suggesting that it promotes tumorigenicity and cancer cell prolif- eration. 9 Furthermore, binding to CDK4 and counteracting the in- hibitory function of the INK4 proteins, including tumor suppres- sors p16 INK4A and p18 INK4B , gankyrin acts as an accelerator for cell cycle progression. 9–11 Gankyrin also binds to MDM2, facili- tating MDM2-p53 interaction and increasing its polyubiquitilation and degradation. 12 These findings suggest the importance of gan- kyrin in the cell cycle control and tumorigenesis. However, the physiological and physiopathological role of gankyrin in the cell cycle progression of normal cells remains unknown. Overexpression of gankyrin has been found ubiquitously in hepa- tocellular carcinoma tissues, liver regeneration and fulminant he- patic failure (FHF), 9 but there are no reports regarding its overex- pression in esophageal cancer. To clarify the clinical significance of gankyrin and evaluate the role of this molecule in the progression of ESCC, the following study was carried out by examining the gankyrin mRNA and protein expression from 11 ESCC cell lines (KYSE series). We then examined gankyrin protein expression in 103 ESCC tumors by immunohistochemistry (IHC), analyzing its clinical significance. In addition, to obtain better understanding of the role of gankyrin in ESCC we used a plasmid vector short-hair- pin RNA (shRNA) expression system in vitro and in vivo, that pro- vides a powerful tool used for inducing the loss of function pheno- types by posttranscriptional gene silencing. 13,14 Material and methods Surgical specimens and cell culturing Frozen esophageal tumor tissues and their corresponding normal tissues were obtained for Real-time PCR from 30 patients with pri- mary ESCC who underwent surgery at Kyoto University Hospital from 1991 to 2004. Paraffin-embedded sections were acquired for IHC from 103 patients with primary ESCC who underwent surgery at Kyoto University Hospital from 1997 to 2003. The tumor charac- teristics are summarized in Table I. The median follow-up time of survival was 30 months. All tumors were confirmed as ESCC by the clinicopathologic department of the hospital. All the cases were diagnosed as formalin-fixed, paraffin-embedded and hematoxilin and eosin (H&E)-stained representative specimens. These were clas- sified according to the fifth edition of the pathological tumor-node- metastasis (TNM) classification. 15–17 Information on the patients’ gender, age, stage of disease and histological factors was extracted from the medical records. Written informed consent was obtained from all patients regarding the performance of surgery and the use of resected samples for research (the approval numbers of the Insti- tutional Review Board of Kyoto University are 232 and G48). All human esophageal squamous carcinoma cell lines (KYSE series) were established in our laboratory and maintained in RPMI 1640 (Life Technologies, Gaithersburg, MD) and Ham’s F12 (Nissui Pharmaceutical, Tokyo, Japan) with 2% fetal bovine se- rum (FBS). 18 The normal esophageal epithelial cell line NEK2 was also established in our laboratory and maintained in a kerati- nocyte serum-free medium containing 2.5 mg of epidermal growth factor and 25 mg of bovine pituitary extract. 19 HeLa cells were purchased from the American Type Culture Collection (Rockville, MD), cultured in DMEM (Life Technologies) with 10% FCS and used as a positive control. Purification of total cellular mRNA and reverse transcription-PCR Total RNA was extracted from KYSE cell lines and the frozen tissues of ESCC patients by the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocols. 20,21 This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0020-7136/suppmat. Grant sponsor: Japanese Ministry of Education, Culture, Sports, Science and Technology; Grant number: 17390363. *Correspondence to: First Department of Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. Fax: +81-79-845-6581. E-mail: sshimada@hyo-med.ac.jp Received 31 March 2007; Accepted after revision 24 July 2007 DOI 10.1002/ijc.23106 Published online 12 October 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 325–332 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer