ANTICANCER RESEARCH 38 : 939-944 (2018) doi:10.21873/anticanres.12307 High STMN1 Expression Is Associated with Tumor Differentiation and Metastasis in Clinical Patients with Pancreatic Cancer KAZUNOBU SUZUKI 1,2,3* , AKIRA WATANABE 1,2* , KENICHIRO ARAKI 1,2 , TAKEHIKO YOKOBORI 2,3 , NORIHUMI HARIMOTO 1, DORGORMAA GANTUMUR 1, KEI HAGIWARA 1,2 , TAKAHIRO YAMANAKA 1,2 , NORIHIRO ISHII 1,2 , MARIKO TSUKAGOSHI 1,2 , TAKAMICHI IGARASHI 1, NORIO KUBO 1,2 , NAVCHAA GOMBODORJ 4, MASAHIKO NISHIYAMA 3,4 , YASUO HOSOUCHI 5, HIROYUKI KUWANO 2 and KEN SHIRABE 1 Departments of 1Hepatobiliary and Pancreatic Surgery, 2General Surgical Science, 4Molecular Pharmacology and Oncology, Gunma University, Graduate School of Medicine, Maebashi, Japan; 3Research Program for Omics-based Medical Science, Division of Integrated Oncology Research, Gunma University Initiative for Advanced Research, Maebashi, Japan; 5Department of Surgery and Laparoscopic Surgery, Gunma Prefecture Saiseikai-Maebashi Hospital, Maebashi, Japan Abstract. Background: Pancreatic ductal adenocarcinoma metastasis compared to those with low STMN1 (n=75). The (PDAC) is a leading cause of cancer-related deaths proliferation rates and migration ability in Suit2-STMN1 were worldwide. Stathmin 1 (STMN1) suppression was reported to higher than those of Suit2-mock. Conclusion: STMN1 reduce cellular viability and migration potential. However, no evaluation could be a useful progression marker, and STMN1 previous studies have addressed whether STMN1 may be a promising candidate for targeted therapies in PDAC. overexpression is associated with malignant potential in PDAC. Materials and Methods: To clarify the clinical Pancreatic ductal adenocarcinoma (PDAC) comprises 90% significance of STMN1 in PDAC, the STMN1 expression in of all pancreatic cancer cases (1). PDAC is a main cause of 104 PDAC samples was evaluated by immunohistochemistry. mortality, accounting for up to 4% of all cancer-related Moreover, we evaluated the proliferative potential and deaths worldwide. The 5-year survival rate is only 25%, migration ability of pancreatic cancer cell line Suit2 cells while for those with metastatic disease, it is 1% (2). To cure highly expressing STMN1. Results: Cytoplasmic STMN1 were the patients with PDAC, surgical resection is the mainstay higher levels in PDAC than in corresponding non-cancerous of radical treatment and provides patients who have small tissues. PDAC patients with high STMN1 (n=29) were and localized pancreatic cancer with longer survival. significantly associated with poor differentiation and distant However, patients with unresectable, metastatic or recurrent PDAC are unlikely to benefit from surgical resection. In addition, PDAC is often resistant to several treatments with chemotherapy, and radiation (1). Therefore, to improve *These Authors contributed equally to this study. prognosis, it is important to elucidate the mechanisms Correspondence to: Kenichiro Araki, MD, Ph.D., Department of underlying cancer progression in PDAC. Hepatobiliary and Pancreatic Surgery, Gunma University, Graduate It is reported that cancer cell migration, e.g. through tumor School of Medicine, 3-39-22 Showamachi, Maebashi 371-8511, budding, leads to poor prognosis in PDAC (3). Migratory Japan. Tel: +81 0272208224, Fax: +81 0272208230, e-mail: cancer cells often have therapeutic resistance to several agents [email protected] and Takehiko Yokobori, MD, Ph.D., (4), and the factors that regulate migratory ability are suggested Department of General Surgical Science, Gunma University, to be associated with cancer progression and poor prognosis in Graduate School of Medicine, 3-39-22 Showamachi, Maebashi 371- PDAC. Therefore, in order to improve patient prognosis in 8511, Japan. Tel: +81 0272208224, Fax: +81 0272208230, e-mail: [email protected] PDAC, it might be important to target molecular candidates related to migratory ability and cellular viability. Key Words: Pancreatic cancer, STMN1, PDAC, pancreatic ductal Stathmin 1 (STMN1, also known as oncoprotein 18) regulates adenocarcinoma, marker, Suit2 cells. microtubule dynamics by preventing the polymerization of 939 ANTICANCER RESEARCH 38 : 939-944 (2018) tubulin and promoting microtubule destabilization (5). STMN1 replacing the primary antibody with PBS. The immuno- is expressed in various malignancies and its expression is histochemical evaluation of STMN1 expression was confirmed associated with cancer aggressiveness and poor prognosis in independently by two observers. In positive cases, staining was primarily cytoplasmic. The intensity of STMN1 staining was scored cutaneous squamous cell carcinoma (6), non-small cell lung as follows: 0: no staining, 1: weak-to-moderate staining, or 2: strong cancer (7), ovarian carcinoma (8), gastric cancer (9, 10), staining. A score of 0 for staining was considered to be low STMN1 hepatocellular carcinoma (11), colorectal cancer (12), and urinary expression (n=75), while scores of 1 and 2 were considered to be bladder cancer (13). STMN1 suppression in several cancer cell high STMN1 expression (n=29). lines, including PDAC (14), was reported to reduce cellular viability and migration potential. Therefore, it is suggested that Cell culture. The human pancreatic cancer cell line Suit2 was used STMN1 is a fundamental cancer-associated gene and a potential in this study. The cell line was obtained from the JCRB cell bank target for diagnosis and treatment. However, to our knowledge, (Ibaragi, Osaka, Japan). The cells were cultured in RPMI 1640 medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine no previous studies have addressed whether STMN1 serum and 1% penicillin–streptomycin (Invitrogen, Carlsbad, CA, overexpression regulates the migratory ability and viability of USA) in a humidified 5% CO 2 incubator at 37˚C. PDAC cells. The purpose of this study was to clarify the clinical Establishment of a stable STMN1-transfected pancreatic cancer cell significance of STMN1 in clinical PDAC samples, and line. Human STMN1 complementary DNA (cDNA) was generated analyze STMN1 function in a PDAC cell line overexpressing by reverse transcription- polymerase chain reaction (RT-PCR) and STMN1. Therefore, we examined the expression of STMN1 subcloned into PiggyBac Dual Promotor Vector PB513B-1 (System Biosciences, Palo Alto, CA, USA) using Ligation high Ver.2 in clinical PDAC samples by using immunohistochemistry (TOYOBO, Osaka, Japan) according to the manufacturer’s protocol. to evaluate whether the STMN1 expression can be used as Accurate in-frame insertion into the expression vector was cancer marker in patients with PDAC. Moreover, we confirmed by sequencing. Transfection into Suit2 cells was evaluated the proliferation potency and migration ability in performed with PiggyBac Transposase Vector and SBI PureFection Suit2 cells expressing a high level of STMN1. transfection reagents (System Biosciences) according to the manufacturer’s protocol, and Suit2-STMN1 cells were established. Materials and Methods A mock vector-transfected clone (Suit2-mock) was used as a control. Enhanced STMN1 expression was confirmed by RT-PCR and western blot analysis. Clinical samples. We analyzed tumor specimens from 104 patients with PDAC who underwent excision surgery for a primary tumor between 1999 and 2012 at the Department of General Surgical RNA extraction and quantitative real-time RT-PCR. Total RNA was Science of Gunma University School of Medicine and Gunma extracted from Suit2 cells using the miRN-easy Kit (Qiagen, Hilden, Prefecture Saiseikai-Maebashi Hospital, Japan. The patients Germany), and the quantity of isolated RNA was measured using included 56 men and 48 women. The age range was 36-87 years. an ND-10000 spectrophotometer (Nano- Drop Technologies, The tumor stage was classified according to the seventh tumor- Wilmington, DE, USA). Quantitative real- time RT-PCR was node-metastasis (TNM) classification of the Union for International performed using the GoTaq 1- Step RT-qPCR System (Promega, Cancer Control (15) and the Sixth General Rules for the Study of Madison, WI, USA) in a total volume of 20 μl. The program Pancreatic Cancer of Japan Pancreas Society (16). Four patients had included four stages: reverse transcription at 37˚C for 15 min; RT stage I, 84 had stage II, five had stage III and 11 had stage IV inactivation and hot-start activation at 95˚C for 10 min; qPCR, of PDAC at the time of surgery. All patients signed written informed 40 cycles of 95˚C for 10 s, 60˚C for 30 s, and 72˚C for 30 s; and consent forms as required by our institutional guidelines. dissociation at 60-95˚C. The sequences of the primer pairs were as follows: STMN1 forward, 5’-CCGAGAAGAAGATCACCTT- Immunohistochemical staining. Sections (4- μm) of tumor and non- GAA-3’; STMN1 reverse, 5’-GACACGTCCTTCTTTTTGAAG C- cancerous tissue were deparaffinized, rehydrated, and incubated with 3’; beta-actin forward, 5’-CCAACCGCGAGAAGATGA-3’; and fresh 0.3% hydrogen peroxide in methanol for 30 min at room beta-actin reverse, 5’-CCAGAGGCGTACAGGGATAG-3’. temperature to block endogenous peroxidase activity. The sections then were heated in boiled water and Immunosaver (Nishin EM, Protein extraction and western blot analysis. Suit2-mock and Suit2- Tokyo, Japan) at 98˚C for 45 min. Nonspecific binding sites were STMN1 cells were harvested at 80% confluence, and the total blocked by incubation with 10% rabbit pre-immune serum for 30 proteins were extracted using PRO-PREP Protein Extraction min. The sections were then incubated with primary monoclonal