Tumor Biol. DOI 10.1007/s13277-014-1947-4

RESEARCH ARTICLE

SPAG9 is overexpressed in human prostate cancer and promotes cancer cell proliferation Hui Li & Yang Peng & Huiyan Niu & Baogang Wu & Yi Zhang & Yue Zhang & Xue Bai & Ping He

Received: 27 February 2014 / Accepted: 3 April 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014

Abstract Sperm-associated antigen 9 (SPAG9) was recently reported to be overexpressed in several cancers and associated with the malignant behavior of cancer cells. However, the expression pattern of SPAG9 and its clinical significance in human prostate cancer have not been reported. In the present study, we analyzed SPAG9 expression in human prostate cancer tissues by immunohistochemistry and found that SPAG9 was overexpressed in 36.5 % of prostate cancer specimens. There was a significant association between SPAG9 overexpression and tumor stage (p=0.0020) and Gleason score (p=0.0377). Transfection of SPAG9 plasmid was performed in PC-3 cell line and siRNA knockdown was carried out in DU145 cells. Colony formation and MTT showed that SPAG9 overexpression promoted while siRNA knockdown inhibited prostate cancer cell proliferation. In addition, we found that SPAG9 could regulate cyclin D1 and cyclin E protein expression. In conclusion, SPAG9 is overexpressed in human prostate cancers and contributes to prostate cancer cell growth, possibly through cyclin protein regulation. Keywords SPAG9 . Prostate cancer . Proliferation

Introduction Prostate cancer is one of the most prevalent malignancies in men and the second most frequent cause of male cancerrelated death [1]. It is a clinically heterogeneous disease and the incidence is rising. Carcinogenesis and the mechanisms influencing the progression and prognosis of prostate cancer is H. Li : Y. Peng : H. Niu : B. Wu : Y. Zhang : Y. Zhang : X. Bai : P. He (*) Department of Geriatrics, Shengjing Hospital of China Medical University, 36 Sanhao Road, Shenyang 110004, China e-mail: [email protected]

a multistep process, involving both genetic insults to epithelial cells and changes in epithelial–stromal interactions [2–4]. In spite of current therapeutic methods, many patients develop metastases. Therefore, it is important to investigate the molecular mechanisms underlying the progression of prostate cancer to provide better strategies for the prevention and therapy of prostate cancer. Sperm-associated antigen 9 (SPAG9) defines a family of scaffolding proteins that bring MAPKs and their target transcription factors together for the execution of specific signaling pathways [5–9]. Recently, SPAG9 was proposed as a candidate cancer-associated protein in various cancers including renal, breast, thyroid, cervical, lung, colorectal carcinoma, and astrocytoma [10–16]. Furthermore, SPAG9 siRNA treatment inhibited tumor cell proliferation and invasion [10–14, 16]. SPAG9 was reported to enhance lung cancer cell invasion through c-Jun N-terminal kinase (JNK)-mediated upregulation of MMP9. SPAG9 expression was also found to be associated with circulating anti-SPAG9 antibodies in early stages and in low grade of breast cancer [10] and cervical cancer patients [11], suggesting its potential use in early detection of disease. In the present study, we examined SPAG9 expression pattern in human prostate cancer tissues by immunohistochemistry and analyzed its prognostic significance. We also knocked down SPAG9 expression with siRNA and examined its role on cancer cell proliferation and invasion.

Materials and methods Patients and specimens The study protocol was approved by the institutional review board of Shengjing hospital. Specimens were obtained from 148 patients diagnosed with prostate cancer and 15 cases of

Tumor Biol.

benign prostate hyperplasia who underwent resection in the First Hospital of China Medical University between 2005 and 2007. None of the patients had received radiotherapy or chemotherapy before surgical resection. The histological diagnosis and differentiation grade were evaluated for sections stained with hematoxylin and eosin according to the World Health Organization (WHO) classification guidelines. Immunohistochemistry Surgically excised tumor specimens were fixed with 10 % neutral formalin and embedded in paraffin, and 4-μm thick sections were prepared. Immunostaining was performed using the avidin–biotin–peroxidase complex method (Ultrasensitive™, MaiXin, Fuzhou, China). The sections were deparaffinized in xylene, rehydrated with graded alcohol, and then boiled in 0.01-M citrate buffer (pH 6.0) for 2 min in an autoclave. Hydrogen peroxide (0.3 %) was applied to block endogenous peroxide activity and the sections were incubated with normal goat serum to reduce nonspecific binding. Tissue sections were incubated with SPAG9 rabbit polyclonal antibody (1:150 dilution; Abcam, USA). Rabbit immunoglobulin was used as a negative control. Staining for both antibodies was performed at room temperature for 2 h. Biotinylated goat antimouse serum IgG was used as a secondary antibody. After washing, the sections were incubated with streptavidin– biotin conjugated with horseradish peroxidase, and the peroxidase reaction was developed with 3,3′-diaminobenzidine tetrahydrochloride. Counterstaining with hematoxylin was performed and the sections were dehydrated in ethanol before mounting. Two independent blinded investigators examined all tumor slides randomly. Immunostaining of SPAG9 was scored on a semiquantitative scale by evaluating in representative tumor areas. Nuclear immunostaining in tumor cells was considered as positive staining. We counted 400 tumor cells and calculated the percentage of positively stained cells. According to previous reports [16], we counted 400 tumor cells and calculated the percentage of positively stained cells. SPAG9 was scored positive when a specimen was showing >10 % of cancer cells stained for SPAG9 protein.

DharmaFECT1 reagent was used for siRNA transfection (Dharmacon, Lafayette, CO, USA) according to the manufacturer’s instructions. The siGENOME SPAG9 siRNA was purchased from Dharmacon (Dharmacon, Lafayette, CO, USA). siGENOME non-targeting siRNAs were used as a negative control. Plasmid transfection was performed using Attractene reagent (QIAGEN, Chicago, IL, USA). pCMV6-SPAG9 plasmid was purchased from Origene (Origene, CA, USA). Western blot analysis Total proteins from cells were extracted in lysis buffer and quantified using the Bradford method. Samples of 50 μg of protein were separated by SDS-PAGE. Samples were transferred to PVDF membranes (Millipore, Billerica, MA, USA) and incubated overnight at 4 °C with antibody against SPAG9 (1:1000, Abcam, USA), cyclin D1, cyclin E, CDK4, CDK6 (1:1000, Cell signaling), and β-actin (1:500; Santa Cruz). After incubation with peroxidase-coupled antimouse IgG (Santa Cruz) at 37 °C for 2 h, bound proteins were visualized using ECL (Pierce) and detected using a BioImaging System (UVP Inc., Upland, CA, USA). Colony formation and MTT assays For colony formation assay, cells were transfected with plasmid for 48 h and plated into three 6-cm cell culture dishes (1,000 per dish). Cells were incubated for 12 days in medium containing 10 % FBS. Plates were washed with PBS and stained with Giemsa. The number of colonies with more than 50 cells was counted. The colonies were manually counted using a microscope. For MTT assay, 24 h after transfection, cells were plated in 96-well plates at a concentration of approximately 3,000 cells per well and cultured for 5 days. For quantitation of cell viability, 20 μl of 5 mg/ml MTT (thiazolyl blue) solution was added to each well and incubated for 4 h at 37 °C. The medium was removed from each well and the resulting MTT formazan was solubilized in 150 μl of DMSO. Each solution was measured spectrophotometrically at 490 nm.

Cell culture and transfection Statistical analysis REPW-1, PC-3, and DU-145 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10 % fetal calf serum (Invitrogen), 100 IU/ml penicillin (Sigma, St. Louis, MO, USA), and 100 μg/ml streptomycin (Sigma). Cells were grown on sterilized culture dishes and were passaged every 2 days with 0.25 % trypsin (Invitrogen).

SPSS version 11.5 for Windows was used for all statistical analyses. A χ2 test was used to examine possible correlations between SPAG9 expression and clinicopathologic factors. All p values were based on the two-sided statistical analysis and p

SPAG9 is overexpressed in human prostate cancer and promotes cancer cell proliferation.

Sperm-associated antigen 9 (SPAG9) was recently reported to be overexpressed in several cancers and associated with the malignant behavior of cancer c...
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