ONCOLOGY LETTERS 14: 6801-6808, 2017 High levels of DEPDC1B predict shorter biochemical recurrence‑free survival of patients with prostate cancer SHOUMIN BAI1,2*, TING CHEN1,2*, TAO DU2,3*, XIANJU CHEN2,4, YIMING LAI4, XIAOMING MA4, WANHUA WU4, CHUNHAO LIN4, LEYUAN LIU5,6 and HAI HUANG2,4,5 1Department of Radiation Oncology; 2Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation; Departments of 3Obstetrics and Gynecology and 4Urology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong 510120, P.R. China; 5Center for Translational Cancer Research, Texas A&M Institute of Biosciences and Technology; 6Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M University, Houston, TX 77030, USA Received November 4, 2016; Accepted March 28, 2017 DOI: 10.3892/ol.2017.7027 Abstract. DEP domain-containing protein 1B (DEPDC1B) Multivariate analysis using Cox proportional hazards model has been reported to serve important functions in breast cancer revealed that levels of DEPDC1B mRNA were significant and non-small cell lung cancer. However, its involvement in independent predictors of BCR-free survival time of patients the development of prostate cancer (PCa) remains unclear. with PCa. Therefore, the expression of DEPDC1B may be used Therefore, the present study aimed to investigate the expres- as an independent predictor of biochemical recurrence-free sion and clinical significance of DEPDC1B in tumor tissues survival time of patients with PCa. from patients diagnosed with PCa. A total of 80 prostate tissue samples were collected following prostatectomy to generate Introduction a tissue microarray for immunohistochemical analysis of DEPDC1B protein expression. High throughput sequencing of Prostate cancer (PCa) is the most common cancer of the mRNAs from 179 prostate tissue samples, either from patients male urogenital system and the second leading cause of with PCa or from healthy controls, was included in the Taylor cancer-associated mortality in the US (1). In China, the dataset. The expression levels of DEPDC1B in tumor tissues incidence and mortality of PCa have been continually from patients with PCa were revealed to be significantly increasing (2). Although treatment modalities for this cancer increased compared with those in normal prostate tissues have improved, predicting the clinical outcome of PCa (P=0.039). Increased expression of DEPDC1B was signifi- remains difficult (3). Biochemical recurrence (BCR) occurs cantly associated with advanced clinical stage (P=0.006), in ~20% of patients with PCa following radical prostatectomy advanced T stage (P=0.012) and lymph node metastasis or radiotherapy (4). A series of clinical parameters, including (P=0.004). Kaplan-Meier analysis demonstrated that patients serum prostate‑specific antigen (PSA) levels, Gleason score with high levels of DEPDC1B mRNA had significantly and surgical margin status, in various combinations, have been shorter biochemical recurrence (BCR)-free survival times. used to predict the outcome for PCa (5,6). However, the ability of conventional prognostic factors to identify insignificant PCa may be limited (7). Therefore, it is important to identify more novel and sensitive PCa molecular markers that are associated with biological aggressiveness and able to provide valuable Correspondence to: Dr Hai Huang, Department of Urology, Sun information for the diagnosis and treatment of the disease. Yat-Sen Memorial Hospital, Sun Yat-sen University, 107W Yanjiang Located at chromosome 5 (5q12.1), the DEP domain- Road, Guangzhou, Guangdong 510120, P.R. China E-mail: [email protected] containing protein 1B (DEPDC1B) gene encodes a protein containing two structural domains: A DEP domain and a Dr Leyuan Liu, Center for Translational Cancer Research, Texas RhoGAP domain (8-10). The DEP domain enables the protein A&M Institute of Biosciences and Technology, Texas A&M to interact with G protein coupled receptors as well as nega- University, 2121 West Holcombe Boulevard, Houston, TX 77030, tively-charged membrane phospholipids, and the RhoGAP USA E-mail: [email protected] domain is responsible for Rho GTPase signaling (9-11). The precise function of DEPDC1B is uncharacterized. It has been *Contributed equally reported to be associated with regulating cellular activities, including cell growth, movement, differentiation, cell cycle Key words: DEP domain-containing protein 1B, prostate cancer, and reorganization of cytoskeleton (10). Subsequent studies prognosis, progression demonstrated that DEPDC1B is also overexpressed in other types of cancer, including breast cancer (12), oral cancer (13) and non-small cell lung cancer (14), and is a prognostic factor 6802 BAI et al: DEPDC1B AS A PROGNOTIC MARKER OF PROSTATE CANCER that predicts outcomes in patients with non-small cell lung were incubated with the primary antibody against DEPDC1B cancer (14). The potential prognostic value of DEPDC1B in (rabbit polyclonal antibody, cat. no. bs-14278R; BIOSS, Beijing, patients with PCa remains unknown. Therefore, the present China) at a dilution of 1:600 at 4˚C overnight. Following study examined the expression of DEPDC1B in prostate washing (PBS for 5 min, 3 times), peroxidase-labeled polymer tissues, using immunohistochemistry to explore its clinical mouse anti-rabbit antibodies (cat. no. 3678S; Cell Signaling significance. Technology, Inc., NY, USA; 1:20,000; at 37˚C for 1 h or at In the present study, DEPDC1B expression in a tissue 4˚C overnight) and substrate‑chromogen staining (DAB, microarray (TMA) containing 80 samples was examined. In no. 9018, Beijing Zhongshan Golden Bridge Biotechnology order to investigate the expression of DEPDC1B at the mRNA Co., Ltd., Beijing, China) were employed in order to visualize level and perform survival analysis, the clinical informa- the protein. Negative controls were performed by omitting the tion of the Taylor dataset (15), including 150 prostate cancer primary antibody. tissues and 29 normal prostate tissue, was also collected. The association between the relative expression of DEPDC1B and Evaluation of immunostaining results. The intensity of clinicopathological parameters was examined to evaluate its immunostaining was scored separately by two independent clinical significance. In addition, the impact of DEPDC1B experienced pathologists, who were blinded to the clinicopath- expression on the biochemical recurrence (BCR) of patients ological data and clinical outcomes of the patients. The scores with PCa was assessed. of the two pathologists were compared and any discrepancies were resolved through re-examination of the staining by the Materials and methods two pathologists to achieve a consensus score. The immuno- labeling of cancer cells was then evaluated. The number of Patients and tissue samples. For immunohistochemical anal- positive‑staining cells in five representative fields at 400‑fold ysis, a TMA (n=80; catalog no. PR803c), including 73 tumor were counted under an inverted microscope and the percentage tissue samples from patients with PCa, 3 adjacent normal of positive cells was also calculated. According to the antibody prostate tissue samples from patients with PCa and 4 normal specification sheet, cytoplasmic staining was regarded as prostate tissue samples from healthy donors, and detailed clin- positive signals. The semi-quantitative scoring of the expres- ical information were obtained from Alenabio Biotechnology sion intensity in each sample was performed according to a Ltd. (Xi'an, China), a distributor of US Biomax, Inc. (Rockville, previous study and was based on the staining intensity and MD, USA) in China. Patients who received chemotherapy or percentage (16). The staining intensity was visually scored radiotherapy prior to surgery were excluded from the present and stratified according to the following criteria: No staining, study. In order to investigate the expression of DEPDC1B at 0 points; mild staining, 1 point; moderate staining, 2 points the mRNA level and perform survival analysis, the clinical and strong staining, 3 points. The score for the percentage information of the Taylor dataset, including 150 prostate of immunoreactive tumor cells was defined as follows: <5%, cancer tissue samples and 29 normal prostate tissue samples, 0 points; 6-25%, 1 point; 26-50%, 2 points; 51-75%, 3 points was also collected (15). All patients were followed up for and >75%, 4 points. The final immunoreactivity scores (IRS) 13 months or longer. Detailed information on the clinical of each sample was calculated by adding the two scores for the features of all patients and healthy controls in the present immunostaining intensity and immunostaining percentage. study is summarized in Table I. All procedures performed in An IRS score <4 was defined as low expression and ≥4 was studies involving human patients were in accordance with the defined as high expression. ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later Assays of levels of DEPDC1B mRNA and protein in pros‑ amendments or comparable ethical standards. The present tate cell lines. Normal prostate epithelial cell (RWPE1), study is a retrospective study in accordance with ethics review androgen-dependent prostatic carcinoma cell (LNcap) and regulations and was reviewed and approved by the Ethics androgen-independent prostatic carcinoma cells (DU145 Committee, Sun Yat-sen Memorial Hospital, Sun Yat-sen and PC-3) were