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Chen et al. BMC Cancer (2020) 20:289 https://doi.org/10.1186/s12885-020-06776-7 RESEARCH ARTICLE Open Access Overexpression of SLC6A1 associates with drug resistance and poor prognosis in prostate cancer Chaojiang Chen1,2,3†, Zhiduan Cai1,2†, Yangjia Zhuo2†, Ming Xi4†, Zhuoyuan Lin3, Funeng Jiang2, Zezhen Liu1,2, Yueping Wan4, Yu Zheng3, Jianxin Li3, Xing Zhou3, Jianguo Zhu5* and Weide Zhong1,2,4,6* Abstract Background: Solute Carrier Family 6 Member 1 (SLC6A1) has been identified as a cancer-promoting gene in various human cancers, such as clear cell renal cell carcinoma and ovarian cancer. However, its roles in prostate cancer (PCa) has not been fully elucidated. The aim of this study was to investigate the expression and clinical significance of SLC6A1 in PCa tissues and its effect on drug resistance to docetaxel in PCa. Methods: Expression patterns of SLC6A1 protein in PCa tissues were examined by immunohistochemistry based on Tissue microarray. Associations of SLC6A1 protein expression with various clinicopathological features and patients’ prognosis of PCa were also statistically evaluated based on TCGA data. Roles of SLC6A1 deregulation in prostate carcinogenesis and drug resistance was further determined in vitro and in vivo experiments. Results: Based on TCGA Dataset, SLC6A1 expression was markedly higher in patients with high Gleason score, advanced clinical stage and positive biochemical recurrence than those with control features (all P < 0.05). Both unvariate and multivariate analyses demonstrated that SLC6A1 expression was significantly associated with biochemical recurrence-free survival in PCa patients. In addition, enforced expression of SLC6A1 effectively promoted cell proliferation, migration and invasion of PCa cells in vitro. Moreover, the inhibition of SLC6A1 suppressed the tumor growth in vivo. Additionally, immunohistochemical notches of PCNA and MMP-9 in the lowexpression cluster were pointedly lower compared to those of NC group. Finally, the cell viability revealed that the overexpression of SLC6A1 obviously promoted the PCa cell resistant to docetaxel (DTX), and the transplanted tumor in the overexpression group had no significant reduction compared with the untreated group. Conclusions: Our data suggest that SLC6A1 overexpression may be associated with aggressive progression and short biochemical recurrence-free survival of PCa, and may be related to the resistance to docetaxel therapy. Keywords: Prostate cancer, Solute carrier family 6 member 1, Prognosis, Chemotherapy resistance * Correspondence: doctorzhujianguo@163.com; zhongwd2009@live.cn † Chaojiang Chen, Zhiduan Cai, Yangjia Zhuo and Ming Xi contributed equally to this work. 5 Department of Urology, Guizhou Provincial People’s Hospital, The Affiliated Hospital of Guizhou Medical University, Guiyang 550002, China 1 Guangdong Provincial Institute of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Chen et al. BMC Cancer (2020) 20:289 Background Prostate cancer, the fourth common malignancy in Europe [1], is the largest annual increase (26%) of men and the second (9%) in deaths in the United States [2]. Because of PSA screening and MRI examination, prostate cancer can be diagnosed and treated at earliest [3]. Despite of the effective treatments, including castration, radical prostatectomy, and high-dose radiotherapy, 20–30% of patients with prostate cancer may recur biochemically within 5–10 years and progress to hormone-resistant prostate cancer (CRPC) [4–6]. Docetaxel is the first-line medication permitted by FDA for the treatment of CRPC [7]. However, clinical evidence show that only 48% of patients with prostate cancer may respond to the treatment of docetaxel plus prednisone, with an average survival improvement of two and half months over the control group [8]. Therefore, it is of great clinical significance to identify molecular biomarkers which can predict the patients’ response to the treatment of docetaxel, in order to assist in screening the responsive prostate cancer patients to docetaxel and thus potentially benefit individualized therapy of prostate cancer in a daily clinical setting. Ion channel and transporter (ICT), a new type of membrane proteins, has been observed to be expressed in various human cancers, such as prostate cancer, which expressed KCNA3(Potassium voltage-gated channel, Shaker-related subfamily, member 3) [9] and TRPV6(Transient receptor potential cation channel, subfamily V, member 6) [10] in primary tumors. ICT may play a role in regulating the malignancy of cancer cells, including cell proliferation, migration, invasiveness and apoptosis. As a member of ICT,SLC6A1 encodes sodium- and chloride-dependent gamma amino-butyric acid (GABA) carrier (GAT-1), and restores it to presynaptic terminals by eliminating GABA with synaptic cleft [11]. SLC6A1 has been indicated to be involved into several diseases of the nervous system [12], such as epilepsy [13], schizophrenia [14] and hippocampal sclerosis [15]. Notably, the aberrant expression of SLC6A1 at both gene and protein levels have been also found in various human cancers. For example, SLC6A1 expression in mucosa of atrophic gastritis and duodenal metaplasia were up-regulated by more than 10 times as compared with normal gastric mucosa [16],; SLC6A1 overexpression was reported to promote cell growth and metastasis of clear cell renal cell carcinoma(CCRCC) [17] and was related to drug resistance to topotecan of ovarian cancer cell line [18]. To the best of our knowledgement, the expression patterns and the clinical significance of SLC6A1 in prostate cancer, as well as its associations with prostate cancer progression and patients’ response to chemotherapy remain unknown. To address these problems, we here Page 2 of 10 examined the expression patterns of SLC6A1 protein in PCa tissues by immunohistochemistry based on Tissue microarray. Associations of SLC6A1 protein expression with various clinicopathological features and patients’ prognosis of PCa were also statistically evaluated based on TCGA data. Roles of SLC6A1 deregulation in prostate carcinogenesis and drug resistance was further determined in vitro and in vivo experiments. Methods Ethic statement This study was approved by the human study ethics committees at Guangzhou First People’s Hospital and Guangzhou Medical University of P. R. China. All specimens were handled and made anonymous according to the ethical and legal standards. The mice were purchased from experimental animal center of Sun Yat sen University, and were killed by cervical dislocation. All animal experiments in this study were performed in compliance with the guidelines of the Institute for Laboratory Animal Research at Guangzhou Medical University, Guangzhou, P. R. China. Patients and tissue samples Tissue microarrays (TMA) included 50 prostate cancer tissues and 30 non-cancerous prostate tissues obtained from 80 consecutive prostate cancer patients purchased from Xi’an Ailina Biotechnology Co, Ltd. (Xi’an, People’s Republic of China; catalog number: PR807c). Patients who received adjuvant or neoadjuvant hormonal or radiation treatment prior to cancer recurrence were excluded. All hematoxyolin-eosin (H&E)-stained sections from each case were reviewed, and the Gleason score was reassigned according to the current grading recommendation provided by the International Society of Urological Pathology. Immunoreactivity scores (IRS) is the sum of the percentage and the intensity score. Staining was scored in accordance with the percentage and intensity of staining. The scores of staining percentage were as follows: 0 (≤5%), 1 (5–30%), 2 (30–70%), 3 (≥70%); the scores of staining intensity were as follows: 0 (negative), 1 (weak), 2 (medium) and 3 (strong). The final IRS (0–6) is the sum of percentage score plus intensity score. The median value of all SLC6A IRSs was used as a cutoff in this study. In addition, we also collected the TCGA dataset and the Taylor dataset, which both are publicly available datasets including 498 and 113 primary prostate cancer patients with the expression data of SLC6A1 mRNA, respectively. Table 1 summarized the clinicopathological data of prostate cancer patients enrolled in this study and the TCGA dataset, including age, Gleason score, clinical stage, metastasis status and PSA failure status. Chen et al. BMC Cancer (2020) 20:289 Page 3 of 10 Table 1 Associations of SLC6A1 protein expression with various clinicopathological characteristics of PCa patients Clinicopathological Characteristics IRS of SLC6A1 in our cohort (n, %) Case Low (n = 11) SLC6A1 expression in TCGA dataset High (n = 39) P Case Mean ± S.D. 0.070 355 15.05 ± 13.76 143 15.04 ± 10.67 292 12.47 ± 10.40 206 18.69 ± 15.15 P Age(years) < 66 20 7(35.0) 13(65.0) ≥ 66 30 4(13.3) 26(86.7) <8 31 6(19.4) 25(80.6) ≥8 19 5(26.3) 14(73.7)

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