4564 ONCOLOGY LETTERS 15: 4564-4570, 2018 SOX2 and SOX12 are predictive of prognosis in patients with clear cell renal cell carcinoma WEIJIE GU1,2*, BEIHE WANG1,2*, FANGNING WAN1,2*, JUNLONG WU1,2, XIAOLIN LU1,2, HONGKAI WANG1,2, YAO ZHU1,2, HAILIANG ZHANG1,2, GUOHAI SHI1,2, BO DAI1,2 and DINGWEI YE1,2 1Department of Urology, Fudan University Shanghai Cancer Center; 2Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, P.R. China Received October 21, 2016; Accepted September 28, 2017 DOI: 10.3892/ol.2018.7828 Abstract. Sex-determining region Y-box protein (SOX) genes were associated with poor prognosis for OS (log-rank test, all serve an important role in cancer growth and metastasis. P<0.05). SOX2 and SOX12 were identified as independent The present study aimed to determine the predictive ability prognostic factors of OS in clear cell RCC. of SOX and associated genes identified through molecular network in clear cell renal cell carcinoma (RCC). A total of Introduction 505 patients with clear cell RCC from The Cancer Genome Atlas (TCGA) cohorts were collected in this study. The Renal cell carcinoma (RCC) accounts for ~2-3% of all malig- expression profile of SOX and associated genes were obtained nancies worldwide (1). Despite an increasing proportion of from the TCGA RNAseq database. Clinicopathological patients with early stage tumors at diagnosis and the develop- characteristics, including age, gender, tumor grade, stage, ment of novel treatment strategies, a quarter still present with laterality disease-free-survival and overall survival (OS) were locally advanced or metastatic disease, and eventually, one collected. Cox's proportional hazards regression model, as third of patients submitted to surgical nephrectomy develop well as Kaplan-Meier curves were used to assess the relative recurrence, or metastasis (2). The clinical outcomes of RCC factors. Selected genes of SOXs that demonstrated significant vary widely, emphasizing the need for prompt and accurate associations with OS were further validated in 192 patients prognostic stratification. To date, the best prognostic system from the validation cohort. In the univariate Cox regression for overall survival (OS) is the tumor node metastasis (TNM) model, SOX1, SOX2, SOX6, SOX11, SOX12, SOX13, SOX15, staging system in RCC, although it is insufficient to signifi- SOX17 and SOX30 expression were predictive in the prognosis cantly improve the management of patients (3,4). Important of clear cell RCC. Following adjustment for clinical factors, prognostic factors of RCC include tumor size, histological SOX2 [hazard ratio (HR), 1.130; 95% confidence interval (CI), subtype, nuclear grade, local extent of the tumor and evidence 1.002-1.275), SOX12 (HR, 1.379; 95% CI, 1.060-1.793) and of metastatic disease at presentation (5). Therefore, identifica- SOX15 (HR, 1.245; 95% CI, 1.063-1.459) remained statisti- tion of novel reliable predictive and independent prognostic cally significant. Furthermore, POU class 5 homeobox 1 factors is critical for improving therapeutic modalities and for (POU5F1), POU2F1 and nuclear receptor subfamily 5 group prolonging the survival of patients with RCC. A member 1 in the gene cluster network analysis associated Sex-determining region Y (SRY)-box protein (SOX) with SOX2 did not reduce the statistical significance when genes arise from the founding member Sry, the mammalian added to the multivariate analysis. The findings were extended testis-determining factor. Currently, 20 SOX proteins are to the Fudan University Shanghai Cancer Center cohort. known in mammals and characterized by a conserved high The results revealed that high SOX2 and SOX12 expression mobility group DNA-binding domain (6,7). Members of the SOX family are developmental regulators with functions in sex determination, chondrogenesis, hematopoiesis, neural crest development and neurogenesis (8). Numerous findings support the involvement of different SOX genes in cancer Correspondence to: Professor Dingwei Ye, Department development. The expression of SOX genes is reported to be of Oncology, Shanghai Medical College, Fudan University, associated with prognosis in various cancer types, including 270 Dong'an Road, Shanghai 200032, P.R. China lung (9,10), brain (11), hepatocellular (12), gastric (13), E-mail: [email protected] prostate (14) and cervical squamous cell carcinoma (15). *Contributed equally However, the potential role of SOX family members in RCC, and its biological functions on the initiation, progression and Key words: SOX2, SOX12, prognosis, clear cell renal cell outcome of the disease remain unclear. Therefore, the present carcinoma, TCGA study aimed to analyze the prognostic value of SOX genes in patients with RCC using public database and validate these findings in a patient cohort. GU et al: SOX EXPRESSION IS PREDICTIVE OF PROGNOSIS IN CLEAR CELL RCC 4565 Materials and methods 5'-TGG ACA GTT ACG CGC ACA T-3' and reverse, 5'-CGA GTA GGA CAT GCT GTA GGT-3'; SOX12 forward, 5'-AAG AGG Patients and samples. The Cancer Genome Atlas (TCGA) CCG ATG AAC GCA TT-3' and reverse, 5'-TAG TCC GGG TAA database makes available gene expression level and clinical TCC GCC AT-3'; SOX15 forward, 5'-GCG ACT ACC CCG ACT data on RCC from the website of Cancer Genomics Browser ACA AG-3' and reverse, 5'-TTG CAG TGG GAA GAG CCA of University of California Santa Cruz (https://genome-cancer. TA-3'; β-actin forward, 5'-AGC GAG CAT CCC CCA AAG TT-3' ucsc.edu/). Only patients with fully characterized tumors, and reverse, 5'-GGG CAC GAA GGC TCA TCA TT-3'. intact OS, disease-free survival (DFS), and RNAseq infor- mation were included as described previously (16). Patients Statistical analysis. Survival endpoints were mortality due receiving pretreatment were excluded. Clinicopathological to any cause for OS and recurrence at any site for DFS. DFS characteristics, including age, gender, tumor size, TNM stage, and OS rates were calculated based on the Kaplan-Meier tumor grade, stage, laterality, OS and DFS were collected. method, and the curves were compared with log-rank tests. A total of 16 patients whose samples were confirmed with Variables of SOX gene expression with P<0.10 in univariate non-clear cell RCC were excluded (17). Finally, 505 patients and multivariate Cox's proportional hazard models in the confirmed with primary clear cell RCC with detailed SOX TCGA cohort were selected for further study. The final Cox's expression data were included in the current study. Proportional Hazards model, including clinical data and genes Network analysis of genes that were associated with OS in the network associated with OS was performed in TCGA, was performed using the tools from cBioPortal (http://www. and validation cohorts. Categorical data were analyzed using cbioportal.org/public-portal/cgds_r.jsp). Genes were consid- Fisher's exact χ² test. Continuous data were analyzed using a ered in the same network if they were in the same complex or Student's t-test. All the statistical analyses were performed interacted with each other with >12% of changes. Information using SPSS software version 20.0 (IBM Corp., Armonk, NY, from 20 members of the SOX family and the associated genes USA). Two-tailed P<0.05 was considered to indicate a statisti- obtained from the TCGA RNAseq database are listed in cally significant difference. Table IV. In order to validate the prognostic value of these genes, Results a validation cohort from Fudan University Shanghai Cancer Center (FUSCC; Shanghai, China) was established, including Clinical characteristics in TCGA and validation cohorts. In 192 patients with histologically confirmed clear cell RCC the TCGA cohort, the median age of the 505 patients with between February 2009 and June 2012 who underwent radical clear cell RCC was 60.6 years old, ranging between 26 and nephrectomy or nephron sparing nephrectomy. Patient charac- 90 years old. A total of 325 (64.4%) were male patients and teristics parallel to TCGA data, including age, gender, tumor 180 (35.6%) were female patients. Tumor size, TNM stage, size, TNM stage, tumor grade, stage and tumor position were tumor grade, stage and laterality are presented in Table I. The obtained from clinical records. Patients with missing data on median OS of this cohort was 79.5 months and 163 patients the aforementioned variables were excluded. succumbed during the follow up. In the validation cohort, Trained research nurses followed up the cohort by tele- the median age of the 192 patients with clear cell RCC was phone once every 3-6 months, and recorded the events of 55.5 years old, ranging between 25 and 86 years old. A clinical interest, including tumor recurrence, progression and total of 135 (70.3%) were male patients and 57 (29.7%) were metastasis. All tissue samples were collected during surgeries female patients. Tumor size, TNM stage, tumor grade, stage and stored at ‑70˚C in the tissue bank of FUSCC. and tumor position are presented in Table I. The median OS The present study was approved by the institutional review of this cohort was 50.2 months and 42 patients succumbed board of FUSCC and written informed consent was obtained during follow up. from all patients. SOX gene expression and the clinical outcomes in the TCGA, RNA extraction and reverse transcription‑quantitative and validation cohorts. In the univariate Cox's proportion polymerase chain reaction (RT‑qPCR) analysis. For the hazard ratio analysis, age, T stage, metastasis, tumor stage, validation cohort, 192 frozen tissue samples (100 mg) were tumor grade, hemoglobin level, white blood cell count and harvested and ground into a fine powder. Total RNA was platelet count, expression of SOX1, SOX2, SOX6, SOX7, isolated using TRIzol® reagent (cat. no. 15596-026; Invitrogen, SOX11, SOX12, SOX13, SOX15, SOX17, and SOX30 were Thermo Fisher Scientific, Inc.). A PrimeScript RT reagent kit significantly associated with prognosis regarding OS in (cat.