MOLECULAR MEDICINE REPORTS 16: 6803-6813, 2017 Identification of genes associated with castration‑resistant prostate cancer by gene expression profile analysis CHUI GUO HUANG1, FENG XI LI2, SONG PAN3, CHANG BAO XU1, JUN QIANG DAI4 and XING HUA ZHAO1 1Department of Urology, The Second Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan 450014; 2Department of Gastrointestinal Glands Surgery, The First Affiliated Hospital, Guangxi Medical University, Nanning, Guangxi 530000; 3Department of Urology, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan 450014; 4Department of Neurosurgery, The Second Affiliated Hospital, Lanzhou University, Lanzhou, Gansu 730030, P.R. China Received December 16, 2016; Accepted July 17, 2017 DOI: 10.3892/mmr.2017.7488 Abstract. Prostate cancer (CaP) is a serious and common western countries (1). CaP growth is driven by androgens; genital tumor. Generally, men with metastatic CaP can easily therefore, the conventional treatment option is to lower the develop castration-resistant prostate cancer (CRPC). However, levels of male sex hormones (2). Current treatment strate- the pathogenesis and tumorigenic pathways of CRPC remain to gies for CaP include surgery, external beam radiotherapy, be elucidated. The present study performed a comprehensive brachytherapy, chemotherapy and androgen-deprivation analysis on the gene expression profile of CRPC in order to therapy (ADT) (2,3). ADT often involves surgically determine the pathogenesis and tumorigenic of CRPC. The removing the testicles or using drugs to block the andro- GSE33316 microarray, which consisted of 5 non-castrated gens from affecting the body (4). Unfortunately, ADT has samples and 5 castrated samples, was downloaded from the gene a limited role in preventing majority of patients progressing expression omnibus database. Subsequently, 201 upregulated to castration-resistant prostate cancer (CRPC) and patients and 161 downregulated differentially expressed genes (DEGs) with metastatic prostate cancer progress to resistance of were identified using the limma package in R and those genes ADT (4,5). However, previous studies have been unable to were classified and annotated by plugin Mcode of Cytoscape. identity a mainstay cancer therapy. Thus, the mortality of Gene ontology (GO) and Kyoto Encyclopedia of Genes patients with CRPC continues to be very high (6). and Genomes (KEGG) pathway enrichment analyses were Previous studies have investigated novel molecular thera- performed using Database for Annotation, Visualization and peutic targets in CRPC and the influence that tumor metastasis Integrated Discovery and KEGG Orthology Based Annotation has on the biological processes. Activation of the androgen System 2.0 online tools to investigate the function of different receptor (AR) may stimulate tumor progression and prolif- gene modules. The BiNGO tool was used to visualize the level eration in prostate cancer (7), Koivisto et al (8) determined of enriched GO terms. Protein‑protein interaction network was that the activity of cytochrome P450 (CYP) may increase AR constructed using STRING and analyzed with Cytoscape. In gene amplification and continuous production of testosterone; conclusion, the present study determined that aldo‑keto reduc- therefore, the serum androgen levels following castration tase 3, cyclin B2, regulator of G protein signaling 2, nuclear can still contribute to prostate cancer cell growth and resis- factor of activated T‑cells and protein kinase C a may have tance. Seruga et al (9) determined that there are three core important roles in the development of CRPC. genes, including human epidermal growth factor receptor 2, transforming growth factor β (TGF-β) and the kinase of SRC Introduction family that are able to activate AR transduction pathways without androgen to stimulate. Chaux et al (10) suggested that Prostate cancer (CaP), as the most common genital neoplasm, the loss or mutation of tensin homolog have been implicated holds the highest incidence among men in the majority of in unlike properties of aggressive prostate cancer, including increasing the risk of biochemical relapse, reducing the time to metastasis and increasing the death rate in high‑risk cohorts of men. However, the molecular mechanisms with oncogenes or tumor suppressors that modulate the levels of critical proteins Correspondence to: Dr Xing Hua Zhao, Department of Urology, The Second Affiliated Hospital, Zhengzhou University, 2 Jingba remain unclear and their relevance in human disease and Road, Zhengzhou, Henan 450014, P.R. China therapy require further investigation. E-mail: [email protected] In order to provide novel mechanistic insights associ- ated with possible endogenous metastatic pathways in an Key words: castration-resistant prostate cancer, protein-protein androgen-deprived environment, the present study used the interaction network, differentially expressed genes, module analysis data from the gene expression profile available provided by Sun et al (11) that used the microarray of human pros- tate cancer xenograft model-LuCaP35 to analyze the gene 6804 HUANG et al: POTENTIAL BIOMARKERS OF CASTRATION-RESISTANT PROSTATE CANCER Table I. Top 10 upregulated and downregulated genes. A, Upregulated Gene symbol logFC Adjusted P-value F3 4.863582313 2.91x10-3 HAGLROS 4.847973534 9.32x10-6 GIMAP7 4.839624566 4.72x10-3 CEL 4.793641494 5.79x10-4 GPAT3 4.585119916 3.68x10-3 FAM3D 4.358475955 2.36x10-3 PDGFRL 3.929026478 1.09x10-3 RGS2 3.899872367 3.01x10-3 UPK3A 3.868345004 5.79x10-4 SI 3.843651777 7.42x10-4 B, Downregulated Gene symbol logFC Adjusted P-value MYBPC1 -4.622857598 7.75x10-3 LAMA1 ‑4.215584491 1.45x10-3 S100P ‑4.16482084 9.77x10-3 LEFTY1 ‑4.151682649 8.22x10-3 DEFB1 ‑3.863896718 1.59x10-3 LOX ‑3.400271316 1.06x10-3 DHRS2 ‑3.381711553 2.59x10-3 CCK ‑3.328128227 1.53x10-3 KRT19 ‑3.323100902 6.53x10-3 ANGPYL4 -3.29177361 9.74x10-3 FC, fold-change. Figure 1. Hierarchical clustering heat maps of the DEGs. The horizontal axis represents sample names, with GSM823845, GSM823846, GSM823847, GSM823851 and GSM823852 being the castration samples, and GSM823844, GSM823848, GSM823849, GSM823850 and GSM823853 the non-castration samples. The vertical axis indicates the clusters of DEGs: Red color stands Figure 2. Top 30 hub genes in from the constructed protein-protein inter- for an expression level above the mean and green color stands for the expres- action network. The horizontal axis represents degree. The vertical axis sion lower than the mean. DEGs, differentially expressed genes. indicated hub genes. MOLECULAR MEDICINE REPORTS 16: 6803-6813, 2017 6805 Figure 3. Protein‑protein interaction network constructed from the DEGs. Red circles indicate upregulated DEGs and green circles indicate downregulated DEGs. DEGs, differentially expressed genes. expression changes between 5 non-castrated and 5 castrated prostate cancer xenograft model-LuCaP35, included 5 controlled men. The current study identified 201 upregulated differen- samples from non-castrated men (GSM823844, GSM823848, tially expressed genes (DEGs) and 161 downregulated DEGs GSM823849, GSM823850, GSM823853) and 5 samples from using a stricter threshold of false discovery rate (FDR)<0.05 castrated men (GSM823845, GSM823846, GSM823847, and |log2 fold-change (FC)|>1.5. The visual protein-to-protein GSM823851, GSM823852). interaction (PPI) network was constructed using Cytoscape and the modules were identified with the Mcode plugin. Differentially expressed gene analysis. The series matrix file The present study used Gene Ontology (GO) and Kyoto was downloaded and a log2 transformation was performed. Encyclopedia of Genes and Genomes (KEGG) pathway to All sample data was normalized using the limma package analyze the potential interactions and functions of DEGs. in R software version 3.3.0 (https://www.r-project.org/) (12). In conclusion, the present study identified key genes which The DEGs were obtained with thresholds of |logFC|>1.5 and may have the potential to be biomarkers of tumorigenesis in P<0.05, using linear models and empirical Bayes methods for patients with CRPC. assessing differential expression in microarray experiments. Finally, a clustering analysis was performed using the DEGs Materials and methods and a heatmap of different groups including castrated and non-castrated was constructed (Fig. 1). Microarray data. The transcription profile of GSE33316 was downloaded from the National Center for Biotechnology Construction of PPI network and module analysis. In order Information Gene Expression Omnibus (GEO) database to predict protein interactions, which include physical and (www.ncbi.nlm.nih.gov/geo/) (11). The data used a human functional associations the present study used the Search Tool 6806 HUANG et al: POTENTIAL BIOMARKERS OF CASTRATION-RESISTANT PROSTATE CANCER Figure 4. Topology parameters of protein‑protein interaction networks. (A) Average clustering coefficient distribution. (B) Closeness centrality. (C) Neighborhood connectivity distribution. (D) Node‑degree distribution. (E) Shortest path length distribution. (F) Topological coefficients. for the Retrieval of Interacting Genes (STRING) to construct Results the PPI network for DEGs (minimum required interaction score >0.4) (13). In addition, Cytoscape software version 3.4.0 Identification of DEGs. Using microarray expression (http://cytoscape.org/download_old_versions.html) was used profiling from GEO database, the present study identified for visualization of the PPI networks. Following