CDC6 mRNA Expression Is Associated with the Aggressiveness of Prostate Cancer

BACKGROUND: Cell division cycle 6 (CDC6) is an essential regulator of DNA replication and plays important roles in the activation and maintenance of the checkpoint mechanisms in the cell cycle. CDC6 has been associated with oncogenic activities in human cancers; however, the clinical significance of CDC6 in prostate cancer (PCa) remains unclear. Therefore, we investigated whether the CDC6 mRNA expression level is a diagnostic and prognostic marker in PCa. METHODS: The study subjects included 121 PCa patients and 66 age-matched benign prostatic hyperplasia (BPH) patients. CDC6 expression was evaluated using real-time polymerase chain reaction and immunohistochemical (IH) staining, and then compared according to the clinicopathological characteristics of PCa. RESULTS: CDC6 mRNA expression was significantly higher in PCa tissues than in BPH control tissues (P = 0.005). In addition, CDC6 expression was significantly higher in patients with elevated prostate-specific antigen (PSA) levels (> 20 ng/mL), a high Gleason score, and advanced stage than in those with low PSA levels, a low Gleason score, and earlier stage, respectively. Multivariate logistic regression analysis showed that high expression of CDC6 was significantly associated with advanced stage (≥ T3b) (odds ratio [OR], 3.005; confidence interval [CI], 1.212–7.450; P = 0.018) and metastasis (OR, 4.192; CI, 1.079–16.286; P = 0.038). Intense IH staining for CDC6 was significantly associated with a high Gleason score and advanced tumor stage including lymph node metastasis stage (linear-by-linear association, P = 0.044 and P = 0.003, respectively). CONCLUSION: CDC6 expression is associated with aggressive clinicopathological characteristics in PCa. CDC6 may be a potential diagnostic and prognostic marker in PCa patients.

Generation of Whole-Genome Sequencing Data for Comparing Primary and Castration-Resistant Prostate Cancer

Because castration-resistant prostate cancer (CRPC) does not respond to androgen deprivation therapy and has a very poor prognosis, it is critical to identify a prognostic indicator for predicting high-risk patients who will develop CRPC. Here, we report a dataset of whole genomes from four pairs of primary prostate cancer (PC) and CRPC samples. The analysis of the paired PC and CRPC samples in the whole-genome data showed that the average number of somatic mutations per patients was 7,927 in CRPC tissues compared with primary PC tissues (range, 1,691 to 21,705). Our whole-genome sequencing data of primary PC and CRPC may be useful for understanding the genomic changes and molecular mechanisms that occur during the progression from PC to CRPC.

Metabolic Pathway Signatures Associated with Urinary Metabolite Biomarkers Differentiate Bladder Cancer Patients from Healthy Controls

PURPOSE: Our previous high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry study identified bladder cancer (BCA)-specific urine metabolites, including carnitine, acylcarnitines, and melatonin. The objective of the current study was to determine which metabolic pathways are perturbed in BCA, based on our previously identified urinary metabolome. MATERIALS AND METHODS: A total of 135 primary BCA samples and 26 control tissue samples from healthy volunteers were analyzed. The association between specific urinary metabolites and their related encoding genes was analyzed. RESULTS: Significant alterations in the carnitine-acylcarnitine and tryptophan metabolic pathways were detected in urine specimens from BCA patients compared to those of healthy controls. The expression of eight genes involved in the carnitine-acylcarnitine metabolic pathway (CPT1A, CPT1B, CPT1C, CPT2, SLC25A20, and CRAT) or tryptophan metabolism (TPH1 and IDO1) was assessed by RT-PCR in our BCA cohort (n=135). CPT1B, CPT1C, SLC25A20, CRAT, TPH1, and IOD1 were significantly downregulated in tumor tissues compared to normal bladder tissues (p<0.05 all) of patients with non-muscle invasive BCA, whereas CPT1B, CPT1C, CRAT, and TPH1 were downregulated in those with muscle invasive BCA (p<0.05), with no changes in IDO1 expression. CONCLUSION: Alterations in the expression of genes associated with the carnitine-acylcarnitine and tryptophan metabolic pathways, which were the most perturbed pathways in BCA, were determined.

Increased Expression of Herpes Virus-Encoded hsv1-miR-H18 and hsv2-miR-H9-5p in Cancer-Containing Prostate Tissue Compared to That in Benign Prostate Hyperplasia Tissue / 대한배뇨장애요실금학회지

PURPOSE: Previously, we reported the presence of virus-encoded microRNAs (miRNAs) in the urine of prostate cancer (CaP) patients. In this study, we investigated the expression of two herpes virus-encoded miRNAs in prostate tissue. METHODS: A total of 175 tissue samples from noncancerous benign prostatic hyperplasia (BPH), 248 tissue samples from patients with CaP and BPH, and 50 samples from noncancerous surrounding tissues from these same patients were analyzed for the expression of two herpes virus-encoded miRNAs by real-time polymerase chain reaction (PCR) and immunocytochemistry using nanoparticles as molecular beacons. RESULTS: Real-time reverse transcription-PCR results revealed significantly higher expression of hsv1-miR-H18 and hsv2-miRH9- 5p in surrounding noncancerous and CaP tissues than that in BPH tissue (each comparison, P<0.001). Of note, these miRNA were expressed equivalently in the CaP tissues and surrounding noncancerous tissues. Moreover, immunocytochemistry clearly demonstrated a significant enrichment of both hsv1-miR-H18 and hsv2-miR-H9 beacon-labeled cells in CaP and surrounding noncancerous tissue compared to that in BPH tissue (each comparison, P<0.05 for hsv1-miR-H18 and hsv2- miR-H9). CONCLUSIONS: These results suggest that increased expression of hsv1-miR-H18 and hsv2-miR-H95p might be associated with tumorigenesis in the prostate. Further studies will be required to elucidate the role of these miRNAs with respect to CaP and herpes viral infections.

Erratum: Increased Expression of Herpes Virus-Encoded hsv1-miR-H18 and hsv2-miR-H9-5p in Cancer-Containing Prostate Tissue Compared to That in Benign Prostate Hyperplasia Tissue / 대한배뇨장애요실금학회지

In this article, a part of fund and grant supports was omitted unintentionally.

Urinary MicroRNAs of Prostate Cancer: Virus-Encoded hsv1-miRH18 and hsv2-miR-H9-5p Could Be Valuable Diagnostic Markers / 대한배뇨장애요실금학회지

PURPOSE: MicroRNAs (miRNAs) in biological fluids are potential biomarkers for the diagnosis and assessment of urological diseases such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The aim of the study was to identify and validate urinary cell-free miRNAs that can segregate patients with PCa from those with BPH. METHODS: In total, 1,052 urine, 150 serum, and 150 prostate tissue samples from patients with PCa or BPH were used in the study. A urine-based miRNA microarray analysis suggested the presence of differentially expressed urinary miRNAs in patients with PCa, and these were further validated in three independent PCa cohorts, using a quantitative reverse transcriptionpolymerase chain reaction analysis. RESULTS: The expression levels of hsa-miR-615-3p, hsv1-miR-H18, hsv2-miR-H9-5p, and hsa-miR-4316 were significantly higher in urine samples of patients with PCa than in those of BPH controls. In particular, herpes simplex virus (hsv)-derived hsv1-miR-H18 and hsv2-miR-H9-5p showed better diagnostic performance than did the serum prostate-specific antigen (PSA) test for patients in the PSA gray zone. Furthermore, a combination of urinary hsv2-miR-H9-5p with serum PSA showed high sensitivity and specificity, providing a potential clinical benefit by reducing unnecessary biopsies. CONCLUSIONS: Our findings showed that hsv-encoded hsv1-miR-H18 and hsv2-miR-H9-5p are significantly associated with PCa and can facilitate early diagnosis of PCa for patients within the serum PSA gray zone.

Urinary Nucleic Acid TSPAN13-to-S100A9 Ratio as a Diagnostic Marker in Prostate Cancer

The potential use of urinary nucleic acids as diagnostic markers in prostate cancer (PCa) was evaluated. Ninety-five urine samples and 234 prostate tissue samples from patients with PCa and benign prostatic hyperplasia (BPH) were analyzed. Micro-array analysis was used to identify candidate genes, which were verified by the two-gene expression ratio and validated in tissue mRNA and urinary nucleic acid cohorts. Real-time quantitative polymerase chain reaction (qPCR) was used to measure urinary nucleic acid levels and tissue mRNA expression. The TSPAN13-to-S100A9 ratio was selected to determine the diagnostic value of urinary nucleic acids in PCa (P = 0.037) and shown to be significantly higher in PCa than in BPH in the mRNA and nucleic acid cohort analyses (P < 0.001 and P = 0.013, respectively). Receiver operating characteristic (ROC) analysis showed that the area under the ROC curve was 0.898 and 0.676 in tissue mRNA cohort and urinary nucleic acid cohort, respectively. The TSPAN13-to-S100A9 ratio showed a strong potential as a diagnostic marker for PCa. The present results suggest that the analysis of urine supernatant can be used as a simple diagnostic method for PCa that can be adapted to the clinical setting in the future.

Novel Combination Markers for Predicting Survival in Patients with Muscle Invasive Bladder Cancer: USP18 and DGCR2

We performed gene expression profiling in bladder cancer patients to identify cancer-specific survival-related genes in muscle invasive bladder cancer (MIBC) patients. Sixty-two patients with MIBC were selected as the original cohort and another 118 MIBC patients were chosen as a validation cohort. The expression of USP18, DGCR2, and ZNF699 genes were measured and we analyzed the association between gene signatures and survival. USP18 and DGCR2, were significantly correlated to cancer-specific death (P=0.020, P=0.007, respectively). Cancer-specific survival in the low USP18 or DGCR2 expression group was significantly longer than the high expression group (P=0.018, P=0.006, respectively). In multivariate Cox regression analysis, a combination of USP18 and DGCR2 mRNA expression levels were significant risk factors for cancer-specific death (HR, 2.106; CI, 1.043-4.254, P=0.038). Overall survival and cancer-specific survival rates in the low-combination group were significantly longer than those in the high-expression group (P=0.001, both). In conclusion, decreased expressions of USP18 and DGCR2 were significantly associated with longer cancer-specific survival, and also the combination of two genes was correlated to a longer survival for MIBC patients. Thus, the combination of USP18 and DGCR2 expression was shown to be a reliable prognostic marker for cancer-specific survival in MIBC.

GSTM1 Tissue Genotype as a Recurrence Predictor in Non-muscle Invasive Bladder Cancer

Tissue genotyping is more useful approach than using blood genomic DNA, which can reflect the effects of the somatic mutations in cancer. Although polymorphisms in glutathione S-transferase (GST) have been associated with the risk of bladder cancer (BC) development, few reports provide information about the prognosis of BC. We investigated glutathione S-transferase mu (GSTM1) and glutathione S-transferase theta (GSTT1) genotypes using genomic DNA from primary 165 BC tissue samples to assess the association with disease prognosis. DNA samples from tumor were analyzed by multiplex polymerase chain reaction (PCR). The results were compared with clinicopathological parameters. The prognostic significance of the GSTs was evaluated by Kaplan-Meier and multivariate Cox regression model. Kaplan-Meier estimates revealed significant differences in time to tumor recurrence according to the GSTM1 tissue genotype (P = 0.038) in non-muscle invasive bladder cancer (NMIBC). Multivariate Cox regression analysis also revealed that the tissue GSTM1 genotype (hazards ratio [HR]: 0.377, P = 0.031) was an independent predictor of bladder tumor recurrence in NMIBC. This identification of GSTM1 tissue genotype as a prognosticator for determining recurrence in NMIBC should prove highly useful in a clinical setting.

Clinical Significance of the Expression of Gli2 and Gli3 in Bladder Cancer / 대한비뇨기과학회지

PURPOSE: The HedgehogGli(HHGli) signaling pathway controls many aspects of tissue patterning, cell proliferation, differentiation and regeneration, and regulates the number of cells in various organs. Inappropriate and uncontrolled activation of the HHGli signaling pathway has been demonstrated in a variety of human cancers. The Gli1, Gli2, and Gli3 genes encoding the Gli family transcription factors play a role as HH effectors. This study examined the significance in Gli2 and Gli3 expression in human bladder cancer. MATERIALS AND METHODS: The tumor tissues were obtained from 144 patients with a primary bladder cancer. The mRNA levels of Gli2, and Gli3 were examined using a real-time polymerase chain reaction(PCR) assay in 144 tumor specimens, and immunohistochemical staining was performed on 127 tumor paraffin blocks. The relationships between their expression and the pathological or clinical characteristics, such as tumor stage, grade, recurrence and progression were also analyzed. RESULTS: Gli2 mRNA expression was higher in the invasive bladder tumors than in the superficial bladder tumors(p<0.001) but, there was no difference in Gli3 mRNA expression according to the tumor stage and grade. The multivariate Cox regression model revealed that Gli2 mRNA expression(hazards ratio(HR): 2.329, 95% confidence interval(CI): 1.043- 5.202, p=0.039) was the only strong predictor of superficial bladder tumor recurrence. Kaplan-Meier analysis also showed identical results (log-rank test, p=0.043). CONCLUSIONS: The enhanced expression of Gli2 mRNA was strongly correlated with the recurrence of superficial bladder cancer. These results suggest that Gli2 may be a useful marker for assessing the recurrence of superficial bladder cancer in human bladder cancers.

Expression and Correlation of Cyclooxygenase-2, Angiogenin and Vascular Endothelial Growth Factor in Human Transitional Cell Carcinoma / 대한비뇨기과학회지

PURPOSE: The role of cyclooxygenase (COX)-2 on the tumorigenesis and progression of human solid organ tumor is unknown, but recent studies have supported an important role for COX-2 in various cancers. Tumor development and progression depend on angiogenesis, which is mediated by several angiogenic factors, including angiogenin and vascular endothelial growth factor (VEGF). This study was performed to evaluate the expressions of COX-2, angiogenin and VEGF in the tumor tissues of patients with urothelial carcinomas. The correlation of these factors was also evaluated. MATERIALS AND METHODS: The quantification for the expressions of COX-2, angiogenin and VEGF mRNA was assessed in 171 human bladder tumor tissues and 34 normal bladder mucosa, using quantitative competitive polymerase chain reaction (QC-PCR). The expression levels of COX-2, angiogenin and VEGF mRNA in the bladder tumor and normal bladder mucosa were compared using Student's t-tests. The correlation of these factors was also assessed by a simple linear regression analysis. RESULTS: The COX-2, angiogenin and VEGF mRNA expressions in the bladder tumor tissues were significantly higher than those in the normal bladder mucosae of the controls. COX-2 expression had a significant correlation with that of VEGF, but not with that of angiogenin. CONCLUSIONS: This study showed that COX-2, angiogenin and VEGF were expressed in human bladder tumors. These finding support that these factors might play an important role in the tumorigenesis of human bladder tumors. These data suggest that angiogenesis, mediated by VEGF, may be involved in the neoplastic growth of urothelial carcinomas by COX-2.

Stress-Activated Protein Kinase/c-Jun NH2 Terminal Kinase (SAPK/JNK) Expression in Transitional Cell Carcinoma of the Urinary Bladder / 대한비뇨기과학회지

PURPOSE: The SAPK/JNK group of MAP (mitogen-activated protein) kinases is known to regulate cellular proliferation, apoptosis and tissue morphogenesis. This study was performed to assess the clinical usefulness of the SAPK/JNK for a bladder tumor. MATERIALS AND METHODS: Ninety-five bladder tumors and 23 normal bladder mucosa were included in this study. Expression of the phosphorylated and unphosphorylated forms of JNK1 and 2 were examined using western blot analysis. The relationship between JNK expression and the bladder tumor stage, grade, recurrence, survival and P53 expression level were analyzed. RESULTS: The unphosphorylated JNK1 level was higher in bladder tumors than in normal bladder mucosa. With respect to the stage, the absolute and relative values of the phosphorylated JNK1 were higher in superficial tumors than in invasive tumors, while those of unphosphorylated JNK1 were higher in invasive tumors. The phosphorylated JNK1 level also had a negative correlation with the tumor grade and recurrence. A positive correlation existed between the p53 expression level and the absolute values of unphosphorylated JNK1 and 2. CONCLUSIONS: Among the 3 isoforms, JNK1 plays a role in the development of a bladder tumor. Higher expressions of the inactive forms in a bladder tumor than in a normal control and the active forms in a low stage and grade tumor might support the hypothesis that the loss of JNK1 activation may contribute to tumorigenesis.

Polymorphisms of Human 8-Oxoguanine Glycosylase1 (hOGG1) in Bladder Tumor / 대한비뇨기과학회지

Purpose: Repairing damaged DNA has been shown to be involved in an increased susceptibility to cancer development and prevention. Therefore, the genetic polymorphisms of the hOGG1 gene associated with the gene repair mechanism were investigated. In this study, the possible association of genetic polymorphisms in hOGG1 with bladder tumor risk was examined. MATERIALS AND METHODS: The hospital based, case-control investigation was carried out in 168 primary bladder tumor patients and 672 controls. The DNA extracted from the blood and tissue samples was analyzed by SSCP, PCR-based restriction fragment length polymorphism (RFLP) and direct DNA sequencing in order to characterize the genetic polymorphism of hOGG1. RESULTS: Two polymorphic sites in hOGG1 were found. A polymorphism at codon 326 (1a type) in exon 7 was associated with an amino acid exchange. Another polymorphic site at codon 324 (1b type) in exon 6 was silent. The association between the polymorphism at codon 326 and the risk of the bladder tumor was examined by age-sex adjusted analysis. The distribution of the hOGG1 codon 326 genotypes in the controls (Ser/Ser, 18.9%; Ser/Cys, 54.0%; Cys/Cys, 27.1%) was significantly different from that in the bladder tumor patients (26.2%, 51.8% and 22.0%, respectively) (p=0.034, adjusted OR=0.652, 95% CI=0.44-0.97). In particular, bladder tumor risk in Korean males under 40 years old was approximately 6 times higher than in males over 40 years old (p=0.015, adjusted OR=0.165, 95% CI=0.04-0.75). Furthermore, frequent mutations of codon 326 in the hOGG1 gene in tumor tissues (23.6%) might occur during tumorigenesis. CONCLUSIONS: The data suggests that polymorphism at codon 326 of hOGG1 gene might affect tumorigenesis of a bladder tumor.

Effects of Genotype of Tumor Necrosis Factor Alpha Promoter on Bladder Tumor Grade / 대한비뇨기과학회지

Purpose: Several polymorphic sites have been reported in the tumor necrosis factor alpha (TNF-alpha) promoter. Comparative studies on TNF-alpha production with promoter genotype have reported variable results, particularly in the 308 promoter region. Therefore, genetic polymorphism of TNF-alpha promoter region ( 308) was investigated to determine if it was associated with bladder tumor. MATERIALS AND METHODS: The DNA from 113 and 109 respective blood samples of bladder tumor patients and controls was analyzed by a PCR-based restriction fragment length polymorphism (RFLP) method to characterize the genetic polymorphism of the 308 region of the TNF-alpha promoter. TNF-alpha expression was also checked by measuring the mRNA and protein content using a quantitative-competitive PCR and ELISA method, respectively. RESULTS: The difference in the genetic variations of TNF-alpha promoter did not exist between the bladder tumor patients and the control group (p=0.259). The tumor grade was significantly related to the GA genotype (p=0.04). The mRNA levels of TNF-alpha in the GA genotype were significantly higher than those in the GG genotype in bladder tumors (p=0.022). The TNF-alpha serum levels in the GA genotype were significantly higher than those in the GG genotype regardless of whether there was a bladder tumor. In addition, the TNF-alpha serum levels in bladder tumor patients were significantly higher than the controls in either the GG or GA type (GG type, p=0.001; GA type, p=0.009). CONCLUSIONS: The GA genotype of the TNF-alpha promoter region ( 308) had a significant impact on TNF-alpha production and is related to a higher-grade tumor compared to the GG genotype. The TNF-alpha serum levels in the bladder tumor patients were significantly higher than in the controls. This suggests that TNF-alpha might be involved in the tumorigenesis of the bladder.