Detection of human papillomavirus (HPV) DNA prevalence and p53 codon 72 (Arg72Pro) polymorphism in prostate cancer in a Greek group of patients.

Prostate cancer is the most common neoplasm found in males and the second most frequent cause of cancer-related mortality in males in Greece. Among other pathogens, the detection frequency of human papillomavirus (HPV) has been found to be significantly increased in tumor tissues among patients with sexually transmitted diseases (STDs), depending on the geographical distribution of each population studied. The present study focused on the detection of HPV and the distribution of Arg72Pro p53 polymorphism in a cohort of healthy individuals, as well as prostate cancer patients. We investigated the presence of HPV in 50 paraffin-embedded prostate cancer tissues, as well as in 30 physiological tissue samples from healthy individuals by real-time PCR. Furthermore, the same group of patients was also screened for the presence of the Arg72Pro polymorphism of the p53 gene, a p53 polymorphism related to HPV. Out of the 30 control samples, only 1 was found positive for HPV (3.33 %). On the contrary, HPV DNA was detected in 8 out of the total 50 samples (16 %) in the prostate cancer samples. The distribution of the three genotypes, Arg/Arg, Arg/Pro, and Pro/Pro, was 69.6, 21.7, and 8.7 % in the cancer patients and 75.0, 17.86, and 7.14 % in healthy controls, respectively. No statistically significant association was observed between the HPV presence and the age, stage, p53 polymorphism status at codon 72, or PSA. The increased prevalence of HPV detected in the prostate cancer tissues is in agreement with that reported in previous studies, further supporting the association of HPV infection and prostate cancer.

Expression of miRNAs involved in angiogenesis, tumor cell proliferation, tumor suppressor inhibition, epithelial-mesenchymal transition and activation of metastasis in bladder cancer.

PURPOSE: miRNAs are noncoding RNAs that posttranscriptionally regulate gene expression. Altered expression and function have been observed in bladder cancer. We analyzed the expression profile of a group of miRNAs involved in bladder cancer angiogenesis, tumor cell proliferation, tumor suppressor inhibition, epithelial-mesenchymal transition and metastasis activation. Prognostic and diagnostic value, and validated targets were further examined. MATERIALS AND METHODS: Using quantitative real-time polymerase chain reaction 77 bladder cancer cases and 77 matched tumor associated normal samples were investigated to determine the expression of miR-10b, 19a, 19b, 21, 126, 145, 205, 210, 221, 296-5p and 378. The relationship between miRNA expression, patient survival and tumor pathological features was also examined. RESULTS: miR-10b, 19a, 126, 145, 221, 296-5p and 378 were significantly down-regulated in bladder cancer compared to adjacent normal urothelium. miR-145 was the most down-regulated microRNA of this group. miR-19b, 21, 205 and 210 showed no significant difference between the 2 tissue types. High miR-21 expression correlated with worse overall patient survival (p = 0.0099). Multivariate analysis revealed that miR-21, 210 and 378 may serve as independent prognostic factors for overall patient survival (p = 0.005, 0.033 and 0.012, respectively). miR-21 and 378 may serve as independent prognostic factors for recurrence (p = 0.030 and 0.031, respectively). miR-145, 221, 296-5p and 378 showed the best combined ROC curves for specificity and sensitivity. miRWalk analysis was used to identify validated miRNA target genes. Further Gene Ontology enrichment revealed the main classes of biological functions of these validated targets. CONCLUSIONS: Most miRNAs analyzed are down-regulated in bladder cancer. They may serve as candidate biomarkers for diagnostic and prognostic purposes in the future.

Altered metabolic pathways in clear cell renal cell carcinoma: A meta-analysis and validation study focused on the deregulated genes and their associated networks.

Clear cell renal cell carcinoma (ccRCC) is the predominant subtype of renal cell carcinoma (RCC). It is one of the most therapy-resistant carcinomas, responding very poorly or not at all to radiotherapy, hormonal therapy and chemotherapy. A more comprehensive understanding of the deregulated pathways in ccRCC can lead to the development of new therapies and prognostic markers. We performed a meta- analysis of 5 publicly available gene expression datasets and identified a list of co- deregulated genes, for which we performed extensive bioinformatic analysis coupled with experimental validation on the mRNA level. Gene ontology enrichment showed that many proteins are involved in response to hypoxia/oxygen levels and positive regulation of the VEGFR signaling pathway. KEGG analysis revealed that metabolic pathways are mostly altered in ccRCC. Similarly, Ingenuity Pathway Analysis showed that the antigen presentation, inositol metabolism, pentose phosphate, glycolysis/gluconeogenesis and fructose/mannose metabolism pathways are altered in the disease. Cellular growth, proliferation and carbohydrate metabolism, were among the top molecular and cellular functions of the co-deregulated genes. qRT-PCR validated the deregulated expression of several genes in Caki-2 and ACHN cell lines and in a cohort of ccRCC tissues. NNMT and NR3C1 increased expression was evident in ccRCC biopsies from patients using immunohistochemistry. ROC curves evaluated the diagnostic performance of the top deregulated genes in each dataset. We show that metabolic pathways are mostly deregulated in ccRCC and we highlight those being most responsible in its formation. We suggest that these genes are candidate predictive markers of the disease.

New miRNA profiles accurately distinguish renal cell carcinomas and upper tract urothelial carcinomas from the normal kidney.

BACKGROUND: Upper tract urothelial carcinomas (UT-UC) can invade the pelvicalyceal system making differential diagnosis of the various histologically distinct renal cell carcinoma (RCC) subtypes and UT-UC, difficult. Correct diagnosis is critical for determining appropriate surgery and post-surgical treatments. We aimed to identify microRNA (miRNA) signatures that can accurately distinguish the most prevalent RCC subtypes and UT-UC form the normal kidney. METHODS AND FINDINGS: miRNA profiling was performed on FFPE tissue sections from RCC and UT-UC and normal kidney and 434 miRNAs were significantly deregulated in cancerous vs. the normal tissue. Hierarchical clustering distinguished UT-UCs from RCCs and classified the various RCC subtypes among them. qRT-PCR validated the deregulated expression profile for the majority of the miRNAs and ROC analysis revealed their capability to discriminate between tumour and normal kidney. An independent cohort of freshly frozen RCC and UT-UC samples was used to validate the deregulated miRNAs with the best discriminatory ability (AUC>0.8, p<0.001). Many of them were located within cytogenetic regions that were previously reported to be significantly aberrated. miRNA targets were predicted using the miRWalk algorithm and ingenuity pathway analysis identified the canonical pathways and curated networks of the deregulated miRNAs. Using the miRWalk algorithm, we further identified the top anti-correlated mRNA/miRNA pairs, between the deregulated miRNAs from our study and the top co-deregulated mRNAs among 5 independent ccRCC GEO datasets. The AB8/13 undifferentiated podocyte cells were used for functional assays using luciferase reporter constructs and the developmental transcription factor TFCP2L1 was proved to be a true target of miR-489, which was the second most upregulated miRNA in ccRCC. CONCLUSIONS: We identified novel miRNAs specific for each RCC subtype and UT-UC, we investigated their putative targets, the networks and pathways in which they participate and we functionally verified the true targets of the top deregulated miRNAs.

Gene expression is highly correlated on the chromosome level in urinary bladder cancer.

OBJECTIVE: Chromosome correlation maps display correlations between gene expression patterns on the same chromosome. Our goal was to map the genes on chromosome regions and to identify correlations through their location on chromosome regions. MATERIALS AND METHODS: Following microarray analysis we used Ingenuity Pathway Analysis (IPA) to construct gene networks of the co-deregulated genes in bladder cancer. Chromosome mapping, mathematical modeling and data simulations were performed using the WebGestalt and Matlab(®) softwares. RESULTS: The top deregulated molecules among 129 bladder cancer samples were implicated in the PI3K/AKT signaling, cell cycle, Myc-mediated apoptosis signaling and ERK5 signaling pathways. Their most prominent molecular and cellular functions were related to cell cycle, cell death, gene expression, molecular transport and cellular growth and proliferation. Chromosome correlation maps allowed us to detect significantly co-expressed genes along the chromosomes. We identified strong correlations among tumors of Tα-grade 1, as well as for those of Tα-grade 2, in chromosomes 1, 2, 3, 7, 12 and 19. Chromosomal domains of gene co-expression were revealed for the normal tissues, as well. The expression data were further simulated, exhibiting an excellent fit (0.7 < R(2) < 0.9). The simulations revealed that along the different samples, genes on same chromosomes are expressed in a similar manner. CONCLUSIONS: Gene expression is highly correlated on the chromosome level. Chromosome correlation maps of gene expression signatures can provide further information on gene regulatory mechanisms. Gene expression data can be simulated using polynomial functions.

Role of the angiogenic components, VEGFA, FGF2, OPN and RHOC, in urothelial cell carcinoma of the urinary bladder.

The objective of this study was to analyze the expression profile of the angiogenic components, vascular endothelial growth factor-A (VEGFA), basic fibroblast growth factor-2 (FGF2), osteopontin (OPN) and ras homolog gene family, member C (RHOC), in urothelial cell carcinoma (UCC) of the urinary bladder and to examine their role as candidate diagnostic biomarkers. Using qPCR, 77 samples of UCC of the urinary bladder and 77 matched tumor-associated normal samples were investigated to determine the expression of the four angiogenic components. The correlation between gene expression, patient survival and pathological features of the tumors was also examined. The VEGFA and OPN transcript levels were greater in the bladder cancer tissue than in the normal urothelium (P<0.001). Patients with higher VEGFA mRNA levels showed a tendency towards shorter cancer-specific survival. OPN levels showed a gradual increase, the lowest levels being found in non-invasive carcinoma and the highest in muscle invasive tumors. Elevated OPN levels indicated poor prognosis in connection with advanced disease stage (P<0.001). Both superficially invasive and muscle invasive tumors had significantly higher FGF2 levels compared to the control tissues (P=0.018 and P=0.050, respectively). Moreover, FGF2 was significantly higher in the metastatic vs. the non-metastatic tumors (P=0.0097). FGF2 levels exhibited a trend towards a correlation with worse patient survival. RHOC mRNA levels were higher in muscle invasive compared to superficially invasive tumors, as well as in grade III vs. grade I/II tumors. Furthermore, we detected worse overall survival for patients with high RHOC expression levels. VEGFA and FGF2 exhibited the best linear combination in the ROC curves for specificity and sensitivity. Thus, VEGFA and FGF2 may serve as candidate biomarkers for diagnostic purposes. Higher OPN expression may be used as a potential biomarker to predict patient survival relative to advanced tumor stage. However, further studies are required to investigate its role in urinary bladder carcinogenesis.

Spotlight on differentially expressed genes in urinary bladder cancer.

INTRODUCTION: We previously identified common differentially expressed (DE) genes in bladder cancer (BC). In the present study we analyzed in depth, the expression of several groups of these DE genes. MATERIALS AND METHODS: Samples from 30 human BCs and their adjacent normal tissues were analyzed by whole genome cDNA microarrays, qRT-PCR and Western blotting. Our attention was focused on cell-cycle control and DNA damage repair genes, genes related to apoptosis, signal transduction, angiogenesis, as well as cellular proliferation, invasion and metastasis. Four publicly available GEO Datasets were further analyzed, and the expression data of the genes of interest (GOIs) were compared to those of the present study. The relationship among the GOI was also investigated. GO and KEGG molecular pathway analysis was performed to identify possible enrichment of genes with specific biological themes. RESULTS: Unsupervised cluster analysis of DNA microarray data revealed a clear distinction in BC vs. control samples and low vs. high grade tumors. Genes with at least 2-fold differential expression in BC vs. controls, as well as in non-muscle invasive vs. muscle invasive tumors and in low vs. high grade tumors, were identified and ranked. Specific attention was paid to the changes in osteopontin (OPN, SPP1) expression, due to its multiple biological functions. Similarly, genes exhibiting equal or low expression in BC vs. the controls were scored. Significant pair-wise correlations in gene expression were scored. GO analysis revealed the multi-facet character of the GOIs, since they participate in a variety of mechanisms, including cell proliferation, cell death, metabolism, cell shape, and cytoskeletal re-organization. KEGG analysis revealed that the most significant pathway was that of Bladder Cancer (p = 1.5×10(-31)). CONCLUSIONS: The present work adds to the current knowledge on molecular signature identification of BC. Such works should progress in order to gain more insight into disease molecular mechanisms.

Identification of common differentially expressed genes in urinary bladder cancer.

BACKGROUND: Current diagnosis and treatment of urinary bladder cancer (BC) has shown great progress with the utilization of microarrays. PURPOSE: Our goal was to identify common differentially expressed (DE) genes among clinically relevant subclasses of BC using microarrays. METHODOLOGY/PRINCIPAL FINDINGS: BC samples and controls, both experimental and publicly available datasets, were analyzed by whole genome microarrays. We grouped the samples according to their histology and defined the DE genes in each sample individually, as well as in each tumor group. A dual analysis strategy was followed. First, experimental samples were analyzed and conclusions were formulated; and second, experimental sets were combined with publicly available microarray datasets and were further analyzed in search of common DE genes. The experimental dataset identified 831 genes that were DE in all tumor samples, simultaneously. Moreover, 33 genes were up-regulated and 85 genes were down-regulated in all 10 BC samples compared to the 5 normal tissues, simultaneously. Hierarchical clustering partitioned tumor groups in accordance to their histology. K-means clustering of all genes and all samples, as well as clustering of tumor groups, presented 49 clusters. K-means clustering of common DE genes in all samples revealed 24 clusters. Genes manifested various differential patterns of expression, based on PCA. YY1 and NFκB were among the most common transcription factors that regulated the expression of the identified DE genes. Chromosome 1 contained 32 DE genes, followed by chromosomes 2 and 11, which contained 25 and 23 DE genes, respectively. Chromosome 21 had the least number of DE genes. GO analysis revealed the prevalence of transport and binding genes in the common down-regulated DE genes; the prevalence of RNA metabolism and processing genes in the up-regulated DE genes; as well as the prevalence of genes responsible for cell communication and signal transduction in the DE genes that were down-regulated in T1-Grade III tumors and up-regulated in T2/T3-Grade III tumors. Combination of samples from all microarray platforms revealed 17 common DE genes, (BMP4, CRYGD, DBH, GJB1, KRT83, MPZ, NHLH1, TACR3, ACTC1, MFAP4, SPARCL1, TAGLN, TPM2, CDC20, LHCGR, TM9SF1 and HCCS) 4 of which participate in numerous pathways. CONCLUSIONS/SIGNIFICANCE: The identification of the common DE genes among BC samples of different histology can provide further insight into the discovery of new putative markers.

Implication of RAF and RKIP genes in urinary bladder cancer.

RKIP has been shown to regulate the RAS-RAF-MEK-ERK kinase cascade acting as modulator of apoptosis and metastasis in prostate cancer. Our goal was to examine the expression of the RAF (A-RAF, B-RAF and RAF-1) and RKIP genes in urinary bladder cancer. Microarray analysis and qPCR was employed to investigate the expression of RAF and RKIP, in 30 patients with transitional cell carcinoma (TCC) of the urinary bladder vs. the corresponding levels of adjacent normal tissue. Computational analysis was also performed on Gene Expression Omnibus (GEO) datasets, to unravel differences in the expression of RAF or RKIP between tumor and control samples, and between superficial and muscle invasive tumors. Microarray analysis revealed >2-fold expression of BRAF and RKIP in T2, T3, grade III tumors vs. controls. B-RAF over-expression was verified by qPCR in pT1, grade III tumors vs. their normal counterparts (p = 0.016). qPCR revealed a significant RKIP reduction in TCC vs. normal tissue (p = 0.002 and p < 0.001 for T1, grade II and Ta-T1, grade III, respectively); All RAF genes were positively correlated among each other (A-RAF/B-RAF, p = 0.003; A-RAF/RAF-1, p < 0.001; B-RAF/RAF-1, p = 0.050), whereas B-RAF was negatively correlated with RKIP in TCC (p = 0.050). Further computational analysis revealed different expression profiles for the genes of interest, among muscle invasive carcinomas, superficial TCCs, cystectomy specimens and normal tissue. The reduced RKIP mRNA levels in TCC and the elevated levels of B-RAF in pT1, grade III tumors vs. normal tissue, corroborate that these genes are involved in the pathogenesis of urinary bladder cancer.