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

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

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 = 1.5x10(-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

Transcription Factor Binding Motifs, Chromosome mapping and Gene Ontology analysis in Cross-platform microarray data from bladder cancer.

<p>We have previously analyzed the gene expression profile in urinary bladder cancer and determined the differentially expressed (DE) genes between cancer and healthy tissue. We aimed: 1) To identify the over-represented Transcription Factor Binding Motifs (TFBMs) in the promoters of the DE genes. 2) To map the DE genes on the chromosomal regions. 3) To gain more insight into the DE gene functions, using Gene Ontology (GO) analysis. We investigated the TFBMs in the Transcription Element Listening System Database (TELiS). The TRANSFAC TF database was used for the identification of TF binding sites. The Gene Ontology Tree Machine, WebGestalt web-tool and the Matlab ® (The Mathworks Inc.) computing environments were used for chromosome mapping. GO analysis was performed using the eGOn online tool. The WebGestalt web-tool was used for gene function classifications. Relations of the DE genes and the transcription factor binding motifs were further investigated using the Pubgene Ontology Database. The glucocorticoid receptor (GR) was predicted as one of the TFs in the common gene set. In order to find which gene was most commonly represented among the TFs, we plotted the incidence of each gene as a function of the times of appearance within the predicted TFs. The gene BMP4 (bone morphogenetic protein 4; ID: 652) exhibited the higher number of binding sites for the predicted TFs. The majority of the chromosomes in BC had inactivated (down-regulated) genes, compared to the normal tissue. However, two genes were significantly over-expressed: CDC20 (in chromosome 1) and HCCS (in chromosome X). Three main functions were outlined by GO for the DE genes: a) circulatory system regulation, b) reproductive organ and sex development, and c) catecholamine metabolism. This enrichment showed that the predicted gene set has more than a dual role. Through this study, we were able to identify several important factors that warrant further investigation both as prognostic markers and as therapeutic targets for bladder cancer. Such approaches may provide a better insight into tumorigenesis and tumor progression.</p

Expression Profile of CYP1A1 and CYP1B1 Enzymes in Colon and Bladder Tumors

<div><p>Background</p><p>The cytochrome P450 CYP1A1 and CYP1B1 enzymes are involved in carcinogenesis via activation of pro-carcinogenic compounds to carcinogenic metabolites. CYP1A1 and CYP1B1 have shown elevated levels in human tumors as determined by qRT-PCR and immunohistochemical studies. However studies that have examined CYP1 expression by enzyme activity assays are limited. </p> <p>Results</p><p>In the current study the expression of CYP1A1 and CYP1B1 was investigated in a panel of human tumors of bladder and colorectal origin by qRT-PCR and enzyme activity assays. The results demonstrated that 35% (7/20) of bladder tumors and 35% (7/20) of colon tumors overexpressed active CYP1 enzymes. CYP1B1 mRNA was overexpressed in 65% and 60% of bladder and colon tumors respectively, whereas CYP1A1 was overexpressed in 65% and 80% of bladder and colon tumors. Mean mRNA levels of CYP1B1 and CYP1A1 along with mean CYP1 activity were higher in bladder and colon tumors compared to normal tissues (p<0.05). Statistical analysis revealed CYP1 expression levels to be independent of TNM status. Moreover, incubation of tumor microsomal protein in 4 bladder and 3 colon samples with a CYP1B1 specific antibody revealed a large reduction (72.5 ± 5.5 % for bladder and 71.8 ± 7.2% for colon) in catalytic activity, indicating that the activity was mainly attributed to CYP1B1 expression. </p> <p>Conclusions</p><p>The study reveals active CYP1 overexpression in human tumors and uncovers the potential use of CYP1 enzymes and mainly CYP1B1 as targets for cancer therapy. </p> </div

Correlation of CYP1 enzyme activity levels with tumor stage.

<p>Group pairs were compared using Mann-U-Whitney test. Bars depict the mean values. Scatterplot depicting CYP1 activity levels in tumor samples of different TNM status and normal samples of bladder and colorectal origin. Statistical significance was set at p < 0.05. Arrows and horizontal lines indicate groups compared with statistical tests.</p

Correlation of CYP1 mRNA expression levels with tumor stage.

<p>Group pairs were compared using Mann-U-Whitney test. Bars depict the mean values. Scatterplot depicting mRNA levels of CYP1A1 and CYP1B1 genes in tumor samples of different TNM status and normal samples of (A) bladder and (B) colorectal origin. Statistical significance was set at p < 0.05. Arrows and horizontal lines indicate groups compared with statistical tests.</p