<p>We have previously analyzed the gene expression profile in urinary bladder cancer and de... more <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
<p>INTRODUCTION AND AIMS: Renal cell carcinoma (RCC) is composed of various distinct subtyp... more <p>INTRODUCTION AND AIMS: Renal cell carcinoma (RCC) is composed of various distinct subtypes, the most prevalent of which are clear cell (ccRCC), papillary (papRCC) and chromophobe (chRCC). MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nt size, and modulate differentiation, growth, apoptosis and proliferation of cells. More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites, frequently amplified or deleted in human cancer. Recognition of miRNAs that are differentially expressed (DE) between RCC and normal tissue may help to identify those miRNAs that are involved in this human malignancy.</p> <p>METHODS: MiRNA profiling was performed on 28 FFPE tissues of ccRCC, chRCC, papRCC and 20 normal cases. Results of DE miRNAs were validated using qRT-PCR and in-situ hybridization (ISH). The ability to discriminate between RCC subtypes and normal samples was characterized by ROC curves. The chromosomal distribution of the DE miRNAs was detected and compared with reported genomic alterations in each subtype. The miRNA targets were predicted using miRWalk. Enriched gene sets were grouped in functional categories by IPA and GO analysis.</p> <p>RESULTS: We identified 434 DE miRNAs in all kidney tumours and built a molecular signature that accurately classified RCC subtypes among them. Ten miRNAs (miR-10b-5p, miR-1257, miR-1303, miR-23c, miR-3171, miR-4270, miR-514b-3p, miR-515-5p, miR-620 and miR-98) were co-deregulated among RCC subtypes, whereas 270 miRNAs were identified uniquely in ccRCC, 33 in papRCC and 5 in chRCC, respectively. The expression of the most DE miRNAs was validated using qRT-PCR. The miRNAs exhibited deregulation patterns that agreed with previously reported chromosomal gains and losses, in each subtype. ISH for miR-25-5p revealed that its differential expression is cancer-cell associated. The top Canonical pathways of the putative gene targets of the deregulated miRNAs, included Molecular Mechanisms of Cancer (p=1.72E-04); PPARα/RXRα Activation (p=2.15E-03); Bladder Cancer Signaling (p=3.57E-03); Cell Cycle: G1/S Checkpoint Regulation (p=5.37E-03) and Estrogen-mediated S-phase Entry (p=6.25E-03). The major gene networks and their associated functions included Tissue Development, Cancer; Tumor Morphology, Cellular Movement; Cell Cycle, Cellular Growth and Proliferation, Cellular Development; and Cell Death and Survival, Tumor Morphology, Cell Morphology. VHL, HIF1A, EPAS1, SMARCB1, TP53 and PAWR constituted the most central nodes in these networks.</p> <p>CONCLUSIONS: Our comprehensive study highlights the dynamic role of miRNAs in the three most common renal cell carcinoma subtypes.</p
The epithelial to mesenchymal transition (EMT) is a powerful process in tumor invasion, metastasi... more The epithelial to mesenchymal transition (EMT) is a powerful process in tumor invasion, metastasis, and tumorigenesis and describes the molecular reprogramming and phenotypic changes that are characterized by a transition from polarized immotile epithelial cells to motile mesenchymal cells. It is now well known that miRNAs are important regulators of malignant transformation and metastasis. The aberrant expression of the miR-200 family in cancer and its involvement in the initiation and progression of malignant transformation has been well demonstrated. The metastasis suppressive role of the miR-200 members is strongly associated with a pathologic EMT. This review describes the most recent advances regarding the influence of miRNAs in EMT and the control they exert in major signaling pathways in various cancers. The ability of the autocrine TGF-β/ZEB/miR-200 signaling regulatory network to control cell plasticity between the epithelial and mesenchymal state is further discussed. Var...
Journal of B.U.ON. : official journal of the Balkan Union of Oncology
To investigate the expression of RhoA, RhoB, RhoC, Rac1 and Cdc42 kinases in urothelial cell carc... more To investigate the expression of RhoA, RhoB, RhoC, Rac1 and Cdc42 kinases in urothelial cell carcinoma (UCC) of the urinary bladder and determine the expression profile of 107 Rho-associated genes, including GTPases, GDIs, GAPs and GEFs. Rho expression was investigated using microarrays, qPCR and Western blotting in 77 UCC specimens with paired normal urothelium. Computational analysis was also performed on Gene Expression Omnibus datasets. Further microarray analysis was carried out for the expression profiling of the Rho-associated genes. RhoB mRNA and protein levels were significantly lower in UCC, suggesting a tumour-suppressor role. On the contrary, mRNA of RhoC and protein levels of RhoA, RhoC and Cdc42, respectively, were significantly higher in UCC vs. normal tissue. High Cdc42 mRNA levels correlated with worse overall survival (p=0.027), whereas high RhoB mRNA levels correlated both with better overall (p=0.0258) and cancer-specific (p=0.0272) survival. Computational analys...
Biological systems are characterized by their potential for dynamic adaptation. One of the challe... more Biological systems are characterized by their potential for dynamic adaptation. One of the challenges for systems biology approaches is their contribution towards the understanding of the dynamics of a growing cell population. Conceptualizing these dynamics in tumor models could help us understand the steps leading to the initiation of the disease and its progression.In vitromodels are useful in answering this question by providing information over the spatiotemporal nature of such dynamics. In the present work, we used physical quantities such as growth rate, velocity, and acceleration for the cellular proliferation and identified the fractal structures in tumor cell proliferation dynamics. We provide evidence that the rate of cellular proliferation is of nonlinear nature and exhibits oscillatory behavior. We also calculated the fractal dimensions of our cellular system. Our results show that the temporal transitions from one state to the other also follow nonlinear dynamics. Furth...
Although it is well established that ras genes contribute to tumourigenesis either through the ac... more Although it is well established that ras genes contribute to tumourigenesis either through the accumulation of mutations or by aberrant expression in a wide range of human cancers, little is known regarding their involvement in human nasal polyps (NPs). In the present study, the occurrence of mutations in codons 11 and 12 of the ras family genes was examined by PCR/RFLP and direct sequencing in 23 human NPs and their adjacent turbinates, as well as in turbinates from 13 control subjects. Moreover, the expression pattern of ras mRNA levels was assessed in NP specimens and compared to adjacent and control tissues. K-ras codon 11 and 12 mutations were detected in 17 and 35% of NPs, respectively, and were found in the adjacent inferior turbinate (AIT) (22 and 16%, respectively) and adjacent middle turbinates (AMT) (16 and 26%, respectively). K-and H-ras expression levels were elevated, whereas N-ras mRNA levels were lower in NPs and adjacent turbinates as compared to the control tissues. K-ras mRNA levels were up-regulated in advanced-stage polyps (P=0.037), while N-ras levels were found elevated in small polyps (P=0.046). Statistically significant negative correlations between K-and N-ras expression profiles arose in NPs and AITs (P=0.009 and 0.003, respectively). This, to our knowledge, is the first report on ras mutations and expression analysis in NPs. Our findings suggest a potential key role for activated members of ras family genes in terms of their contribution to the development of NPs as well as to the hypertrophy of adjacent turbinates.
Proceedings of the National Academy of Sciences of the United States of America, Jan 17, 2015
Breast cancer type 1 susceptibility protein (BRCA1) has a multitude of functions that contribute ... more Breast cancer type 1 susceptibility protein (BRCA1) has a multitude of functions that contribute to genome integrity and tumor suppression. Its participation in the repair of DNA double-strand breaks (DSBs) during homologous recombination (HR) is well recognized, whereas its involvement in the second major DSB repair pathway, nonhomologous end-joining (NHEJ), remains controversial. Here we have studied the role of BRCA1 in the repair of DSBs in switch (S) regions during immunoglobulin class switch recombination, a physiological, deletion/recombination process that relies on the classical NHEJ machinery. A shift to the use of microhomology-based, alternative end-joining (A-EJ) and increased frequencies of intra-S region deletions as well as insertions of inverted S sequences were observed at the recombination junctions amplified from BRCA1-deficient human B cells. Furthermore, increased use of long microhomologies was found at recombination junctions derived from E3 ubiquitin-protein...
Clear cell renal cell carcinoma (ccRCC) is the predominant subtype of renal cell carcinoma (RCC).... more 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 an...
<p>We have previously analyzed the gene expression profile in urinary bladder cancer and de... more <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
<p>INTRODUCTION AND AIMS: Renal cell carcinoma (RCC) is composed of various distinct subtyp... more <p>INTRODUCTION AND AIMS: Renal cell carcinoma (RCC) is composed of various distinct subtypes, the most prevalent of which are clear cell (ccRCC), papillary (papRCC) and chromophobe (chRCC). MicroRNAs (miRNAs) are small non-coding RNAs of approximately 22 nt size, and modulate differentiation, growth, apoptosis and proliferation of cells. More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites, frequently amplified or deleted in human cancer. Recognition of miRNAs that are differentially expressed (DE) between RCC and normal tissue may help to identify those miRNAs that are involved in this human malignancy.</p> <p>METHODS: MiRNA profiling was performed on 28 FFPE tissues of ccRCC, chRCC, papRCC and 20 normal cases. Results of DE miRNAs were validated using qRT-PCR and in-situ hybridization (ISH). The ability to discriminate between RCC subtypes and normal samples was characterized by ROC curves. The chromosomal distribution of the DE miRNAs was detected and compared with reported genomic alterations in each subtype. The miRNA targets were predicted using miRWalk. Enriched gene sets were grouped in functional categories by IPA and GO analysis.</p> <p>RESULTS: We identified 434 DE miRNAs in all kidney tumours and built a molecular signature that accurately classified RCC subtypes among them. Ten miRNAs (miR-10b-5p, miR-1257, miR-1303, miR-23c, miR-3171, miR-4270, miR-514b-3p, miR-515-5p, miR-620 and miR-98) were co-deregulated among RCC subtypes, whereas 270 miRNAs were identified uniquely in ccRCC, 33 in papRCC and 5 in chRCC, respectively. The expression of the most DE miRNAs was validated using qRT-PCR. The miRNAs exhibited deregulation patterns that agreed with previously reported chromosomal gains and losses, in each subtype. ISH for miR-25-5p revealed that its differential expression is cancer-cell associated. The top Canonical pathways of the putative gene targets of the deregulated miRNAs, included Molecular Mechanisms of Cancer (p=1.72E-04); PPARα/RXRα Activation (p=2.15E-03); Bladder Cancer Signaling (p=3.57E-03); Cell Cycle: G1/S Checkpoint Regulation (p=5.37E-03) and Estrogen-mediated S-phase Entry (p=6.25E-03). The major gene networks and their associated functions included Tissue Development, Cancer; Tumor Morphology, Cellular Movement; Cell Cycle, Cellular Growth and Proliferation, Cellular Development; and Cell Death and Survival, Tumor Morphology, Cell Morphology. VHL, HIF1A, EPAS1, SMARCB1, TP53 and PAWR constituted the most central nodes in these networks.</p> <p>CONCLUSIONS: Our comprehensive study highlights the dynamic role of miRNAs in the three most common renal cell carcinoma subtypes.</p
The epithelial to mesenchymal transition (EMT) is a powerful process in tumor invasion, metastasi... more The epithelial to mesenchymal transition (EMT) is a powerful process in tumor invasion, metastasis, and tumorigenesis and describes the molecular reprogramming and phenotypic changes that are characterized by a transition from polarized immotile epithelial cells to motile mesenchymal cells. It is now well known that miRNAs are important regulators of malignant transformation and metastasis. The aberrant expression of the miR-200 family in cancer and its involvement in the initiation and progression of malignant transformation has been well demonstrated. The metastasis suppressive role of the miR-200 members is strongly associated with a pathologic EMT. This review describes the most recent advances regarding the influence of miRNAs in EMT and the control they exert in major signaling pathways in various cancers. The ability of the autocrine TGF-β/ZEB/miR-200 signaling regulatory network to control cell plasticity between the epithelial and mesenchymal state is further discussed. Var...
Journal of B.U.ON. : official journal of the Balkan Union of Oncology
To investigate the expression of RhoA, RhoB, RhoC, Rac1 and Cdc42 kinases in urothelial cell carc... more To investigate the expression of RhoA, RhoB, RhoC, Rac1 and Cdc42 kinases in urothelial cell carcinoma (UCC) of the urinary bladder and determine the expression profile of 107 Rho-associated genes, including GTPases, GDIs, GAPs and GEFs. Rho expression was investigated using microarrays, qPCR and Western blotting in 77 UCC specimens with paired normal urothelium. Computational analysis was also performed on Gene Expression Omnibus datasets. Further microarray analysis was carried out for the expression profiling of the Rho-associated genes. RhoB mRNA and protein levels were significantly lower in UCC, suggesting a tumour-suppressor role. On the contrary, mRNA of RhoC and protein levels of RhoA, RhoC and Cdc42, respectively, were significantly higher in UCC vs. normal tissue. High Cdc42 mRNA levels correlated with worse overall survival (p=0.027), whereas high RhoB mRNA levels correlated both with better overall (p=0.0258) and cancer-specific (p=0.0272) survival. Computational analys...
Biological systems are characterized by their potential for dynamic adaptation. One of the challe... more Biological systems are characterized by their potential for dynamic adaptation. One of the challenges for systems biology approaches is their contribution towards the understanding of the dynamics of a growing cell population. Conceptualizing these dynamics in tumor models could help us understand the steps leading to the initiation of the disease and its progression.In vitromodels are useful in answering this question by providing information over the spatiotemporal nature of such dynamics. In the present work, we used physical quantities such as growth rate, velocity, and acceleration for the cellular proliferation and identified the fractal structures in tumor cell proliferation dynamics. We provide evidence that the rate of cellular proliferation is of nonlinear nature and exhibits oscillatory behavior. We also calculated the fractal dimensions of our cellular system. Our results show that the temporal transitions from one state to the other also follow nonlinear dynamics. Furth...
Although it is well established that ras genes contribute to tumourigenesis either through the ac... more Although it is well established that ras genes contribute to tumourigenesis either through the accumulation of mutations or by aberrant expression in a wide range of human cancers, little is known regarding their involvement in human nasal polyps (NPs). In the present study, the occurrence of mutations in codons 11 and 12 of the ras family genes was examined by PCR/RFLP and direct sequencing in 23 human NPs and their adjacent turbinates, as well as in turbinates from 13 control subjects. Moreover, the expression pattern of ras mRNA levels was assessed in NP specimens and compared to adjacent and control tissues. K-ras codon 11 and 12 mutations were detected in 17 and 35% of NPs, respectively, and were found in the adjacent inferior turbinate (AIT) (22 and 16%, respectively) and adjacent middle turbinates (AMT) (16 and 26%, respectively). K-and H-ras expression levels were elevated, whereas N-ras mRNA levels were lower in NPs and adjacent turbinates as compared to the control tissues. K-ras mRNA levels were up-regulated in advanced-stage polyps (P=0.037), while N-ras levels were found elevated in small polyps (P=0.046). Statistically significant negative correlations between K-and N-ras expression profiles arose in NPs and AITs (P=0.009 and 0.003, respectively). This, to our knowledge, is the first report on ras mutations and expression analysis in NPs. Our findings suggest a potential key role for activated members of ras family genes in terms of their contribution to the development of NPs as well as to the hypertrophy of adjacent turbinates.
Proceedings of the National Academy of Sciences of the United States of America, Jan 17, 2015
Breast cancer type 1 susceptibility protein (BRCA1) has a multitude of functions that contribute ... more Breast cancer type 1 susceptibility protein (BRCA1) has a multitude of functions that contribute to genome integrity and tumor suppression. Its participation in the repair of DNA double-strand breaks (DSBs) during homologous recombination (HR) is well recognized, whereas its involvement in the second major DSB repair pathway, nonhomologous end-joining (NHEJ), remains controversial. Here we have studied the role of BRCA1 in the repair of DSBs in switch (S) regions during immunoglobulin class switch recombination, a physiological, deletion/recombination process that relies on the classical NHEJ machinery. A shift to the use of microhomology-based, alternative end-joining (A-EJ) and increased frequencies of intra-S region deletions as well as insertions of inverted S sequences were observed at the recombination junctions amplified from BRCA1-deficient human B cells. Furthermore, increased use of long microhomologies was found at recombination junctions derived from E3 ubiquitin-protein...
Clear cell renal cell carcinoma (ccRCC) is the predominant subtype of renal cell carcinoma (RCC).... more 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 an...
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Papers by A. Zaravinos