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Coordinated activation of candidate proto-oncogenes and cancer testes antigens via promoter demethylation in head and neck cancer and lung cancer.

Smith IM, Glazer CA, Mithani SK, Ochs MF, Sun W, Bhan S, Vostrov A, Abdullaev Z, Lobanenkov V, Gray A, Liu C, Chang SS, Ostrow KL, Westra WH, Begum S, Dhara M, Califano J - PLoS ONE (2009)

Bottom Line: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported.We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS.Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.

ABSTRACT

Background: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported. We used an integrative method to analyze expression in primary head and neck squamous cell carcinoma (HNSCC) and pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC.

Methodology/principal findings: We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS. Aberrant BORIS expression correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC, induced coordinated proto-oncogene specific promoter demethylation and expression in non-tumorigenic cells, and transformed NIH3T3 cells.

Conclusions/significance: Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.

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Related in: MedlinePlus

Integrative epigenetic screening strategy and strategy for validation of targets.(a) Initially, minimally-transformed cell lines were treated with 5-aza-deoxycytidine and TSA to unmask epigenetically silenced genes. In order to correlate epigenetic unmasking with meaningful upregulated cancer-specific genes, we performed a comparative epigenetic approach with Cancer Outlier Profiling Analysis (COPA) using 49 tumors and 19 normal tissues that had been characterized on the Affymetrix U133A mRNA expression microarray platform. Genes (by probeset) were ranked first by degree of upfold regulation with 5-aza/TSA treatment and second by COPA upregulation at the 90th percentile. The product of these ranks was used to rank all targets and a significance threshold (α = 0.005) was chosen resulting in 106 genes of which the top 26 genes were evaluated. In order to not exclude genes outside the U133A platform, we also considered all other genes in the U133 Plus 2.0 platform on the sole basis of 5-aza/TSA upfold regulation. Genes were subsequently screened by presence of CpG islands using MethPrimer and all genes were validated by bisulfite sequencing of tumor and normal tissues, and QRT-PCR of cell lines and primary tumors. Of the integrative targets 7/26 passed our validation, while 2/46 of the non-integrative targets passed. Functional experiments were then conducted on these genes. (b) Representative COPA graph of MAGEA3 demonstrating the statistical approach to finding candidate overexpressed oncogenes. Difference in tumor (n = 49) versus normal (n = 19) expression was significant, p value<0.001 measured by Mann-Whitney U test. (c) Promoter demethylation causes transcriptional upregulation. Upregulation after treatment with 5-aza/TSA is shown in cell lines as measured by QRT-PCR. The ratio of 5-aza/TSA treated expression to baseline is shown for C19ORF28, H19, TKLT1, GPR17, GRIN1, MAGEA2, MAGEA3/6, MAGEA4, MAGEA11. Each gene demonstrated significant upregulation by 5-aza/TSA treatment in at least one cell-line. Error bars show SE.
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pone-0004961-g001: Integrative epigenetic screening strategy and strategy for validation of targets.(a) Initially, minimally-transformed cell lines were treated with 5-aza-deoxycytidine and TSA to unmask epigenetically silenced genes. In order to correlate epigenetic unmasking with meaningful upregulated cancer-specific genes, we performed a comparative epigenetic approach with Cancer Outlier Profiling Analysis (COPA) using 49 tumors and 19 normal tissues that had been characterized on the Affymetrix U133A mRNA expression microarray platform. Genes (by probeset) were ranked first by degree of upfold regulation with 5-aza/TSA treatment and second by COPA upregulation at the 90th percentile. The product of these ranks was used to rank all targets and a significance threshold (α = 0.005) was chosen resulting in 106 genes of which the top 26 genes were evaluated. In order to not exclude genes outside the U133A platform, we also considered all other genes in the U133 Plus 2.0 platform on the sole basis of 5-aza/TSA upfold regulation. Genes were subsequently screened by presence of CpG islands using MethPrimer and all genes were validated by bisulfite sequencing of tumor and normal tissues, and QRT-PCR of cell lines and primary tumors. Of the integrative targets 7/26 passed our validation, while 2/46 of the non-integrative targets passed. Functional experiments were then conducted on these genes. (b) Representative COPA graph of MAGEA3 demonstrating the statistical approach to finding candidate overexpressed oncogenes. Difference in tumor (n = 49) versus normal (n = 19) expression was significant, p value<0.001 measured by Mann-Whitney U test. (c) Promoter demethylation causes transcriptional upregulation. Upregulation after treatment with 5-aza/TSA is shown in cell lines as measured by QRT-PCR. The ratio of 5-aza/TSA treated expression to baseline is shown for C19ORF28, H19, TKLT1, GPR17, GRIN1, MAGEA2, MAGEA3/6, MAGEA4, MAGEA11. Each gene demonstrated significant upregulation by 5-aza/TSA treatment in at least one cell-line. Error bars show SE.

Mentions: Concurrently, we performed a comparative epigenetic approach utilizing Cancer Outlier Profiling Analysis (COPA) using 49 primary HNSCC and 19 normal mucosal tissues assayed for mRNA expression on the Affymetrix U133A mRNA expression microarray platform (16,383 probe sets) compiled from prior work and public sources of expression (oncomine.org). COPA is particularly useful to determine differences in expression for particular genes in subsets of primary tumor samples, with improved performance compared to statistical tools that rely on median or average expression difference between two datasets [14]. We calculated COPA at the 90th percentile for our final rankings of all 16,383 features of the arrays, as this resulted in the most pronounced differences in expression with our sample size. Statistical significance of the expression differences in the COPA diagrams were measured by Mann-Whitney U test (Figure 1B).


Coordinated activation of candidate proto-oncogenes and cancer testes antigens via promoter demethylation in head and neck cancer and lung cancer.

Smith IM, Glazer CA, Mithani SK, Ochs MF, Sun W, Bhan S, Vostrov A, Abdullaev Z, Lobanenkov V, Gray A, Liu C, Chang SS, Ostrow KL, Westra WH, Begum S, Dhara M, Califano J - PLoS ONE (2009)

Integrative epigenetic screening strategy and strategy for validation of targets.(a) Initially, minimally-transformed cell lines were treated with 5-aza-deoxycytidine and TSA to unmask epigenetically silenced genes. In order to correlate epigenetic unmasking with meaningful upregulated cancer-specific genes, we performed a comparative epigenetic approach with Cancer Outlier Profiling Analysis (COPA) using 49 tumors and 19 normal tissues that had been characterized on the Affymetrix U133A mRNA expression microarray platform. Genes (by probeset) were ranked first by degree of upfold regulation with 5-aza/TSA treatment and second by COPA upregulation at the 90th percentile. The product of these ranks was used to rank all targets and a significance threshold (α = 0.005) was chosen resulting in 106 genes of which the top 26 genes were evaluated. In order to not exclude genes outside the U133A platform, we also considered all other genes in the U133 Plus 2.0 platform on the sole basis of 5-aza/TSA upfold regulation. Genes were subsequently screened by presence of CpG islands using MethPrimer and all genes were validated by bisulfite sequencing of tumor and normal tissues, and QRT-PCR of cell lines and primary tumors. Of the integrative targets 7/26 passed our validation, while 2/46 of the non-integrative targets passed. Functional experiments were then conducted on these genes. (b) Representative COPA graph of MAGEA3 demonstrating the statistical approach to finding candidate overexpressed oncogenes. Difference in tumor (n = 49) versus normal (n = 19) expression was significant, p value<0.001 measured by Mann-Whitney U test. (c) Promoter demethylation causes transcriptional upregulation. Upregulation after treatment with 5-aza/TSA is shown in cell lines as measured by QRT-PCR. The ratio of 5-aza/TSA treated expression to baseline is shown for C19ORF28, H19, TKLT1, GPR17, GRIN1, MAGEA2, MAGEA3/6, MAGEA4, MAGEA11. Each gene demonstrated significant upregulation by 5-aza/TSA treatment in at least one cell-line. Error bars show SE.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654921&req=5

pone-0004961-g001: Integrative epigenetic screening strategy and strategy for validation of targets.(a) Initially, minimally-transformed cell lines were treated with 5-aza-deoxycytidine and TSA to unmask epigenetically silenced genes. In order to correlate epigenetic unmasking with meaningful upregulated cancer-specific genes, we performed a comparative epigenetic approach with Cancer Outlier Profiling Analysis (COPA) using 49 tumors and 19 normal tissues that had been characterized on the Affymetrix U133A mRNA expression microarray platform. Genes (by probeset) were ranked first by degree of upfold regulation with 5-aza/TSA treatment and second by COPA upregulation at the 90th percentile. The product of these ranks was used to rank all targets and a significance threshold (α = 0.005) was chosen resulting in 106 genes of which the top 26 genes were evaluated. In order to not exclude genes outside the U133A platform, we also considered all other genes in the U133 Plus 2.0 platform on the sole basis of 5-aza/TSA upfold regulation. Genes were subsequently screened by presence of CpG islands using MethPrimer and all genes were validated by bisulfite sequencing of tumor and normal tissues, and QRT-PCR of cell lines and primary tumors. Of the integrative targets 7/26 passed our validation, while 2/46 of the non-integrative targets passed. Functional experiments were then conducted on these genes. (b) Representative COPA graph of MAGEA3 demonstrating the statistical approach to finding candidate overexpressed oncogenes. Difference in tumor (n = 49) versus normal (n = 19) expression was significant, p value<0.001 measured by Mann-Whitney U test. (c) Promoter demethylation causes transcriptional upregulation. Upregulation after treatment with 5-aza/TSA is shown in cell lines as measured by QRT-PCR. The ratio of 5-aza/TSA treated expression to baseline is shown for C19ORF28, H19, TKLT1, GPR17, GRIN1, MAGEA2, MAGEA3/6, MAGEA4, MAGEA11. Each gene demonstrated significant upregulation by 5-aza/TSA treatment in at least one cell-line. Error bars show SE.
Mentions: Concurrently, we performed a comparative epigenetic approach utilizing Cancer Outlier Profiling Analysis (COPA) using 49 primary HNSCC and 19 normal mucosal tissues assayed for mRNA expression on the Affymetrix U133A mRNA expression microarray platform (16,383 probe sets) compiled from prior work and public sources of expression (oncomine.org). COPA is particularly useful to determine differences in expression for particular genes in subsets of primary tumor samples, with improved performance compared to statistical tools that rely on median or average expression difference between two datasets [14]. We calculated COPA at the 90th percentile for our final rankings of all 16,383 features of the arrays, as this resulted in the most pronounced differences in expression with our sample size. Statistical significance of the expression differences in the COPA diagrams were measured by Mann-Whitney U test (Figure 1B).

Bottom Line: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported.We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS.Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.

ABSTRACT

Background: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported. We used an integrative method to analyze expression in primary head and neck squamous cell carcinoma (HNSCC) and pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC.

Methodology/principal findings: We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS. Aberrant BORIS expression correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC, induced coordinated proto-oncogene specific promoter demethylation and expression in non-tumorigenic cells, and transformed NIH3T3 cells.

Conclusions/significance: Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.

Show MeSH
Related in: MedlinePlus