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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

Transient transfection of target genes in minimally transformed oral keratinocytes.(a) Transient transfection of an H19 construct into OKF6-Tert-1R cells (at day 4, 41.4%±15% growth increase). (b) Transient transfection of an MAGEA2 construct into OKF6-Tert-1R cells (at day 3, 72.7%±26% growth increase). (c) Transient transfection of an TKTL1 construct into OKF6-Tert-1R cells(at day 4, 50.1%±38% growth increase). (d) Transient transfection of MAGEA4 construct into OKF6-Tert-1R cells (at day 4, 203%±17% growth increase). For (e) we developed a quantitative assay for measuring unmethylated promoters, termed Quantitative Unmethylation-Specific PCR (QUMSP). QUMSP percentage of C19ORF28, GRIN1, H19, MAGEA11, MAGEA2, MAGEA3/6, GPR17, and TKTL1 was conducted in a separate cohort of head and neck cancer patients using 25 tumors and 11 upper aerodigestive mucosal samples to assay promoter demethylation. Statistically significant differences were found in GRIN1, MAGEA11, MAGEA2. Next promoter demethylation was considered as a cause of mRNA expression increases seen in the expO dataset. (f) shows the QUMSP results for an independent cohort of 14 lung normals and 13 lung tumor patients. Significant differences in QUMSP were found in H19, MAGEA11, MAGEA2, and MAGEA3/6.
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pone-0004961-g003: Transient transfection of target genes in minimally transformed oral keratinocytes.(a) Transient transfection of an H19 construct into OKF6-Tert-1R cells (at day 4, 41.4%±15% growth increase). (b) Transient transfection of an MAGEA2 construct into OKF6-Tert-1R cells (at day 3, 72.7%±26% growth increase). (c) Transient transfection of an TKTL1 construct into OKF6-Tert-1R cells(at day 4, 50.1%±38% growth increase). (d) Transient transfection of MAGEA4 construct into OKF6-Tert-1R cells (at day 4, 203%±17% growth increase). For (e) we developed a quantitative assay for measuring unmethylated promoters, termed Quantitative Unmethylation-Specific PCR (QUMSP). QUMSP percentage of C19ORF28, GRIN1, H19, MAGEA11, MAGEA2, MAGEA3/6, GPR17, and TKTL1 was conducted in a separate cohort of head and neck cancer patients using 25 tumors and 11 upper aerodigestive mucosal samples to assay promoter demethylation. Statistically significant differences were found in GRIN1, MAGEA11, MAGEA2. Next promoter demethylation was considered as a cause of mRNA expression increases seen in the expO dataset. (f) shows the QUMSP results for an independent cohort of 14 lung normals and 13 lung tumor patients. Significant differences in QUMSP were found in H19, MAGEA11, MAGEA2, and MAGEA3/6.

Mentions: We then performed transient transfections to evaluate and/or confirm growth-promoting effects of these nine targets that show tumor-specific promoter hypomethylation. Although H19 codes for a nontranslated RNA transcript, the H19 product appears to induce growth in lung and breast cancer cell lines [16] and may induce drug resistance in hepato-cellular carcinoma [17]. Figure 3A shows results obtained by transient transfection of an H19 construct into OKF6-Tert-1R cells. At four days, there was a 41.4% (±15%) increase in growth over the transfected empty vector. The MAGE family consists of related family members that are known to be upregulated in a variety of tumor types[18], but have recently been implicated in inducing transcriptional reprogramming in tumor cells[19]. MAGEA2 induced a 72.7% (±26%) increase in growth at day three (Figure 3B). MAGEA4 transfection induced a 203% (±17%) increase in growth (Figure 3D). Functional growth differences were tested, but not found for C19ORF28.


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)

Transient transfection of target genes in minimally transformed oral keratinocytes.(a) Transient transfection of an H19 construct into OKF6-Tert-1R cells (at day 4, 41.4%±15% growth increase). (b) Transient transfection of an MAGEA2 construct into OKF6-Tert-1R cells (at day 3, 72.7%±26% growth increase). (c) Transient transfection of an TKTL1 construct into OKF6-Tert-1R cells(at day 4, 50.1%±38% growth increase). (d) Transient transfection of MAGEA4 construct into OKF6-Tert-1R cells (at day 4, 203%±17% growth increase). For (e) we developed a quantitative assay for measuring unmethylated promoters, termed Quantitative Unmethylation-Specific PCR (QUMSP). QUMSP percentage of C19ORF28, GRIN1, H19, MAGEA11, MAGEA2, MAGEA3/6, GPR17, and TKTL1 was conducted in a separate cohort of head and neck cancer patients using 25 tumors and 11 upper aerodigestive mucosal samples to assay promoter demethylation. Statistically significant differences were found in GRIN1, MAGEA11, MAGEA2. Next promoter demethylation was considered as a cause of mRNA expression increases seen in the expO dataset. (f) shows the QUMSP results for an independent cohort of 14 lung normals and 13 lung tumor patients. Significant differences in QUMSP were found in H19, MAGEA11, MAGEA2, and MAGEA3/6.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654921&req=5

pone-0004961-g003: Transient transfection of target genes in minimally transformed oral keratinocytes.(a) Transient transfection of an H19 construct into OKF6-Tert-1R cells (at day 4, 41.4%±15% growth increase). (b) Transient transfection of an MAGEA2 construct into OKF6-Tert-1R cells (at day 3, 72.7%±26% growth increase). (c) Transient transfection of an TKTL1 construct into OKF6-Tert-1R cells(at day 4, 50.1%±38% growth increase). (d) Transient transfection of MAGEA4 construct into OKF6-Tert-1R cells (at day 4, 203%±17% growth increase). For (e) we developed a quantitative assay for measuring unmethylated promoters, termed Quantitative Unmethylation-Specific PCR (QUMSP). QUMSP percentage of C19ORF28, GRIN1, H19, MAGEA11, MAGEA2, MAGEA3/6, GPR17, and TKTL1 was conducted in a separate cohort of head and neck cancer patients using 25 tumors and 11 upper aerodigestive mucosal samples to assay promoter demethylation. Statistically significant differences were found in GRIN1, MAGEA11, MAGEA2. Next promoter demethylation was considered as a cause of mRNA expression increases seen in the expO dataset. (f) shows the QUMSP results for an independent cohort of 14 lung normals and 13 lung tumor patients. Significant differences in QUMSP were found in H19, MAGEA11, MAGEA2, and MAGEA3/6.
Mentions: We then performed transient transfections to evaluate and/or confirm growth-promoting effects of these nine targets that show tumor-specific promoter hypomethylation. Although H19 codes for a nontranslated RNA transcript, the H19 product appears to induce growth in lung and breast cancer cell lines [16] and may induce drug resistance in hepato-cellular carcinoma [17]. Figure 3A shows results obtained by transient transfection of an H19 construct into OKF6-Tert-1R cells. At four days, there was a 41.4% (±15%) increase in growth over the transfected empty vector. The MAGE family consists of related family members that are known to be upregulated in a variety of tumor types[18], but have recently been implicated in inducing transcriptional reprogramming in tumor cells[19]. MAGEA2 induced a 72.7% (±26%) increase in growth at day three (Figure 3B). MAGEA4 transfection induced a 203% (±17%) increase in growth (Figure 3D). Functional growth differences were tested, but not found for C19ORF28.

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