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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

BORIS transfection studies.(a) BORIS expression correlates with expression of target genes in HNSCC (QRT-PR) heat map (Pearson correlation) (b) Transient transfection of BORIS construct into NIH-3T3 and OKF6-Tert1R cell lines. BORIS overexpression resulted in increased cell growth in the 3T3 cell line (at day 3, 77%±34% growth increase) and in the OKF6-Tert1R cell line (at day 3, 161%±78% growth increase). Cell growth over baseline calculated by dividing values by day 1's Calcein signal. (c) Anchorage independent growth was assayed after transfection with empty vector (EV), CTCF, and BORIS at various concentrations of doxycycline, with representative colony (below). (d) QUMSP of nine targets of interest after transfection with empty vector (untreated) and BORIS construct (treated) in presence of 0.0625 µg/mL of doxycycline. (e) Fold increase Quantitiative RT-PCR of nine targets of interest after BORIS transfection normalized to values after transfection with empty vector.
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pone-0004961-g007: BORIS transfection studies.(a) BORIS expression correlates with expression of target genes in HNSCC (QRT-PR) heat map (Pearson correlation) (b) Transient transfection of BORIS construct into NIH-3T3 and OKF6-Tert1R cell lines. BORIS overexpression resulted in increased cell growth in the 3T3 cell line (at day 3, 77%±34% growth increase) and in the OKF6-Tert1R cell line (at day 3, 161%±78% growth increase). Cell growth over baseline calculated by dividing values by day 1's Calcein signal. (c) Anchorage independent growth was assayed after transfection with empty vector (EV), CTCF, and BORIS at various concentrations of doxycycline, with representative colony (below). (d) QUMSP of nine targets of interest after transfection with empty vector (untreated) and BORIS construct (treated) in presence of 0.0625 µg/mL of doxycycline. (e) Fold increase Quantitiative RT-PCR of nine targets of interest after BORIS transfection normalized to values after transfection with empty vector.

Mentions: The obvious presence of several MAGE genes among our targets prompted us to study upstream regulatory pathways of known cancer-testis antigens. BORIS and CTCF are a unique cognate pair of transcriptional factors involved in epigenetic regulation that share an identical DNA-binding domain. BORIS is transcriptionally silenced in most normal tissues, but expressed in normal embryonic, germ cell, and cancer tissues. We determined if expression of BORIS correlated with candidate proto-oncogene expression in a separate cohort of 36 primary HNSCC. Figure 7A presents a heat map constructed from median normalized, qRT-PCR expression data of our proto-oncogenes, sorted by BORIS expression. In these 36 cancers, BORIS overexpression was significantly correlated to overexpression of 6/9 proto-oncogenes including: MAGEA3/6 (p = 0.0017), MAGEA4 (p = 0.04), MAGEA11 (p<0.001), GPR17 (p = 0.01), and C19ORF28 (p = 0.001). To further examine the correlation of BORIS expression with our target genes in solid cancers, we analyzed the expO dataset data for 1041 human tumors of a wide variety of tissue sources and histologies. Significant positive correlation of BORIS expression with expression of each of our nine proto-oncogenes was noted: GRIN1 (p<0.001), C19ORF28 (p<0.001), H19 (p<0.001), MAGEA11 (p<0.001), MAGEA2 (p<0.001), MAGEA3/6 (p = 0.003), MAGE4 (p<0.001), TKTL1 (p<0.001), GPR17 (p<0.001), (Figure S2). Although BORIS transcripts are usually undetectable in normal cells, we determined that 59% of all tumors have a BORIS level that exceeds the median expression of all genes, and 90% of tumors have a BORIS expression level >25% of median expression value for all genes, indicating that aberrant BORIS expression is a common event in human cancer.


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)

BORIS transfection studies.(a) BORIS expression correlates with expression of target genes in HNSCC (QRT-PR) heat map (Pearson correlation) (b) Transient transfection of BORIS construct into NIH-3T3 and OKF6-Tert1R cell lines. BORIS overexpression resulted in increased cell growth in the 3T3 cell line (at day 3, 77%±34% growth increase) and in the OKF6-Tert1R cell line (at day 3, 161%±78% growth increase). Cell growth over baseline calculated by dividing values by day 1's Calcein signal. (c) Anchorage independent growth was assayed after transfection with empty vector (EV), CTCF, and BORIS at various concentrations of doxycycline, with representative colony (below). (d) QUMSP of nine targets of interest after transfection with empty vector (untreated) and BORIS construct (treated) in presence of 0.0625 µg/mL of doxycycline. (e) Fold increase Quantitiative RT-PCR of nine targets of interest after BORIS transfection normalized to values after transfection with empty vector.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654921&req=5

pone-0004961-g007: BORIS transfection studies.(a) BORIS expression correlates with expression of target genes in HNSCC (QRT-PR) heat map (Pearson correlation) (b) Transient transfection of BORIS construct into NIH-3T3 and OKF6-Tert1R cell lines. BORIS overexpression resulted in increased cell growth in the 3T3 cell line (at day 3, 77%±34% growth increase) and in the OKF6-Tert1R cell line (at day 3, 161%±78% growth increase). Cell growth over baseline calculated by dividing values by day 1's Calcein signal. (c) Anchorage independent growth was assayed after transfection with empty vector (EV), CTCF, and BORIS at various concentrations of doxycycline, with representative colony (below). (d) QUMSP of nine targets of interest after transfection with empty vector (untreated) and BORIS construct (treated) in presence of 0.0625 µg/mL of doxycycline. (e) Fold increase Quantitiative RT-PCR of nine targets of interest after BORIS transfection normalized to values after transfection with empty vector.
Mentions: The obvious presence of several MAGE genes among our targets prompted us to study upstream regulatory pathways of known cancer-testis antigens. BORIS and CTCF are a unique cognate pair of transcriptional factors involved in epigenetic regulation that share an identical DNA-binding domain. BORIS is transcriptionally silenced in most normal tissues, but expressed in normal embryonic, germ cell, and cancer tissues. We determined if expression of BORIS correlated with candidate proto-oncogene expression in a separate cohort of 36 primary HNSCC. Figure 7A presents a heat map constructed from median normalized, qRT-PCR expression data of our proto-oncogenes, sorted by BORIS expression. In these 36 cancers, BORIS overexpression was significantly correlated to overexpression of 6/9 proto-oncogenes including: MAGEA3/6 (p = 0.0017), MAGEA4 (p = 0.04), MAGEA11 (p<0.001), GPR17 (p = 0.01), and C19ORF28 (p = 0.001). To further examine the correlation of BORIS expression with our target genes in solid cancers, we analyzed the expO dataset data for 1041 human tumors of a wide variety of tissue sources and histologies. Significant positive correlation of BORIS expression with expression of each of our nine proto-oncogenes was noted: GRIN1 (p<0.001), C19ORF28 (p<0.001), H19 (p<0.001), MAGEA11 (p<0.001), MAGEA2 (p<0.001), MAGEA3/6 (p = 0.003), MAGE4 (p<0.001), TKTL1 (p<0.001), GPR17 (p<0.001), (Figure S2). Although BORIS transcripts are usually undetectable in normal cells, we determined that 59% of all tumors have a BORIS level that exceeds the median expression of all genes, and 90% of tumors have a BORIS expression level >25% of median expression value for all genes, indicating that aberrant BORIS expression is a common event in human cancer.

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