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AP-2alpha induces epigenetic silencing of tumor suppressive genes and microsatellite instability in head and neck squamous cell carcinoma.

Bennett KL, Romigh T, Eng C - PLoS ONE (2009)

Bottom Line: It was found to repress transcription of the tumor suppressor gene C/CAAT Enhancer Binding Protein alpha (C/EBPalpha), and its binding site correlated with upstream methylation of the C/EBPalpha promoter.These findings are significant because they suggest AP-2alpha plays a role not only in epigenetic silencing, but also in genomic instability.This intensifies the potential level of regulation AP-2alpha has through transcriptional regulation.

View Article: PubMed Central - PubMed

Affiliation: Genomic Medicine Institute, Lerner Research Institute and Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Activator protein 2 alpha (AP-2alpha) is involved in a variety of physiological processes. Increased AP-2alpha expression correlates with progression in various squamous cell carcinomas, and a recent publication found AP-2alpha to be overexpressed in approximately 70% of Head and Neck Squamous Cell Carcinoma (HNSCC) patient samples. It was found to repress transcription of the tumor suppressor gene C/CAAT Enhancer Binding Protein alpha (C/EBPalpha), and its binding site correlated with upstream methylation of the C/EBPalpha promoter. Therefore, we investigated the potential for AP-2alpha to target methylation to additional genes that would be relevant to HNSCC pathogenesis.

Principal findings: Stable downregulation of AP-2alpha stable by shRNA in HNSCC cell lines correlated with decreased methylation of its target genes' regulatory regions. Furthermore, methylation of MLH1 in HNSCC with and without AP-2alpha downregulation revealed a correlation with microsatellite instability (MSI). ChIP analysis was used to confirm binding of AP-2alpha and HDAC1/2 to the targets. The effects of HDAC inhibition was assessed using Trichostatin A in a HNSCC cell line, which revealed that AP-2alpha targets methylation through HDAC recruitment.

Conclusions: These findings are significant because they suggest AP-2alpha plays a role not only in epigenetic silencing, but also in genomic instability. This intensifies the potential level of regulation AP-2alpha has through transcriptional regulation. Furthermore, these findings have the potential to revolutionize the field of HNSCC therapy, and more generally the field of epigenetic therapy, by targeting a single gene that is involved in the malignant transformation via disrupting DNA repair and cell cycle control.

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Related in: MedlinePlus

Decreased MSI following AP-2α downregulation in HNSCC cell lines SCC22B and SCC17as.a) 9 MSI markers were used with DNA from SCC22B and SCC17as cells with and without AP-2α downregulation, and the products were analyzed on an 8% PAGE gel. b) ChIP analysis using an AP-2α antibody in SCC22B cells with and without AP-2α downregulation. ChIP eluate was then utilized for quantitative PCR using MLH1 promoter primers. c) Bisulfite sequencing analysis of 3 additional DNA repair genes in WT SCC22B cells. Because no methylation was observed in the WT cells, it was unnecessary to analyze methylation in AP-2α downregulated cells. Solid circles represent methylated CpGs; whereas, open circles represent unmethylated CpG sites.
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pone-0006931-g003: Decreased MSI following AP-2α downregulation in HNSCC cell lines SCC22B and SCC17as.a) 9 MSI markers were used with DNA from SCC22B and SCC17as cells with and without AP-2α downregulation, and the products were analyzed on an 8% PAGE gel. b) ChIP analysis using an AP-2α antibody in SCC22B cells with and without AP-2α downregulation. ChIP eluate was then utilized for quantitative PCR using MLH1 promoter primers. c) Bisulfite sequencing analysis of 3 additional DNA repair genes in WT SCC22B cells. Because no methylation was observed in the WT cells, it was unnecessary to analyze methylation in AP-2α downregulated cells. Solid circles represent methylated CpGs; whereas, open circles represent unmethylated CpG sites.

Mentions: Methylation of MLH1, a DNA repair gene, has been previously shown to correlate with microsatellite instability in colon, gastric, and endometiral cancers [20], [21], [22]. Therefore, MSI was compared in SCC22B, SCC17as, SCC11B, HCT116, and HT29 cells with and without AP-2α downregulation. Interestingly, MSI primers yielded fewer products in both SCC22B and SCC17as AP-2α-downregulated cells compared to the control cells (Figure 3a) (SCC22B = Bat25, D18S487, D17S250, D2S123, Bat40, and D18S56; SCC17as = Bat25, D18S487, D2S123, D18S67, and Bat40; D18S56 did not amplify in SCC17as). This suggests a potential decrease in MSI in these cells correlating with decreased AP-2α (Figure 3a). As expected, the cell lines that exhibited no change in MLH1 methylation (SCC11B, HT29, and HCT116) in the region tested did not reflect any changes in MSI with AP-2α downregulation (data not shown).


AP-2alpha induces epigenetic silencing of tumor suppressive genes and microsatellite instability in head and neck squamous cell carcinoma.

Bennett KL, Romigh T, Eng C - PLoS ONE (2009)

Decreased MSI following AP-2α downregulation in HNSCC cell lines SCC22B and SCC17as.a) 9 MSI markers were used with DNA from SCC22B and SCC17as cells with and without AP-2α downregulation, and the products were analyzed on an 8% PAGE gel. b) ChIP analysis using an AP-2α antibody in SCC22B cells with and without AP-2α downregulation. ChIP eluate was then utilized for quantitative PCR using MLH1 promoter primers. c) Bisulfite sequencing analysis of 3 additional DNA repair genes in WT SCC22B cells. Because no methylation was observed in the WT cells, it was unnecessary to analyze methylation in AP-2α downregulated cells. Solid circles represent methylated CpGs; whereas, open circles represent unmethylated CpG sites.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2734430&req=5

pone-0006931-g003: Decreased MSI following AP-2α downregulation in HNSCC cell lines SCC22B and SCC17as.a) 9 MSI markers were used with DNA from SCC22B and SCC17as cells with and without AP-2α downregulation, and the products were analyzed on an 8% PAGE gel. b) ChIP analysis using an AP-2α antibody in SCC22B cells with and without AP-2α downregulation. ChIP eluate was then utilized for quantitative PCR using MLH1 promoter primers. c) Bisulfite sequencing analysis of 3 additional DNA repair genes in WT SCC22B cells. Because no methylation was observed in the WT cells, it was unnecessary to analyze methylation in AP-2α downregulated cells. Solid circles represent methylated CpGs; whereas, open circles represent unmethylated CpG sites.
Mentions: Methylation of MLH1, a DNA repair gene, has been previously shown to correlate with microsatellite instability in colon, gastric, and endometiral cancers [20], [21], [22]. Therefore, MSI was compared in SCC22B, SCC17as, SCC11B, HCT116, and HT29 cells with and without AP-2α downregulation. Interestingly, MSI primers yielded fewer products in both SCC22B and SCC17as AP-2α-downregulated cells compared to the control cells (Figure 3a) (SCC22B = Bat25, D18S487, D17S250, D2S123, Bat40, and D18S56; SCC17as = Bat25, D18S487, D2S123, D18S67, and Bat40; D18S56 did not amplify in SCC17as). This suggests a potential decrease in MSI in these cells correlating with decreased AP-2α (Figure 3a). As expected, the cell lines that exhibited no change in MLH1 methylation (SCC11B, HT29, and HCT116) in the region tested did not reflect any changes in MSI with AP-2α downregulation (data not shown).

Bottom Line: It was found to repress transcription of the tumor suppressor gene C/CAAT Enhancer Binding Protein alpha (C/EBPalpha), and its binding site correlated with upstream methylation of the C/EBPalpha promoter.These findings are significant because they suggest AP-2alpha plays a role not only in epigenetic silencing, but also in genomic instability.This intensifies the potential level of regulation AP-2alpha has through transcriptional regulation.

View Article: PubMed Central - PubMed

Affiliation: Genomic Medicine Institute, Lerner Research Institute and Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio, United States of America.

ABSTRACT

Background: Activator protein 2 alpha (AP-2alpha) is involved in a variety of physiological processes. Increased AP-2alpha expression correlates with progression in various squamous cell carcinomas, and a recent publication found AP-2alpha to be overexpressed in approximately 70% of Head and Neck Squamous Cell Carcinoma (HNSCC) patient samples. It was found to repress transcription of the tumor suppressor gene C/CAAT Enhancer Binding Protein alpha (C/EBPalpha), and its binding site correlated with upstream methylation of the C/EBPalpha promoter. Therefore, we investigated the potential for AP-2alpha to target methylation to additional genes that would be relevant to HNSCC pathogenesis.

Principal findings: Stable downregulation of AP-2alpha stable by shRNA in HNSCC cell lines correlated with decreased methylation of its target genes' regulatory regions. Furthermore, methylation of MLH1 in HNSCC with and without AP-2alpha downregulation revealed a correlation with microsatellite instability (MSI). ChIP analysis was used to confirm binding of AP-2alpha and HDAC1/2 to the targets. The effects of HDAC inhibition was assessed using Trichostatin A in a HNSCC cell line, which revealed that AP-2alpha targets methylation through HDAC recruitment.

Conclusions: These findings are significant because they suggest AP-2alpha plays a role not only in epigenetic silencing, but also in genomic instability. This intensifies the potential level of regulation AP-2alpha has through transcriptional regulation. Furthermore, these findings have the potential to revolutionize the field of HNSCC therapy, and more generally the field of epigenetic therapy, by targeting a single gene that is involved in the malignant transformation via disrupting DNA repair and cell cycle control.

Show MeSH
Related in: MedlinePlus