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Promoter methylation-mediated silencing of β-catenin enhances invasiveness of non-small cell lung cancer and predicts adverse prognosis.

Miao Y, Wang L, Zhang X, Xu X, Jiang G, Fan C, Liu Y, Lin X, Yu J, Zhang Y, Wang E - PLoS ONE (2014)

Bottom Line: The methylation status in SPC (methylated) and A549 (unmethylated) was confirmed by bisulfite sequencing PCR. 5-Aza-dC treatment inhibited invasiveness of SPC but not A549.Dual-luciferase assays indicated that 5-aza-dC treatment caused no significant increase in Wnt signaling activity compared with Wnt3a treatment.Subsequent immunohistochemistry results further confirmed that β-catenin promoter hypermethylation correlated with loss of immunoreactive protein expression, positive lymph node metastasis, high TNM stage and poor prognosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The First Hospital and College of Basic Medical Sciences of China Medical University, Shenyang, China.

ABSTRACT
β-Catenin plays dual role in adhesion complex formation and the Wnt signaling pathway. Although β-catenin expression appears to be upregulated and Wnt signaling pathway is activated in the majority of cancers, its expression level seems to be lost in non-small cell lung cancer (NSCLC). We previously reported that the promoter of β-catenin was hypermethylated in two NSCLC cell lines. In the current study, we expanded our analysis for the methylation status of β-catenin promoter region and its protein expression in seven NSCLC cell lines and a series of 143 cases of primary human lung cancer with adjacent non-neoplastic tissues. Quantitative methylation specific PCR (qMSP) analysis showed methylation of β-catenin promoter region in five NSCLC cell lines, with increased β-catenin protein levels upon 5'-Aza-2'-deoxycytidine (5-aza-dC) treatment. The methylation status in SPC (methylated) and A549 (unmethylated) was confirmed by bisulfite sequencing PCR. 5-Aza-dC treatment inhibited invasiveness of SPC but not A549. Immunofluorescence analysis showed membranous β-catenin expression was lost in SPC and could be re-established by 5-aza-dC, while Wnt3a treatment led to nuclear translocation of β-catenin in both SPC and A549. Dual-luciferase assays indicated that 5-aza-dC treatment caused no significant increase in Wnt signaling activity compared with Wnt3a treatment. The effect of demethylation agent in SPC can be reversed by β-catenin depletion but not E-cadherin depletion which indicated that the methylation mediated β-catenin silencing might enhance NSCLC invasion and metastasis in an E-cadherin independent manner. Subsequent immunohistochemistry results further confirmed that β-catenin promoter hypermethylation correlated with loss of immunoreactive protein expression, positive lymph node metastasis, high TNM stage and poor prognosis. The present study implicates β-catenin promoter hypermethylation in the mechanism of epigenetic changes underlying NSCLC metastasis and progression, thus indicating the potential of β-catenin as a novel epigenetic target for the treatment of NSCLC patients.

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Analysis of methylation status of β-catenin promoter in NSCLC cell lines.(A) Quantitative MSP analysis of the β-catenin promoter region in NSCLC cell lines. (B) Promoter region of β-catenin gene: Position of CpG islands and primer design are indicated by lines and arrows. (C) Methylation of one of the 19 CG-dinucleotides in region −614 bp to −270 bp of both A549 and SPC. Filled circles represent methylated CG sites, unfilled circles represent unmethylated CG sites.
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pone-0112258-g001: Analysis of methylation status of β-catenin promoter in NSCLC cell lines.(A) Quantitative MSP analysis of the β-catenin promoter region in NSCLC cell lines. (B) Promoter region of β-catenin gene: Position of CpG islands and primer design are indicated by lines and arrows. (C) Methylation of one of the 19 CG-dinucleotides in region −614 bp to −270 bp of both A549 and SPC. Filled circles represent methylated CG sites, unfilled circles represent unmethylated CG sites.

Mentions: The β-catenin promoter methylation levels were tested in lung cancer cell lines. As shown in Figure 1A, five cell lines (LH7, BE1, SPC, LTE and H1299) exhibited high levels of methylation, while two cell lines (HBE and A549) were unmethylated. Analysis of the CTNNB1 gene promoter region −1,124 – 11,114 bp in NSCLC cell lines revealed the presence of two CpG islands at positions −1,124 – 876 and 10,676 – 11,114, respectively. These sequences contained 189 single CG sites, 19 of which were CG-dinucleotides. Using the sodium bisulfite genomic sequencing method, we designed primers to analyze the first CpG islands in a 1,331 base pair region (−614–717) containing part of the β-catenin promoter region across its transcriptional start site in the SPC and A549 cell lines. Multiple methylation sites were identified in SPC cells, whereas only a few 5-methylcytosine residues were observed in several sequenced clones derived from A549 cells (Fig. 1B and 1C).


Promoter methylation-mediated silencing of β-catenin enhances invasiveness of non-small cell lung cancer and predicts adverse prognosis.

Miao Y, Wang L, Zhang X, Xu X, Jiang G, Fan C, Liu Y, Lin X, Yu J, Zhang Y, Wang E - PLoS ONE (2014)

Analysis of methylation status of β-catenin promoter in NSCLC cell lines.(A) Quantitative MSP analysis of the β-catenin promoter region in NSCLC cell lines. (B) Promoter region of β-catenin gene: Position of CpG islands and primer design are indicated by lines and arrows. (C) Methylation of one of the 19 CG-dinucleotides in region −614 bp to −270 bp of both A549 and SPC. Filled circles represent methylated CG sites, unfilled circles represent unmethylated CG sites.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4232381&req=5

pone-0112258-g001: Analysis of methylation status of β-catenin promoter in NSCLC cell lines.(A) Quantitative MSP analysis of the β-catenin promoter region in NSCLC cell lines. (B) Promoter region of β-catenin gene: Position of CpG islands and primer design are indicated by lines and arrows. (C) Methylation of one of the 19 CG-dinucleotides in region −614 bp to −270 bp of both A549 and SPC. Filled circles represent methylated CG sites, unfilled circles represent unmethylated CG sites.
Mentions: The β-catenin promoter methylation levels were tested in lung cancer cell lines. As shown in Figure 1A, five cell lines (LH7, BE1, SPC, LTE and H1299) exhibited high levels of methylation, while two cell lines (HBE and A549) were unmethylated. Analysis of the CTNNB1 gene promoter region −1,124 – 11,114 bp in NSCLC cell lines revealed the presence of two CpG islands at positions −1,124 – 876 and 10,676 – 11,114, respectively. These sequences contained 189 single CG sites, 19 of which were CG-dinucleotides. Using the sodium bisulfite genomic sequencing method, we designed primers to analyze the first CpG islands in a 1,331 base pair region (−614–717) containing part of the β-catenin promoter region across its transcriptional start site in the SPC and A549 cell lines. Multiple methylation sites were identified in SPC cells, whereas only a few 5-methylcytosine residues were observed in several sequenced clones derived from A549 cells (Fig. 1B and 1C).

Bottom Line: The methylation status in SPC (methylated) and A549 (unmethylated) was confirmed by bisulfite sequencing PCR. 5-Aza-dC treatment inhibited invasiveness of SPC but not A549.Dual-luciferase assays indicated that 5-aza-dC treatment caused no significant increase in Wnt signaling activity compared with Wnt3a treatment.Subsequent immunohistochemistry results further confirmed that β-catenin promoter hypermethylation correlated with loss of immunoreactive protein expression, positive lymph node metastasis, high TNM stage and poor prognosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The First Hospital and College of Basic Medical Sciences of China Medical University, Shenyang, China.

ABSTRACT
β-Catenin plays dual role in adhesion complex formation and the Wnt signaling pathway. Although β-catenin expression appears to be upregulated and Wnt signaling pathway is activated in the majority of cancers, its expression level seems to be lost in non-small cell lung cancer (NSCLC). We previously reported that the promoter of β-catenin was hypermethylated in two NSCLC cell lines. In the current study, we expanded our analysis for the methylation status of β-catenin promoter region and its protein expression in seven NSCLC cell lines and a series of 143 cases of primary human lung cancer with adjacent non-neoplastic tissues. Quantitative methylation specific PCR (qMSP) analysis showed methylation of β-catenin promoter region in five NSCLC cell lines, with increased β-catenin protein levels upon 5'-Aza-2'-deoxycytidine (5-aza-dC) treatment. The methylation status in SPC (methylated) and A549 (unmethylated) was confirmed by bisulfite sequencing PCR. 5-Aza-dC treatment inhibited invasiveness of SPC but not A549. Immunofluorescence analysis showed membranous β-catenin expression was lost in SPC and could be re-established by 5-aza-dC, while Wnt3a treatment led to nuclear translocation of β-catenin in both SPC and A549. Dual-luciferase assays indicated that 5-aza-dC treatment caused no significant increase in Wnt signaling activity compared with Wnt3a treatment. The effect of demethylation agent in SPC can be reversed by β-catenin depletion but not E-cadherin depletion which indicated that the methylation mediated β-catenin silencing might enhance NSCLC invasion and metastasis in an E-cadherin independent manner. Subsequent immunohistochemistry results further confirmed that β-catenin promoter hypermethylation correlated with loss of immunoreactive protein expression, positive lymph node metastasis, high TNM stage and poor prognosis. The present study implicates β-catenin promoter hypermethylation in the mechanism of epigenetic changes underlying NSCLC metastasis and progression, thus indicating the potential of β-catenin as a novel epigenetic target for the treatment of NSCLC patients.

Show MeSH
Related in: MedlinePlus