Limits...
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.

Show MeSH

Related in: MedlinePlus

β-catenin expression and promoter methylation in normal lung tissues and lung cancer tissues.(A) β-catenin expression in normal lung epithelium with strong membranous staining. Membranous β-catenin expression was lost in lung squamous cell carcinoma (B) and lung adenocarcinoma (C). Scale bar = 20 µm. (D) Quantitative methylation results of q-MSP. Data represent mean±SD. The Mann-Whitney U-test was used to determine the statistical significance. **P<0.001. (E) Survival curves of patients with or without β-catenin promoter methylation. Mantel's log-rank test was used to determine statistical significance.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4232381&req=5

pone-0112258-g004: β-catenin expression and promoter methylation in normal lung tissues and lung cancer tissues.(A) β-catenin expression in normal lung epithelium with strong membranous staining. Membranous β-catenin expression was lost in lung squamous cell carcinoma (B) and lung adenocarcinoma (C). Scale bar = 20 µm. (D) Quantitative methylation results of q-MSP. Data represent mean±SD. The Mann-Whitney U-test was used to determine the statistical significance. **P<0.001. (E) Survival curves of patients with or without β-catenin promoter methylation. Mantel's log-rank test was used to determine statistical significance.

Mentions: We next investigated the methylation status of β-catenin promoter in clinical tissue samples. β-Catenin was expressed predominantly on the cell membrane in normal lung tissue samples (83.9%, 120/143; Fig. 4A), with reduced membranous expression in NSCLC tissues (18.9%; 27/143; Fig. 4B, C). Subsequently, the β-catenin promoter methylation levels were quantitated by real-time methylation-specific PCR. Using a hypermethylation value of greater than 4% as a cutoff, hypermethylation of β-catenin (mean ± SD, 8.60%±11.41%; range, 0–100%) was found in 74 (51.7%) of the 143 NSCLC specimens, which was significantly higher than that in adjacent normal lung tissue (13.3%, 19/143; mean ± SD, 2.66%±2.63%; range, 0–100%; P<0.001; Fig. 4D). Taken together, our study suggested that β-catenin methylation significantly correlated with loss of membranous β-catenin expression (P = 0.001).


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)

β-catenin expression and promoter methylation in normal lung tissues and lung cancer tissues.(A) β-catenin expression in normal lung epithelium with strong membranous staining. Membranous β-catenin expression was lost in lung squamous cell carcinoma (B) and lung adenocarcinoma (C). Scale bar = 20 µm. (D) Quantitative methylation results of q-MSP. Data represent mean±SD. The Mann-Whitney U-test was used to determine the statistical significance. **P<0.001. (E) Survival curves of patients with or without β-catenin promoter methylation. Mantel's log-rank test was used to determine statistical significance.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112258-g004: β-catenin expression and promoter methylation in normal lung tissues and lung cancer tissues.(A) β-catenin expression in normal lung epithelium with strong membranous staining. Membranous β-catenin expression was lost in lung squamous cell carcinoma (B) and lung adenocarcinoma (C). Scale bar = 20 µm. (D) Quantitative methylation results of q-MSP. Data represent mean±SD. The Mann-Whitney U-test was used to determine the statistical significance. **P<0.001. (E) Survival curves of patients with or without β-catenin promoter methylation. Mantel's log-rank test was used to determine statistical significance.
Mentions: We next investigated the methylation status of β-catenin promoter in clinical tissue samples. β-Catenin was expressed predominantly on the cell membrane in normal lung tissue samples (83.9%, 120/143; Fig. 4A), with reduced membranous expression in NSCLC tissues (18.9%; 27/143; Fig. 4B, C). Subsequently, the β-catenin promoter methylation levels were quantitated by real-time methylation-specific PCR. Using a hypermethylation value of greater than 4% as a cutoff, hypermethylation of β-catenin (mean ± SD, 8.60%±11.41%; range, 0–100%) was found in 74 (51.7%) of the 143 NSCLC specimens, which was significantly higher than that in adjacent normal lung tissue (13.3%, 19/143; mean ± SD, 2.66%±2.63%; range, 0–100%; P<0.001; Fig. 4D). Taken together, our study suggested that β-catenin methylation significantly correlated with loss of membranous β-catenin expression (P = 0.001).

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