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Methylation of Wnt7a is modulated by DNMT1 and cigarette smoke condensate in non-small cell lung cancer.

Tennis MA, Vanscoyk MM, Wilson LA, Kelley N, Winn RA - PLoS ONE (2012)

Bottom Line: The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity.We treated H157 and H1299 NSCLC cell lines with 5-Aza-2'-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway.When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado, United States of America. Meredith.tennis@gmail.com

ABSTRACT
Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2'-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC.

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Treatment with 5 Aza reduces Wnt7a methylation.A) 157 and 1299 cells were treated with 5 Aza for 2 hours and Wnt7a was measured by QPCR compared to untreated controls. Error bars are the SEM of triplicate experiments. B) 157 and 1299 cells were treated with 5 Aza for 1, 2, and 4 hours and compared to untreated cells. Wnt7a protein expression was measured by immunoblot with β-actin as a loading control. C) 157 and 1299 cells were transiently transfected with Fzd9 and PPRE luciferase and 48 hours after were treated with 5 Aza for 2 hours. Luciferase activity was measured and treated cells were compared to an empty control. Error bars are the SE of triplicate experiments. D) 1299 cells were treated with 5 Aza for 2 hours and percent methylation of the Wnt7a promoter was measured by the Methyl-Profiler system. Treated cells were compared to an untreated control. Error bar is the SE of triplicate experiments.
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pone-0032921-g003: Treatment with 5 Aza reduces Wnt7a methylation.A) 157 and 1299 cells were treated with 5 Aza for 2 hours and Wnt7a was measured by QPCR compared to untreated controls. Error bars are the SEM of triplicate experiments. B) 157 and 1299 cells were treated with 5 Aza for 1, 2, and 4 hours and compared to untreated cells. Wnt7a protein expression was measured by immunoblot with β-actin as a loading control. C) 157 and 1299 cells were transiently transfected with Fzd9 and PPRE luciferase and 48 hours after were treated with 5 Aza for 2 hours. Luciferase activity was measured and treated cells were compared to an empty control. Error bars are the SE of triplicate experiments. D) 1299 cells were treated with 5 Aza for 2 hours and percent methylation of the Wnt7a promoter was measured by the Methyl-Profiler system. Treated cells were compared to an untreated control. Error bar is the SE of triplicate experiments.

Mentions: As methylation of Wnt7a had not been studied previously, we wanted to verify that methylation of the promoter region would lead to inactivation of the promoter. A Wnt7a promoter luciferase was treated with SssI, HpaII, and HhaI methylases to determine the effect of methylation on the Wnt7a promoter region. SssI affects all CpG sites, HpaII only the CpG within 5″CCGG, and HhaI only the CpG within 5′GCGC. Transfection of the Wnt7a promoter luciferase into B2B and HBEC cell lines demonstrated decreased luciferase activity after treatment with all three methylases compared to an untreated Wnt7a promoter luciferase, indicating that even limited methylation inactivates the Wnt7a promoter (Fig. 2C). We had identified two NSCLC cell lines with methylation of the Wnt7a promoter region, H157 and H1299, and wanted to determine the effect of treatment with a demethylating agent, 5-aza-2′ deoxycytidine (5 Aza). Cells were treated with 6 uM 5 Aza for 1–4 hours and expression of Wnt7a was measured by QPCR and western blot. Exposure to 5 Aza led to increased expression of Wnt7a mRNA and protein (Fig. 3A and B). As expected, 5 Aza also led to decreased methylation of the Wnt7a promoter region (Fig. 3D). Wnt7a is known to signal through its receptor Fzd9 to downstream target PPARγ, so we treated H157 and H1299 cells with 5 Aza, along with transient transfection of Fzd9, and analyzed the activity of the Wnt7a signaling pathway [19]. The PPAR response element luciferase (PPRE), which is activated by PAPRγ, demonstrated increased activity after cells with methylated Wnt7a were exposed to 5 Aza and expression of Fzd9 (Fig. 3C). These data indicate that pharmaceutical treatment of NSCLC cells can reverse the methylated state of the Wnt7a promoter.


Methylation of Wnt7a is modulated by DNMT1 and cigarette smoke condensate in non-small cell lung cancer.

Tennis MA, Vanscoyk MM, Wilson LA, Kelley N, Winn RA - PLoS ONE (2012)

Treatment with 5 Aza reduces Wnt7a methylation.A) 157 and 1299 cells were treated with 5 Aza for 2 hours and Wnt7a was measured by QPCR compared to untreated controls. Error bars are the SEM of triplicate experiments. B) 157 and 1299 cells were treated with 5 Aza for 1, 2, and 4 hours and compared to untreated cells. Wnt7a protein expression was measured by immunoblot with β-actin as a loading control. C) 157 and 1299 cells were transiently transfected with Fzd9 and PPRE luciferase and 48 hours after were treated with 5 Aza for 2 hours. Luciferase activity was measured and treated cells were compared to an empty control. Error bars are the SE of triplicate experiments. D) 1299 cells were treated with 5 Aza for 2 hours and percent methylation of the Wnt7a promoter was measured by the Methyl-Profiler system. Treated cells were compared to an untreated control. Error bar is the SE of triplicate experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3293913&req=5

pone-0032921-g003: Treatment with 5 Aza reduces Wnt7a methylation.A) 157 and 1299 cells were treated with 5 Aza for 2 hours and Wnt7a was measured by QPCR compared to untreated controls. Error bars are the SEM of triplicate experiments. B) 157 and 1299 cells were treated with 5 Aza for 1, 2, and 4 hours and compared to untreated cells. Wnt7a protein expression was measured by immunoblot with β-actin as a loading control. C) 157 and 1299 cells were transiently transfected with Fzd9 and PPRE luciferase and 48 hours after were treated with 5 Aza for 2 hours. Luciferase activity was measured and treated cells were compared to an empty control. Error bars are the SE of triplicate experiments. D) 1299 cells were treated with 5 Aza for 2 hours and percent methylation of the Wnt7a promoter was measured by the Methyl-Profiler system. Treated cells were compared to an untreated control. Error bar is the SE of triplicate experiments.
Mentions: As methylation of Wnt7a had not been studied previously, we wanted to verify that methylation of the promoter region would lead to inactivation of the promoter. A Wnt7a promoter luciferase was treated with SssI, HpaII, and HhaI methylases to determine the effect of methylation on the Wnt7a promoter region. SssI affects all CpG sites, HpaII only the CpG within 5″CCGG, and HhaI only the CpG within 5′GCGC. Transfection of the Wnt7a promoter luciferase into B2B and HBEC cell lines demonstrated decreased luciferase activity after treatment with all three methylases compared to an untreated Wnt7a promoter luciferase, indicating that even limited methylation inactivates the Wnt7a promoter (Fig. 2C). We had identified two NSCLC cell lines with methylation of the Wnt7a promoter region, H157 and H1299, and wanted to determine the effect of treatment with a demethylating agent, 5-aza-2′ deoxycytidine (5 Aza). Cells were treated with 6 uM 5 Aza for 1–4 hours and expression of Wnt7a was measured by QPCR and western blot. Exposure to 5 Aza led to increased expression of Wnt7a mRNA and protein (Fig. 3A and B). As expected, 5 Aza also led to decreased methylation of the Wnt7a promoter region (Fig. 3D). Wnt7a is known to signal through its receptor Fzd9 to downstream target PPARγ, so we treated H157 and H1299 cells with 5 Aza, along with transient transfection of Fzd9, and analyzed the activity of the Wnt7a signaling pathway [19]. The PPAR response element luciferase (PPRE), which is activated by PAPRγ, demonstrated increased activity after cells with methylated Wnt7a were exposed to 5 Aza and expression of Fzd9 (Fig. 3C). These data indicate that pharmaceutical treatment of NSCLC cells can reverse the methylated state of the Wnt7a promoter.

Bottom Line: The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity.We treated H157 and H1299 NSCLC cell lines with 5-Aza-2'-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway.When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Colorado School of Medicine, Aurora, Colorado, United States of America. Meredith.tennis@gmail.com

ABSTRACT
Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2'-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC.

Show MeSH
Related in: MedlinePlus