Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer

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

Treatment with 5 Aza reduces Wnt7a methylation.

<p>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.</p

DNMT1 modulates Wnt7a promoter methylation.

<p>A) 1299 cells were transiently transfected with DNMT1 shRNA and compared to empty and shRNA negative control-transfected cells. DNMT1 and Wnt7a mRNA expression was measured by QPCR and presented as fold-change compared to the empty control. Error bars are the SE of triplicate experiments. DNMT1 shRNA and a shRNA negative control were stably transfected into 1299 cells and DNMT1 and Wnt7a protein were measured by immnoblot with a β-actin loading control. B) Transiently transfected 1299 cells were analyzed for Wnt7a promoter methylation using pyrosequencing. DNMT1 shRNA and a negative shrNA control transfected cells were compared to empty cells. Error bars are the SE of triplicate experiments. C) 1299 cells with transient DNMT1 shRNA and/or Wnt7a shRNA expression were compared to an empty control and negative shRNA control in a clonogenicity assay. Cells were stained after 4 days of growth, photographed, and counted for cloning efficiency. Error bars are the SE of duplicate experiments.</p

Wnt7a is methylated in NSCLC cell lines and tissues.

<p>A) B2B and HBEC cells were treated with 10 uM NNK for 2 hours and Wnt7a expression was measured by QPCR compared to an untreated control. Error bars are the SEM of triplicate experiments. Percent methylation in B2B and HBEC cells was analyzed using the Methyl Profiler system after 2 hr 10 uM NNK treatment compared to untreated cells. B) Mean percent methylation of the Wnt7a promoter was measured by pyrosequencing in NSCLC tissue and compared to matched normal adjacent lung tissue by paired t-test. Mean percent methylation of B2B, H157, and H1299 cell lines was measured by pyrosequencing. C) A Wnt7a promoter luciferase was modified by SssI, HpaII, and HhaI methylating enzymes and transiently transfected into B2B and HBEC cell lines. Fold-change in luciferase activity was measured compared to unmodified Wnt7a promoter luciferase activity. Error bars are the SE of triplicate experiments.</p

Effects of Wnt7a loss in lung epithelial cells.

<p>A) Expression of Wnt7a was measured by QPCR in HBEC and B2B cells after stable transfection with Wnt7a shRNA or a negative shRNA. Data is presented as fold-change in Wnt7a expression normalized to GAPDH. Error bars are the SE of triplicate experiments. Wnt7a knock down was also confirmed by western blot with a β-actin loading control. B) Cell proliferation was measured in B2B cells expressing Wnt7a shRNA or a negative shRNA. Cells were analyzed in a 6-day growth assay. C) B2B cells with Wnt7a shRNA were analyzed in a 3D culture assay using Matrigel and observed after 5 days of growth for changes in morphology compared to a negative shRNA control. B2B cells with Wnt7a shRNA were also analyzed in a scratch migration assay and observed at 24 hours for movement into the introduced scratch compared to a negative shRNA control. Arrows indicate edges of the scratch. Pictures represent triplicate experiments.</p

CSC treatment leads to Wnt7a methylation.

<p>A) HBEC and B2B cells were treated with 1% CSC in cell growth media for 12 weeks and Wnt7a expression was measured by QPCR. Error bars are the SE of triplicate experiments. B) Expression of DNMT1 was measured by western blot in B2B cells treated with 1% CSC for 12 weeks. Loading control is β-actin. C) HBEC and B2B cells treated with 1% CSC for 12 weeks were analyzed for Wnt7a promoter methylation compared to untreated controls using the Methyl-Profiler system. Error bars are the SE of triplicate experiments.</p