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

Meredith A Tennis,Robert A Winn,Michelle M Vanscoyk,Lora A Wilson,Nicole Kelley,Sumitra Deb

doi:10.1371/journal.pone.0032921

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.

Highlights

Lung cancer continues to be the leading cause of cancer death, with minimal options available for treatment of tumors typically diagnosed at late stages
We have demonstrated that loss of Wnt7a is a significant event for lung epithelial cells and that this loss is caused by methylation through DNMT1 activity in a subset of NSCLC
We have previously demonstrated that Wnt7a expression alters the transformed characteristics of NSCLC cells

Summary

Introduction

Lung cancer continues to be the leading cause of cancer death, with minimal options available for treatment of tumors typically diagnosed at late stages. We are interested in the role of noncanonical Wnt signaling in non-small lung cancer (NSCLC), the tumor suppressive activities of Wnt7a and its receptor Frizzled 9 (Fzd). Wnt7a is highly expressed in embryonic lung and serves to maintain a normal epithelial phenotype in the adult lung [1,2]. Previous studies have demonstrated that Wnt7a is frequently lost in NSCLC and that restoration of Wnt7a expression leads to decreased transformed growth in NSCLC cell lines [2]. NSCLC cells with reexpression of Wnt7a have decreased proliferation, decreased anchorage independent growth, and restoration of an epithelial phenotype [2]. Wnt7a is located on chromosome 3p25, which is a ‘‘hotspot’’ for deletion, we suspected that Wnt7a expression in NSCLC can be regulated by an epigenetic mechanism [3]

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