Abstract B18: Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to early steps of transformation
Authors: Vaz Michelle , Hwang Stephen Y. , Patil Ashwini , Easwaran Hariharan , Baylin Stephen B. 
Published: May 2016

Abstract
Chronic inflammation has been shown to play a significant role in mediating early epigenetic changes that contribute to the development of cancer. Prolonged exposure to inflammatory stimuli such as cigarette smoke causes genetic and epigenetic alterations in the lung, thereby contributing to the development of lung cancer. However, the full timing, and precise mechanisms leading to the evolution of these epigenetic alterations have not been well defined. The goal of this study is to model the progressive chromatin and DNA methylation alterations which associate with key gene expression changes during the progression to lung cancer.
Cdk-4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in medium with or without cigarette smoke condensate (CSC) for 15 months and genome-wide DNA methylation, expression, chromatin changes plus binding to chromatin of key epigenetic regulators and cell phenotypic features are examined over time. At each time point, non-exposed cultured cells are also examined to match their “aging” on plastic changes to the CSC-specific changes.
By 10 months, exposure to CSC induces distinct phenotypic changes in which cells become less translucent and have features of EMT. Preceding these changes, exposure acutely causes, within 10 days, DNA methyltransferase 1 (DNMT1) and EZH2 to become tightly bound to chromatin. Over the first month of exposure, EZH2 binding decreases while DNMT1 remains tightly bound to chromatin after 15 months. Progressive, but stochastically variable global DNA methylation changes begin by 6 months and are observed over the entire time course of the study. As early as 6 months, two types of increases in DNA methylation emerge at unmethylated CpG-island promoters, those specific to CSC treated cells and those which evolve in the “aging” cells. The latter changes often increase further with time in the CSC exposed cells. The early CSC-specific methylated genes are biased to homeobox genes, transcription factors and developmental genes. Increased methylation of key tumor suppressor genes is observed beginning at 10 months of exposure at a time when the CSC exposed cells begin to clone in soft agar. This change is suggestive of transformation but the cells do not yet form tumors when explanted into immunodeficient mice. The genes that get methylated at this time point are biased to those regulating the Hedgehog, Wnt and mitogen-activated protein kinase (MAPK) signaling pathways. Gene expression studies show that activation of MAP-kinase and KRAS pathway dominate by the time point the above transformation changes have ensued. The CSC specific DNA methylation changes seem to track best in TCGA with patterns for lung adenocarcinoma, although there is definite overlap with those for squamous cell lung cancer. ChIP-seq analyses reveal important chromatin changes that precede the above methylation changes. Promoters of genes methylated by CSC during the course of the treatment show an initial recruitment of EZH2 at their promoters following CSC exposure, which begins at 10 days and then decreases with time. However, in the untreated “aging” cells, recruitment of EZH2 increases with time and remains dominant for the genes that become DNA hypermethylated with CSC exposure, which have limited EZH2 binding. We thus define a model for lung cancer evolution due to CSC exposure wherein key molecular changes appear, and switch regulatory features with time, to evolve a more co-ordinated pattern of cancer-associated epigenetic features which may help drive inflammation induced pulmonary carcinogenesis.
Citation Format: Michelle Vaz, Stephen Y. Hwang, Ashwini Patil, Hariharan Easwaran, Stephen B. Baylin. Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to early steps of transformation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B18.