Abstract 2773: Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to transformation
Authors: Vaz Michelle P. , Hwang Stephen Y. , Patil Ashwini , Phallen Jillian , Murphy Lauren , Zahnow Cynthia A. , Velculescu Victor E. , Easwaran Hariharan , Baylin Stephen B. 
Published: July 2016

Abstract
Herein, we define a model for how cigarette smoke and chronic inflammation induce human lung cancer via evolution of a co-ordinated pattern of progressive cancer-associated epigenetic abnormalities which prime cells for addiction to a single key genetic alteration, KRAS mutation. Non-clonogenic, non-tumorigenic, epigenetically stable human bronchial epithelial cells (HBEC) were exposed to cigarette smoke condensate (CSC) for 15 months. Earlier studies have established a requirement for the simultaneous disruption of three oncogenes to fully transform these cells. Genome-wide DNA methylation, expression, chromatin changes, as well as binding to chromatin of key epigenetic regulators and cell phenotypic features were examined over time in exposed versus non-exposed cells. CSC exposure acutely causes, within 10 days, a change we have previously associated with DNA damage, tightening of DNA methyltransferase 1 (DNMT1) and EZH2 to chromatin. While the EZH2 binding decreases with prolonged exposure, DNMT1 remains tightly bound to chromatin. Chronic exposure causes progressive, but stochastically variable, global DNA methylation changes which begin by six months and progress over the time course of the study to include hypermethylation of gene promoters which are frequent in human lung cancer. ChIP-seq analyses reveal, preceding the above methylation changes, that promoters of such methylated genes have an initial recruitment of EZH2, which begins at 10 days and then decreases with time. In contrast, recruitment of EZH2 increases with time and remains dominant for these same genes in the non-exposed controls. Following 10 months of exposure, CSC treated cells begin to clone in soft agar, often a feature of transformation, yet do not form tumors in immunodeficient mice. At this time point, gene expression studies show the top signaling pathway change is strong activation of MAP-kinase and KRAS pathways. Yet genome-wide, exome sequencing reveals no known lung cancer driver gene mutations. Remarkably, overexpression of mutant KRAS alone now markedly enlarges the soft agar colonies, and the cells are now fully transformed and form tumors in mice. Our study reveals, in a chronic cigarette exposure model relevant to the time course for evolution of KRAS mutant human lung adenocarcinoma, a key initial role for smoking induced epigenetic changes which facilitate addiction to the oncogene.
Citation Format: Michelle P. Vaz, Stephen Y. Hwang, Ashwini Patil, Jillian Phallen, Lauren Murphy, Cynthia A. Zahnow, Victor E. Velculescu, Hariharan Easwaran, Stephen B. Baylin. Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to transformation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2773.