Abstract 4993: Nicotine induced EMT and metastasis of human NSCLC : Role of beta-arrestin-1

Abstract

Abstract Cigarette smoking is a major risk factor in the development of non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers. Nicotine, an active component of tobacco smoke has been found to induce proliferation, invasion and epithelial-mesenchymal transition in NSCLC cell lines and promote the metastasis of NSCLC in mice. Nicotine induces cell proliferation utilizing a scaffolding protein, β-arrestin-1, which translocates to nucleus and associates with E2F1 transcription factor in response to nicotine stimulation. Here we demonstrate that mesenchymal markers such as vimentin and fibronectin are E2F1 regulated and β-arrestin-1 is involved in regulating nicotine induced expression of these genes. Vimentin and fibronectin promoters were E2F responsive and E2F1 could be detected on these promoters by chromatin immunoprecipitation (ChIP) assays. Depletion of β-arrestin-1 resulted in down-regulation of vimentin and fibronectin expression as well as inhibition of nicotine induced invasion of cells. Quantitative RT-PCR conducted on patient samples revealed a significant correlation between the levels of β-arrestin-1 and the expression of these genes. A microarray analysis conducted on nicotine stimulated parental A549 cells and those lacking β-arrestin-1 showed that about 290 genes were upregulated in the absence of β-arrestin-1, while 787 genes were downregulated. These included genes for transcription factors, growth factor receptors and signaling molecules. We further analyzed the global association of β-arrestin-1 in the genomic region upon nicotine stimulation by ChIP-sequencing and found that β-arrestin-1 is recruited on the promoters of many genes that regulate EMT such as ZEB2 (Zinc Finger E-box Binding Homeobox-2) as well as other regulatory pathways. ChIP assays conducted on NSCLC cell lines revealed the association of β-arrestin-1 on ZEB1 (Zinc Finger E-box Binding Homeobox-1) and ZEB2 promoters. Depletion of β-arrestin-1 in A549 cells resulted in the downregulation of ZEB1 and ZEB2. Additionally, in an orthotopic lung cancer model in SCID-beige mice, implantation of A549-luciferase stable cells lacking β-arrestin-1 showed a decrease in primary tumor growth and also resulted in significantly lower levels of nicotine-induced metastasis when compared to controls. Taken together, these data suggest that β-arrestin-1 contributes to nicotine induced progression and metastasis of NSCLC, especially in patients exposed to tobacco smoke. Further, β-arrestin-1 and its associated molecules might be targeted for the development of novel agents to combat NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4993. doi:10.1158/1538-7445.AM2011-4993