Li, Xueli

Abstract Genome Wide Association Studies (GWAS) are designed to identify single nucleotide polymorphisms (SNPs) mapping to genomic regions associated with a particular trait or disease. Currently, 19 genomic loci associated with risk for estrogen receptor negative breast cancer have been identified. However, for most of these loci the mechanisms driving susceptibility have remained elusive. The 2p23.2 locus, defined by SNP rs67073037 (p = 4.76 x 10-9), contains 79 SNPs exhibiting genome-wide significant associations as well as four candidate genes: SPDYA, TRMT61B, WDR43, and FAM179A. Post-GWAS functional analysis revealed two candidate causal regulatory elements: an enhancer-like element defined by SNP rs44017214 located in the first intron of the WDR43 gene; and a segment in the 3’-UTR region of WDR43 defined by SNPs rs11680458 and rs11331880 which are predicted to have allele specific binding of non-coding microRNAs miR-548 and miR-376, respectively. We validated allele specific transcription factor binding to rs44017214 using competitive binding of USF1/USF2 to the risk allele in both MCF10A and CAL51 cell lines in Electrophoretic Mobility Shift Assays (EMSA). Additionally, rs44017214 was removed using CRISPR-cas9, and expression of neighboring genes was evaluated by qRT-PCR. No change in expression was found for any gene in a 2 Mb region surrounding the SNP, with the exception of PLB1. This suggests that the enhancer element in the intron of WDR43 has PLB1 as its target. To assess the miRNA binding site impact on WDR43 expression the pMIR-REPORT Luciferase allele-specific vector was co-transfected with miR-141/200, miR548, or miR-376 in MCF10A cells. The rs1131880 allele C in the WDR43 3’-UTR lead to the suppression of luciferase expression by miR-376 compared to the G allele. This study reveals two independent regulatory mechanisms within the 2p23.2 locus; (i) modulation of enhancer activity by allele specific binding of transcription factors USF1/USF2 to rs4401714, and (ii) decreasing the expression of WDR43 through the formation of a miR-376 binding site at the 3’-UTR of the WDR43 gene. Elucidating the functional significance of these common variants in the novel susceptibility loci 2p23.2 furthers our knowledge of the etiology of estrogen receptor negative breast cancer. Citation Format: Carly M. Harro, Gustavo Mendoza-Fandino, Nicholas T. Woods, Xueli Li, Fergus J. Couch, Alvaro N. Monteiro. Two distinct regulatory mechanisms underlie estrogen receptor negative breast cancer susceptibility at the 2p23.2 locus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1306. doi:10.1158/1538-7445.AM2017-1306

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, the major addictive component of tobacco smoke, can induce proliferation, invasion, and epithelial-to-mesenchymal transition (EMT) in NSCLC cell lines and promote metastasis of NSCLC in mice. Here, we demonstrate that the scaffolding protein β-arrestin-1 is necessary for nicotine-mediated induction of mesenchymal genes vimentin and fibronectin as well as EMT regulators ZEB1 and ZEB2. Nicotine induced changes in cell morphology and ablate tight junctions consistent with EMT; β-arrestin-1, but not β-arrestin-2, was required for these changes. β-Arrestin-1 promoted the expression of the mesenchymal genes, as well as ZEB1 and ZEB2, through the mediation of the E2F1 transcription factor; this required Src kinase activity. Stimulation of multiple NSCLC cell lines with nicotine led to enhanced recruitment of β-arrestin-1 and E2F1 on vimentin, fibronectin, and ZEB1 and ZEB2 promoters. Furthermore, there was significantly more β-arrestin-1 and E2F1 associated with these promoters in human NSCLC tumors, and β-arrestin-1 levels correlated with vimentin and fibronectin levels in human NSCLC samples. A549-luciferase cells lacking β-arrestin-1 showed a significantly reduced capacity for tumor growth and metastasis when orthotopically implanted into the lungs of SCID-beige mice. Taken together, these studies reveal a novel role for β-arrestin-1 in the growth and metastasis of NSCLC. Cancer Res; 75(6); 1009–20. ©2015 AACR.