title: ARRB1-mediated regulation of E2F target genes in nicotine-induced growth of lung tumors.
abstract: BACKGROUND
  Nicotine induces the proliferation of non-small cell lung cancer (NSCLC) cells via nicotinic acetylcholine receptors and the arrestin, β1 (ARRB1) protein. However, whether ARRB1 translocates to the nucleus upon nicotinic acetylcholine receptor activation and how it regulates growth of human NSCLCs are not known.
  
  
  METHODS
  We investigated nuclear localization of ARRB1 in human NSCLC cell lines (A549 and H1650), normal lung cell lines (NHBE and SAEC), and lung cancer tissue microarray. A549 cells were transfected with ARRB1-specific short hairpin RNA (A549-sh) to knockdown ARRB1 expression, or with empty vector (A549-EV), to examine the role of ARRB1 in the mitogenic and antiapoptotic effects of nicotine, binding of ARRB1 to E2F transcription factors, and the role of ARRB1 in nicotine-induced expression of E2F-regulated survival and proliferative genes cell division cycle 6 homolog (CDC6), thymidylate synthetase (TYMS), and baculoviral IAP repeat-containing 5 (BIRC5). Real-time polymerase chain reaction was performed for quantitative analysis of mRNA expression. Chromatin immunoprecipitation assays were performed on A549 cells and fresh-frozen human NSCLC tumors (n = 8) to examine the binding of ARRB1, E1A binding protein (EP300), and acetylated histone 3 (Ac-H3) on the E2F-regulated genes. All statistical tests were two-sided.
  
  
  RESULTS
  Nicotine induced the nuclear translocation of ARRB1 in NSCLC and normal lung cells, and lung tumor tissues from smokers showed an increased nuclear localization. The mitogenic and antiapoptotic effects of nicotine were reduced in A549-sh cells. Nuclear ARRB1 bound to E2F transcription factors in normal lung cells, NSCLC cells, and tumors. Nicotine treatment induced a statistically significant increased expression of E2F-regulated genes in A549-EV but not in A549-sh cells; the maximum difference being observed in BIRC5 (A549-EV vs A549-sh, mean fold-increase in mRNA level upon nicotine treatment = 20.7-fold, 95% confidence interval = 19.2- to 22.2-fold, vs mean = 0.8-fold, 95% confidence interval= 0.78- to 0.82-fold, P < .001). Furthermore, nicotine induced the binding of ARRB1, EP300, and Ac-H3 on E2F-regulated genes.
  
  
  CONCLUSIONS
  Nicotine induced the nuclear translocation of ARRB1 and showed increased expression of proliferative and survival genes, thereby contributing to the growth and progression of NSCLCs.
authors.affiliation: Department of Oncologic Sciences, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
authors.forename: Piyali
authors.initials: P
authors.lastname: Dasgupta
authors.forename: Wasia
authors.initials: W
authors.lastname: Rizwani
authors.forename: Smitha
authors.initials: S
authors.lastname: Pillai
authors.forename: Rebecca
authors.initials: R
authors.lastname: Davis
authors.forename: Sarmistha
authors.initials: S
authors.lastname: Banerjee
authors.forename: Kevin
authors.initials: K
authors.lastname: Hug
authors.forename: Mark
authors.initials: M
authors.lastname: Lloyd
authors.forename: Domenico
authors.initials: D
authors.lastname: Coppola
authors.forename: Eric
authors.initials: E
authors.lastname: Haura
authors.forename: Srikumar P
authors.initials: SP
authors.lastname: Chellappan
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D019390:Arrestins / Q000378:metabolism*; D002289:Carcinoma, Non-Small-Cell Lung / Q000209:etiology / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D050684:E2F Transcription Factors / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008168:Lung / Q000166:cytology / Q000378:metabolism; D008175:Lung Neoplasms / Q000209:etiology / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D018613:Microscopy, Confocal; D009538:Nicotine / Q000009:adverse effects*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D012907:Smoking / Q000009:adverse effects; D015854:Up-Regulation; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins
chemical_list: C000605253:ARRB1 protein, human; D019390:Arrestins; D050684:E2F Transcription Factors; D012333:RNA, Messenger; D000071559:beta-Arrestin 1; D000071557:beta-Arrestins; D009538:Nicotine