title: Overexpression and activation of the alpha9-nicotinic receptor during tumorigenesis in human breast epithelial cells.
abstract: BACKGROUND
  Large epidemiological cohort studies in the United States have indicated that active and passive smoking are associated with increased breast cancer risk. However, there was no direct evidence of an effect of tobacco carcinogens on the cellular molecules involved in breast tumorigenesis.
  
  
  METHODS
  Reverse transcription-polymerase chain reaction was used to determine the expression of all of the nicotinic acetylcholine receptor (nAChR) subunits in 50 human breast cancer samples and to determine the expression of the alpha9-nAChR subunit in 276 surgical and laser capture microdissected breast tumor vs normal tissue pairs. Stable MDA-MB-231 breast cancer cell lines were established in which expression of the alpha9-nAChR subunit was inhibited using short interfering RNA. MCF-10A normal human breast epithelial cells were established in which the alpha9-nAChR subunit could be conditionally overexpressed by removal of doxycycline from the culture fluid. Cell proliferation and soft agar assays and tumor growth in nude mice were used as measures of cell transformation. All statistical tests were two-sided.
  
  
  RESULTS
  In 186 (67.3%) of the 276 paired samples, alpha9-nAChR mRNA was expressed at (mean 7.84-fold) higher levels in breast cancers than in surrounding normal tissue. Stable expression of alpha9-nAChR short interfering RNA in MDA-MB-231 cells attenuated nicotine-stimulated proliferation and growth in soft agar and reduced tumor volume when the cells were introduced as xenografts in SCID mice (n = 5 mice per group; mean tumor volume at 6 weeks treatment in mice injected with Si alpha9 cells = 995.6 mm(3), in mice injected with parental cells = 2993.2 mm(3), difference = 1997.6 mm(3), 95% confidence interval [CI] = 1705 to 2290.2 mm(3), P = .009). Long-term treatment of MCF-10A normal breast epithelial cells with either nicotine or its active metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, triggered precancerous transformation as defined by soft agar assay. Inducible overexpression of alpha9-nAChR in MCF-10A cell xenografts in nude mice substantially increased tumor growth (n = 5 mice per group; DOX+, mean tumor volume without nicotine vs with nicotine = 266.2 vs 501.6 mm(3), difference = 235.4 mm(3), 95% CI = 112.7 to 358 mm(3), P = .009; DOX-, mean tumor volume without nicotine vs with nicotine = 621.2 vs 898.6 mm(3), difference = 277.4 mm(3), 95% CI = 98.1 to 456.7 mm(3), P = .016; mean tumor volume in the presence of nicotine, DOX+ vs DOX- = 501.6 vs 898.6 mm(3), difference = 397 mm(3), 95% CI = 241.3 to 552.6 mm(3), P = .009).
  
  
  CONCLUSIONS
  The alpha9-nAChR is important for nicotine-induced transformation of normal human breast epithelial cells.
authors: Graduate Institute of Biomedical Technology, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei, Taiwan.
authors: Chia-Hwa
authors: CH
authors: Lee
authors: Ching-Shui
authors: CS
authors: Huang
authors: Ching-Shyang
authors: CS
authors: Chen
authors: Shih-Hsin
authors: SH
authors: Tu
authors: Ying-Jan
authors: YJ
authors: Wang
authors: Yu-Jia
authors: YJ
authors: Chang
authors: Ka-Wai
authors: KW
authors: Tam
authors: Po-Li
authors: PL
authors: Wei
authors: Tzu-Chun
authors: TC
authors: Cheng
authors: Jan-Show
authors: JS
authors: Chu
authors: Li-Ching
authors: LC
authors: Chen
authors: Chih-Hsiung
authors: CH
authors: Wu
authors: Yuan-Soon
authors: YS
authors: Ho
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001940:Breast / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004847:Epithelial Cells / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008819:Mice, Nude; D018613:Microscopy, Confocal; D034741:RNA, Small Interfering / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012307:Risk Factors; D012907:Smoking / Q000009:adverse effects* / Q000378:metabolism*; D014028:Tobacco Smoke Pollution / Q000009:adverse effects; D014183:Transplantation, Heterologous; D015854:Up-Regulation
chemical_list: D034741:RNA, Small Interfering; D011978:Receptors, Nicotinic; D014028:Tobacco Smoke Pollution; C443079:nAChR alpha9