Overexpression and activation of the alpha9-nicotinic receptor during tumorigenesis in human breast epithelial cells.
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.
Lee
Chia-Hwa
CH
Graduate Institute of Biomedical Technology, Taipei Medical University, No. 250 Wu-Hsing Street, Taipei, Taiwan.
Huang
Ching-Shui
CS
Chen
Ching-Shyang
CS
Tu
Shih-Hsin
SH
Wang
Ying-Jan
YJ
Chang
Yu-Jia
YJ
Tam
Ka-Wai
KW
Wei
Po-Li
PL
Cheng
Tzu-Chun
TC
Chu
Jan-Show
JS
Chen
Li-Ching
LC
Wu
Chih-Hsiung
CH
Ho
Yuan-Soon
YS
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
D034741:RNA, Small Interfering; D011978:Receptors, Nicotinic; D014028:Tobacco Smoke Pollution; C443079:nAChR alpha9