Smoking-induced breast cancer formation have been discussed for many years.However, the molecular mechanisms is still uncertained.In this study, reverse-transcription polymerase chain reaction (RT-PCR) was used to determine the expression of all of the nicotinic acetylcholine receptor (nAChR) subunits in 50 human breast cancer samples,and to determine expression of the α9-nAChR subunit in 276 surgical and laser capture microscope-dissected breast tumor vs normal tissue pairs.Stable MDA-MB-231 breast cancer cell lines were established in which expression of the α9-nAChR subunit was inhibited using short interfering RNA (siRNA).MCF-10A normal human breast epithelial cells were established in which the cα9-nAChR subunit could be conditionally overexpressed by removal of doxycycline from the culture fluid.In 186 of 276 (67.3%) of paired samples, α9-nAChR mRNA was expressed at (mean 7.84-fold) higher levels in breast cancers than in surrounding normal tissue.Stable expression of α9-nAChR siRNA 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 cα9 cells =995.6 mm3, in mice injected with parental cells =2993.2 mm3, difference =1997.6 mm3, 95% CI =1705 to 2290.2 mm3, P =.009).Long-term treatment of MCF-10A normal breast epithelial cells with either nicotine or its active metabolite, 4-(methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK), triggered precancerous transformation as defined by soft agar assay.Inducible overexpression of α9-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 mm3 vs 501.6 mm3, difference =235.4 mm3, 95% CI =112.7 to 358 mm3,P =.009;DOX-, mean tumor volume without nicotine vs with nicotine =621.2 mm3 vs 898.6 mm3, difference =277.4 mm3, 95% CI =98.1 to 456.7 mm3;P =.016 [AU: These data differ from those presented on page 28.Please clarify.Ans: We correct the data on page 28.Thank you for your effort.];mean tumor volume in the presence of nicotine, DOX+ vs DOX-=501.6 mm3 vs 898.6 mm3, difference =397 mm3, 95% CI =241.3 to 552.6 mm3, P =.009).All these results demonstrated that the α9-nAChR is important for nicotine-induced transformation of normal human breast epithelial cells.