Overexpression and activation of the α9-nicotinic receptor during tumorigenesis in human breast epithelial cells

Chia Hwa Lee, Ching Shui Huang, Ching Shyang Chen, Shih Hsin Tu, Ying Jan Wang, Yu Jia Chang, Ka Wai Tam, Po Li Wei, Tzu Chun Cheng, Jan Show Chu, Li Ching Chen, Chih Hsiung Wu, Yuan Soon Ho

Research output: Contribution to journalArticle

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Abstract

BackgroundLarge 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. MethodsReverse 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 α9-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 α9-nAChR subunit was inhibited using short interfering RNA. MCF-10A normal human breast epithelial cells were established in which the α9-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.ResultsIn 186 (67.3%) of the 276 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 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 α9 cells = 995.6 mm3, in mice injected with parental cells = 2993.2 mm3, difference = 1997.6 mm3, 95% confidence interval [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)-1-(3-pyridyl)-1-butanone, 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 vs 501.6 mm 3, difference = 235.4 mm3, 95% CI = 112.7 to 358 mm 3, P =. 009; DOX-, mean tumor volume without nicotine vs with nicotine = 621.2 vs 898.6 mm3, difference = 277.4 mm3, 95% CI = 98.1 to 456.7 mm3, P =. 016; mean tumor volume in the presence of nicotine, DOX+ vs DOX-= 501.6 vs 898.6 mm3, difference = 397 mm3, 95% CI = 241.3 to 552.6 mm3, P =. 009).ConclusionThe α9-nAChR is important for nicotine-induced transformation of normal human breast epithelial cells.

Original languageEnglish
Pages (from-to)1322-1335
Number of pages14
JournalJournal of the National Cancer Institute
Volume102
Issue number17
DOIs
Publication statusPublished - Sep 8 2010
Externally publishedYes

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Nicotinic Receptors
Nicotine
Carcinogenesis
Breast
Epithelial Cells
Tumor Burden
Breast Neoplasms
Confidence Intervals
Agar
Heterografts
Nude Mice
Small Interfering RNA
Growth
Tobacco Smoke Pollution
SCID Mice
Doxycycline
Carcinogens
Tobacco
Epidemiologic Studies
Neoplasms

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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Overexpression and activation of the α9-nicotinic receptor during tumorigenesis in human breast epithelial cells. / Lee, Chia Hwa; Huang, Ching Shui; Chen, Ching Shyang; Tu, Shih Hsin; Wang, Ying Jan; Chang, Yu Jia; Tam, Ka Wai; Wei, Po Li; Cheng, Tzu Chun; Chu, Jan Show; Chen, Li Ching; Wu, Chih Hsiung; Ho, Yuan Soon.

In: Journal of the National Cancer Institute, Vol. 102, No. 17, 08.09.2010, p. 1322-1335.

Research output: Contribution to journalArticle

@article{c467007e2a6b46d5938fc3ce500b308b,
title = "Overexpression and activation of the α9-nicotinic receptor during tumorigenesis in human breast epithelial cells",
abstract = "BackgroundLarge 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. MethodsReverse 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 α9-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 α9-nAChR subunit was inhibited using short interfering RNA. MCF-10A normal human breast epithelial cells were established in which the α9-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.ResultsIn 186 (67.3{\%}) of the 276 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 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 α9 cells = 995.6 mm3, in mice injected with parental cells = 2993.2 mm3, difference = 1997.6 mm3, 95{\%} confidence interval [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)-1-(3-pyridyl)-1-butanone, 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 vs 501.6 mm 3, difference = 235.4 mm3, 95{\%} CI = 112.7 to 358 mm 3, P =. 009; DOX-, mean tumor volume without nicotine vs with nicotine = 621.2 vs 898.6 mm3, difference = 277.4 mm3, 95{\%} CI = 98.1 to 456.7 mm3, P =. 016; mean tumor volume in the presence of nicotine, DOX+ vs DOX-= 501.6 vs 898.6 mm3, difference = 397 mm3, 95{\%} CI = 241.3 to 552.6 mm3, P =. 009).ConclusionThe α9-nAChR is important for nicotine-induced transformation of normal human breast epithelial cells.",
author = "Lee, {Chia Hwa} and Huang, {Ching Shui} and Chen, {Ching Shyang} and Tu, {Shih Hsin} and Wang, {Ying Jan} and Chang, {Yu Jia} and Tam, {Ka Wai} and Wei, {Po Li} and Cheng, {Tzu Chun} and Chu, {Jan Show} and Chen, {Li Ching} and Wu, {Chih Hsiung} and Ho, {Yuan Soon}",
year = "2010",
month = "9",
day = "8",
doi = "10.1093/jnci/djq300",
language = "English",
volume = "102",
pages = "1322--1335",
journal = "Journal of the National Cancer Institute",
issn = "0027-8874",
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TY - JOUR

T1 - Overexpression and activation of the α9-nicotinic receptor during tumorigenesis in human breast epithelial cells

AU - Lee, Chia Hwa

AU - Huang, Ching Shui

AU - Chen, Ching Shyang

AU - Tu, Shih Hsin

AU - Wang, Ying Jan

AU - Chang, Yu Jia

AU - Tam, Ka Wai

AU - Wei, Po Li

AU - Cheng, Tzu Chun

AU - Chu, Jan Show

AU - Chen, Li Ching

AU - Wu, Chih Hsiung

AU - Ho, Yuan Soon

PY - 2010/9/8

Y1 - 2010/9/8

N2 - BackgroundLarge 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. MethodsReverse 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 α9-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 α9-nAChR subunit was inhibited using short interfering RNA. MCF-10A normal human breast epithelial cells were established in which the α9-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.ResultsIn 186 (67.3%) of the 276 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 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 α9 cells = 995.6 mm3, in mice injected with parental cells = 2993.2 mm3, difference = 1997.6 mm3, 95% confidence interval [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)-1-(3-pyridyl)-1-butanone, 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 vs 501.6 mm 3, difference = 235.4 mm3, 95% CI = 112.7 to 358 mm 3, P =. 009; DOX-, mean tumor volume without nicotine vs with nicotine = 621.2 vs 898.6 mm3, difference = 277.4 mm3, 95% CI = 98.1 to 456.7 mm3, P =. 016; mean tumor volume in the presence of nicotine, DOX+ vs DOX-= 501.6 vs 898.6 mm3, difference = 397 mm3, 95% CI = 241.3 to 552.6 mm3, P =. 009).ConclusionThe α9-nAChR is important for nicotine-induced transformation of normal human breast epithelial cells.

AB - BackgroundLarge 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. MethodsReverse 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 α9-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 α9-nAChR subunit was inhibited using short interfering RNA. MCF-10A normal human breast epithelial cells were established in which the α9-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.ResultsIn 186 (67.3%) of the 276 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 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 α9 cells = 995.6 mm3, in mice injected with parental cells = 2993.2 mm3, difference = 1997.6 mm3, 95% confidence interval [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)-1-(3-pyridyl)-1-butanone, 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 vs 501.6 mm 3, difference = 235.4 mm3, 95% CI = 112.7 to 358 mm 3, P =. 009; DOX-, mean tumor volume without nicotine vs with nicotine = 621.2 vs 898.6 mm3, difference = 277.4 mm3, 95% CI = 98.1 to 456.7 mm3, P =. 016; mean tumor volume in the presence of nicotine, DOX+ vs DOX-= 501.6 vs 898.6 mm3, difference = 397 mm3, 95% CI = 241.3 to 552.6 mm3, P =. 009).ConclusionThe α9-nAChR is important for nicotine-induced transformation of normal human breast epithelial cells.

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