Nicotine-modulated gene expression profiles in MCF7 breast cancer cell line and involvement of estrogen in CHRNA5 expression

Breast cancer, highly heterogeneous in nature, has been classified into multiple molecular subtypes based on hormone receptor status and also possess variable genetic and environmental etiologies. Prognosis and therapy of breast cancer depends on the presence or absence of these molecular markers. Nicotine, the major psychoactive addictive component in tobacco smoke, has been associated with multiple cancers because of its ability to increase cell proliferation, migration, and angiogenesis, and to decrease apoptosis. Nicotine binds to cholinergic receptors made up of multiple subunits, one of which, the alpha 5 (CHRNA5) whose polymorphisms have recently been implicated in nicotine addiction and lung cancer as functional. Microarray datasets provide genomic and functional information on the whole transcriptome when exposed to a certain treatment or under different pathological conditions allowing for molecular classification. The association between nicotine use and breast cancer has been controversial and to our knowledge no high-throughput expression profiling of breast cancer cells exposed to nicotine exists in the literature. In the present study, we determined that 1 uM nicotine affects cell proliferation in MCF7 cells measured by MTT assay only under serum starvation (0.1% Serum) condition at days 5 or 7 but not earlier. Similarly, effects on the protein levels of selected molecular markers with roles in proliferation and/or apoptosis, i.e., CyclinE, bcl-xl, and p53 were affected under serum starved conditions in more pronounced ways. Effects of nicotine at the transcriptome level were studied in MCF7 cells when exposed to 1uM Nicotine using Affymetrix Human HGU133 plus 2 arrays under the 10% serum levels. Our findings indicated that nicotine affects multiple pathways including MAPK, focal adhesion, and apoptosis although the magnitude of changes was mild. Preliminary analyses performed under serumstarvation indicated that starvation resulted in drastic changes in MCF7 transcriptome, some of which can be reversed, by 1uM nicotine. CHRNA5 expression was highly modulated by serum levels. Multiple microarray datasets on breast cancer cell lines and primary tumors in GEO were re-analyzed to assess the dependency of CHRNA5 expression on estrogen. Our findings were:

1. CHRNA5 expression increased in the presence of estrogen in a dose- and time-dependent fashion; 2) CHRNA5 was found to be a likely secondary target of estrogen; 3) CHRNA5 expression was higher in ER negative and/or Grade 3 breast cancer patients, implicating CHRNA5 with prognosis; 4) DNA replication and cell cycle genes seemed to be highly correlated with CHRNA5 expression; 5) the coexpressed genes could predict ER and Grade status of primary tumors with high accuracies. In conclusion, cholinergic signaling as modulated by nicotine through acetylcholine receptors might have a role in breast cancer etiology. CHRNA5 represents a novel candidate for breast cancer diagnostic and prognostic studies.