Human non-small cell lung tumors and cells derived from normal lung express both estrogen receptor alpha and beta and show biological responses to estrogen.

Lung cancer is becoming increasingly common in women and in the United States accounts for more female cancer deaths annually than breast cancer. Many epidemiological studies have provided evidence that women are more susceptible than men to the adverse effects of tobacco smoke. These observations suggest the possible role of estrogens in lung carcinogenesis. We report here the expression of mRNA for estrogen receptor alpha (ERalpha) and beta (ERbeta) in cultured human non-small cell lung cancer cells, cultured lung fibroblasts, and primary cultures of normal bronchial epithelium. Western analysis of ERalpha suggested that the main protein expressed in lung tumor cells is a variant, probably attributable to alternative splicing. Protein for ERbeta was found to be a mixture of full-length as well as alternatively spliced variants. beta-Estradiol produced a proliferative response in vitro in both normal lung fibroblasts and cultured non-small cell lung tumor cells. This effect was also observed in vivo. In this regard, beta-estradiol stimulated growth of the non-small cell lung tumor line, H23, grown as tumor xenografts in SCID mice. This effect was blocked by fluvestrant (ICI 182,780). In paraffin sections of non-small cell lung tumors, ERbeta immunoreactivity was localized to the nucleus, whereas ERalpha immunoreactivity was mainly localized to the cytoplasm, suggesting that both nuclear and cytoplasmic signaling may be involved in estrogenic responses in the lung. To show that the ERs found in the lung are functional, we demonstrated that beta-estradiol stimulated transcription of an estrogen response element-luciferase construct transfected in non-small cell lung tumor cell lines. Antiestrogens blocked this effect. Treatment of lung fibroblasts with beta-estradiol also increased secretion of hepatocyte growth factor by 2-fold. These results suggest that estrogen signaling plays a biological role in both the epithelium and the mesenchyme in the lung and that estrogens could potentially promote lung cancer, either through direct actions on preneoplastic or neoplastic cells or through indirect actions on lung fibroblasts. Additionally, it is possible that antiestrogens may have therapeutic value to treat or prevent lung cancer.

Nicotine signals through muscle-type and neuronal nicotinic acetylcholine receptors in both human bronchial epithelial cells and airway fibroblasts.

BACKGROUND: Non-neuronal cells, including those derived from lung, are reported to express nicotinic acetylcholine receptors (nAChR). We examined nAChR subunit expression in short-term cultures of human airway cells derived from a series of never smokers, ex-smokers, and active smokers. METHODS AND RESULTS: At the mRNA level, human bronchial epithelial (HBE) cells and airway fibroblasts expressed a range of nAChR subunits. In multiple cultures of both cell types, mRNA was detected for subunits that constitute functional muscle-type and neuronal-type pentomeric receptors. Two immortalized cell lines derived from HBE cells also expressed muscle-type and neuronal-type nAChR subunits. Airway fibroblasts expressed mRNA for three muscle-type subunits (alpha1, delta, and epsilon) significantly more often than HBE cells. Immunoblotting of HBE cell and airway fibroblast extracts confirmed that mRNA for many nAChR subunits is translated into detectable levels of protein, and evidence of glycosylation of nAChRs was observed. Some minor differences in nAChR expression were found based on smoking status in fibroblasts or HBE cells. Nicotine triggered calcium influx in the immortalized HBE cell line BEAS2B, which was blocked by alpha-bungarotoxin and to a lesser extent by hexamethonium. Activation of PKC and MAPK p38, but not MAPK p42/44, was observed in BEAS2B cells exposed to nicotine. In contrast, nicotine could activate p42/44 in airway fibroblasts within five minutes of exposure. CONCLUSIONS: These results suggest that muscle-type and neuronal-type nAChRs are functional in airway fibroblasts and HBE cells, that prior tobacco exposure does not appear to be an important variable in nAChR expression, and that distinct signaling pathways are observed in response to nicotine.

Nicotine activates cell-signaling pathways through muscle-type and neuronal nicotinic acetylcholine receptors in non-small cell lung cancer cells.

Nicotinic acetylcholine receptors (nAChR) are expressed on non-neuronal cell types, including normal bronchial epithelial cells, and nicotine has been reported to cause Akt activation in cultured normal airway cells. This study documents mRNA and protein expression of subunits known to form a muscle-type nAChR in non-small cell lung cancer (NSCLC) cell lines. In one NSCLC examined, mRNA and protein for a heteropentamer neuronal-type alpha3beta2 nAChR was detected in addition to a muscle-type receptor. Protein for the alpha5 nAChR was also detected in NSCLC cells. Although, mRNA for the alpha7 nAChR subunit was observed in all cell lines, alpha7 protein was not detectable by immunoblot in NSCLC cell extracts. Immunohistochemistry (IHC) of NSCLC primary tissues from 18 patients demonstrated protein expression of nAChR alpha1 and beta1 subunits, but not alpha7 subunit, in lung tumors, indicating preferential expression of the muscle-type receptor. In addition, the beta1 subunit showed significantly increased expression in lung tumors as compared to non-tumor bronchial tissue. The alpha1 subunit also showed evidence of high expression in lung tumors. Nicotine at a concentration of 10 microM caused phosphorylation of mitogen-activated protein kinase (MAPK) (p44/42) that could be inhibited using nAChR antagonists. Inhibition was observed at 100 nM alpha-bungarotoxin (alpha-BTX) or 10 microM hexamethonium (HEX); maximal inhibition was achieved using a combination of alpha-BTX and HEX. Akt was also phosphorylated in NSCLC cells after exposure to nicotine; this effect was inhibited by the PI3K inhibitor LY294002 and antagonists to the neuronal-type nAChR, but not to the muscle-type receptor. Nicotine triggered influx of calcium in the 273T NSCLC cell line, suggesting that L-type calcium channels were activated. 273T cells also showed greater activation of p44/42 MAPK than of Akt in response to nicotine. Cultures treated with nicotine and the EGFR tyrosine kinase inhibitor gefitinib showed a significant increase in the number of surviving cells compared to gefitinib alone. These data indicate that the muscle-type nAChR, rather than the alpha7 type, is highly expressed in NSCLC and leads to downstream activation of the p44/42 MAPK pathway. Neuronal-type receptors are also present and functional, as evidenced by antagonist studies, although, the expression levels are lower than muscle-type nAChR. They also lead to downstream activation of MAPK and Akt. Nicotine may play a role in regulating survival of NSCLC cells and endogenous acetylcholine released locally in the lung and/or chronic nicotine exposure might play a role in NSCLC development. In addition, exposure of NSCLC patients to nicotine through use of nicotine replacement products or use of tobacco products may alter the efficacy of therapy with EGFR inhibitors.