SCLC is a highly aggressive type of lung cancer that expresses neuroendocrine markers such as 5-hytroxytryptamine (5-HT, serotonin), mammalian bombesin (MB), calcitonin, neuron specific enolase, and others. SCLC does not harbor activating point mutations in K-ras, but frequently demonstrates amplification of c-myc and mutations in the retinoblastoma and p53 genes . The majority of SCLCs are though to have derived from the epithelial lining cells of large airways while a small population of SCLCs may derive from small airway epithelial cells. SCLC initially responds well to conventional cancer therapy but relapses frequently and progresses rapidly with extensive metastasis to extrapulmonary organs. Of all histological lung cancer types, SCLC shows the closest association with smoking, and a diagnosis of SCLC is very rare in non-smokers [52, 53]. Ionizing radiation and exposure to chloromethyl ethers are additional risk factors .
A potential role for nAChRs in smoking-associated lung carcinogenesis was first suggested in 1989 when it was shown that nicotine and NNK stimulated the proliferation of SCLC cells and that this response was blocked by antagonists for nAChRs but not by an antagonist for muscarinic AChRs [14, 54]. These reports were followed by publications in 1990 and 1994 which documented a nicotine-induced reversal of apoptosis in response to opioids in a large panel of SCLC cell lines as well as non-SCLC cell lines [17, 55]. An additional report in 1993 showed that nicotine stimulated the proliferation of human SCLC cell lines via stimulation of a serotonergic autocrine loop . Collectively, these initial findings suggested that nicotine itself may contribute to the development of smoking-associated lung cancer by interaction with nAChR-mediated proliferative and apoptotic signaling pathways. In addition, the data with NNK indicated that the extreme potency of NNK as a pulmonary carcinogen might be linked to its ability to function as an agonist for nAChRs. Radio receptor assays showed a high Bmax in saturation binding assays with the selective antagonist for nAChRs comprised of homomeric a7 subunits, a-bungarotoxin (a-BTX), indicative of high levels of expression of this receptor in human SCLC cell lines as opposed to lung adenocarcinoma cell lines which demonstrated low or non-detectable binding of a-BTX . These findings were extended by recent investigations that showed expression of mRNA for the a7nAChR in a large panel of cell lines derived from different types of human lung cancers and in immortalized human small airway epithelial cells while significant amounts of receptor protein were only detected in SCLC cell lines . Taken together, these findings indicate that the a7nAChr is expressed in many lung cell types but demonstrates particularly high levels of expression in SCLC cells. Radio receptor assays assessing the relative binding affinities of nicotine and NNK in competition with a-BTX identified NNK as a ligand with unprecedented high affinity for the a7nAChR. Analysis of these binding data by non-linear regression in fact showed that the affinity of NNK for the a7nAChR was about 1,300 times greater than that of nicotine . Stimulation of the a7nAChr by NNK significantly increased cell number and DNA synthesis in SCLC cells, a response blocked by the a7nAChR antagonist, a-BTX. Flow cytometric analysis showed a significant increase in intracellular Ca2+ in response to 1 nM NNK and this effect was blocked by a-BTX , thus confirming NNK as an agonist for the a7nAChR. In conjunction with earlier reports, these findings clearly established an important regulatory role of the a7nAChr in SCLC. Recent studies have revealed the expression of the a7nAChRs in a wide variety of cell types in the monkey lung [58, 59]. In vitro studies with immortalized human bronchial epithelial cells and human small airway epithelial cells have additionally shown that stimulation by nicotine or NNK of this receptor activated the serine/threonine kinase AKT, an effect resulting in the attenuation of apoptosis induced by etoposide, radiation or hydrogen peroxide, as well as the induction of a transformed phenotype . However, the concentrations of nicotine (10-100 |xm) or NNK (1 |xm) required to elicit these effects were significantly higher than those reported in studies with SCLC cell lines (1 |xm nicotine, 1 nM NNK). At these high concentrations nicotine as well as NNK may bind non-selectively to cellular targets other than the a7nAChR and the reported activation of AKT may have involved non-nAChR receptors. Another laboratory additionally reported the activation of NFkB and up regulation of cyclin D1 in human bronchial epithelial cells NHBE and human small airway epithelial cells exposed to NNK at concentrations ranging from 0.5-10 |M . Again, these concentrations are considerably higher than those required to stimulate mitogenic signaling in SCLC cells and may have involved non-nicotinic receptor types. Convincing support for an important role of a7nAChR stimulation in the growth regulation of SCLC and the putative cell of origin of this cancer, PNEC, came from a number of in vitro studies which showed that binding of nicotine (1 |M) or NNK (100 pM) to the a7nAChR resulted in phosphorylation of protein kinase c (PKC), Raf-1, ERK1/2 and c-myc [15, 62]. Another laboratory also reported activation of ERK1/2 in response to nicotine in human SCLC cell lines and showed that this effect was mediated by nicotinic receptor-mediated release of serotonin, which is an autocrine growth factor for these cells [16, 63]. Recent investigations additionally have reported a7nAChR-mediated NNK-induced functional cooperation between Bcl2 and c-myc that inhibited apoptosis while stimulating cell proliferation of human SCLC cell lines . In addition, NNK phosphorylated and m-calpains in human SCLC cells in an ERK1/2 and Ca2+-dependent manner, resulting in the induction of cell migration and invasion, and these effects were abrogated by the a7nAChR antagonist a-BTX . It is of note that the concentration of NNK required to elicit these effects reported by all three laboratories were extremely low (100 pM), thus underlining the very high-affinity of NNK for the a7nAChR which is over expressed in SCLC cells. These findings are in accord with the frequent expression of amplified c-myc in SCLC . In addition, it has been shown that the PKC/Raf-1/ERK1/2 signaling cascade is also stimulated by autocrine growth factors for SCLC, including the neuropeptide growth factors bradykinin, vasopressin, bombesin, neurotensin and galanin as well as serotonin and acetylcholine [63, 66]. In turn, it was shown that the release of some of these growth factors was triggered by the influx of Ca2+ caused by stimulation of the a7nAChR . Interestingly, recent studies in human SCLC cells have shown that the P-adrenergic agonist isoproterenol significantly reduced NNK-induced ERK1/2 activation . These findings suggest that P-adrenoreceptors may have inhibitory function on the growth of this cancer type and that p-adrenergic agonists may be suitable adjuvants to prevent the relapse of SCLC after conventional cancer therapy.
Collectively, these data suggest that the major signaling pathway that regulates SCLC growth, apoptosis and invasiveness includes Ca2+ influx, activation of PKC, Raf-1, ERK1/2, Bcl2, c-myc as well as calpains and that binding of agonists to the a7nAChR initiate the activation of these pathways while (3-adrenergic signaling may have inhibitory effects.
In addition to the summarized direct effects of nicotinic agonists on lung cancer cells, it has been recently discovered that nicotine stimulates angiogene-sis and enhances the neovascularization of lung tumors . In fact, studies in xenographs from Lewis lung cancer cells even showed that exposure to side stream smoke (the experimental equivalent of second hand smoke) increased tumor size and angiogenesis and that this effect was inhibited by the broad-spectrum antagonist for neuronal nAChRs, mecamylamine .
It has been shown that PNECs, the putative origin of SCLC, express a receptor protein that senses hypoxia and triggers the release of serotonin (5-hydroxytryptamine, 5-HT) and bombesin (MB) via influx of Ca2+ . In addition to modulating bronchial smooth muscle tone and respiration, 5-HT and MB act as autocrine growth factor for PNECs and SCLC. The diseased lung with impaired pulmonary ventilation thus typically demonstrates hyperplasia of PNECs . In addition, uranium mining and other sources of exposure to radon that cause interstitial pulmonary fibrosis are documented risk factors for the development of SCLC . We therefore hypothesized that stimulation of the oxygen sensing receptor in PNECs by impaired pulmonary ventilation would facilitate the development of a neuroendocrine type of lung cancer in animals exposed to the nicotinic agonists nicotine or NNK as well as diethyl-nitrosamine (DEN) which has structural similarities with acetylcholine (fig. 1). We induced mild pulmonary interstitial fibrosis in Syrian golden hamsters by maintaining the animals in an environment of 60% oxygen. These animals developed multiple foci of hyperplastic PNECs with positive immunoreactivity for 5-HT. Hamsters that were additionally given multiple subcutaneous injections of NNK  or DEN  developed multiple neuroendocrine lung tumors at a high incidence. Similar to most human SCLCs, these tumors expressed the neuroendocrine markers 5-HT, calcitonin, bombesin and neuron specific enolase, they lacked activating point mutations in K-ras while over-expressing c-myc . Due to their relatively small size and well-differentiated morphological appearance, these experimentally induced tumors were classified as atypical carcinoids even though they demonstrated functional and molecular features of SCLC. Hamsters with hyperoxia-induced pulmonary interstitial fibrosis and treated with multiple subcutaneous injections of nicotine developed a low but significant incidence of lung tumors with focal areas of positive immunoreactivity to the neuroendocrine markers 5-HT and neuron specific enolase . Collectively, these findings support the hypothesis that the diseased lung with impaired pulmonary oxygenation and resulting hyper-plasia of PNECs is more susceptible for the development of neuroendocrine lung cancers upon simultaneous exposure to the nAChR agonists nicotine,
NNK or DEN. In vitro experiments corroborated this interpretation by demonstrating that SCLC or PNEC cells maintained in an environment of high CO2 at the expense of O2 showed induction of ERK1/2 activation  and enhanced the proliferation response to nicotine or NNK , suggesting sensitization of the a7nAChR. Taken together, these findings suggest that the a7nAChR and the hypoxia receptor cooperate in the regulation of SCLC (fig. 3).
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