Our observation that the nAChR α3, α5 and β4 subunit genes are over-expressed in SCLC is particularly intriguing in light of the recent genome wide association studies implicating the CHNRA5/A3/B4 gene locus in lung cancer susceptibility (20
). Over-expression of the clustered nAChR genes in lung cancer cells supports the notion that these genes play a role independent of the nicotine addiction pathway. Extrapolating on data gained from work in the nervous system and our own observations, the possible nAChR subtypes that can form in SCLC include α3β2, α3β4, α3β4α5, and α3β2β4α5 (39
). These subtypes are believed to be involved in ganglionic neurotransmission in the peripheral nervous system (40
). A thorough investigation of functional nAChR subtypes in lung cancer has yet to be done but there is evidence that specific subtypes mediate distinct processes. For example, α3-containing nAChR subtypes have been implicated in nicotine-mediated activation of the Akt pathway (41
) whereas the α7 subtype is thought to mediate nicotine-induced angiogenesis and NNK-induced apoptotic inhibition (4
). α7 nAChRs also have high calcium permeability and binding of NNK results in calcium influx, which triggers signaling pathways that result in cell proliferation, increased cell migration, apoptotic inhibition, and angiogenesis (6
). These two examples indicate the need to identify all of the precise nAChR subtypes in lung cancer cells as this may be important for design of targeted therapeutics given the unique pharmacological and functional properties of each nAChR subtype.
As nAChRs are the cognate receptors for nicotine and NNK, their activation is likely the first step in signal transduction cascades involving these ligands. Persistent activation of cancer-promoting pathways has been shown to result from nicotine and NNK exposure and may facilitate SCLC development (42
). While these pathways remain to be completely elucidated, they appear to involve the mitogen activated kinases ERK1 and ERK2, protein kinase C (PKC), the serine/threonine kinase RAF1 and the transcription factors FOS, JUN and MYC (6
). In addition, exposure to nicotine has also been shown to reduce the efficacy of anti-cancer agents by inhibiting apoptosis (44
). Pharmacological approaches suggest that these effects are mediated at least in part by homomeric α7 nAChRs (6
) but the role of other nAChR subtypes cannot be ruled out due to the lack of specificity of currently available pharmacological agents.
That nAChRs may function in SCLC is not totally unexpected given their important role in the nervous system. SCLC is believed to develop from pulmonary neuroendocrine cells. As the name suggests, these cells share properties with neurons such as the expression of ion channels and neuropeptides and have been referred to as paraneurons (45
From a regulatory standpoint, the over-expression of the clustered nAChR genes also yields some interesting insights. Several laboratories have previously identified regulatory features shared by these genes (36
). Based on these studies, it is believed that expression of the clustered nAChR genes results from interactions between ubiquitously expressed and cell-type-specific transcription factors with cis-acting regulatory elements located within or near the cluster. To date, only one cell-type-specific factor, Sox10, has been identified and shown to regulate nAChR gene expression (54
). Sox10 activates the promoters of the clustered genes in neuronal cell lines but not in non-neuronal cells. However, we have observed that Sox10 is not expressed in any of the lung cancer cell lines we used in this study (data not shown). This suggests that other transcription factors must be involved in the expression of nAChR genes in lung cancer. As mentioned above, the transcription factor ASCL1 is an interesting candidate given its role in SCLC (31
). ASCL1 is also known to activate neuroendocrine differentiation markers while suppressing putative tumor suppressor genes (56
). In addition, ASCL1 is required for the proper development of peripheral sympathoadrenal tissues, the same tissues where the clustered nAChR genes are abundantly expressed (57
The knockdown experiments presented here indicate that ASCL1 robustly regulates the expression of the α3 and β4 genes while α5 gene expression was, at most, modestly affected. These regulatory differences are likely due to the fact that each gene has its own promoter. Hence, although the three genes share common regulatory elements, each gene may have additional mechanisms that allow fine-tuning of its specific expression. Moreover, the α5 gene is transcribed in the opposite direction as the α3 and β4 genes raising the possibility that transcription factors that bind to the α3 and β4 promoters may be differentially utilized by the α5 promoter and vice versa. Nevertheless, the effect of ASCL1 on nAChR subunit gene expression in SCLC appears to be specific for the clustered subunit genes, as expression of the α7 and β2 genes was not affected by ASCL1 knockdown. In contrast, ASCL1 knockdown does not reduce the expression of the clustered subunit genes in NSCLC whereas it increases the expression of the β2 gene, suggesting cell-type specificity of ASCL1 regulation.
Control of nAChR gene expression by ASCL1 may provide a mechanism for the role of nicotine in lung cancer. Nicotine has been shown to induce cellular processes that may lead to the development of cancer including activation of cell proliferation and survival pathways (6
). Acetylcholine, the endogenous ligand for nAChRs, is also thought to act as an autocrine growth factor in lung cancer cells (58
). Over-expression of their cognate receptors via transcriptional control by ASCL1 may thereby potentiate the effects of these ligands, providing a mechanism by which cigarette smoking can promote the growth and aggressiveness of SCLC.