Connections of nicotine to cancer · signals (reviewed in REF. 11). The overall cellular response to stimulation with ACh is determined by the delicate balance between these signals

Along with increased consumption of products containing tobacco or nicotine, we have come to realize that nicotine itself can be genotoxic and tumour-promoting. Carcinogenesis is a continuous process that is thought to be initiated by a fac-tor that induces a mutation, followed by other factors that stimulate the growth of the mutated cell. Tobacco smoke contains approximately 5,000 different molecules, and more than 60 of them are known car-cinogens1,2. In addition to nicotine, its tis-sue metabolite cotinine and two tobacco nitrosamines, Nʹ‑nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), can also have carcinogenic effects owing to binding to nicotinic acetyl-choline receptors (nAChRs) on non-neuronal cells. NNN is formed from a reaction between nicotine and related tobacco alkaloids by nitrosation in mammals3. Classic studies indi-cated that unlike NNN, NNK does not derive from tissue metabolism of nicotine4,5.

The alkaloid nicotine was first isolated from the tobacco plant Nicotiana tabacum (Solanaceae) in 1828 by German chemists

Posselt and Riemann. Nowadays, nicotine-containing products are used for both recreational and medical (BOX 1) needs. One of the key trends is creating novel smoke-less products using modern technologies of nicotine delivery, and some of these deliver higher doses of nicotine than cigarettes. Electronic cigarettes (eCigs) are rapidly gaining acceptance in helping people to quit smoking, reducing cigarette consumption, relieving tobacco withdrawal symptoms (for example, because of smoking restrictions) and allowing people to continue having a ‘smoking’ experience but with reduced health risks6. However, there is uncertainty about the place of eCigs in tobacco control, and more research is needed to clearly establish their overall benefits and harms7. Because eCigs do not burn tobacco, they do not emit smoke. Rather, the user inhales and exhales nicotine vapour. This substantially lowers environmental concentrations of tobacco carcinogens8. However, there is no standard definition of eCigs, and different manufacturers incorporate a range of ingredients. For instance, the total level

of nicotine in the inhaled vapour gener-ated by 20 series of 15 puffs can vary from 0.5 mg to 15.4 mg. There is also very limited published data regarding the potential toxic effects of non-nicotine chemicals incor-porated in eCigs, which can also include hazardous substances, as well as the known carcinogens NNN and NNK9. As will be discussed in this Opinion article, current research provides reasons to be concerned about the safety of nicotine, be it in tobacco products, eCigs or medicines. Of particu-lar concern are eCigs in which nicotine is heated with various undocumented ingredi-ents, thereby increasing the potential for the generation of carcinogens.

ACh is a ubiquitous chemical in life, and it is best known for its role in neurotrans-mission. Increasingly, a wider role for ACh in other aspects of cell functions is being recognized. It has become evident that ACh that is locally released by non-neuronal cells can regulate tissue homeostasis in autocrine and paracrine manners through many biological effects on different cell types (reviewed in REF. 10). Knowledge is rapidly growing about non-neuronal functions of ACh that is mediated by the muscarinic and the nicotinic classes of cholinergic recep-tors outside of the neural system. When nAChRs were discovered on epithelial cells lining the mucocutaneous and aerodiges-tive epithelia, as well as on many other types of non-neuronal cells (reviewed in REF. 10), it became apparent that on its way to the ‘pleasure’ centres of the central nervous system (CNS), nicotine hits and potentially damages multiple non-neuronal targets (BOX 2). Activation of the nAChRs in non-neuronal cells can affect cell prolif-eration, growth arrest and apoptosis. ACh can differentially activate specific subtypes of nAChRs that are uniquely expressed by different cell types or the same cell at different stages of its differentiation, and this diferential activation can lead to both growth-promoting and growth-inhibiting signals (reviewed in REF. 11). The overall cellular response to stimulation with ACh is determined by the delicate balance between these signals. A switch of the predominant nAChR subtype that is expressed on the cell membrane occurs during malignant

O P I N I O N

Connections of nicotine to cancerSergei A. Grando

Abstract | This Opinion article discusses emerging evidence of direct contributions of nicotine to cancer onset and growth. The list of cancers reportedly connected to nicotine is expanding and presently includes small-cell and non-small-cell lung carcinomas, as well as head and neck, gastric, pancreatic, gallbladder, liver, colon, breast, cervical, urinary bladder and kidney cancers. The mutagenic and tumour-promoting activities of nicotine may result from its ability to damage the genome, disrupt cellular metabolic processes, and facilitate growth and spreading of transformed cells. The nicotinic acetylcholine receptors (nAChRs), which are activated by nicotine, can activate several signalling pathways that can have tumorigenic effects, and these receptors might be able to be targeted for cancer therapy or prevention. There is also growing evidence that the unique genetic makeup of an individual, such as polymorphisms in genes encoding nAChR subunits, might influence the susceptibility of that individual to the pathobiological effects of nicotine. The emerging knowledge about the carcinogenic mechanisms of nicotine action should be considered during the evaluation of regulations on nicotine product manufacturing, distribution and marketing.

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© 2014 Macmillan Publishers Limited. All rights reserved

transformation (reviewed in REFS 12,13), which indicates that the effects of autocrine or paracrine ACh on cancer cells might dif-fer from its effects on non-malignant cells, even if they are situated next to each other in the same tissue. This is also true for the pharmacological action of nicotine, which has a higher nAChR-binding affinity than ACh and can therefore displace ACh from nAChRs. By interfering with ACh signal-ling in non-neuronal tissues, nicotine pro-duces a plethora of pathobiological effects, some of which might contribute to health problems such as cancer. For instance, it has been shown that nicotine is selectively concentrated in malignant gallbladder tis-sue compared with normal gallbladder tissue, which suggests an association with gallbladder cancer14. The list of cancers that may be initiated and/or promoted by nicotine is expanding and presently includes small-cell and non-small-cell lung carci-nomas, as well as head and neck, gastric, pancreatic, gallbladder, liver, colon, breast, cervical, urinary bladder and kidney can-cers (see REFS 14–19). Furthermore, recent data provide cumulative evidence that single nucleotide polymorphisms (SNPs) in genes encoding nAChR subunits influ-ence the susceptibility of individuals to lung (reviewed in REF. 18), oesophageal20, gastric21 and cervical16 cancers (BOX 3).

This Opinion article discusses numerous mechanisms by which nicotine might con-tribute to the onset and expansion of cancer. The data accumulated in the literature indi-cate that nicotine has the ability to damage the genome, disrupt cellular metabolic pro-cesses, amplify oncogenes, inactivate tumour suppressor genes and promote a cancer-supporting microenvironment. It should be clarified that although one meta-analysis22 did not find an increased risk of oropharyn-geal cancer in users of Scandinavian snus — a smokeless tobacco product that is not heated or inhaled — there are more studies showing an increased risk than those show-ing no risk (reviewed in REF. 23). There is also risk for cancer of the oesophagus, stomach and pancreas in snus users23.

Mutagenic effects of nicotineThe carcinogenic tissue metabolites of nicotine. Nicotine is metabolized in the liver, primarily by the cytochrome P450 enzymes CYP2A6 and CYP2B6, and 70–80% of the nicotine that is absorbed from the gastro-intestinal tract is converted into cotinine. Cotinine shows tumour-promoting effects, as evidenced by abnormal cell proliferation, reac-tivation of telomerase, suppression of apop-tosis, facilitated tumour growth in the Lewis lung cancer mouse model and accelerated development of lung adenomas induced by

NNK in the A/J mouse strain24,25. Other pri-mary metabolites include nicotine Nʹ-oxide, nornicotine, nicotine isomethonium ion, 2-hydroxy nicotine and nicotine glucuro-nide that apparently are not carcino genic per se26. NNN is a strong carcinogen that can induce tumours both locally and systemically (reviewed in REF. 27). It increases the prolif-erative potential of cells, is anti-apoptotic and promotes anchorage-independent growth and the production of tumours in nude mice, which can be abolished by nAChR antago-nists28,29. This effect of nAChR antagonists is consistent with the ability of NNN to bind to nAChRs (reviewed in REF. 30).

Nicotine-induced mutagenesis. For a very long time, it had been in doubt whether or not nicotine can induce cancer per se (reviewed in REF. 31). However, results of the recent studies demonstrating the genotoxic effect of nicotine on cultured epithelial cells32–34 and its tumorigenic action in A/J mice35 indicate that it may. The genotoxic effects of physiological doses of nicotine were documented by the chromo-some aberr ation test and the sister chro-matid exchange assay with isolated human epithelial cells and lymphocytes32–34. In these studies, the genotoxic effects were triggered by the activation of cell surface nAChRs, which led to increased levels of reactive oxygen species (ROS). In addition to activat-ing cell surface nAChRs, nicotine can also freely permeate epithelial cells and elicit its pathobiological effects owing to DNA dam-age by ROS that are activated through intra-cellular pathways34,36–42. However, in contrast to tobacco nitrosamines that cause KRAS and TP53 mutation43, the targets of nicotine genotoxicity remain unidentified.

Despite a large number of in vitro studies, the tumorigenic potential of long-term sys-temic administration of nicotine has not been fully evaluated. In an initial proof-of-concept study, we injected A/J mice subcutaneously with the dose of nicotine lethal to 50% of animals tested (LD50), 3 mg per kg, five times per week for 24 months, yielding an average dose of 2.1 mg per kg per day35. This dose is approximately equal to the dose of nicotine consumed by a regular Scandinavian snus user, who usually receives from 60 mg to 150 mg of nicotine per day23. Of note, an eCig cartridge filled with the ‘XXX-High Density’ nicotine solution contains 48 mg nicotine 192 (on average, one eCig cartridge is consumed per day by a user). The nicotine LD50 dose in humans is 6.5–13 mg per kg44. We observed that 78.6% of experimental, but not control, A/J mice developed leiomyosarcoma or

Box 1 | Medical use of nicotine

Nicotine-containing products are predominantly used for smoking cessation to replace nicotine and alleviate dependence and withdrawal symptoms. Nicotine products include vaporized nicotine, nicotine gum, delayed release oral forms, nasal spray, liquid rectal enemas, transdermal patches and topical cream. Nicotine is absorbed through the skin and mucous membranes in a dose-dependent manner and has a half-life of about 2 hours164. In recent years, however, the medical use of nicotine has been expanded; new uses exploit its anti-inflammatory and wound-healing-promoting effects (reviewed in REF. 165). The therapeutic effects of nicotine-containing products are best characterized in ulcerative colitis and recurrent aphthous stomatitis, both of which are epidemiologically related to smoking. It is well documented that the incidence of these diseases is lower in tobacco users and that the negative association with smoking is dose- and time-dependent166–169. Furthermore, cessation of smoking is associated with disease recurrence, which can, however, be relieved by returning to smoking. Nicotine seems to be the key mediator of these responses, because its administration inhibits the inflammation that is associated with these diseases170,171. A similar situation is observed in patients with Behçet’s disease172,173 — an immune-mediated small-vessel vasculitis that manifests as mucocutaneous ulcerations — and uveitis. The anti-inflammatory effects of nicotine-containing products have been well documented in various clinical and experimental settings. For example, nicotine facilitates healing of both cutaneous174–176 and oral171,173,177 ulcers in humans, as well as skin blisters in rats178 and excisional skin wounds in mice100. Interestingly, systemic use of nicotine facilitates healing of foot ulcers in Buerger’s disease174, which is common in smokers. It was therefore concluded that Buerger’s disease may be caused by something other than nicotine present in tobacco smoke and that nicotine on its own has the opposite (healing) effect. There are also anecdotal reports of the successful use of systemic or topical nicotine to treat some other mucocutaneous ulcerative diseases (for example, pyoderma gangrenosum) and inflammatory diseases of blood vessels (for example, malignant atrophic papulosis (also known as Degos disease) and erythema nodosum), subcutaneous lymph nodes (that is, Kimura’s disease) and hair follicles (eosinophilic pustular folliculitis), as well as an immune-mediated oral inflammatory disease such as lichen planus that is resistant to other treatments (reviewed in REF. 165).

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rhabdomyosarcoma. Rhabdomyosarcoma can spontaneously develop in A/J mice, but leiomyosarcoma does not45,46, which indi-cates that the leiomyosarcoma development was specific to experimental treatment. It could result from the nAChR-mediated and/or non-receptor actions of nicotine, as well as from its endogenously produced metabolites. As these mice were not treated with tobacco but were treated with pure nicotine, it is presumed that NNK could not have been present4,5. Although NNN could derive from tissue metabolism of nicotine, its contribution to leiomyosarcoma develop-ment seems unlikely because although it has been documented that A/J mice exposed to NNN have more than 80% lung tumour incidence47, no lung malignancy was seen in A/J mice treated with nicotine35. Thus, it can be concluded that neither NNN nor NNK caused sarcomas in A/J mice treated with nicotine and that the tumorigenic action was implemented by nicotine per se. Perhaps the A/J mice developed tumours in soft tissue instead of the lung because the subcutane-ous route of administration and the rapid metabolism of nicotine (~2 hours) prevented it from reaching the respiratory epithelium. Although the subcutaneous route is not used by recreational nicotine-containing products, the above results have salient implications, because they provide the first evidence for a carcinogenic potential of nicotine in vivo. Through its genotoxic action, nicotine could trigger malignant transformation and then facilitate growth of transformed cells via its tumour-promoting activities.

These initial studies showing nicotine-dependent chromosome aberration in vitro and leiomyosarcoma development in A/J mice justify further investigations of the direct mutagenic and tumorigenic actions of nicotine.

nAChR-mediated nicotine actionsnAChRs. The nAChRs are classical repre-sentatives of a large superfamily of ligand-gated ion channel proteins. Channel opening is regulated by the binding of ACh or nico-tinic agonists, such as nicotine. Nicotine-containing products can amplify signalling through nAChRs, because nicotine binds to these receptors with a higher affinity than ACh. Furthermore, the upregulation of nAChR expression by nicotine48 can also amplify the physiological stimulation of cells with autocrine or paracrine ACh. As cancer cells both produce ACh and express nAChRs49, these receptors may be involved in tumorigenesis, even without stimulation with nicotine. The ACh-gated channels are

pentamers that comprise different combina-tions of α1–α10, β1–β4, γ, δ and ε subunits (FIG. 1a), wherein each subunit has four puta-tive transmembrane-spanning domains and a similar topological structure (FIG. 1b). The nAChRs are composed of ACh-binding α-subunits and ‘structural’ subunits. Each of the α7, α8 (not found in humans) and α9 subunits can form homomeric nAChR chan-nels (hereafter referred to as α7 nAChRs or α9 nAChRs). As depicted in FIG. 1a, the het-eromeric pentameric protein channels can be composed of various combinations of α1, α2, α3, α4, α5, α6, β1, β2, β3 and β4 subunits, for example, α3(β2/β4)±α5 and α9 can form a heteromeric channel with α10. The ‘muscle’-type nAChR is composed of two α1 subunits, β1, γ and δ in developing muscle, and the γ-subunit is replaced by the ε-subunit in mature muscle50. Other nAChR types are also called ‘neuronal’ nAChRs because they were originally thought to be expressed exclu-sively by neurons. The differences in subunit composition determine the functional and pharmacological characteristics of the ion channels that are formed. For instance, the addition of α5 — an auxiliary subunit that forms functional ion channels only when it is co-expressed with both other α-subunits and β-subunits — to a doublet of α3 and either β2 or β4 modifies the pharmacological and bio-physical properties of nAChR and increases Ca2+ permeability51. Non-neuronal cells can express almost all known nAChR subunits and form functional nAChRs. Cotinine, like nicotine, can bind to and activate cellular nAChRs, such as those containing α4 and β2, α3 and β2, or α6 and β2 (REFS 52,53).

The receptor subtypes and downstream signalling pathways vary between normal and malignant cells and depend on the cell type, current cell cycle state and environ-mental exposures (for example, to tobacco smoke)54,55. For instance, different types of non-small-cell lung carcinoma cell lines feature unique nAChR repertoires, with some adenocarcinoma and squamous cell carcinoma lines expressing the muscle-type nAChR composed of α1, β1, δ and ε subunits and other cell lines expressing the neuronal-type α3-, α7- and α9-containing nAChRs56,57. Chronic exposure of cultured human cuta-neous and oral keratinocytes to either pure nicotine or tobacco extract alters the expres-sion of nAChR subunits54,58–60, which often leads to the acquisition of receptors that are characteristic of cancer cells55,56,61. The number of nAChRs also has an important role, since it has been shown that cancer progression is associated with overexpres-sion of nAChRs. In particular, nicotine upregulates expression of the α7 subunit, which leads to more α7 nAChRs; these are known to mediate many pathobiological effects of nicotine and tobacco nitrosamines (reviewed in REF. 62). Some nAChR subtypes are selectively overexpressed in various can-cers, regardless of the smoking status of the person from which the sample was derived (for example, α7 nAChRs in lung cancer and α9 nAChRs in breast cancer), and inhibi-tion of the expression of nAChR subunits (for example, α9) attenuates nicotine- or tobacco nitrosamine-induced cell prolif-eration in vitro and/or in vivo (reviewed in REFS 13,15).

Box 2 | The non-neuronal cholinergic system

Acetylcholine (ACh) has emerged as a candidate regulatory molecule (‘cytotransmitter’) in numerous biological processes that are intimately connected to each other, including proliferation, differentiation, apoptosis, adhesion and migration of non-neuronal cells (reviewed in REF. 90). ACh is present in bacteria, blue-green algae, yeast, fungi, protozoa and primitive plants179, which indicates that ACh has been functioning as a signalling molecule in non-neuronal cells for about 3 billion years, whereas its neuronal function spans only a relatively short period of about 0.5 billion years. Indeed, free ACh is found in almost all types of mammalian non-neuronal tissues at the levels equivalent to or even exceeding those in the neural system180. The concentration of free ACh is a result of its synthesis by choline acetyltransferase and hydrolysis by acetylcholinesterase, both of which are present in non-neuronal cells. The extracellular pool of ACh is replenished by the secretion of ACh-containing vesicles that fuse with plasma membrane and thus release free ACh, whereas the intracellular pool primarily contains free cytoplasmic ACh. The environmental, neural, endocrine and paracrine stimuli affect ACh metabolism and signalling in non-neuronal cells, which helps to adjust tissue homeostasis to a new environmental condition and sends signals back to the central nervous system and endocrine organs. It is therefore well-accepted that the non-neuronal ACh axis is an example of the more general neuroendocrine-like mechanisms that mediate peripheral responses to environmental factors and of evolutionary conservation of neuroendocrine systems in the periphery. Appreciation of these facts indicates that ACh does not operate exclusively in the neural system, as implied by the term ‘neurotransmitter’, and sets a stage for exciting discoveries of new functions of non-neuronal ACh in the physiological control of homeostasis and in human diseases.




Ionic signalling events triggered by opening of the nAChR channels. The ligand-gated nAChR channels mediate the influx of Na+ and Ca2+, and the efflux of K+. In neurons, activation of nAChRs leads to cell membrane depolarization that allows influx of Ca2+ through voltage-sensitive calcium channels. Although voltage-gated Ca2+ channels also occur in non-neuronal cells such as keratino-cytes63, Ca2+ entry following nicotinergic stimulation occurs through nAChR chan-nels, and the magnitude of Ca2+ influx var-ies among different receptor subtypes. The homomeric channels composed of α7 or α9 subunits, as well as the heteromeric nAChRs containing α9 and α10, have the greatest Ca2+ permeability64,65. The Ca2+ ions that enter cells through nAChRs raise the con-centration of intracellular free Ca2+ (REF. 66). However, experiments with several types of non-neuronal cells showed that nicotinergic effects can also be elicited in the absence of Na+ or Ca2+ entry67,68. Therefore, the down-stream signalling from nAChR expressed in non-neuronal cells apparently proceeds via both ionic and non-ionic pathways, and each type of signalling may be required to elicit specific biological responses to stimulation of the cellular nicotinergic signalling network.

Non-ionic signalling events triggered by the nAChR-coupled protein kinases. Activation of nAChRs in non-neuronal cells elicits the

non-ionic signalling events that regulate protein phosphorylation and dephosphory-lation, which is a novel function of nAChR subunit proteins in non-excitable cells. Experimental results revealed substantial variations of the enzymatic cascades that can be triggered owing to binding of nicotine to nAChRs expressed in non-neuronal cells. The mode of interaction of nAChRs with the signal transduction molecules varies depend-ing on both the type of non-neuronal cell targeted by nicotine and the type of nAChR subunit involved. The nAChR-coupled sig-nalling kinases may be activated (through phosphorylation or dephosphorylation) owing to conformational changes of nAChR subunits and/or associated proteins (FIG. 1c). The downstream signalling from nAChRs can activate phospholipase C (PLC), pro-tein kinase C (PKC) isoforms, PI3K, AKT, JUN N-terminal kinase (JNK), SRC, Janus kinase 2 (JAK2), RAC, RHO, p38 MAPK and the RAS–RAF–MEK–ERK pathway57–60,69–77. Ca2+/calmodulin-dependent protein-kinase II (CaMKII) may be activated owing to both ionic (that is, Ca2+ influx) and non-ionic (that is, liberation of intracellular Ca2+) signalling events. For example, activation of the α3-containing nAChR in keratinocytes can result in activation of PKC; activation of nAChR containing both α3 and α5 subunits leads to CaMKII and p38 MAPK activa-tion; activation of α7 nAChR can activate

p38 MAPK, AKT, RAS–RAF–MEK–ERK and JAK2; and activation of α9-containing nAChR leads to the activation of PLC, SRC, epidermal growth factor receptor (EGFR), PKC, RAC and RHO. Indeed, these effectors participate in signal transduction pathways downstream of other types of cell surface receptors, such as EGFR, and can therefore mediate intracellular signal crosstalk.

The nAChR proteins can physically asso-ciate with both protein kinases and protein tyrosine phosphatases in large multimeric complexes78. For example, JAK2 binds to α7 upon stimulation of nAChR with nicotine, which leads to phosphorylation and activa-tion of JAK2 and subsequent activation of PI3K15,79,80. The α7 subunit can also physically and functionally associate with SRC-family kinases81 (SFKs), whereas the subunits α3, α4, α5 and β2 show a positive interaction with the G protein subunits Goα and Gβγ

82. Associated proteins can modulate the ampli-tude of nicotinergic signalling (FIG. 1c). SFKs and putative SRC-associated phosphotyrosine phosphatases (PPTPs) regulate the activity of AChRs, with SRC eliciting signalling events that emanate from nAChR and with PPTPs counteracting this effect81. Of particular interest is an observation that nicotinergic upregulation of mitogenesis through α7 nAChR involves β-arrestin-mediated activa-tion of the SRC and RB–RAF1 pathways83. However, pharmacological activation of α7 nAChRs stimulates a tyrosine phosphatase that can inactivate SRC84, thereby leading to negative-feedback regulation. There are many other targets of nAChR-coupled signal-ling cascades, including transcription factors controlling gene expression (see FIG. 1d for an example), metabolic pathway components and structural components of the cyto-skeleton (reviewed in REF. 85), but the exact receptor-mediated mechanisms of signal induction in each particular situation remain to be elucidated.

A study of nicotinergic regulation of α2 integrin expression in keratinocytes77 has established a novel paradigm of α7 nAChR-mediated coordination of ionic and non-ionic signalling events. In this case, nicotine was able to simultaneously alter gene expression and induce changes in the cytoskeleton that were required for execu-tion of a particular step in cell migration. The RAF–MEK–ERK cascade upregulating the α2 integrin was activated owing to both Ca2+-dependent recruitment of CaMKII and PKC, as well as Ca2+-independent activa-tion of RAS. In turn, the PI3K-mediated activation of RHO-associated protein kinase (ROCK) was elicited owing to both Ca2+

Box 3 | Genetic predisposition to aberrant signalling through nAChRs

It has been shown that although smoking is associated with lung carcinoma in the general population, single nucleotide polymorphisms (SNPs) in nicotinic acetylcholine receptors (nAChRs) give an additional increased risk of lung cancer after adjustment for smoking status181. Classic genome-wide association (GWA) studies had shown that SNPs in the CHRNA5–CHRNA3–CHRNB4 gene cluster on the chromosome 15q25 encoding α5, α3 and β4 nAChR subunits are associated with both nicotine dependence and lung cancer182. The major limitation of the GWA studies was their focus on the SNP that is associated with nicotine addiction, which initially dampened enthusiasm for pursuing this relationship, because it was assumed that higher nicotine addiction would correlate with higher cancer rates. Later on, however, it was shown that the association of nAChR SNPs with lung cancer can be independent of smoking status183. Furthermore, CHRNA3 SNPs were found to be associated not only with an increased risk for lung cancer but also with a poor prognosis and a larger tumour size, especially in smokers184,185. However, negative sex- and race- or ethnicity-dependent association with risk for lung cancer was found for certain variants in the 15q25 region, which contains CHRNA5, and in the 8p11 region, which contains the CHRNB3 and CHRNA6 genes, encoding β3 and α6 nAChR subunits, respectively186,187. Our case–control study revealed both positive and negative associations of CHRNA9 SNPs with the risk for lung cancer188. Although the N442 α9 variant both decreased transformation of bronchial cells and was negatively associated with risk for lung cancer, the S442 α9 variant both facilitated cell transformation and was associated with an increased risk for lung cancer188. The structural differences between the N442 and S442 α9 subunit variants might alter the ligand-binding abilities of the assembled of nAChR channels and influence cell response. Thus, it became apparent that some nAChR subtypes (for example, the N442 α9 variant and possibly α5-containing nAChRs189) might function as tumour suppressors, whereas the others (for example, the S442 α9 variant and possibly α7 nAChR62) might function as tumour promoters. The molecular mechanism affecting the susceptibility of individuals to cancer due to specific genetic variations of nAChR remains to be elucidated.




Nucleus

Nature Reviews | Cancer

β1α1 δACh

ACh

ACh

Muscle-type: (α1)2, β1, δ and γ or ε

β2, β3or β4

α2, α3, α4, α5 or α6; or β2, β3 or β4α2, α3,

α4 or α6 α7 or α9

Neuronal-type: (α2, α3, α4 or α6)2–3, (β2 and/or β3 and/or β4)2–3 ± α5 (α9)2–3 (α10)2–3

Subunits: (α7 or α9)5

α1γ or εβ2, β3or β4

α2, α3α4 or α6

a Human heteromeric nAChRs Human homomeric nAChRs b

M1 M2 M3 M4

P

P

P

P

c d

COOHNH2

nAChR

Nicotine

Na+ and Ca2+

Upstream: regulation of gene expression Downstream: regulation of nAChRactivity or internalization by controllingits accessibility for phosphorylation

Upstream: regulation of nAChR activity by phosphorylation or dephosphorylationDownstream: activationof signalling cascades

Anchoring (stabilizing)proteins (e.g. rapsyn)

Adaptor (scaffolding)proteins (e.g. β-arrestin)SFKs and PPTPs

Upstream: interaction of nAChR with cytoskeleton unitsDownstream: nAChR clustering, trafficking and degradation

RASRAF

MEK

ERK

JAK2

STAT3 STAT3

UpregulationGene expression

α7 nAChR

K+

entry-dependent involvement of CaMKII and Ca2+-independent activation of JAK2. Thus, different signals emanating from activated α7 nAChRs can activate common effector molecules and end points. The biological importance of the cooperative signalling from α7 nAChRs that simultaneously upregulates α2 integrin and activates ROCK stems from the fact that both of these effector systems are crucial for the initiation of cell migration, which may have a role in cancer cell invasion of surrounding tissues and metastasis.

Regulation of gene expression through nAChRs. Both ionic events and non-ionic signalling cascades triggered by nAChRs can lead to changes in gene expression86,87. Classic studies with the neuron-like SH-SY5Y neuroblastoma cell line showed that the activation of nAChRs modulates the expression of a diverse set of genes that may be broadly categorized into four groups: transcription factors, protein pro-cessing factors, RNA-binding proteins and plasma membrane-associated proteins88.

Subsequent studies with non-neuronal cells gave similar results, indicating that regula-tion of gene expression is a general biological function of nAChRs. For instance, 118 genes were upregulated and 97 were downregu-lated by nicotine in the human macrophage-like cell line U937 (REF. 89). In keratinocytes, the activation of nAChRs altered the expres-sion of the genes encoding proteins involved in signal transduction, cell cycle regulation, apoptosis, and cell–cell and cell–substrate adhesion (reviewed in REFS 11,90). The genes

Figure 1 | Structure and function of nAChRs. a | Subunit composition of heteromeric and homomeric nicotinic acetylcholine receptor (nAChR) subtypes. The ligand binding sites are located at the interface between two subunits. The ‘structural’ subunit, which is not involved in ligand bind-ing, is highlighted in beige. b | Membrane topology of the ligand-binding α nAChR subunit. The four hydrophobic transmembrane domains are des-ignated M1–M4. The ligand-binding site is located on the amino terminus. The phosphorylation sites for SRC family kinases (SFKs) reported by Charpantier et al.190 are designated as “P”. c | Possible nAChR-associated proteins and their biological effects. Current research indicates coupling of nAChRs to anchoring (stabilizing) and adaptor (scaffolding) proteins, as well as to protein kinases or phosphatases. The downstream signalling that regulates the phosphorylation status of signalling and structural pro-teins and can alter gene expression is triggered by conformational changes of nAChR subunit proteins upon binding of a ligand, such as ACh, nicotine or its congener. The upstream signalling that can alter stoichiom-etry, cell-membrane number, or topology, and function of nAChRs is medi-ated by the aforementioned nAChR-associated proteins in response to

various inside-out signals. These binary systems can mediate crosstalk of nAChR with other types of cell surface receptors. d | Cooperation of the RAS–RAF–MEK–ERK and Janus kinase 2 (JAK2)–signal transducer and acti-vator of transcription 3 (STAT3) pathways downstream of α7 nAChR. Stimulation of α7 nAChRs by either ACh or nicotine leads to alterations in gene expression owing to transactivation of STAT3, which occurs through two complementary downstream signalling pathways. Activation of RAS–RAF–MEK–ERK increases levels of STAT3 protein by upregulating STAT3 gene transcription, and activation of JAK2 leads to phosphorylation of STAT3, which allows subsequent translocation of STAT3 dimers to the nucleus to alter expression of other genes. PPTPs, phosphotyrosine phos-phatases. Part d is adapted with permission from The Federation of American Societies for Experimental Biology, from Receptor-mediated tobacco toxicity: cooperation of the Ras/Raf 1/MEK1/ERK and JAK 2/STAT 3 pathways downstream of α7 nicotinic receptor in oral keratinocytes. Arredondo, J., Chernyavsky, A. I., Jolkovsky, D. L., Pinkerton, K. E. & Grando, S. A. 20, 2006; permission conveyed through Copyright Clearance Center, Inc.59.




that were upregulated by nicotine through α3-containing nAChRs in keratinocytes included signal transducer and activator of transcription 1 (STAT1)58, whereas those that were upregulated through α7 nAChR included STAT3 and GATA2 (REF. 60). Nicotine also upregulated the expression levels of the cell cycle and cell differen-tiation markers Ki67, proliferating cell nuclear antigen (PCNA), p21, cyclin D1, p53, filaggrin and loricrin, which can alter the dynamic equilibrium between cell growth and maturation towards an early and excessive squamatization91.

Recent analysis of the effects of nicotine on the transcriptome of non-malignant breast epithelial cells revealed altered expression of a large number of genes involved in cellular and metabolic processes, including many genes linked to cancer92. In human malignant MDA-MB-231 breast cells, nicotine increased the expression of BCL-2, which is anti- apoptotic, and increased long-term cell sur-vival via the SRC–AKT signalling pathway, revealing a regulatory network governed by the interaction of nicotine and nAChR that may integrate mitogenic signals for breast cancer development75. In a rat model of blad-der cancer, nicotine upregulated the expres-sion of mutant p53 that was deprived of its tumour-suppressor ability93. In the human colorectal cancer cell lines Caco-2 and HCT-8, nicotine increased AKT phosphorylation and the expression of PI3K, PKC, ERK1, ERK2, survivin, and BCL-2, which was mediated by activation of α7 nAChRs and associated with an increase in cell proliferation and a decrease in apoptosis94. Intraperitoneal administration of nicotine to A/J mice with NNK-initiated lung cancer decreased survival probability and reduced the overall expression of sirtuin 1 — a tumour suppressor — in the lung95.

Taking into consideration the tumour-promoting potential of autocrine or paracrine ACh (reviewed in REF. 96), the nicotine-induced genomic events that lead to the upregulation of nicotinergic signalling path-way components are of particular interest. In human bronchioalveolar carcinoma cells, nicotine upregulated choline acetyltrans-ferase and vesicular ACh transporter, thereby increasing production and secretion of ACh97. These genomic effects of nicotine were medi-ated by α7-, α3- and β2-, and β3-containing nAChRs. In human squamous cell lung and nasopharyngeal carcinoma cells, nicotine upregulated the expression of α7 (REFS 74,98). These recent observations are in keeping with earlier reports of the nicotine-dependent modulation of nAChR expression in epithelial cells, wherein the α3-containing nAChRs are

replaced by α7 nAChRs66,91. This is not sur-prising, because although most cellular recep-tors are downregulated by agonists, chronic stimulation of nAChRs results in a paradoxi-cal upregulation of α7, and also α5-containing nAChR channels that also contain α3 and β2 (REFS 58,99). In keratinocytes, the transition from nAChR containing α3 and either β2 or β4 to nAChR also containing α5 predomi-nantly involves PKC; a further transition from nAChR containing α3, β2 or β4 and α5 to the α7 nAChR involves CaMKII and p38 MAPK; and the self-upregulation of α7 nAChRs involves the p38 MAPK–AKT pathway and JAK2 (REF. 60). The transcription factor GATA2 has a key role in mediating positive-feedback-mediated upregulation of the α7 subunit. This change in nAChR subtypes pro-gressively increases Ca2+ influx, which is asso-ciated with changes in the expression of genes encoding proteins that regulate cell state and function58,91. Therefore, nicotine-dependent changes in the nAChR repertoire may be a novel pathophysiological mechanism of nicotine toxicity in non-neuronal cells.

Synergy of nicotine signalling with that of growth factors and hormones. It is well estab-lished that nicotine can accelerate wound healing by synergizing with and mimicking the effects of various growth factors100–102. Nicotine can upregulate the expression of fibroblast growth factor 1 (FGF1), FGF1 receptor, FGF2 and vascular endothelial growth factor (VEGF)74,103–108, which may help to explain some of its tumorigenic actions. Stimulation of nAChR activates the bovine FGF2 gene via signalling to induce tyrosine phosphorylation of several proteins, includ-ing promoter-binding factors108. Accordingly, nAChR inhibition reduces FGF2 upregu-lation109. In turn, FGF2 and insulin-like growth factor I (IGF-I) can alter the nAChR expression level and clustering110,111, which can modify biological effects of nicotine. However, nicotine has been shown to inhibit the production and release of transforming growth factor-β (TGFβ) — in some but not all cell types112,113 — which can help tumour cells to escape from the anti-proliferative effect of this growth factor114. In human nasopharyn-geal carcinoma cells, nicotine upregulated the expression of VEGF but suppressed that of pigment epithelium-derived factor (PEDF) — a multifunctional secreted protein that has anti-angiogenic, anti-proliferative, and neuro-trophic functions — and this led to a substan-tial increase of the ratio of VEGF/PEDF74. Several studies also showed that the down-stream signalling from nAChRs activated by nicotine involves phosphorylation and

activation of EGFR75,115, and this pathway might be involved in breast cancer develop-ment75. However, growth factor signalling pathways can alter the composition and expression of nAChRs in a cell. For example, signalling by FGF2, insulin and IGF-I, as well as oestrogen–oestrogen receptors, alters the nAChR expression level and clustering, thereby modifying the biological effects of autocrine or paracrine ACh, as well as nico-tine110,111,116. Nicotine also upregulates the production of adrenaline, which can stimulate growth of tumour cells through its G protein-coupled receptors117.Taken together, these observations provide strong evidence of the synergistic action between growth fac-tor receptors and nAChRs, which might be required to elicit the biological and tumori-genic effects of nicotine and its metabolites following their activation of nAChRs.

Tumour promotion and spreadingCancer cell survival and protection from apoptosis. It has been shown that nicotine increases the survival of cancer cells, which-may depend on the p53 status of cancer cells94,118,119. Nicotine can also increase telo-merase activity via the PI3K–AKT pathway downstream of nAChRs, thereby inhibiting cell senescence and death120. It has been doc-umented that nicotine abolishes cancer cell apoptosis that is induced by chemo therapy and radiotherapy, which can decrease sensitivity and facilitate resistance to these therapies121–125.

Recent research indicates that the nicotin-ergic regulation of cell survival and death is more complex than originally thought and involves anti-apoptotic activities of nicotine that permeates cells. Functional nAChRs of both α7 and non-α7 subtypes are expressed on the mitochondrial outer membrane in non-neuronal cells, and activation of mito-chondrial α7 nAChR prevents cytochrome c release, thereby blocking the initial step of intrinsic apoptosis126,127. The recent demon-stration that the intracellular nAChRs inhibit the opening of mitochondrial permeability transition pores128 is in keeping with earlier reports about anti-apoptotic effects of nico-tine in mitochondria129,130. In the cytosol, nicotine is insensitive to the regulatory action of intracellular acetylcholinesterase, which functions to hydrolyse ACh in the cytosol131, and nicotine can thereby shift the equilib-rium to favour the anti-apoptotic activities of mitochondrial nAChRs. Elucidation of the molecular events following activation of mito-chondrial nAChRs should shed light on the mechanisms facilitating the survival of transformed cells that are exposed to nicotine.




Proliferation. It has been documented that nAChRs, particularly those of the α7 subtype, can mediate nicotine-dependent upregulation of proliferative and survival genes that con-tribute to the growth and progression of lung cancer cells in vitro and in vivo74,83,132,133. Even a short-term exposure to nicotine activates mitogenic signalling pathways involving PKC, ERK and AKT118. In the chick chorioallantoic membrane tumour implant model, nicotine doubled the growth rate of breast, colon and lung cancers104. It also increased both the numbers and the size of tobacco nitrosamine-initiated lung tumours in A/J mice95,118. Interestingly, exposure to NNK upregulates α7 nAChR in the lung of cancer-susceptible A/J mice, but not the C3H mouse strain, which is resistant to NNK-induced tumours134. An increase of nAChR numbers may allow malignant cells to bind to higher than normal amounts of autocrine or paracrine ACh (or nicotine) to facilitate their rapid growth.

Metastasis. Pharmacological stimulation of nAChRs correlates with metastatic dissemi-nation of primary tumour cells (reviewed in REF. 13). In particular, nicotine promoted the metastasis to the liver of human pancreatic adenocarcinoma cells that were orthotopically implanted into severe-combined immuno-deficient (SCID) mice135, as well as two head and neck squamous cell carcinoma cell lines subcutaneously injected into nude mice136. In A/J mice, nicotine increased the growth and metastasis of NNK-induced lung tumours61, which indicates that it can contribute to can-cer progression by facilitating the outgrowth of cells with genetic damage. The pro-invasive action of nicotine may require stabilization and activation of hypoxia-inducible fac-tor 1α (HIF1α) — an oxygen-sensitive trans-criptional activator — as well as synergistic cooperation of AKT and MAPK pathways137. Notably, in vivo studies showing substantially more metastasis to various distant organs in mice with orthotopically implanted pan-creatic cancer cells and exposed to cigarette smoke (versus sham controls) demonstrated that metastatic cells abundantly express α7 nAChR138. Indeed, nicotine-dependent migration of pancreatic cancer cells in vitro can be regulated through α7 nAChRs138.

The pro-metastatic activity of nicotine on cancer cells may stem from its ability to elicit the epithelial–mesenchymal transition (EMT) — a mechanism by which cells lose their epithelial characteristics and acquire more migratory mesenchymal properties — and stimulate migration and invasion of tumour cells. In various human cancer cell lines, nicotine induced changes in gene expression

that were consistent with EMT, such as reduc-tion of the epithelial markers E-cadherin and zona occludens 1 (ZO1), and a concomitant increase in levels of the mesenchymal proteins vimentin and fibronectin139. Indeed, nicotine enhanced in vitro migration of the colon cancer cells DLD-1 and SW480 in a dose-dependent manner140. Overall, pro-invasive activities of nicotine in cancer are in keeping with its well-established ability to stimulate cell migration and the epithelialization stage of wound healing100,101, which thereby provides a model of nAChR-facilitated metastasis.

The process of cell migration starts with detachment of a migratory cell from neighbouring cells (scattering) followed by directional (chemotaxis) or random migration. Experiments with epidermal keratinocytes showed that the physiological control of distinct phases of cell migration is predominantly mediated by particular subtypes of nAChRs69–71. Signalling through α9-containing nAChRs is crucial for the initiation of cell migration71. Stimulation of α9-containing nAChR upregulates phospho-rylation of the focal adhesion proteins focal adhesion kinase (FAK) and paxillin, as well as phosphorylation of the intercellular junc-tion proteins β-catenin and desmoglein 3, thereby disengaging cell–cell attachment units, whereas the inhibition of α9 nAChRs inter-feres with phosphorylation of adhesion and cytoskeletal proteins and colony scattering71. In in vitro migration assays, the stimulation of α9 nAChRs was associated with the activation of PLC, SRC, EGFR, PKC, RAC and RHO, whereas inhibition of this receptor interfered with the phosphorylation of adhesion and cytoskeletal proteins71. In turn, α7 nAChR directs cell chemotaxis towards the concentra-tion gradient of an agonist such as ACh or nicotine, and this process is associated with increased expression of α2 integrin59,69,70. The signalling pathway of α7-dependent chemo-taxis includes intracellular Ca2+, CaMKII, con-ventional isoforms of PKC, PI3K, RAC and

cell division control protein 42 (CDC42)70. The α3β2-containing nAChR regulates ran-dom cell migration via the PKCδ and RHOA-dependent signalling cascades70.

Tumour-supporting environmentAngiogenesis or neovascularization. There is overwhelming evidence that nicotine induces pathological angiogenesis that facilitates tumour survival and spreading (reviewed in REFS 141,142). In both the Lewis lung cancer model and colon cancer xeno-grafts in mice, nicotine enhanced tumour growth in association with an increase in tumour vascularity143,144. Accordingly, the α7 nAChR antagonist MG624 inhibited angio-genesis of human small-cell lung cancer cells in two in vivo models — the chick chorio-allantoic membrane model and the nude mouse model109. In the chick chorioallantoic membrane model of angiogenesis, nicotine induced endothelial cell tube formation in a concentration-dependent manner, and this was blocked by an inhibitor of ERK1 and ERK2 and by antibodies to αVβ3 integrin104. Taken together, these observations indicate that nicotine promotes endothelial cell pro-liferation and migration, thereby mimicking the effect of traditional angiogenic growth factors (such as VEGF), which suggests that blockade of autocrine or paracrine nicotin-ergic pathways could be helpful for arresting excessive tumour angiogenesis142.

Tissue (stromal) remodelling. Nicotine can contribute to the tumour-promoting environment by facilitating stromal matrix reorganization and/or degradation, as well as secretion of extracellular matrix proteins. Fibroblasts are the primary cellular compo-nent of the connective tissue and seem to be a major target of nicotine, enabling nicotine to promote a pro-tumorigenic environment. Factors that are secreted by tobacco-exposed fibroblasts increased proliferation and inva-siveness of immortalized but not malignant epithelial cells145. These effects might be mediated, at least in part, by nicotine or its metabolites activating fibroblast nAChRs, because mecamylamine — an antagonist of nAChRs — abolished nicotine-dependent upregulation of the dermal matrix proteins collagen type Iα1 and elastin, as well as matrix metalloproteinase 1 in fibroblasts146. Taken together, the results of experiments with cells exposed to ether whole tobacco145 or pure nicotine146 suggest that nicotine can not only initiate tumour-promoting changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a pro-tumorigenic stromal environment.

The data accumulated in the literature indicate that nicotine has the ability to damage the genome, disrupt cellular metabolic processes, amplify oncogenes, inactivate tumour suppressor genes and promote a cancer-supporting microenvironment.




Tumour cellNucleus

Nature Reviews | Cancer

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Cell membrane nAChR

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Nicotine Nicotine Nicotine

Anti-apoptotic

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GenotoxicMitochondrialnAChRROS

b Tumour progression Invasion and metastasis Resistance to apoptosis(chemoresistance)

α7 nAChR α9 nAChR

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RAS–RAF–MEK–ERK

STAT expression

STAT phosphorylation

Regulation of gene expression

Cancer cell growth and proliferation

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Ca2+ Ca2+

PLC, PKC, SRC, EGFR,RAC, RHO or ROCK

Phosphorylation or dephosphorylationof adhesion and cytoskeletal proteins

Assembly or disassembly of cell–cell and cell–matrix adhesion complexes

Cancer cell detachment, migration and re-attachment

CAMKII

α3-containingnAChR

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PI3K–AKTBCL-2 and NF-κBexpression

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Inhibition of programmed cell death

mPTP

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Protection from cancer immunosurveillance. Nicotine might allow tumour cells to over-come immunosurveillance, which might reduce cancer formation via immune-mediated clearance of transformed cells. Nicotine can inhibit antitumour immunity through various mechanisms. It has been reported that exposure to nicotine adversely affects dendritic cells, a cell type that has an important role in anticancer immuno-surveillance (reviewed in REF. 147), and that this compromises host response to a foreign agent148. A general immunosuppressive effect of nicotine was shown by decreased interleukin-2 (IL-2) production in mitogen-stimulated human peripheral blood mono-nuclear cells149. Nicotine also increases the number of regulatory T (TReg) cells, reduces that of T helper 17 (TH17) cells and inhibits IL-17 production in mice150,151; these changes might reduce anticancer immunosurveil-lance152. Most importantly, nicotine inhibits cytotoxic activity of natural killer (NK) cells153,154 — the effectors of innate immunity, which are directly involved in cancer immu-nosurveillance (reviewed in REF. 155). For example, the NK cell-mediated lysis of Yac-1 lymphoma cells in an in vitro co-culture was reduced by nicotine156. The inhibitory effect of nicotine on NK cells can be abrogated by the deficiency of β2-containing nAChR154.

Conclusions and perspectivesNicotine can contribute to cancer in various ways through its genotoxic effects, as well as by facilitating tumour cell survival, growth, metastasis, resistance to chemotherapy or radiotherapy, and creating a tumour-supporting environment, which might promote tumours that are initiated by other factors. The multiple mechanisms of nico-tine carcinogenicity can result from both its nAChR-mediated and non-receptor effects. The tumour-promoting effects are apparently mediated by the nAChRs expressed on the cell membrane and mitochondria, whereas the genotoxic effects probably result from elevation of ROS levels (FIG. 2a). The receptor-mediated effects of nicotine are implemented through the synergistic ionic and non-ionic signalling events (FIG. 2b). The genotoxic effects of nicotine synergize with those of other tobacco carcinogens, and certain hor-mones and growth factors synergize with nicotine to induce other tumour-promoting effects. Future research should identify rela-tive contributions of different mutagenic and tumour-promoting pathways that are acti-vated by nicotine in various types of cancers. Therapeutic strategies that target nAChRs may restore normal physiological functions

and reactivate apoptosis in malignant cells. Elucidation of the nicotinergic pathways of tumorigenesis that are activated in an indi-vidual cancer patient may therefore shift the current clinical research paradigm towards the use of pharmacological inhibitors of the pathway operating in a particular individual. Pharmacological inhibition of nicotinic sig-nalling may have applications in anticancer

therapy, as silencing of the expression of nAChR subunits and treatment with nAChR antagonists produce antitumour effects both in vitro and in vivo97,157–163. Therefore, it is cur-rently believed that nAChRs might be a novel drug target for the prevention and treatment of cancer12,13,15,62. The translational impor-tance, however, goes beyond tobacco-related cancers, because nAChRs are also activated

Figure 2 | Hypothetical schemes of several carcinogenic mechanisms of nicotine action. a | The synergy of distinct carcinogenic pathways of nicotine action. The mechanism of action of nicotine comprises combined and synergistic growth-promoting and anti-apoptotic effects mediated by the nicotinic acetylcholine receptors (nAChRs) expressed on the cell membrane and mitochondria, respectively, as well as the genotoxic action of reactive oxygen species (ROS). b | The principle path-ways of downstream signalling from major nAChR subtypes that can mediate pathobiological effects of nicotine. Although components and ordering of the signalling cascades shown have been identified in a series of mechanistic studies involving human keratinocytes58,59,69–71,77,84, the same pathways are known to operate in tobacco- or nicotine-related cancers (reviewed in REFS 17,85,87,191). CaMKII, Ca2+/calmodulin-dependent protein-kinase II; EGFR, epidermal growth factor receptor; JAK2, Janus kinase 2; mPTP, mitochondrial permeability transition pore; NF-κB, nuclear factor-κB; PKC, pro-tein kinase C; PLC, phospholipase C; ROCK, RHO-associated protein kinase; STAT, signal transducer and activator of transcription.




by autocrine or paracrine ACh and thereby control the delicate balance of cell prolifera-tion and death that separates the normal state of cells from neoplasia. The pharmacological modulation of nAChRs can affect both ion flow through the channels and activation of various kinases or signalling pathways.

Individuals who carry certain nAChR variants are at a higher risk of develop-ing cancer. The results that show opposite effects of nAChR variants on cell growth and resistance to oncogenic transformation (BOX 3) indicate that certain SNPs in nAChRs modify the response of an individual to nico tine. The mutated receptors are con-stantly stimulated by physiological concen-trations of autocrine or paracrine ACh, but nicotine and other chemicals that can activate nAChRs, including the tobacco carcinogen NNK, can increase aberrant signalling from these receptors, thereby facilitating neoplastic transformation. Hence, nicotine may repre-sent a ‘second hit’ that increases the risk for survival and propagation of the transformed cells. Therefore, nicotine-containing products should be avoided by not only cancer sur-vivors and cancer patients currently receiv-ing chemotherapy or radiotherapy but also healthy individuals carrying these nAChR subunit SNPs. Elucidation of the specific mechanisms by which individual genetic vari-ations of nAChRs modify predisposition to cancer development could guide personalized approaches to cancer prevention.

The emerging knowledge about a direct connection of nicotine to cancer should be considered in the development and evalu-ation of regulations on nicotine product manufacturing, distribution and marketing. Future studies should determine the dose- and time-dependent effects and establish whether the route of administration of nicotine (that is, skin patch versus inhalation versus oral versus enema) affects its possible carcinogenic activity. Development of sensi-tive in vitro and in vivo assays of mutagenic and tumour-promoting activities of nicotine should help to standardize the evaluation of different nicotine-containing products.

Sergei A. Grando is at the Departments of Dermatology and Biological Chemistry, and Cancer

Center and Research Institute, University of California, Irvine, California 92782, USA.

e‑mail: [email protected] doi:10.1038/nrc3725

Published online 15 May 2014

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Connections of nicotine to cancer · signals (reviewed in REF. 11). The overall cellular response to stimulation with ACh is determined by the delicate balance between these signals