Cell signaling and epigenetic regulation of nicotine-induced carcinogenesis

Abstract

Nicotine, a naturally occurring tobacco alkaloid responsible for tobacco addiction, has long been considered non-carcinogenic. However, emerging evidence suggests that nicotine may possess carcinogenic properties in mice and could be a potential carcinogen in humans. This review aims to summarize the potential molecular mechanisms underlying nicotine-induced carcinogenesis, with a specific focus on epigenetic regulation and the activation of nicotinic acetylcholine receptors (nAChRs) in addition to genotoxicity and excess reactive oxygen species (ROS). Additionally, we explore a novel hypothesis regarding nicotine’s carcinogenicity involving the downregulation of stem-loop binding protein (SLBP), a critical regulator of canonical histone mRNA, and the polyadenylation of canonical histone mRNA. By shedding light on these mechanisms, this review underscores the need for further research to elucidate the carcinogenic potential of nicotine and its implications for human health.

Graphical Abstract

Introduction

Nicotine is one of the naturally occurring tobacco alkaloids, acting as an addictive chemical in tobacco and maintaining smoking behaviors (Benowitz et al., 2009; Fowler et al., 2020). Nicotine was first isolated from tobacco plants in 1828, and its chemical empirical formula was described by Melsens in 1843. It was first synthesized by Pictet and Crepieux in 1893. The pH of a tobacco cigarette is around 5.5, and nicotine at this pH is largely positively charged. Nicotine absorption occurs through the oral cavity, skin, lung, urinary bladder, and gastrointestinal tract (Han et al., 2023; Tutka et al., 2005).The absorption of nicotine through the alveoli of the lung is the principal route for smokers who inhale, while the principal route of absorption for smokers who do not inhale and for smokeless tobacco users is through the oral mucosa (Catassi and Fasano, 2008; Hoag et al., 2022). Once smoke enters the body, nicotine is absorbed rapidly and distributed widely. More than 80% of nicotine is metabolized into cotinine by CYP2A6, UDP-glucuronosyltransferase, and a flavin-containing monooxygenase in the liver (Benowitz et al., 2009; Fowler et al., 2020).

Previously, nicotine exposure was considered a promotor that activates molecular pathways involved in tumorigenesis in in vitro experiments, rather than an established carcinogen in animal and human studies (Milgram et al., 1991; Price and Martinez, 2019). Authoritative documents in the past decade have stated that nicotine is not considered a complete carcinogen (US 2014, FDA and RCP). It is believed that carcinogens associated with tobacco combustion may account for the causal between smoking and cancer (Jethwa and Khariwala, 2017; Pemberton, 2018; Preston-Martin, 1991). Consequently, many non-combustible tobacco products have been evaluated for their risk of cancer development. Snus, a moist smokeless tobacco product for oral use, is popular in Scandinavian countries due to its lower exposure to tobacco carcinogens while delivering equivalent nicotine levels. Araghi et al. (Araghi et al., 2017) conduced a pooled analysis of nine cohort studies with 424,152 male participants to examine the association between Swedish snus use and pancreatic cancer development. They failed to find conclusive evidence of an association between snus use and the development of pancreatic cancer, further supporting the notion that constituents of tobacco combustion, other than nicotine, are the causal agents for increased pancreatic cancer risk in smokers.

Each puff of tobacco smoke contains over 70 identified carcinogens, including 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine, in addition to nicotine (Doukas et al., 2022; Li and Hecht, 2022). It is well-known that NNN is formed through the nitration of nicotine, resulting from the reaction of nicotine with tobacco alkaloids in mammals (Konstantinou et al., 2018). However, there is no in vivo evidence demonstrating that NNK can be derived from the tissue metabolism of nicotine, even though it forms through the nitrosation of nicotine. As a result, nicotine replacement therapy (NRT), which includes chewing gum, transdermal patches, and intranasal spray, has been advocated to reduce smoking due to its lower risk of carcinogenesis associated with combustible tobacco and its well-established safety (Le Houezec et al., 2011; Wadgave and Nagesh, 2016). More recently, electronic-cigarettes (E-cigs), designed to deliver nicotine through aerosols without combustion, have been developed as an alternative to traditional cigarettes. It has been suggested that E-cig aerosols contain no carcinogens and are less harmful than tobacco smoke, as fewer toxic substances have been observed in E-cig vapor (Marques et al., 2021; Rom et al., 2015). Since their introduction in the U.S., E-cigs have gained rapid acceptance among youth tobacco users, never-smokers, and individuals attempting smoking cessation, despite limited knowledge regarding their biomedical effects (Fadus et al., 2019; Hajek et al., 2014).

Recent studies have demonstrated that nicotine and E-cigs have diverse harmful effects on health, including cancer (Bracken-Clarke et al., 2021; Gould, 2023; Merecz-Sadowska et al., 2020; Mravec et al., 2020; Price and Martinez, 2019; Sahu et al., 2023). Evidence from an increasing number of in vitro and in vivo studies suggests that nicotine itself can cause cancer, at least in animal models (Grando, 2014; Lee et al., 2018; Mishra et al., 2015; Sanner and Grimsrud, 2015; Tang et al., 2019). For example, Tang and colleagues showed that long-term exposure to nicotine aerosols generated from E-cigs induced lung adenocarcinoma and bladder urothelial hyperplasia in mice (Tang et al., 2019). Although E-cig smoke dose not contain incomplete combustion byproducts, tobacco nitrosamines, or nicotine nitrosation products, DNA damage has been detected in the lung, bladder, and heart of mice exposed to E-cig aerosols (Lee et al., 2018).

In this review, we will provide a brief overview of the evidences supporting the carcinogenicity of nicotine. Additionally, we will review the potential molecular mechanisms underlying the carcinogenic effects of nicotine. Our focus will be on the cell signaling pathways related to the activation of nicotinic acetylcholine receptors (nAChRs), as well as the recent advances in our understanding of the epigenetic regulation of nicotine-induced carcinogenesis.

1. Nicotine and carcinogenesis

1.1. In vitro evidence of nicotine carcinogenesis

Multiple studies have demonstrated that nicotine itself can promote the proliferation of cancer cells (Cardinale et al., 2012; Dasgupta et al., 2009; Puisieux et al., 2014) and induce the transformation of epithelial cells (Chang and Singh, 2019; Fararjeh et al., 2019; Martinez-Garcia et al., 2008; Sun et al., 2022a). Acute treatment of normal human bronchial epithelial (NHBE) cells with recurrent doses of 500 μM nicotine led to a transition of these cells towards a neuronal-like phenotype, accompanied by the loss of epithelial cell markers (Martinez-Garcia et al., 2008). Another study reported that nicotine treatment attenuated apoptosis caused by anti-cancer drugs and UV irritation, while inducing a transformed phenotype characterized by the loss of contact inhibition and dependence on exogenous growth factors (West et al., 2003).

In addition, long-term exposure to low-dose nicotine was found to induce cell transformation in the HBL-100 breast epithelial cell line, which is normally non-tumorigenic (Fararjeh et al., 2019). Our previous study also demonstrated that both a single dose of 750 μM nicotine and long-term treatment with 50 μM nicotine facilitated the anchorage-independent growth of BEAS-2B immortalized human lung bronchial epithelial cells, probably through downregulation of stem-loop binding protein (SLBP) and subsequent polyadenylation of canonical histone H3.1 mRNA (Sun et al., 2022a).

Nicotine treatment has also been shown to induce the neoplastic transformation in kidney epithelial cell lines, such as HK-2 cells, by affecting genes related to cellular reprogramming, redox status, and growth signaling pathways (Chang and Singh, 2019). Furthermore, daily exposure to nicotine for 2 weeks significantly increased the spheroid formation of pancreatic cancer stem cells derived from pancreatic ductal adenocarcinoma by upregulating the production of the sonic hedgehog (SHH) pathway (Marechal et al., 2015). Nicotine has also been found to enhance the malignant potential of pancreatic cancer stem cells through the SHH pathway (Al-Wadei et al., 2016).

Moreover, nicotine treatment has been shown to enhance stemness and epithelial-mesenchymal transition (EMT) in human umbilical cord mesenchymal stem cells (hUC-MSCs), which, in turn, promotes migration and proliferation of lung cancer A549 cells and breast cancer MCF-7 cells, as well as and tumor formation of A549 in nude mice (Li et al., 2018). These in vitro experimental findings collectively suggest that nicotine has the ability to induce cell transformation and enhance carcinogenic properties of different types of cancer cells, likely through various mechanisms.

1.2. Tumorigenesis effects of nicotine in rodents

Animal models have provided additional evidence supporting the association between nicotine exposure and tumorigenesis. In one study, oral administration of 52 ppm of nicotine for rats and 514 ppm for mice over a 4-week period induced urothelial hyperplasia in both species, which was suppressed by the NADPH oxidase inhibitor (Dodmane et al., 2014; Suzuki et al., 2018; Suzuki et al., 2021). Female A/J mice that received subcutaneous injections of nicotine hydrogen tartrate at a dose of 3 mg/kg/day, 5 days per week for 2 years, developed neoplasms originating from the uterus or skeletal muscle, with examination of the uterine neoplasms revealing leiomyosarcoma (Galitovskiy et al., 2012). These experiments suggest that chronic nicotine exposure can induce the development of muscle sarcoma in mice (Galitovskiy et al., 2012).

Strong evidence of nicotine carcinogenesis was demonstrated in a study by Tang et al. (Tang et al., 2019). They showed that 22.5% of mice exposed to e-cig aerosols generated by heating nicotine-containing e-cigs for 54 weeks developed lung adenocarcinomas, while 57.5% of mice developed bladder urothelial hyperplasia (Tang et al., 2019). In contrast, an extensively rare incidence of tumors and hyperplasia was observed in mice exposed to e-cigs without nicotine, implicating nicotine as a potential lung and bladder carcinogen in mice.

Several studies in rodents have suggested that nicotine promotes tumor progression and the metastasis. Davis et al. reported that nicotine administration caused a marked increase in the size of implanted Line1 tumors in BALB/c mice and dramatically increased metastasis of these tumors to the lungs (Davis et al., 2009). Additionally, administration of nicotine resulted in an increase in the size and number of tumors induced by the carcinogen NNK in the lungs, suggesting that nicotine facilitates tumor growth and metastasis in immunocompetent mice. Another study showed that when exposed to nicotine, xenografted tumors originating from head and neck squamous cell carcinoma (HNSCC) cells exhibited an escalation in lymph node metastasis. However, when nAChRs were inhibited, the process of lymph node metastasis was suppressed, underscoring the significance of nAChR in nicotine-facilitated cancer metastasis (Shimizu et al., 2019).

Using a patient-derived pancreatic cancer xenograft model, Delitto et al. demonstrated that nicotine administration promotes the growth and metastasis of pancreatic cancer (Delitto et al., 2016). Additionally, a study involving xenografts in BALB/c nude mice showed significantly higher tumor uptake in the nicotine-injected group (Kumari et al., 2018). Collectively, these data suggest that nicotine acts as a carcinogen and promotes tumor progression and metastasis in mice.

1.3. Tumorigenesis effects of nicotine in human studies

While nicotine replacement therapy (NRT) products are considered free of toxicants and carcinogens present in tobacco and cigarette smoke (Stepanov et al., 2006), there have been concerns about the potential endogenous formation of NNN through the direct reaction of nitrosation of nicotine and its metabolite nornicotine (Hukkanen et al., 2005). Stepanov et al. (Stepanov et al., 2009) observed occasional significant increases in urinary biomarkers of the carcinogen NNN in some users of nicotine gum or lozenges (oral NRT) compared to baseline smoking levels in the same subjects after they quit smoking (Stepanov et al., 2009). This finding suggests the possible endogenous formation of this carcinogen from nicotine or its presence in NRT products, rasing concerns about the long-term cancer risk for NRT users.

While there is no sufficient evidence to support the carcinogenic effect of nicotine exposure in human, several recent studies have provided evidence for an association between nicotine exposure and metastasis. Wu et al. (Wu et al., 2020) examined 281 lung cancer patients with distant metastasis and found that nicotine promoted brain metastasis by altering the polarization of M2 microglia, increasing the secretion of IGF-1 and CCL20, and thereby promoting tumor progression and stemness (Wu et al., 2020). In another study, Tyagi et al. demonstrated that nicotine promotes breast cancer metastasis (Tyagi et al., 2021). They found that nicotine plays a critical role in the formation of a pre-metastatic niche within the lungs by recruiting pro-tumor N2-neutrophils. The pre-metastatic niche facilitated the release of STAST3-activate lipocalin 2 (LCN2), leading to the EMT of tumor cells and facilitating colonization and metastatic outgrowth. Wang et al. reported that nicotine downregulates OTU domain-containing protein 3 (OTUD3), leading to the degradation of ZFP36 protein and suppression of VEGF-C mRNA decay. This process results in the generation of VEGF-C, tumor-induced lymphangiogenesis, and lymphatic metastasis in human esophageal cancer (Wang et al., 2021b).

2. Possible molecular mechanisms of carcinogenesis induced by nicotine

2.1. Genotoxicity

Multiple studies have demonstrated the genotoxic effects of nicotine using the chromosome aberration (CA) test, sister chromatid exchange (SCE) assay, and other techniques (Ginzkey et al., 2013; Ginzkey et al., 2009; Ginzkey et al., 2012; Trivedi et al., 1990). For example, treatment of human gingival fibroblasts (HGF) with nicotine led to a significant increase in micronucleus (MN) frequency. Interestingly, HGF cells exposed to nicotine showed normal cell cycle progression and proliferation ability without mitotic delay or cell loss, suggesting that DNA lesions occurred while cells continued to divide (Argentin and Cicchetti, 2004). These findings suggest the potential carcinogenicity of nicotine itself.

Nicotine, as a small molecule, can freely permeate epithelial cells and exert its carcinogenic effects by inhibiting DNA repair activity and DNA damage (Guo et al., 2005; Lee et al., 2018; Tang et al., 2019). Guo et al. (Guo et al., 2005) demonstrated that chronic exposure to nicotine for 8 weeks disrupted the G1 checkpoint and caused DNA damage through increased production of ROS in rat lung epithelial cells. However, the initiation of the gene amplication process requires the loss of p53, as observed under p53-deficient conditions in response to long-term nicotine exposure (Guo et al., 2005). This finding suggests that the loss of p53 plays a role in the establishment of oncogenic transformation in the context of nicotine exposure. Similar effects of nicotine on creating a microenvironment favorable for tumorigenesis in lung epithelium were reported by Zhang et al (Zhang et al., 2020).

Tang and colleagues recently reported that mice exposed to nicotine-containing E-cig aerosols developed lung adenocarcinomas and bladder urothelial hyperplasia (Tang et al., 2019). They found that nicotine and its nitrosation product NNK can induce mutagenic DNA adducts, namely γ-hydroxy-1,N²-propano-deoxyguanosines (γ-OH-PdG) and O⁶-methyldeoxyguanosines (O⁶-medG), in cultured human bronchial epithelial and urothelial cells. Moreover, nicotine and NNK treatments not only inhibited nucleotide excision repair (NER) and base excision repair (BER) activities but also reduced the expression of DNA repair proteins XPC and hOGG1/2 in the lung (Lee et al., 2018). They further found that nicotine and NNK reduce DNA repair proteins and DNA repair activity, as well as facilitate mutational susceptibility and transformation of cultured human bronchial epithelial and urothelial cells (Lee et al., 2018). These results implicate that nicotine could be a carcinogen for the lungs and potentially the bladder in mice.

Immunostaining for γ-H2AX, which specifically assesses the formation of DNA double-strand breaks (DSBs), showed that the number of γ-H2AX foci significantly increased in several cell lines upon nicotine exposures (Yu et al., 2016). Moreover, a significantly greater number of DSBs was observed with nicotine-containing e-cigs compared to nicotine-free e-cigs, indicating that nicotine can cause and facilitate the formation of DSBs.

It is known that tobacco workers who are transdermally exposed to nicotine by absorption of the alkaloid via leaves may develop green tobacco sickness (GST). DNA damage in nicotine-exposed workers was detected using the single-cell gel electrophoresis (SCGE) assay, which is based on the determination of DNA migration in an electric field and reflects single- and double-strand DNA breaks and apurinic sites. This result indicates that occupational nicotine exposure is associated with DNA damage and may lead to adverse long-term effects that are causally related to the instability of the genetic material, such as cancer (Alves et al., 2020).

2.2. Generation of ROS

ROS refers to a number of reactive partially reduced oxygen metabolites, including free radicals such as hydroxyl and superoxide anion-free radicals, and their non-radical derivatives such as H₂O₂. They are necessary for basic physiological cellular functions as signaling molecules (Lennicke and Cocheme, 2021). ROS levels should be maintained within physiological limits to prevent oxidative stress in cells and tissues. Excess generation of ROS has been closely associated with cancer (Wang et al., 2014). Nicotine has been shown to induce ROS production in many cell types (Barr et al., 2007). NADPH oxidases, involving proteins of the NOX family, are a major intracellular source of ROS in cancer cells (Edderkaoui et al., 2011), and their activation can be triggered by nicotine (Asano et al., 2012). Nicotine has been found to causes DNA mutations potentially mediated by oxidative stress, which is related to cancer-associated genes in normal human lung epithelial cells (Bavarva et al., 2014).

Zhang and colleagues (Zhang et al., 2020) examined the effects of nicotine on redox state in human and murine lung epithelial cells. They found that nicotine caused a transient and high-level ROS increase in the cells. Furthermore, the exposure to nicotine over an extended period resulted in the induction of ER stress, triggering the activation of the unfolded protein response (UPR). However, prolonged nicotine treatment alone could not initiate cell transformation unless p53 was inhibited. Overall, the concomitant conditions of oxidative stress and inhibition of p53 induced by nicotine creates a microenvironment that favors tumorigenesis in lung epithelium (Zhang et al., 2020).

Using the human adenocarcinoma cell line HT-29, Pelissier-Rota et al. found that nicotine induced mitochondrial ROS production. The generation of ROS was associated with a disruption of the mitochondrial membrane potential and endoplasmic reticulum stress induced by nicotine treatment (Pelissier-Rota et al., 2015). This, in turn, led to caspase-3 activation and the upregulation of cyclooxygenase-2 (Cox-2) and prostaglandin E2 (PGE2) in a PI3K-dependent manner.

In a study by Lian et al. (Lian et al., 2022) using the gastric cancer cell line AGS cells, nicotine was shown to stimulate IL-8 expression through ROS-mediated MAPK signal pathway (Lian et al., 2022). AP-1, a downstream transcript factor of MAPK cascade pathway (Guo et al., 2021), was found to be phosphorylated and exhibited increased binding activity to the promoter of IL-8 upon nicotine treatment. Pretreatment with ROS scavenger N-acetyl cysteine (NAC) reversed the effects of the transcription activity of AP-1 and the expression of IL-8 by blocking the aberrant MAPK cascading (Lian et al., 2022). IL-8 has been shown to increase angiogenesis and tumorigenesis of human gastric cancer cells in an animal model (Kitadai et al., 1999). Furthermore, Lian et al. (Lian et al., 2022) found IL-8 expression induced by nicotine could affect endothelial cell proliferation by treating EA.hy926 endothelial cells with the nicotine-treated conditioned medium derived from AGS cells.

Nicotine-induced oxidative stress is associated with the development of renal cell carcinoma (Chow et al., 2010). Firstly, chronic nicotine exposure induces stem cell-like sphere formation and EMT changes during neoplastic transformation in HK-2 human kidney epithelial cells. An increased level of intracellular ROS was detected by the DCFH-DA assay, while representative antioxidative markers (MnSOD, GPX-1, CAT, GSTP1, NRF2, and SOD1) were also upregulated in HK-2 cells. Nicotine-induced cell growth and morphological changes, including the formation of spheres and floating cells, were reversed by NAC treatment. Moreover, the expressions of EMT marker proteins ZO-1 and Vimentin were slightly restored with NAC treatment (Chang and Singh, 2019).

2.3. Activation of nAChRs

2.3.1. Overview of nAChRs

Nicotine-containing products exhibits their diverse functions through agonistic interaction with various subtypes of nAChRs, as nicotine binds to these receptors with higher affinity than acetylcholine. nAChRs belong to the superfamily of homologous Cys-loop ion channel receptors, consisting nine α subunits (α2 to α10) and three β subunits (β2-β4). The assembly of five subunits, either as homopentamers (α7 or α9) or heteropentamers (α2-α6 with β2-β4), generates many distinctive subtypes that share a common basic structure but possess specific pharmacological and functional properties (Zoli et al., 2018). These receptors form a central ion channel.

All nAChRs subunits share homologous structure, consisting of a large extracellular domain, four transmembrane regions (M1-M4) organized in α-helices, a large cytoplasmic domain between M3 and M4, and a short extracellular C-terminal tail. nAChRs are expressed on numerous neuronal and non-neuronal cells. Nearly all nAChR subtypes can permeate small cations such as Na⁺, K⁺ and Ca²⁺. When exogenous nicotine, acting as an agonist, binds to nAChRs, it transiently permeates these small cations, thereby regulating the opening of the channel (Dani, 2015). The upregulation of nAChRs expression by nicotine can also enhance the physiological signaling of cells (Grando, 2014). Consequently, membrane depolarization occurs, leading to the activation of voltage-operated calcium channels and subsequent calcium overload (Schuller, 1994). Calcium influx triggers the secretion of mitogenic factors and activates signaling cascades involved in cell proliferation, apoptotic inhibition, migration, and angiogenesis (Cattaneo, 1993; Schuller, 1994, 2009).

2.3.2. Activation of nAChRs in cancer cells and tumor tissues

Initially, it was widely believed that the expression of nAChRs in central and peripheral nervous system is associated with smoking dependence and addiction (Benowitz, 2008). However, it has been demonstrated that the upregulation and activation nAChRs play a direct role in cancer progression. Prolonged nicotine exposure has been found to increase the number of binding sites and binding activity of nAChRs in non-neuronal cells (Serov et al., 2021). Studies have shown that the upregulation of various nAChRs subunits in different non-neuronal cells induced by nicotine exhibits similar concentration dependence to that observed in brain receptors (Brown et al., 2013; Peng et al., 1994). This upregulation may not require an ion flow through the ion channel, as prolonged exposure to nicotine leads to the permanent inability of most receptors to open their channels in response to nicotine binding due to the accumulation of chronically desensitized receptors (Peng et al., 1994).

Converging evidence suggests that the biological activity associated with the carcinogenesis of nAChRs in non-neuronal cells can be attributed to both ion channel-dependent and ion channel-independent events. In the ion channel-independent events, the binding activity of nicotine modulates the transient opening of the ion channels. The ion channel-independent events also involve the activation of protein kinases, second messengers, and transcription factors mediated by upregulation of nAChRs (Chernyavsky et al., 2010). Furthermore, the mechanisms of nAChR upregulation appear to vary in different tissues and cell types.

2.3.2.1. Nicotine-induced activation of nAChRs and lung cancer

Previous studies have shown that the α7 subunit is the primary nAChR involved in mediating nicotine-induced tumor progression in human lung cancer (Song et al., 2008). The expression of α7-nAChR has been observed in various cell types, including normal human bronchial epithelial cells (NHBEs), small airway epithelial cells (SAECs), and non-small-cell lung cancer (NSCLC) cell lines (Brown et al., 2013; Lam et al., 2007; West et al., 2003). Unlike in neuronal cells, where nicotine-induced upregulation of nAChR subunits occurs through post-translational mechanisms involving nAChR assembly, maturation, turnover, and inhibition of receptor degradation (Govind et al., 2009), the upregulation of α7-nAChR in lung SCC-L cells involves transcriptional mechanisms targeting the binding of GATA4 or GATA6 to Sp1 on the α7-nAChR promoter (Brown et al., 2013). The transcription factor Sp1 has the ability to directly interact with the transcription factors belonging to the GATA family, thereby exerting control over gene expression (Zhou et al., 2008). Arredondo et al. (Arredondo et al., 2008) also demonstrated that nicotine can increase levels of α7-nAChR in oral keratinocytes by upregulating GATA2 expression, which interacts with Sp1.

Nicotine exhibits a high affinity for the α7-nAChR in lung cancer cells. It promotes the growth and metastasis of lung and pancreatic cancers in mouse xenograft models, primarily through the α7 subunit of nAChRs. Both nicotine and equivalent E-cig extracts enhance sphere formation, self-renewal, and induce epithelial-mesenchymal transition (EMT) in lung adenocarcinoma A549 cell lines by induction of Sox2 expression through the α7-nAChR-Yap1-E2F1 axis. Upon nicotine binding to α7-nAChR, the scaffolding protein β-arrestin-1 (β-arr-1) is recruited and activates Src kinase (p-Src). Yes-associated protein 1 (Yap1), a direct phosphorylation target of Src, then co-localizes with the transcription factor E2F1, which associates with the promoter of Sox2. The embryonic stem cell transcription factor Sox2 is crucial for the self-renewal of SP cells from lung adenocarcinomas (Schaal et al., 2018).

EMT is the process by which epithelial cells undergo multiple biochemical changes to acquire a mesenchymal phenotype with increased invasive capacity (Lamouille et al., 2014). Nicotine has also recently been shown to induce the EMT in a various cancers, including breast, lung and colon (Chen et al., 2018; Lee et al., 2016; Wei et al., 2011; Zhang et al., 2016), and nicotine-induced EMT and metastasis of human lung cancers depend on α7-nAChR (Pillai and Chellappan, 2012; Singh et al., 2011). Therefore, it is conceivable that lung tumors with high levels of α7-nAChR may exhibit enhanced vascularization and increased susceptibility to metastasis (Khodabandeh et al., 2022). Pretreatment with a specific inhibitor of α7-nAChR could mitigate the cell proliferation, invasive, and migratory effects induced by nicotine in the lung, suggesting a potential therapeutic strategy for lung cancer (Maouche et al., 2013; Medjber et al., 2015). Activation of α7-nAChRs induced by nicotine has also been shown to upregulate the expression of PPARβ/δ in H1838 NSCLC cells through PI3K/mTOR signaling pathway (Sun et al., 2009). PPARβ/δ is a member of the ligand-dependent transcription factor superfamily known as nuclear hormone receptor. Activation of PPARβ/δ has been linked to an elevated incidence of lung cancer in humans (Kim et al., 2006). The expression of PPARβ/δ was altered by pretreatment with α-Bungarotoxin (α-Btx) and PUN282987, a specific inhibitor and selective agonist of α7-nAChR (Sun et al., 2009). The transcription factor Sp1 and AP2, downstream of the PI3K/mTOR pathway, mediated the effects of nicotine on PPARβ/δ expression (Sun et al., 2009). α-Neurotoxins are basic peptides comprised of 60–70 amino acids that form a three-fingers snake structure. Binding of long-chain neurotoxins, such as α-Btx, to α7-AChR effectively blocks its biological actions induced by small molecule agonists, including nicotine and NNK (daCosta et al., 2015). α-Btx can bind to α7-AChR at each of its five agonist binding sites, and a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, even if four binding sites remain unoccupied by α-Btx and accessible to the agonist (Moise et al., 2002). Pretreatment with α-Btx reversed non-small cell lung cancer (NSCLC) cell proliferation induced by nicotine (Fan and Wang, 2017). Human Ly-6/uPAR-related protein-1 (SLURP-1) is an allosteric negative modulator of the α7-nAChR, sharing structural homology with α-Neurotoxins, and acts as an auto/paracrine regulator of physiological processes in epithelial cells. Elevated SLURP-1 concentration in the blood of cancer patients correlates with a favorable prognosis for patient survival outcome. Recombinant SLURP-1 selectively suppresses nicotine-induced proliferation of A549 cells by abolishing the upregulation of α7-nAChR (Shulepko et al., 2020).

Nicotine can freely permeate cells and directly stimulate mitochondrial nAChRs, which have been identified on the mitochondrial outer membrane of lung cells (Lykhmus et al., 2014). The functional ligand-binding sites of nAChRs have also been found in mitochondria isolated from BEC and SW900 cells, and increased expression of α7 mt-nAChR is associated with the malignant transformation of lung cells (Chernyavsky et al., 2015). The upregulation of α7 mt-nAChR induced by nicotine may contribute to the inhibition of apoptosis by inhibiting mPTP opening in lung cells and blocking the release of Cytochrome c (CytC) (Chernyavsky et al., 2015). CytC is a key component that binds form apoptosomes in the cytosol and initiates caspase activation (Martinou and Youle, 2011). Therefore, nicotine exhibits its tumorigenic activities through two distinct mechanisms: cell proliferation by activating α7-nAChR and anti-apoptotic effects by prevention the opening of mitochondrial permeability transition pores through activation of mt-nAChRs (Chernyavsky et al., 2015). Given the protective effect of nicotine on cell apoptosis, it is conceivable that nAChR antagonists could be employed as standalone agents or in conjunction with established chemotherapeutic drugs to combat associated cancers (Grozio et al., 2008).

α5-nAChR is also closely associated with nicotine-related lung cancer (Sun and Ma, 2015b). Nicotine can induce the activation of α5-nAChR, leading to the activation of the ERK1/2 and PI3K/AKT signaling pathways, increased migration, and invasion of A549 cell. Silencing α5-nAChR through siRNA suppresses A549 cell migration and invasion by upregulating E-cadherin (Ma et al., 2014; Sun and Ma, 2015a). The molecular mechanism involves the activation of JAK2/STAT3/Jab1 signaling pathway, which is suppressed by silencing of α5-nAChR in A549 and H1299 cells (NSCLC cells). Additionally, there are four STAT3-response elements for the promoter of α5-nAChR. The upregulation of α5-nAChR and cell proliferation induced by nicotine can be suppressed by silencing STAT3 expression. A feedback loop between α5-nAChR and STAT3 may contribute to nicotine-induced tumor cell proliferation. Phosphorylated STAT3 on Tyr705 translocates into the nucleus and subsequently activates the expression of Jab1 (Chen et al., 2020b; Sun et al., 2017; Zhang et al., 2017). Jab1 is a tumor oncogene involved in various human cancers, and its effects on cell transformation and tumorigenesis are evident in regulating cell proliferation, cell cycle control, angiogenesis, apoptosis, invasion, and DNA damage repair (Wang et al., 2016).

2.3.2.2. Nicotine-induced activation of nAChRs and pancreatic cancer

Pancreatic cancer has been strongly correlated with smoking, particularly the component nicotine, which initiates pancreatic cancers through its mutagenic properties (Ben et al., 2020a). Nicotine can stimulate EMT in pancreatic ductal adenocarcinoma Panc-1 and BxPC-3 cells and xenograft tumors through a hypoxia-inducible factor (HIF)1A/Yap1 positive feedback loop. The Yap pathway plays a modulating role in tumorigenesis, and upregulation of Yap1 is associated with cell proliferation and tumor growth (Zhang et al., 2009).

Upon nicotine treatment, HIF1A can bind to the hypoxia-responsive elements of the Yap1 promoter, promoting Yap1 nuclear localization and transactivation (Ben et al., 2020a). HIF1A drives tumor cells towards an invasive phenotype by altering microenvironment of solid tumors (Schito, 2018). Furthermore, the upregulations of HIF1A and YAP1 expression induced by nicotine is mediated by the activation of α7-nAChR (Ben et al., 2020a). α7-nAChR also mediates the proliferation, migration, and invasion of pancreatic cancer cells through the phosphorylation of the PI3K/AKT signal pathway. Atypical protein kinase C (aPKC) is downstream of PI3K signaling and plays a crucial role in carcinogenesis by modulating epithelial cell polarity, proliferation, and survival (Hanaki et al., 2016). Inhibiting α7-nAChR through siRNA and antagonists can prevent nicotine-induced PI3K/AKT phosphorylation and aPKC activation (Hanaki et al., 2016). Additionally, a significant finding in pancreatic epithelial ductal cells (HPNE) demonstrated that nicotine can promote proliferation and inhibit apoptosis by stimulating aberrant hypermethylation of the tumor suppressor gene PENK. Pretreatment with the demethylating drug 5-Aza reversed nicotine-induced cell proliferation and anti-apoptotic effects in HPNE cells. The hypermethylation of PENK was mediated by the upregulation of the α7-nAChR/p38 MAPK signaling pathway in HPNE cells (Jin et al., 2018).

2.3.2.3. Nicotine-induced activation of nAChRs and breast cancer

Analysis of α9-nAChR expression in clinical breast tumor tissues has indicated its importance in tumor carcinogenesis, with higher expression observed in advanced-stage breast cancer compared with early-stage cancer. The mRNA levels of α9-nAChR were upregulated nearly 8-fold in primary tumors and nonmalignant breast tissue obtained from patients, higher than in surrounding normal tissues (Lee et al., 2010). The α9-nAChR subunit is ubiquitously expressed in normal (nonmalignant) human breast cell lines (MCF-10A and HBL-100) as well as human breast cancer cell lines (MDA-MB-231, MDA-MB-453, AU-565, BT-483, and MCF-7) compared to the α5 or α10 subunits.

Treatment with low dose (10 μM) of nicotine for a long duration (2 months) significantly increased the number of transformed colonies in the MDA-MB-231 cell line compared to those treated with α9-nAChR siRNA. Injection of MDA-MB-231 cells with reduced α9-nAChR expression into SCID mice treated with nicotine (10 mg/mL) in their drinking significantly inhibited tumor growth. The authors also investigated the effects of overexpression or activation of α9-nAChR on the transformation of MCF-10A cells exposed to nicotine. In contrast to reduced expression, overexpression of α9-nAChR resulted in more transformed colonies and larger xenografts. Confocal microscopy revealed the membranous colocalization of α9-nAChR protein with caveolin, a protein essential for the formation of cavelike membrane structures, in MCF-7 breast cancer cells. Moreover, the binding activity of [³H]-nicotine was significantly inhibited in MDA-MB-231 cells with reduced α9-nAChR expression. These results indicate that breast cancer cell transformation induced by nicotine could be mediated through the endogenous α9-nAChR receptor (Lee et al., 2010).

The tumor microenvironment is of paramount importance in both the onset and advancement of tumors. The presence of nicotine may synergize with a high-fat diet (HFD) to promote breast tumor growth by inducing an anti-inflammatory microenvironment, which can also be regulated by nAChRs. In an experiment where the breast cell line HCC70 and HCC1806 were xenografted into immunodeficient mice, coexposure to nicotine and HFD resulted in larger tumor size compared to nicotine exposure alone. The key mechanisms underlying tumorigenesis were associated with the upregulation of anti-inflammatory cytokines IL13 and the phosphorylation of p38 MAPK/ERK1/2. Additionally, the xenograft growth could be significantly inhibited by a non-specific antagonist of nAChRs and SB203580, a p38 kinase inhibitor (Jimenez et al., 2020).

It has been reported that nicotine induces EMT in MCF7 and MDA-MB-231 cells and promotes the activation of fibroblasts (Chen et al., 2018). Conditioned medium from nicotine-activated fibroblasts (WI38 cells) had a greater impact on promoting the EMT of breast cancer cells. Nicotine can induce the production and secretion of connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β in fibroblasts, which in turn enhance breast cancer migration. The activation in fibroblasts by nicotine is mediated by TAZ, the transducer of the Hippo pathway, and pretreatment with the TAZ inhibitor verteporfin suppresses the expression of CTGF and TGF-β. The activation of TAZ is regulated by α7-nAChR-mediated AKT signaling cascade in fibroblasts (Chen et al., 2018).

2.3.2.4. Nicotine-induced activation of nAChRs and other cancers

A recent study by Pucci et al. (Pucci et al., 2021) revealed that nicotine-activated α7- and α9-nAChR-mediated signaling pathways contribute to the aggressive behavior of high-grade human glioblastomas. Nicotine increased the proliferation rate of U87MG and GBM5 cells by suppressing the activation of α7- and α9-nAChR, subsequently inhibiting the phosphorylation of the anti-apoptotic AKT and pro-proliferative ERK signal pathways. The proliferation of GBM cells can be blocked by inhibiting α7- and α9-nAChR using both siRNA and antagonists (Pucci et al., 2021).

In the case of cholangiocarcinoma (CCA) tumors, nicotine exposure was found to accelerate tumor growth in a xenograft mouse model and enhance the proliferation of CCA cells in vitro through the upregulation of α7-nAChR (Martínez et al., 2017). In CCA Mz-ChA-1 cells, nicotine exposure led to a rapid increase in α7-nAChR-dependent p-ERK1/2 signaling within 3 minutes. Silencing a7-nAChR using shRNA significantly reduced nicotine-induced cell proliferation. Interestingly, treatment with PNU282987, an agonist for a7-nAChR, increased the tumor growth of CCA xenograft (Martínez et al., 2017).

Gene set enrichment analysis (GSEA) revealed a strong correlation between cigarette smoking and the cancer-initiating cells (CIC) signature in human esophageal squamous cell carcinoma (ESCC). Nicotine exposure increased the number and size of ESCC tumorspheres in a 3D culture of KYSE270 cells by upregulating the mRNA levels of a7-nAChR, which is associated with CIC properties in ESCC patients. Knockdown and overexpression a7-nAChR in two ESCC cells (KYSE270 and TE1) resulted in reduced or enhanced activation of JAK2/STAT3/SOX2 signaling pathway, respectively (Wang et al., 2021a).

The carcinogenic effects of nicotine on oral cancer initiation are related to the inhibition of cell apoptosis and promotion of cell survival through α7-nAChR-mediated signaling (Hsu et al., 2020b; Nishioka et al., 2019b; Wang et al., 2017b). Nicotine binding to α7nAChR on keratinocytes triggers Ras/Raf-1/MEK1/ERK cascade, promoting anti-apoptosis and pro-proliferative effects (Sharma et al., 2022). In vitro studies have shown that nicotine inhibits apoptosis in oral cancer (Xu et al., 2007; Zhang et al., 2012). The inhibition of apoptosis by nicotine in oral cancer cells is dependent on Prx1 through α7-nAChR, but not α3-nAChR (Wang et al., 2017a). Prx1, a member of the thiol-specific peroxidases family, plays diverse roles, including H₂O₂ scavenger, redox signal transducer, molecular chaperoning, and particularly acts as an oncogene (He et al., 2013). α-BTX significantly reduces nicotine-induced overexpression of α7-nAChR and Prx1 and activates apoptosis of oral cancer cells (Wang et al., 2017a). In oral squamous cell carcinoma (OSCC) cells YD8 and OEC-M1, nicotine exhibits a dose- and time-dependent survival effect. Moreover, nicotine may reduce the effectiveness of the chemotherapy drug cisplatin by activating the α7-nAChR. Inhibiting the expression of α7-nAChR using shRNA and the selective antagonist methyllycaconitine (MLA) reverses cell proliferation and anti-apoptosis induced by nicotine (Hsu et al., 2020a). α7-nAChR also mediates the activation of EGFR and its downstream ERK and PI3K/AKT signal pathway in nicotine-induced cell growth of HSC-2 cells. The phosphorylation of T308, rather than S473, of AKT is found to play a key role in nicotine-mediated cell growth (Nishioka et al., 2019a).

Besides the mentioned cancer types, nicotine has been found to activate the p38 MAPK and PI3K/AKT signaling pathways in human colorectal cancer cells through the upregulation of nAChRs (Xiang et al., 2016). Matrix metalloproteinases (MMPs) are a group of pericellular proteases that can degrade components of the extracellular matrix (ECM) (Sevenich and Joyce, 2014). Overexpression of MMPs has been associated with tumor aggressiveness and metastatic potential in various cancers (Lazar et al., 2010). Pretreatment with hexamethonium, an α3-nAChR antagonist, was found to attenuate the stimulatory effect of nicotine on MMP-1, −2 and −9 expression levels in the LoVo colorectal cancer cells (Xiang et al., 2016). Table 1 compiles information on nicotine-induced activation of nAChRs across various cancer types, including details on the animal model, dosage, duration, cancerous effects, and any employed mode of reversal.

Table 1:

Nicotine-induced activation of nAChRs in carcinogenesis

Subtypes	Cancer type	Animal model/cell lines	Nicotine dose and duration	Cancerous effect	Intervention strategies	Reference
α7	Lung cancer	A549 and H1650 NSCLC cell lines	2 μM nicotine or equivalent nicotine extracted from E-cigs treatment for 10 days	Self-renewal of SP cells and maintenance of stem cell properties	siRNAs of α7-nAChR, E2F1 and YAP1	(Schaal et al., 2018)
α7	Lung cancer	HAECs and AC, SCC NSCLC cells	1 or 10 μM nicotine treatment for 4–6 days (HAECs) or 8–14 days (AC and SCC)	Cell proliferation and invasion	α-BTX	(Medjber et al., 2015)
α7	Lung cancer	A549 and H1838 NSCLC cell lines	0.5 μM nicotine treatment for 72 h	Cell proliferation	α-BTX, EP4 inhibitor (AH23848) and siRNA; inhibitors of PI3K, JNK and PKC	(Fan and Wang, 2017)
α7	Lung cancer	H1838 NSCLC cell lines	0.1 μM nicotine treatment for 24 h	Cell proliferation	siRNAs of α7-nAChR, PPARβ/δ; AP-2α and Sp1; α-BTX, LY294002 and rapamycin	(Sun et al., 2009)
α7	Lung cancer	A549 NSCLC cell lines	10 nM nicotine treatment for 24 h	Cell proliferation	Recombinant SLURP-1 (r SLURP-1)	(Shulepko et al., 2020)
α7	Lung cancer	SW900 NSCLC cell lines and normal BEC cells	10 μM nicotine treatment for 45 mins (BEC) and 0.1 μM nicotine treatment for 24 h (SW900)	Cell proliferation	α-BTX, and siRNA of α7-nAChR	(Chernyavsky et al., 2015)
α5	Lung cancer	A549 NSCLC cell lines	1.0 μM nicotine treatment for 24 h	Cell migration and invasion	siRNA of α5-nAChR	(Sun and Ma, 2015)
α5	Lung cancer	A549 NSCLC cell lines	1.0 μM nicotine treatment for 16 h	Cell proliferation	siRNA of α5-nAChR	(Ma et al., 2014)
α5	Lung cancer	A549 and H1299 NSCLC cell lines, and tumour xenografts	1.0 and 10 μM nicotine treatment for 16 h	Cell migration and invasion	siRNA of α5-nAChR	(Chen et al., 2020b)
α5	Lung cancer	A549 and H1299 NSCLC cell lines	1.0 μM nicotine treatment for 16 h	Cell proliferation	siRNA of α5-nAChR	(Zhang et al., 2017)
α7	Pancreatic cancer	Panc-1 and BxPC-3 PC cell lines	1.0 μM nicotine treatment for 24 h	EMT	siRNAs of α7-nAChR, YAP-1 and HIF1A	(Ben et al., 2020a)
α7	Pancreatic cancer	Panc-1 and BxPC-3 PC cell lines	0.1 μM nicotine treatment for 72 h	Cell migration, invasion and proliferation	siRNAs of α7-nAChR and aPKC	(Hanaki et al., 2016)
α7	Pancreatic cancer	HPNE and PC cell lines (AsPc-1 and Panc-1)	10 and 100 nM nicotine treatment for 72 h	Cell proliferation	siRNAs of α7-nAChR, DNMT3A and DNMT3B; 5-Aza; MAA; SB203580	(Jin et al., 2018)
α9	Breast cancer	MDA-MB-231 and MCF-10A cell lines	10 μM nicotine treatment for 2 months	Cell transformation	siRNAs of α9-nAChR	(Lee et al., 2010)
α9	Breast cancer	Xenografts of SCID mice from nicotine-treated MCF-10A cells	10 mg/mL nicotine in drinking water for 6 weeks following 10 μM nicotine treatment for 2 months	Larger tumor size	siRNAs of α9-nAChR	(Lee et al., 2010)
α7	Breast cancer	Xenografts of immunodeficient mice from nicotine-treated HCC1806 and HCC70 breast cancer cell lines	300 nM nicotine treatment by intraperitoneal injection twice/day, 0.75 mg/kg/mice/injection for 8 weeks alone or with HFD feeding	Larger tumor size	MEC; SB203580	(Jimenez et al., 2020)
α7	Breast cancer	MCF-7 and MDA-MB-231 cell lines	10 μM nicotine treatment for 72 h	EMT	Verteporfin, the inhibitor of TAZ; α-BTX	(Chen et al., 2018)
α7 and α9	Glioblastomas	U87MG and GBM5 cell lines	50 nM (U87MG) and 100 nM (GBM5) nicotine treatment for 72 h or 6 days	Cell proliferation	MLA; α-BTX; siRNAs of α7- and α9-nAChR	(Pucci et al., 2021)
α7	CCA	HIBEpic, and Mz-ChA-1, HuCCT-1 and CCLP-1 CCA cell lines	5–20 μM nicotine treatment for 48 h	Cell proliferation	siRNA of α7-nAChR	(Martínez et al., 2017)
α7	CCA	Xenografts of mice from nicotine-treated Mz-ChA-1 cell lines	50 and 200 μM nicotine administration for 38 days	Tumor proliferation and EMT	shRNA of α7-nAChR	(Martínez et al., 2017)
α7	ESCC	KYSE270 ESCC cell lines 3D culture	10 μM nicotine treatment for 48 h	Tumor-initiating cell properties and ESCC sphere formation	Dextromethorphan, a non-competitive inhibitor of α7-nAChR	(Wang et al., 2021a)
α7	Oral cancer	OEC-M1 and YD8 OSCC cell lines	1 μM nicotine treatment for 24 h	Cell survival and chemotherapy drug resistance	MLA	(Hsu et al., 2020)
α7	Oral cancer	HSC-2 OSCC cell lines	1 μM nicotine treatment for 24 h	Cell proliferation and migration	α-BTX; MCA	(Nishioka et al., 2019)
α7	Oral cancer	DOK cell lines and C57BL/6 mice	1 μM nicotine treatment for 7 days (in vitro); 5% nicotine treatment for 16 weeks through smeared on tongue mucosa of mice	Apoptosis inhibition and progression of tongue precancerous lesions	α-BTX; shRNA of Prx1	(Wang et al., 2017)
N/A	Colorectal cancer	LOVO and SW620 colorectal cancer cell lines	0.1, 1 and 10 μM nicotine treatment for 48 h	Cell invasion	Hexamethonium, non-selective antagonist	(Xiang et al., 2016)

Notes: NSCLC: non-small-cell lung cancer; α-BTX: a specific inhibitor of α7-nAChR; MAA, antagonist of α7-nAChR; SP cells: side population cells PC: pancreatic cancer; HAEC cells: normal human airway epithelial cells; HPNE cell lines: human pancreatic epithelial duct cell lines; MDA-MB-231: human mammary gland epithelial adenocarcinomas; MCF-10A: human normal mammary gland epithelial fibrocystic cell lines; HFD: high fat diet; MEC: a non-specific antagonist of nAChRs; SB203580: inhibitor of p38 signal pathway; MLA: antagonist of α7- and α9-nAChR nAChR; CCA: Cholangiocarcinoma; ESCC: esophageal squamous cell carcinoma; OSCC: oral squamous cell carcinoma; MCA: a non-selective antagonist of nAChRs; DOK cell lines: human dysplastic oral keratinocyte cell lines.

2.4. Epigenetic mechanisms

Epigenetic mechanisms, which broadly refer to histone modifications, DNA methylation, noncoding RNA expression, and histone variant incorporation, play important roles in cancer initiation, development and invasion (Baylin and Jones, 2016; Garcia-Martinez et al., 2021; Xie et al., 2021).

2.4.1. Histone modifications and nicotine carcinogenesis

Several studies indicated that histone modifications may have a critical role in smoking-induced cancer development (Hussain et al., 2009; Liu et al., 2010). Trimethylation of histone H3 at lysine 27 (H3K27me3) is a transcriptionally repressive histone mark that is correlated with tumor development and progression (Yoo and Hennighausen, 2012). The ubiquitously transcribed TPR gene on the X chromosome (UTX) has been identified as one of the histone demethylases that specifically targets H3K27me3 (van Haaften et al., 2009). Nicotine exposure has been shown to alter H3K27me3 levels and the expression of its associated demethylase, UTX, in the kidney cancer cell line 786-O (Guo et al., 2014).

EZH2, a histone methyltransferase, catalyzes the modification of H3K27me3, leading to the suppression of gene expression. Upregulation of EZH2 plays an oncogenic role in breast carcinoma cells and is associated with the aggressiveness of breast cancer (Behrens et al., 2013; Chase and Cross, 2011). Nicotine exposure has been found to induce the overexpression of EZH2 in normal breast epithelial cells (MCF-10A) as well as in breast carcinoma cells (T47D, MCF-7, MDA-MB-231 and MDA-MB-453). This effect appears to be direct, as luciferase assays that have shown that the EZH2 promoter is activated upon nicotine treatment in MDA-MB-231 cells. Notably, silencing of EZH2 through siRNA or its inhibitor DZNepA abolished nicotine-induced upregulation of EMT genes and tumor invasion-related genes in breast carcinoma cells and inhibited nicotine-induced tumor growth (Kumari et al., 2018).

Furthermore, the expression of histone methyltransferase 1 (HMT1) was found to be increased in HK-2 human kidney epithelial cells exposed to 1 μM nicotine. Intriguingly, while nicotine treatment induced the formation of stem cell-like spheres in HK-2 cells, treatment with trichostatin A, an HDAC inhibitor, partially suppressed nicotine-induced stemness. This suggest that changes in histone acetylation status may contribute to nicotine-induced cell transformation (Chang and Singh, 2019).

2.4.2. DNA methylation and nicotine carcinogenesis

Aberrant DNA methylation is another consistent epigenetic alteration observed in cancer (Holliday and Pugh, 1975). The widespread recognition of hypermethylation in the promoter regions of tumor suppressor genes has established it as a prominent biomarker with implications across various types of cancer (Bharti et al., 2022; Dong et al., 2019). Jin et al. reported that nicotine exposure induces hypermethylation of the promoter region of tumor suppressor gene proenkephalin (PENK) and inhibits its expression in pancreatic ductal epithelial cell lines (Jin et al., 2018). The PENK gene encodes met-enkephalin and has been shown to inhibit tumor cell proliferation in various cancer cell lines (Maneckjee and Minna, 1994; Zagon et al., 1999). The hypermethylation of the PENK gene was regulated by nicotine-induced upregulation of DNA methyltransferases DNMT3A and DNMT3B through the activation of α7nAChR and the MAPK signaling pathway (Jin et al., 2018).

The fragile histidine triad (FHIT) gene, recognized as a tumor suppressor gene, assumes a vital role in the initial stage of carcinogenesis. Soma et al. demonstrated that nicotine treatment induced methylation of the FHIT gene in human esophageal squamous epithelial cells (HEECs), which was associated with a lower FIHT protein and upregulation of DNMT3a (Soma et al., 2006).

Nicotine is also associated with hypomethylation of nicotine-responsive gene(s). Wang et al. showed that nicotine exposure decreased the DNA methylation level of the α7nAChR promoter region in esophageal squamous cell carcinoma (ESCC) cells. The hypomethylation of the α7nAChR promoter was associated with nicotine-induced upregulation of α7nAChR expression and subsequent activation of the JAK2/STAT3 signaling pathway, as well as cancer-initiating cells (CIC) properties. Interestingly, metformin, which has been shown to possess antineoplastic property toward ESCC, downregulated α7nAChR expression by antagonizing nicotine-mediated hypomethylation of α7nAChR, thereby suppressing nicotine-induced CIC traits (Wang et al., 2021a).

In another study, Chang and Singh reported abnormal expression of histone methyltransferases DNMT3a and DNMT3b following exposure of HK-2 cells to nicotine. Moreover, treatment of HK-2 cells with 5-aza-C, the inhibitor of DNA methyltransferases, diminished nicotine-induced formation of stem-cell like spheres (Chang and Singh, 2019). Notably, the antioxidant N-acetyl cysteine (NAC) reversed nicotine-induced aberrant expression of DNMT3a and DNMT3B, as well as the stemness of HK-2 cells, indicating that the changes in DNA methylation are associated with the nicotine-induced generation of ROS and morphological changes during cell transformation.

Peng et al. examined the whole-genome DNA methylation status of white blood cells in male ApoE−/− mice after 14 weeks of e-cig exposure with different nicotine concentrations (Peng et al., 2022). Their results indicated significant changes in 8,985 CpGs by nicotine in a dose-dependent manner, suggesting that nicotine is involved in extensive alteration of DNA methylation. The gene set enrichment analysis showed the activation of MAPK, which is known to correlate with upregulation of cytokine expression. Additionally, gene promoter analysis using GO exhibited involvement of two pathways related to mitochondrial gene expression and mitochondrial translation.

2.4.3. miRNAs and nicotine carcinogenesis

MicroRNAs (miRNAs), a class of small non-coding RNAs, have demonstrated their capacity to govern diverse cellular processes linked to the development of cancer (Mohr and Mott, 2015). Their influence pattern involves regulating the expression of protein-coding oncogenes and tumor suppressors, directly acting as oncogenes and tumor suppressors, and modulation of EMT (Iorio and Croce, 2009). Among these, the miR200 family plays an important role in the regulation of EMT. Nicotine exposure reduced miR-200c expression in colorectal cancer cells, resulting in enhanced cell proliferation, migration, invasion, and EMT. Overexpression of miR-200c attenuated the pro-metastatic effects of nicotine (Lei et al., 2019). Moreover, nicotine was found to promote NSCLC cell proliferation and EMT by downregulating miR-99b and miR-192 and upregulating the expression of FGFR3 and RB1, respectively (Du et al., 2018). FGFR3 is a member of transmembrane receptor kinase for the FGF family of ligands and plays a vital role in cancer cell proliferation, EMT, migration and invasion (Kang et al., 2017), whereas RB1 is an important oncogene related to the initiation and development of cancers (Bastide et al., 2009). In pancreatic ductal adenocarcinoma (PDAC), nicotine upregulated miR-155-5p, which directly targeted NDFIP1, thereby promoting PDAC cell proliferation and EMT (Ben et al., 2020b). miR-21 was upregulated by nicotine in esophageal cancer cells, and the increased miR-21 promoted EMT through TGF-β (Zhang et al., 2014). Nicotine has been observed to promote EMT and intensify the generation of spheres and tumors with heightened efficacy in head and neck squamous cell carcinoma (HNSCC) cells. This effect is accompanied by upregulating miR-9, an inhibitor of E-cadherin, while concurrently downregulating miR-101, an inhibitor of EZH2 (Yu et al., 2012).

miR-21 as well as miR-16 were also upregulated in AGS cells (a human gastric adenocarcinoma cell-line) upon nicotine stimulation via activation of NF-κB through COX-2/prostaglandin E₂ (PGE₂) signaling (Shin et al., 2011). Nicotine has been shown to increase gastric tumor growth via COX-2 activation and PGE₂ release (Shin et al., 2004). The increased expression of miR-21 and miR-16 promoted gastric cancer cell proliferation (Shin et al., 2011). Moreover, nicotine decreased the expression of miR-218 in NSCLC cells, which led to increased proliferation by directly targeting CDK6 (Liu et al., 2019). Cheng et al. reported that nicotine exposure suppressed the anti-tumor effect of CD8+ T cells by reducing IL2RB and granzyme B levels through the upregulation of miR-629-5p in the lung adenocarcinoma HCC827 cells (Cheng et al., 2021).

Nicotine-mediated deregulation of various miRNAs also play roles in cancer metastasis and chemotherapy resistance. For example, nicotine exposure recruited STAT3-activated N2-neutrophils within the brain pre-metastatic niche and secreted exosomal miR-4466, which promoted stemness and metabolic switching in tumor cells, thereby enabling metastasis (Tyagi et al., 2022). Moreover, Deng et al. found that nicotine negatively regulates miR-296-3p, which targets the oncogenic protein, mitogen-activated protein kinase-activated protein kinase-2 (MK2). Suppression of MK2 downregulated c-Myc and inhibited chemotherapy resistance in nasopharyngeal carcinoma (NPC) cells (Deng et al., 2018).

2.4.4. Histone variant and nicotine carcinogenes

2.4.4.1. Nicotine-induced downregulation of SLBP in vitro and in vivo

Nucleosomes are fundamental building blocks of chromatin, composed of two copies each of histone H2A, H2B, H3, and H4. The expression of replication-coupled canonical histone genes, such as histone H3.1, is largely limited to the S phase, while the replication-independent variant histone genes, such as histone H3.3, are expressed throughout the cell cycle. The mRNAs for canonical histone genes do not possess a typical poly(A) tail. Their 3’ ends are generated by specific processing factors, including stem-loop binding protein (SLBP) and U7 small nuclear ribonucleoprotein (snRNP), that recognize a highly conserved stem-loop structure and a histone downstream element (HDE), respectively (Dominski and Marzluff, 2007; Romeo and Schumperli, 2016).

In our previous study, we observed that the mRNA and protein levels of SLBP were reduced in human lung epithelial BEAS-2B cells following nicotine treatment (Sun et al., 2022b). Furthermore, we demonstrated a significant decrease in SLBP levels in both BEAS-2B cells and normal human bronchial epithelial NHBE cells exposed to nicotine-containing E-cig aerosols, in contrast to nicotine-free aerosols. We also confirmed the lower levels of SLBP expression in lung tissues obtained from female A/J mice exposed to nicotine-containing E-cig aerosols compared to the control group. Importantly, both konckdown of α7-nAChR and treatment of cells with an α7-nAChR inhibitor abolished the nicotine-induced depletion of SLBP, suggesting that the downregulation of SLBP by nicotine is specifically dependent on α7-nAChR.

2.4.4.2. Signal transduction pathways that regulate nicotine-induced SLBP depletion

The PI3K/AKT pathways were activated upon nicotine treatment, as evidenced by the phosphorylation of AKT at S473 in BEAS-2B cells (Sun et al., 2022b). Additionally, the nicotine-induced reduction of SLBP was attenuated by a PI3K inhibitor, indicating that the PI3K/AKT pathway plays an important role in nicotine-mediated downregulation of SLBP.

Phosphorylation of SLBP by cyclin A/CDK1 or CK2 at different residues is critical for SLBP degradation (Dankert et al., 2016). We found that inhibition of CDKs, including CDK1/2, reversed the nicotine-induced depletion of SLBP, suggesting the potential involvement of CDK1/2 in nicotine-induced downregulation of SLBP (Sun et al., 2022b). Interestingly, nicotine-induced phosphorylation of AKT was not affected by CDK1 knockdown but was attenuated by CDK2 knockdown, indicating that CDK1 is involved in nicotine-induced downregulation of SLBP independently of AKT, while CDK2 acts through the activation of the PI3K/AKT pathway. Using the siRNAs, it was found that CK2α1, but not CK2α2, played a major role in regulating SLBP expression following nicotine exposure (Sun et al., 2022b). Furthermore, the nicotine-induced phosphorylation of AKT was attenuated by the CK2α1 knockdown, indicating the possible role of CK2α1 in nicotine-induced activation of the PI3K/AKT pathway. Importantly, the activation of the PI3K/AKT pathway and upregulation of CDK1 and CK2 induced by nicotine were shown to be dependent on α7-nAChR (Sun et al., 2022b). These findings underscore the significance of nAChR and a series of signaling factors in SLBP downregulation, nicotine toxicity, and potential carcinogenicity.

2.4.4.3. Polyadenylation of canonical histone H3.1 mRNA, cell transformation, and displacement of histone variant H3.3

SLBP plays a cruicial role in the proper 3’ end processing of canonical histone mRNAs. Depletion of SLBP results in the generation of polyadenylated mRNA from each of the canonical histone genes (Lanzotti et al., 2002; Sullivan et al., 2001; Sullivan et al., 2009). Therefore, we investigated whether nicotine can induce polyadenylation of canonical histone mRNAs. Exposure of BEAS-2B and NHBE cells to liquid nicotine or nicotine-containing E-cig aerosols increased the levels of polyadenylated H3.1 mRNA. Similar increases in polyadenylated H3.1 mRNA levels were observed in lung tissues of mice exposed to nicotine (Sun et al., 2022b).

It has been demonstrated that the overexpression of polyadenylated H3.1 mRNA facilitates anchorage-independent cell growth (Brocato et al., 2015; Chen et al., 2020a). Considering that nicotine exposure resulted in the polyadenylation of H3.1 mRNA through the reduction of SLBP, the role of SLBP depletion in nicotine-induced cell transformation was examined (Sun et al., 2022b). While the knockdown of SLBP enhanced colony formation in soft agar (Brocato et al., 2015), the overexpression of SLBP prevented nicotine-induced cell transformation, suggesting that the loss of SLBP plays an cruicial role in the nicotine-induced cell transformation, probably through inducing polyadenylation of canonical histone mRNAs (Sun et al., 2022b). Previously, we found that overexpression of polyadenylated H3.1 mRNA led to the displacement of the histone variant H3.3 from critical gene regulatory elements, such as active promoters, enhancers, and insulator regions (Chen et al., 2020a). Therefore, the inhibition of H3.3 assembly might be a significant contributor to nicotine-induced carcinogenesis. Table 2 provides a summary of the epigenetic mechanisms involved in nicotine carcinogenesis.

Table 2:

Epigenetic changes induced by nicotine

Epigenetic markers	Cancer type	Animal model/cell lines	Nicotine dose and duration	Cancerous effect	Intervention strategies	Reference
H3K27me3	Kidney cancer	786-O kidney cancer cell lines	100 and 500 nM nicotine treatment for 24 h	N/A	N/A	(Guo et al., 2014)
H3K27me3	Breast cancer	MCF-10A cells and breast carcinoma cell lines (T47D, MCF-7, MDA-MB-231 and MDA-MB-453); xenograft nude mice	10 μM nicotine treatment for 48 h (in vitro); 0.25 mg/kg nicotine treatment for 11 weeks in mice	Tumor growth	siRNA and inhibitor (DZNepA) of EZH2	(Kumari et al., 2018)
HMT1 and DNMTs	Kidney cancer	HK-2 cells	10 μM nicotine treatment for 6 months followed by further 1 and 10 μM nicotine treatment for 72 h	EMT and stem cell-like sphere formation	Trichostatin A; 5-aza-2’-deoxycytidine; NAC	(Chang and Singh, 2019)
DNMTs	Pancreatic cancer	HPEN cells	10 and 100 nM nicotine treatment for 72 h	Cell proliferation	MAA	(Jin et al., 2018)
DNMTs	ESCC	HEECs cells	100 μM nicotine treatment for 12 weeks	Hypermethylation status	N/A	(Soma et al., 2006)
miRNA-200c	Colorectal cancer	Colorectal cancer cell lines (HT-29 and SW620)	5 μM nicotine treatment for 24 h	Cell proliferation and EMT	N/A	(Lei et al., 2019)
miRNA-99b and miRNA-192	Lung cancer	A549 and NCI-H460 NSCLC cell lines	100 μM nicotine treatment for 48 h	Cell proliferation and EMT	siRNAs of FGFR3 and RB1	(Du et al., 2018)
miRNA-155-5p	Pancreatic cancer	PDAC Panc-1 and SW1990 cell lines; nude BALB/c were subcutaneously with Panc-1 cell lines	0.1 μM and 1 μM nicotine treatment for 24 h (in vitro); 1 mg/kg nicotine treatment for 3 weeks in mice	Cell proliferation, invasion and migration, and EMT; tumor growth	Inhibitors of miRNA-155-5p	(Ben et al., 2020b)
miRNA-9 and miRNA −101	HNSCC	HNSCC cell lines (UMSCC10B and HN-1) and SCID mice from HN-1 cells	3 mM (UMSCC10B) and 1 mM (HN-1) nicotine treatment for 6 weeks (in vitro)	Sphere formation; EMT; tumor growth	N/A	(Yu et al., 2012)
miRNA-16 and miRNA-21	Gastric cancer	AGS cell lines	100 μM and 500 μM nicotine treatment for 5 h	Cell proliferation	siRNAs of EP2 and EP4	(Shin et al., 2011)
miRNA-218	Lung cancer	A549, H661, H460 and H520 NSCLC cell lines	10 μM nicotine treatment for 72 h	Cell proliferation	miRNA-218 mimics	(Liu et al., 2019)
miRNA-629-5p	Lung cancer	HCC827, A549, H1975 and H520 NSCLC cell lines co-cultured with CD8+ T cells and xenografts from ASID mice were co-injection with HCC827 and PBMCs	1 μg/mL nicotine treatment for 24 h	Inhibited PBMCs-derived anti-tumor capacity	N/A	(Cheng et al., 2021)
Exosomal miRNA-4466	Lung cancer brain metastasis	Nude BALB/c mice intraperitoneally injection with LL/2 lung cancer cells	2 mg/kg nicotine treatment for 21 days in mice	Brain metastasis and cancer cell stemness	STAT3 inhibitor	(Tyagi et al., 2022)
miRNA-296-3p	NPC	HONE1 and SUNE1 NPC cell lines	100 μM nicotine treatment for 72 h	Chemotherapy resistance	LY294002; MK2 overexpression	(Deng et al., 2018)
Histone variant	Lung cancer	BEAS-2B cell lines	50 μM nicotine treatment for 4 weeks and 750 μM nicotine treatment for 24 h	Cell transformation	SLBP overexpression, α-BTX, and siRNA of α7-nAChR	(Sun et al., 2022)

Notes: HK-2 cells: human renal proximal tubular epithelial cells; HMT1: histone methyltransferase 1; Trichostatin A: an HDCA inhibitor; NAC: antioxidant, N-acetyl cysteine; HEECs: human esophageal squamous epithelial cells; ESCC: esophageal squamous cell carcinoma; PDAC: pancreatic ductal adenocarcinoma; HNSCC: head and neck squamous cell carcinoma; AGS: human gastric adenocarcinoma cells; PBMCs: peripheral blood mononuclear cells; NPC: nasopharyngeal carcinoma; LY294002: inhibitor of PI3K/AKT signaling; MK2: mitogen-activated protein kinase-activated protein kinase-2; BEAS-2B: Immortalized human lung bronchial epithelial cell lines; SLBP: Stem-loop binding protein.

3. Discussion and conclusion

Nicotine has long been considered non-carcinogenic. However, growing evidence from recent studies suggests that nicotine is a potential carcinogen in mice and may contribute to carcinogenesis and metastasis in humans. Nicotine should no longer be viewed solely as an addictive chemical, considering the key novel findings reported recently. In this study, we summarized the possible molecular mechanisms of nicotine-induced carcinogenesis (Figure 1), including genotoxicity, generation of ROS, activation of nAChR and downstream signaling pathways, and epigenetic regulations, with a focus on nAChR activation and epigenetic mechanisms. Our recently findings demonstrated the depletion of SLBP and polyadenylation of canonical histone mRNAs in response to nicotine exposures, potentially through the activation of α7-nAChR and a series of downstream signal transduction pathways involving PI3K/AKT, CDK1/2, and CK2 (Sun et al., 2022b). The downregulation of SLBP, the acquisition of polyadenylated canonical mRNAs, and the subsequent displacement of histone variants from the critical genomic loci appeared to represent a novel mechanism for nicotine-induced cell transformation and potential carcinogenesis. Further studies are urgently needed to elucidate not only the harmful implications of nicotine and nicotine-containing product exposure in public health, including carcinogenesis, but also the underlying mechanistic insights, especially since E-cigs are currently being widely marketed worldwide.

Figure 1.

Possible molecular mechanisms of nicotine-induced carcinogenesis (created with Figdraw.com).

Highlights.

• Nicotine is a potential carcinogen in mice and humans.

• Epigenetic regulation contributes to nicotine-induced carcinogenesis.

• Nicotine downregulates stem-loop binding protein (SLBP).

• Nicotine induces polyadenylation of canonical histone mRNAs.