The Role of Nitrosamine (NNK) in Breast Cancer Carcinogenesis

Smoking cigarettes is one of the most concerning issues that leads to tobacco-related cancers and can even result in death. Therefore, these issues should be addressed with a great sense of urgency with low-cost and simple approaches. Over the past several years, the scientific community has attempted to find solutions to overcome this issue. Thus, a large number of excellent studies have been reported in this field, and summarizing these results and providing important roadmaps for future studies is currently of great importance. Finding an outstanding solution to address aforementioned issue would be of great value to the community and to the social. Tobacco contains thousands of chemicals, and sixty-nine compounds have been established as human carcinogens; specifically, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the strongest carcinogen among the tobacco-specific nitrosamines. Tobacco carcinogens are also linked to mammary gland pathogenesis and increased risk of developing many cancers, including breast cancer, the most common cancer in women worldwide. This mini-review summarizes the role of NNK and the mechanisms of its receptor, nicotine acetylcholine receptor (nAChR), signaling in breast cancer based on publications identified using the keywords “secondhand smoke (SHS)”, “Nitrosamines” and “breast cancer”. Furthermore, this review considers the risk of NNK to the public in an effort to reduce exposure to SHS in women and their chances of developing breast cancer.


Introduction
The effect of smoking tobacco has been studied in patients with lung cancer or smoking-related cancer, including breast cancer, due to heavy smoking [1,2]. Breast cancer is the most common cancer in women; 1.7 million were diagnosed and 521,000 died worldwide in 2012 [3,4]. Smoking tobacco has been suspected as one of the risk factors which can be regulated for breast [5,6].
However, many recently published studies show that the risk of developing breast cancer is not directly associated with active smoking. Since 1986, an expert panel agency that includes the US Surgeon General and the International Agency for Research on Cancer (IARC) repeatedly concluded that the evidence does not support an association between smoking and the risk for breast cancer [7,8]. Moreover, Hamajima et al. concluded that active smoking and breast cancer did not correlate based on their collated analysis of 53 epidemiological studies that included more than 150,000 cases [7].
On the other hand, secondhand smoke (SHS) (also known as passive smoking, involuntary smoking and environmental tobacco smoke (ETS)) has been identified as a toxic air contaminant since 2006 by Air Resources Board, California Environmental Protection Agency [9]. Non-smokers, who inhaled tobacco gases by being exposed to environmental tobacco smoke, are called passive and secondhand smokers. Throughout life, SHS occurs in the childhood household, in the adulthood household, and at work [10]. The CAREX (carcinogen exposure) database confirmed that in the European Union, 7.5 million workers are exposed to environmental tobacco smoke during~75% of their workday, which is the second-most common environmental hazard after solar radiation (9.1 million workers) (http://www.ttl.fi/Internet/English/ Organization/Collaboration/Carex/default.htm) [11]. Worse than active smoking, the association of SHS with the risk of breast cancer has been contested because the assessment of SHS is more difficult than that of active smoking due to lack of information about a major source of exposure [12,13]. Notably, recent studies of large number of prospective cohorts provided evidence to suggest that breast cancer risk is elevated among women exposed to the highest levels of SHS [14]. In addition, a study by Johnson and Glantz comparing the strength of evidence from epidemiologic studies showed that SHS affects breast cancer more significantly than lung cancer [15].
Tobacco smoke is well known to contain thousands of chemicals, including more than 60 known carcinogens, which cause a variety of diseases and cancers [16]. Out of 60, at least 20 chemicals have been documented to induce mammary tumors in animal studies using rodent models [17] (Table 1). For the last few decades, 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK) has been implicated in the risk of breast cancer development, it remains unclear in breast cancer development, specifically breast cancer induced by SHS [18,19]. Moreover, NNK can reportedly bind to and activate nicotine acetylcholine receptors (nAchRs) [20,21], and cancer cells express nAChRs, suggesting that these receptors play a crucial role in cancer development [22].
Although active and passive smoke likely contain same chemicals but at different amounts, the fact that SHS is significantly more toxic than active smoke is surprising [23,24]. This review focuses on the role of NNK in SHS-triggered breast cancer and the molecular mechanism underlying NNK-induced carcinogenesis.

Tobacco Nitrosoamines
More than 70 tobacco species are known; tobacco is a green and leafy plant that originates in South America and contains nicotine, its main addictive compound, and thousands of other chemicals. Therefore, tobacco is often referred to as a chemical factory. The major chemical components of tobacco are converted to 5300 tobacco smoke compounds, such as nicotine derived N-nitrosamines, polycyclic aromatic hydrocarbons (PAHs), aromatic amines, aldehydes and other inorganic and organic compounds [25]. Roberts classified unburned tobacco and cigarette smoke into a functional group of 25   [27,28]. (Fig. 1). Some of these chemical compounds, which are released when tobacco is burned, are carcinogenic and toxic [29]. Their carcinogenicity has been extensively examined both in vivo and in vitro systems [30][31][32]. Tobacco-specific N-nitrosamines, such as NNK, are derived from tobacco curing and smoking and their levels range from 50 to 200 ng per cigarette [33,34].

Tobacco-Specific Carcinogen Nitrosamine, NNK
As mentioned above, nicotine is an addictive but noncarcinogenic to healthy laboratory animals [33], and nicotine has consequently not been significantly researched in the context of cancer. However, the nitrosation of nicotine and alkaloids results in a Bnicotine-derived nitrosamine ketone^, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) [35], by replacing the N-H moiety with an N-N = O moiety, a change that was first reported in 1978 [30]. Among tobacco-specific nitrosamines, NNK is widely considered the most potent carcinogen to humans for tobacco-induced cancers and was evaluated by the International Agency for Research on Cancer [10,36]. NNK has been detected in cigarettes, cigars, snuff tobacco and mainstream and sidestream tobacco smoke. Nitrosamines affect not only tobacco consumer but also non-smokers, who are exposed to environmental tobacco pollution [37]. Moreover, tobacco-specific nitrosamine, 63-74% of which consists of NNK, is formed from nicotine during smoking [38,39] (Table 2). Nicotine-derived nitrosamines can cause cancers or increase the risk of cancer in animals and humans [40][41][42][43], and humans may be exposed to nitrosamines by inhaling both   [77] Hoffmann et al. [120] NNN: N-nitrosonornicotine, NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNB: N-nitrosoanabasine mainstream and sidestream smoke [44]. Accordingly, carcinogenic NNK and its metabolite are found more in the urine (0.06 to 1.4 pmol/mg creatinine) and blood (42 ± 22 fmol/mL) of smokers than urine (0.02 to 1.2 pmol/mg creatinine) and blood (<8 fmol/mL) of nonsmoker [45,46]. Current technical improvements have also directly detected nitrosaminehemoglobin adducts in the blood of smokers [47]. Notably, raw sidestream smoke includes 20-30-fold more aromatic amines and 30-90% more particulate matter than mainstream smoke [48]. In addition, worldwide use of electronic cigarettes has increased rapidly. It is known that electronic cigarette liquids contain low levels of tobacco-specific nitrosamines but a few studies have tested that vapour from electronic cigarette is not free from NNK. Goniewicz

SHS and Breast Cancer
In fact, true non-smokers may not exist since all individuals are exposed to SHS at some point during their life, either in the childhood household, in the adulthood household, or at work [10]. In other words, most non-active smokers willingly or unwillingly inhale tobacco smoke from the environment. Moreover, previous meta-analysis studies have reported that breast cancer is associated with cigarette smoking because long periods of exposure to SHS increased breast cancer risk [2,51,52]. A.Wesley Horton reported that breast cancer and cigarette consumption are closely related in 20 countries, and the inhalation of tobacco smoke is considered a major environmental risk factor for breast cancer [14]. Furthermore, Chu et al. found that cigarette smoking significantly increases the risk of breast cancer in younger or premenopausal women, and this risk strongly correlates with smoking intensity and duration [53]. Similar studies have been reported in several countries, including Japan, Canada, and the USA [8,[54][55][56]. The main risk of cigarette smoking-associated breast cancer is presumably related to exposure to the potent carcinogens contained in tobacco smoke, including aromatic hydrocarbons, aromatic amines and nitrosamines, which may cause DNA mutations and DNA adducts [57]. Thus, many meta-analyses evaluated causal associations between environmental tobacco smoke and breast cancer and were reviewed by the California Environmental Protection Agency [58]. These studies demonstrated a causal relationship between exposure to SHS and breast cancer [59]. Reynolds et al. reported that high levels of SHS during adulthood may increase the risk of breast cancer in female lifetime non-smokers exhibiting the highest level of cumulative exposure (HR, 1.18; 95% CI, 1.00-1.40) [12]. Furthermore, breast cancer risk was strongly associated with SHS, and SHS exposure is reportedly riskier than active smoking [60]. Schick and Glant investigated unpublished in vivo research data on SHS obtained by the cigarette industry. Interestingly, animal experiments showed that inhaled sidestream smoke is approximately four times more toxic than mainstream cigarette smoke. Sidestream smoke can also damage the respiratory epithelium after 21 days of exposure, and the length of exposure directly correlates with the degree of damage. Overall, the study showed that whole sidestream smoke is more toxic than all of its major constituents combined [61]. Passive exposure is recognized as a high-risk factor because N-nitrosamines and carcinogens found in tobacco are more concentrated in sidestream than in mainstream smoke [10,61]. Brunnemann et al. quantitatively analyzed N-nitrosamines in tobacco, fresh cigarette mainstream smoke and sidestream smoke using a thermal energy analyzer. Specifically, the representative nitrosamines in tobacco smoke, such as dimethylnitrosamine, methylethylnitrosamine, diethylnitrosamine, and nitrosopyrrolidine, were found at low levels (0.1 ng-97 ng) in mainstream smoke and higher concentrations (9~1770 ng) in sidestream smoke [62].
In cohort studies, Luo et al. observed that passive smokers who had never been active smokers had a 32% increased risk of breast cancer [63]. A relationship was also identified between breast cancer and the husband's smoking habit among Korean and Chinese women. Specifically, the risk directly correlated with the duration of the husband's smoking habit [64,65]. In Thailand, sources of passive tobacco exposure include spouses (40.8%), the workplace (30.6%), and public areas (26.3%), and this exposure is an important risk factor for breast cancer among females in urban areas [66]. Moreover, an ecologic analysis indicated that women who live in smoke-free environments had a lower risk of breast cancer [67], and importantly, scientific data show that passive smoke is the most important risk factor for female breast cancer, despite the fact that passive smoke is more diluted than direct smoke [15]. These results suggest that effective preventative methods, such as smokefree public environments, are needed to prevent breast cancer. Since 1985, many research groups have published relationships between breast cancer and SHS, as summarized in Table 3, which shows the results of 10 prospective cohort studies, 16 case-control studies and 7 meta-analyses.

Nicotinic Acetylcholine Receptor (nAChRs) Signaling in Breast Cancer nAChRs
The nicotine contained in tobacco products and its receptor, nAChR, have been well studied due to their numerous effects on the human body [68][69][70]. This receptor family plays a role in the synthesis and release of neurotransmitters in the central nervous system and regulates all involuntary organ functions via the autonomic nervous system [71,72]. The nAChR is a cation-selective channel receptor, and its activation is associated with cell proliferation, anti-apoptosis, and angiogenesis processes [73]. Moreover, many biological reports have suggested that the effects of tobacco exposure are mediated by the actions of nitrosamine and nAChRs [20,74,75]. Cancer related-nAChR function was first described in 1989 using small-cell lung cancer cell lines [76]. Although followup studies have supported that nicotine inhibited apoptosis via nAChRs in lung cancer cell lines, it has not been highlighted until the binding of nicotine-derived nitrosamine to α7nAChR was found because nicotine itself does not cause cancer in laboratory animals [20,30,77]. In 1998, Schuller finally discovered that the nicotine-derived nitrosamine and powerful carcinogen 4-(methylnitrosamino)-1-(3-pyridyl-1 butanone (NNK) is an α7nAChR agonist with a more than 1300-fold higher affinity for the α7nAChR than nicotine. This finding provided a completely new direction in tobacco-associated cancer research [20,76].

nAChRs in Cancers
The neuronal system expresses homo-pentamers (α7-α9 subunits) or hetero-pentamers (α2-α6, α10 subunits with β2-β4 subunits) of nAChRs, unlike the different combinations of nAChR subunits found in muscle (α1 with β1, γ, δ, or ε subunit) [78]. However, both arrangements are reportedly expressed and exhibit opposite functions in cancer cells. α7nAChR most effectively stimulates the growth of cancer cells, whereas α4β2nAChR inhibits growth [79,80]. α7nAChR can bind to NNK with 1300 times higher affinity than to nicotine, whereas heteromeric αβ nAChRs exhibits a high affinity to NNN (~5000 times higher than to nicotine) [20,21]. Notably, long-term exposure to nicotine and nicotinederived nitrosamines, such as habitual tobacco smoking and exposure to SHS, upregulates all nAChRs but selectively leads the desensitization of α4β2nAChR activity, whereas the activity of α7nAChR remains unchanged [7]. Nicotine-derived nitrosamines change the expression and function of the α7nAChR to hyperactivate all its downstream Baccelerator functions^while simultaneously activating desensitized heteromeric nAChRs, which disables their Bbrake^functions. Therefore, smoking is a prominent risk factor for all human cancers [80], and α7nAChR may represent a major receptor subfamily that mediates cancer cell proliferation.
Many studies have demonstrated that nAChR is closely involved in the development of various cancers [81]. The most well-studied nAChR mechanism is that of α7nAChR in tobacco-related non-small cell lung carcinoma (NSCLC). Specifically, Paleari et al. demonstrated that nicotinic receptor antagonists inhibit the growth of NSCLC, accounting for 30% of all primary pulmonary malignancies both in vitro and in vivo. [82]. Moreover, Improgo et al. showed that nAChR, especially α3β4α5 nAChR, plays a crucial role in small-cell lung cancer (SCLC). Using publicly available data of multiple genome-wide association studies (GWAS) in lung cancer, they found that gene variants encoding 3β4α5 nAChR are closely associated with lung cancer and confirmed that α3β4α5 nAChR promotes SCLC growth [22]. Nicotine also reportedly induces the secretion of different types of calpain, a proteolytic enzyme, from non-small cell lung cancer, which can promote the cleavage of various substrates in the extracellular matrix to result in metastasis and tumor progression [83]. In addition to lung cancer, the association of nicotine and nicotine derivatives with various types of nAChRs has been investigated in breast cancer [84,85], colon cancer [86], leukemia [87,88], cervical cancer [89], mesothelioma [90], medulloblastoma [91], and neuroblastoma [92].

nAChR and Breast Cancer
Although α7nAChR is the oncogenic receptor responsible for most oncogenic responses in cancer, α9nAChR has recently become the focus of breast cancer research since α9nAChR is upregulated in estrogen receptor-positive breast cancer cells and stimulates the initiation and progression of breast cancer in coalition with the estrogen receptor [84]. Recent studies have documented that α9nAChR is highly associated with breast cancer [93][94][95]. Specifically, Chen et al. showed that the α9nAChR subunit played an important role in cell growth signaling in human breast cancer [95]. Lee et al. confirmed this finding both in vitro and in vivo to show that the downregulation of α9nAChR results in decreased tumor volume for both the MDA-MB-231 human breast cancer cell line and xenografts in SCID mice [93]. Conversely, the stimulation of the α9nAChR led to breast cancer growth [96].

nAChR Signaling in Cancers
The nAChR is the one of the best-characterized ligand-dependent cation channel receptors. nAChRs are expressed in autonomic ganglia and at the neuromuscular junction, and they play important roles in brain activity by being involved in synaptic and cellular function. Specifically, the opening of cation channel receptors results in the influx of sodium and calcium ions and exit of potassium ions, which causes membrane depolarization. [73].
Typically, nicotine-and NNK-mediated α7nAChR pathway activation has been observed to activate protein kinase A (PKA), the serine/threonine kinase Raf-1, the mitogenactivated kinases ERK1 and ERK2 and the transcription factors FOS, JUN and MYC in SCLC. [18,22]. Moreover, PI3K-AKT and NF-kB activation by nicotine and NNK has been reported in NSCLC cell lines originating from large-cell carcinoma, squamous-cell carcinoma, and adenocarcinoma. Both pathways are known to be involved in cell proliferation [97]. In addition, Dasgupta et al. found that α3β4nAChR-initiated and XIAPand survivin-mediated signaling play an important role in nicotine-induced chemoresistance in NSCLC. Moreover, PI3K-AKT and downstream XIAP or NF-kB signaling promote the anti-apoptotic effects of nicotine in A549 human adenocarcinoma cells, resulting in cell increased survival [98]. In other studies, both Mcl-1 and PPAR βδ proteins were also reported to be involved in nicotine-stimulated survival [99].
Notably, nAChR signaling affects other major characteristics of cancer development, such as angiogenesis, epithelial to mesenchymal transition (EMT) and metastasis. Furthermore, ERK/MAP kinase, PI3K-AKT and NF-kB signaling have been implicated in nicotine-induced and α7nAChRmediated pro-angiogenic effects in endothelial cells. Specifically, Zhang et al. showed that nicotine induced proangiogenic effects by upregulating HIF-1α, and this effect was significantly attenuated by blocking the nAChR pathway, which consists of the Ca2+/calmodulin, Src, protein kinase C(PKC), PI3K-AKT, MAPK-ERK1/2 and mTOR pathways in NSCLC cell lines [100]. Additionally, the α7nAChRmediated signaling pathway has recently been shown to be involved in the nicotine-induced invasion and migration of lung, breast and pancreatic cancer cells [98].

nAChR Signaling in Breast Cancer
Although many studies have shown that nicotine and its derivatives from continuous tobacco smoking, including SHS, stimulate nAChRs to activate various intracellular signaling cascades that directly link to cancer characteristics (most were studied in lung cancer), little is known about the molecular mechanisms that connect nicotine to breast cancer.
In 2008, a signaling cascade involving PKC and cdc42 was identified in nicotine-induced mammary tumor migration. Guo et al. demonstrated that human breast cancer and normal cell lines constitutively express four types of nAChR subunits, and nicotine enhances the mobility of these cells. Thus, they concluded that nicotine initiates a signaling cascade that involves PKC and cdc42 and consequently promotes the migration of mammary epithelial or tumor cells by interacting with its receptor. However, they were not able to identify a receptor specific to breast cancer [83] In 2010, Lee et al. identified α9nAChR as a nicotineresponsible receptor in breast cancer development [84,93]. These studies found that α9nAChR plays a crucial role in human breast cancer cells that is mediated by nicotine and NNK. Moreover, they demonstrated that nicotine-and NNK-induced cancer cell proliferation was inhibited by knocking down α9nAChR using siRNA. By contrast, the over-expression of α9nAChR substantially increased tumor growth in a normal human breast cell line. Subsequently, a molecular pathway initiated by nicotine-mediated α9nAChR activation was studied in vivo and in vitro [101][102][103]. MAPK and PI3K-AKT can be activated by both nicotine and estrogen hormone stimulation. Importantly, the activation of α9nAChR features a strong positive feedback loop via the PI3K-AKT and MAPK pathways and is followed by the activation of AP1 and VDR transcription factors, which can bind to the promoter region of α9nAChR. These signal transduction cascades eventually result in cancer development [104] (Fig. 2). Additionally, acetylcholine and NNN have been demonstrated to exhibit a higher binding affinity to heteropentameric nAChRs (α4β2, α3β2, or α3β4) than to homopentameric nAChRs (α7, α9) [22,[105][106][107].
Indeed, the modulation of α9nAChR activity by nicotine and NNK in breast cells was associated with changes in proliferation, differentiation, migration, adhesion, cell contact, apoptosis, and angiogenesis [81].

Conclusions
We herein showed that nicotine and its derivatives, i.e., NNK, in smoking tobacco products, and SHS are proven risk factors for cancer at all organ sites [108]. Mammary gland cancer is the most common cause of cancer death in females after lung cancer, and breast cancer caused by tobacco is referred to as tobacco-related primary and secondary cancer [109].
Long-term exposure to SHS among younger/primarily premenopausal women who had never smoked increased breast cancer risk by 60-70% because sidestream smoke contains more toxic chemicals than mainstream smoke [110].
The main component of tobacco smoke is nicotine, and it also contains more than 60 carcinogens. Nicotine is addictive but non-carcinogenic to healthy experimental animals, and the primary causes of cancer appear to be nicotine-derived nitrosamine and the powerful carcinogen NNK, which is an α7nAChR agonist with more than 1000-fold higher affinity to the receptor than nicotine. Additionally, it has been well documented that beta-adrenergic receptors and downstream cascades have a role in various cancer cell proliferation in response to NNK [111,112]. Beta-adrenergic receptors, a family of G-protein coupled receptors, initiate multiple signaling cascades, including the adenylyl cyclase/cAMP/PKA/ CREB pathway, which transactivates the epidermal growth factor receptor pathway [113][114][115].
Numerous studies have examined the role of nicotine and its receptors in the development of various cancers, especially lung cancer, but few articles have attempted to elucidate the molecular signaling cascades stimulated by NNK in breast cancer. Nevertheless, α9nAChR was identified as the receptor for nicotine and NNK in breast cells and is involved in nicotine-mediated cell proliferation in conjunction with estrogen hormone stimulation [93][94][95] Although, all evidence provided in this review supports that SHS increases the risk of breast cancer development via NNK and its major target, the α9nAChR, extensive studies are necessary in the future to better understand the mechanism of tobacco-related breast cancer and assess the effect of low-dose exposures, such as that provided by SHS, on the metastatic process. Moreover, published data regarding the potential toxic effects of non-nicotine chemicals incorporated in tobacco smoke, which can also include hazardous substances, are limited. In addition, the measurement of chronic tobacco smoke exposure has become essential to study the metabolic fate of tobacco products and their potential impact on public health, particularly in non-smokers. For example, Pérez-Ortuño developed a method to analyze nicotine, cotinine, NNN, NNK, and NNAL in human hair samples. Although this study was subject to several limitations, such as hair composition (e.g., the color of hair, cosmetic treatments, etc.), they concluded that the detection of NNK instead of nicotine in hair is a very good biomarker of long-term exposure to SHS. Therefore, monitoring the hair NNK levels among nonsmokers irregularly exposed to SHS may serve as a good biomarker of individual carcinogenic risk [116].