Saharnaz Sarlak

Claude Lalou

Nivea Dias

Rodrigue Rossignol
email: rodrigue.rossignol@u-bordeaux.fr

Metabolic reprogramming by tobacco smoke-derived nitrosamines (TSNAs) in cancer

Metabolic reprogramming is a hallmark of cancer and the link between oncogenes activation, tumor supressors inactivation and bioenergetics modulation is well established.

However, numerous carcinogenic environmental factors are responsible for early cancer initiation and their impact of metabolic reprogramming just starts to be deciphered. For instance, it was recently shown that UVB irradiation triggers metabolic reprogramming at the pre-cancer stage with implication for skin cancer detection and therapy. These observations foster the need to study the early changes in tissue metabolism following exposure to other carcinogenic events. According to the International Agency for Research on Cancer (IARC), tobacco smoke is a major class I-carcinogenic environmental factor that contains different carcinogens, but little is known on the impact of tobacco smoke on tissue metabolism and its participation to cancer initiation. In particular, tobacco-smoke derived nitrosamines (TSNAs) play a central role in tobacco-smoke mediated cancer initiation. Here we describe the recent advances that have led to a new hypothesis regarding the link between nitrosamines signaling and metabolic reprogramming in cancer.

Introduction

Cigarette smoke is the predominant form of tobacco consumption, and it is the largest preventable cause of cancer, causing approximately 30% of all cancer deaths [START_REF]How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General[END_REF][START_REF] Pfeifer | Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers[END_REF][START_REF] Hecht | Tobacco smoke carcinogens and lung cancer[END_REF]. Tobacco smoke contains approximately 7000 different types of molecules. Among these compounds, more than 60 are known carcinogens, and several are tumor promoters or co-carcinogens [START_REF]How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease: A Report of the Surgeon General[END_REF].

Carcinogenesis is a continuous process initiated by genetic, epigenetic and metabolic alteration, followed by other factors that stimulate growth, survival and spreading of the cell. The carcinogens contained in tobacco smoke belong to various chemical classes, including polycyclic aromatic hydrocarbons (PAHs) (such as benzo[a]pyrene), tobaccospecific nitrosamines (such as NNK and NNN), aromatic amines (such as 4-aminobiphenyl), aldehydes, phenols, volatile hydrocarbons, nitro compounds, and other organic and inorganic compounds [START_REF] Pfeifer | Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers[END_REF]. Tobacco can be used in different forms: smoking (roll-your-own (RYO) or manufactured cigarettes, cigars that are made of air-cured and fermented tobaccos with a tobacco-leaf wrapper, water pipe, pipe, etc.) or smokeless (moist and dry snuff, chewing tobacco). The main focus of this review is on one of the most carcinogenic components of tobacco smoke, nitrosamines. Tobacco-specific nitrosamines (TSNAs) only exist in tobacco products. During fresh green tobacco air curing, storage and fermentation, nicotine and minor tobacco alkaloids can be converted to TSNAs through nitrosation [START_REF] Ramage | Induction of apoptosis with tobacco smoke and related products in A549 lung epithelial cells in vitro[END_REF][START_REF] Ishii | Tobacco smoke reduces viability in human lung fibroblasts: protective effect of glutathione Stransferase P1[END_REF][START_REF] Hoshino | Cytotoxic effects of cigarette smoke extract on an alveolar type II cell-derived cell line[END_REF]. This group of (Group 1) by the International Agency for Research on Cancer (IARC) [START_REF]Tobacco smoke and involuntary smoking[END_REF]. It is well documented that NNK and NNN can induce several malignancies, including those of the lung, esophageal, liver, pancreatic and nasal cavity [START_REF] Chang | Chronic cigarette smoke extract treatment selects for apoptotic dysfunction and mitochondrial mutations in minimally transformed oral keratinocytes[END_REF][START_REF] Chang | Adenylate kinase 3 sensitizes cells to cigarette smoke condensate vapor induced cisplatin resistance[END_REF][START_REF] Huang | Cigarette smoke induces promoter methylation of single-stranded DNA-binding protein 2 in human esophageal squamous cell carcinoma[END_REF][START_REF] Raja | Chronic exposure to cigarette smoke leads to activation of p21 (RAC1)-activated kinase 6 (PAK6) in non-small cell lung cancer cells[END_REF][START_REF] Nilsson | The molecular basis for induction of human cancers by tobacco specific nitrosamines[END_REF][START_REF] Hecht | Tobacco carcinogens, their biomarkers and tobacco-induced cancer[END_REF][START_REF] Tang | Association between carcinogen-DNA adducts in white blood cells and lung cancer risk in the physicians health study[END_REF]. NNK causes lung tumors in all tested species, and its activity in rats is particularly high [START_REF] Hecht | Biochemistry, biology, and carcinogenicity of tobacco-specific Nnitrosamines[END_REF]. To study the role of cigarette smoke in health in vitro, two different methods are currently used: acute exposure and chronic exposure. Acute exposure can provide general insight about the alterations related to tobacco smoke. Data from these kinds of short-term studies can reveal effects that may constitute the underlying causal chain leading to the ultimate chronic effects. For instance, it has been shown that cigarette smoke can induce apoptosis in cells when applied at higher concentrations [START_REF] Ramage | Induction of apoptosis with tobacco smoke and related products in A549 lung epithelial cells in vitro[END_REF][START_REF] Ishii | Tobacco smoke reduces viability in human lung fibroblasts: protective effect of glutathione Stransferase P1[END_REF][START_REF] Hoshino | Cytotoxic effects of cigarette smoke extract on an alveolar type II cell-derived cell line[END_REF]. Moreover, it has been reported that acute smoking causes proinflammatory states in the lung that can lead to inflammatory diseases such as COPD (chronic obstructive pulmonary disease) and asthma and promote tumorigenesis. In general, acute exposure studies could demonstrate cellular changes similar to cancerous cells [START_REF] Koczulla | Acute and Chronic Effects of Smoking on Inflammation Markers in Exhaled Breath Condensate in Current Smokers[END_REF]. Although acute studies help to understand numerous details about the effects of cigarette smoking, it is still not possible to understand the exact mechanism underlying tobacco smoke as studying some effects (such as cellular transformation) is only feasible with chronic exposure. As a result, to study the molecular alterations, long-term studies are recommended. Until now, several chronic exposure studies have been conducted in vitro, providing valuable information about metabolic changes due to chronic tobacco smoke [START_REF] Advani | Long-Term Cigarette Smoke Exposure and Changes in MiRNA Expression and Proteome in Non-Small-Cell Lung Cancer[END_REF][START_REF] Chang | Chronic cigarette smoke extract treatment selects for apoptotic dysfunction and mitochondrial mutations in minimally transformed oral keratinocytes[END_REF][START_REF] Chang | Adenylate kinase 3 sensitizes cells to cigarette smoke condensate vapor induced cisplatin resistance[END_REF][START_REF] Huang | Cigarette smoke induces promoter methylation of single-stranded DNA-binding protein 2 in human esophageal squamous cell carcinoma[END_REF][START_REF] Raja | Chronic exposure to cigarette smoke leads to activation of p21 (RAC1)-activated kinase 6 (PAK6) in non-small cell lung cancer cells[END_REF]; however, additional chronic studies are needed to better understand the detailed cellular mechanism of tobacco smoke.

The levels of TSNA in smokeless tobacco depends on the preparation used [START_REF] Nilsson | The molecular basis for induction of human cancers by tobacco specific nitrosamines[END_REF] and the variations in TSNA content of cigarette tobacco are also appreciable. For instance, Greek and Turkish types of tobacco are generally characterized by low TSNA contents. Conversely, French Gauloises had 8.6 μg/g of TSNA, whereas the TSNA concentrations in US and German brands range from 1.6-5.5 μg/g [START_REF] Nilsson | The molecular basis for induction of human cancers by tobacco specific nitrosamines[END_REF]. Numerous lines of evidence have established that NNK and NNN induce carcinogenesis by causing DNA adductions and mutations as well as promoting tumor growth through receptor-mediated effects, which are facilitated by associated receptors, such as nicotinic acetylcholine receptor (nAChR) and β-adrenergic receptors (β-AR) [START_REF] Hecht | Human urinary carcinogen metabolites: biomarkers for investigating tobacco and cancer[END_REF][START_REF] Falter | Biomonitoring of hemoglobin adducts: aromatic amines and tobacco-specific nitrosamines[END_REF]. However, additional cancer-driving mechanisms could be stimulated

TSNA-induced carcinogenic genomic alterations

Most carcinogens in tobacco products undergo metabolic activation before they are able to react with DNA. In this process, an unreactive carcinogen is converted to a form that is able to bind to DNA. There are also some carcinogens that can act without any activation process [START_REF] Hecht | Tobacco carcinogens, their biomarkers and tobacco-induced cancer[END_REF]. Cytochrome P450 enzymes (P450s) generally initiate this metabolic activation for foreign compounds, including natural products, pharmaceuticals, and carcinogens. P450s catalyze reactions in which produced metabolites are either excreted in a detoxified form or readily undergo further detoxification reactions. As long as this detoxification process works efficiently, cells are protected. However, some metabolites resulting from this carcinogeninduced metabolic activation are electrophilic (have an electron-deficient center) and can react with DNA. The reaction of these metabolites with DNA can lead to the generation of DNA adducts, which are generally regarded as intermediaries in the mutagenesis process. DNA adducts can be removed by the cellular repair system, but some can also escape from this system and subsequently initiate the carcinogenic process [START_REF] Hecht | Tobacco smoke carcinogens and lung cancer[END_REF][START_REF] Tang | Association between carcinogen-DNA adducts in white blood cells and lung cancer risk in the physicians health study[END_REF]. It has been reported that NNK can be activated through different reactions by cytochrome P450s: α-hydroxylation, pyridine oxidation, and carbonyl reduction [START_REF] Akopyan | Understanding tobacco smoke carcinogen NNK and lung tumorigenesis[END_REF]. Such activation generates the toxic methylating agent methanediazohydroxide which further reacts with DNA to produce N7methylguanine (N7-meG), O 6 -methylguanine (O 6 -meG) and O 4 -methylthymine (O 4 -meT).

During the activation process, the carbonyl group in NNK is also reduced to produce NNAL that will generate glucuronide or undergo methylene hydroxylation (Figure 1). The excretion of NNAL and its glucuronide represents a useful index of exposure in tobacco users [START_REF] Carmella | Metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in smokers' urine[END_REF][START_REF] Hecht | Human urinary carcinogen metabolites: biomarkers for investigating tobacco and cancer[END_REF].

There is also another pathway in which 4-(3-pyridyl)-4-oxobutane-1-diazohydroxide will form, which leads to the introduction of pyridyloxobutyl (POB) adducts in DNA [START_REF] Falter | Biomonitoring of hemoglobin adducts: aromatic amines and tobacco-specific nitrosamines[END_REF][START_REF] Hecht | Hemoglobin adducts as biomarkers of exposure to and metabolic activation of carcinogenic tobacco-specific nitrosamines[END_REF][START_REF] Carmella | Formation of hemoglobin adducts upon treatment of F344 rats with the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine[END_REF].

Oxidative metabolism of NNN generates the same reactive diazohydroxide as obtained upon α-hydroxylation of the terminal methyl group of NNK, thereby introducing POB in proteins and DNA. NNN exhibits a complex pattern of metabolites in urine [START_REF] Stepanov | Tobacco-specific nitrosamines and their pyridine-Nglucuronides in the urine of smokers and smokeless tobacco users[END_REF][START_REF] Hecht | Comprehensive analysis of urinary metabolites of N'nitrosonornicotine[END_REF]. Three important types of DNA damage can be caused by NNK and NNN: nucleotide methylation, pyridyloxobutylations as well as pyridylhydroxybutylation (Figure 1). As mentioned above, DNA phosphate POB adducts are also caused by these TSNAs [START_REF] Haglund | Evidence for phosphate adducts in DNA from mice treated with 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)[END_REF]. The N7-meG is the predominant adduct found in target tissues induced by NNK, followed by O 6 -meG, whereas very low levels of O 4 -meT are present [START_REF] Nilsson | The molecular basis for induction of human cancers by tobacco specific nitrosamines[END_REF]. It has been demonstrated in the lung that by αhydroxylation, NNK can transform into either a methylating agent or a pyridyloxobutylating agent. The methylation pathway, which is critical for NNK-induced lung tumorigenesis (in A/J mice), generates methyl DNA adducts such N7-meG and O 6 -meG (37-40). The pyridyloxobutylating agent produce relatively bulky DNA adducts [START_REF] Hecht | DNA adduct formation from tobacco-specific N-nitrosamines[END_REF]. It has been shown

that NNK pyridyloxobutylation is a potential initiating event of CIN in lung carcinogenesis [START_REF] Herzog | Array CGH analysis reveals chromosomal aberrations in mouse lung adenocarcinomas induced by the human lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone[END_REF]. Chromosome instability (CIN), which is an abnormal increase in the rate of change in chromosome number and structure, is assumed to be a common genomic instability in tumor evolution [START_REF] Loeb | Cancer Cells Exhibit a Mutator Phenotype[END_REF]. It has been documented that NNK is also able to induce extensive genome-wide changes in DNA copy number (or in general chromosomal alterations), indicative of CIN [START_REF] Herzog | Array CGH analysis reveals chromosomal aberrations in mouse lung adenocarcinomas induced by the human lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone[END_REF]. Gross chromosomal alterations have been repeatedly observed in NNK-induced mouse lung adenocarcinomas [START_REF] Herzog | Array CGH analysis reveals chromosomal aberrations in mouse lung adenocarcinomas induced by the human lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone[END_REF][START_REF] Herzog | Carcinogen-specific targeting of chromosome 12 for loss of heterozygosity in mouse lung adenocarcinomas: implications for chromosome instability and tumor progression[END_REF]. In general, the majority of research reports that most DNA adducts derived from cigarette-smoke carcinogens, including nitrosamines, cause miscoding permanent mutations (most frequently G-T and G-A) [START_REF] Loechler | In vivo mutagenesis by O6-methylguanine built into a unique site in a viral genome[END_REF] that can occur in crucial regions of oncogenes or tumor-suppressor genes. These mutations can lead to cancer development by creating disruptions in cell-cycle checkpoints, chromosomal instability and other changes [START_REF] Osada | Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer[END_REF]. Candidate NNK-induced genes with roles in lung carcinogenesis are p53 and retinoblastoma (Rb) tumor suppressor genes, and KRAS oncogene [START_REF] Herzog | Array CGH analysis reveals chromosomal aberrations in mouse lung adenocarcinomas induced by the human lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone[END_REF]. NNK-mediated activation of the C-Myc oncogene could also participate to CIN. Partial evidences demonstrated that, on the one hand, NNK could activate C-Myc through Thr58 and Ser62 phosphorylation in a ERK1/2 and PKC-alpha dependent manner [START_REF] Jin | Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional cooperation of Bcl2 and c-Myc through phosphorylation in regulating cell survival and proliferation[END_REF], and on the other hand, that Myc activation could generate CIN [START_REF] Kuzyk | c-MYC-induced genomic instability[END_REF]. Yet, C-Myc activation is not a major contributor to lung carcinogenesis that depends more on KRAS, suggesting that NNKmediated KRAS activation could contribute to CIN in this type of cancer [START_REF] Jinesh | Molecular genetics and cellular events of K-Ras-driven tumorigenesis[END_REF]. Lastly, gene silencing by promoter hypermethylation is an emerging mechanism that might also be important in tobacco carcinogenesis [START_REF] Baylin | Alterations in DNA methylation: a fundamental aspect of neoplasia[END_REF]. To conclude on the TSNA-induced carcinogenic genomic alterations, one should also consider the importance of the cumulated dose of tobacco-smoke exposure in this phenomenon. Comparison of the genomics and epigenomics alterations in lung tumors from smokers and from never smokers revealed important differences in the mutational status of KRAS and STK11 (LKB1) that showed a higher level of alteration in smokers [START_REF] Subramanian | Molecular profile of lung cancer in never smokers[END_REF]. Given the role of LKB1 in the control of energy metabolism through the AMPK pathway [START_REF] Faubert | AMPK is a negative regulator of the Warburg effect and suppresses tumor growth in vivo[END_REF], bioenergetic differences could be expected in lung tumors of smokers as compared to never smokers. Accordingly, the gene expression profiling of lung and plasma from mice exposed to tobacco smoke during seven days to nine month revealed significant changes in the expression of AMPK-target genes as oxidative phosphorylation enzymes [START_REF] Miller | Gene and metabolite time-course response to cigarette smoking in mouse lung and plasma[END_REF], but the underpinning signaling mechanisms governing these bioenergetic alterations still remain unkown.

Nitrosamine receptors and signaling

Nicotine and its oncogenic derivatives NNK and NNN are thought to promote tumor progression by binding to and activating the two cell surface receptors nicotinic nAChR and β-AR. This activation can lead to the stimulation of multiple downstream tumor-promoting signal transduction pathways [START_REF] Warren | Nicotine and lung cancer[END_REF] discussed below. The two main branches of TNSAs signaling, namely β-AR and nAChR interwinned pathways, play a role in the reprogramming of energy metabolism and could thus be involved in the bioenergetic reprogramming of precancer and cancer cells, as discussed below. At first, we provide a rapid description of β-AR and nAChR signaling pathways.

Adrenergic receptors (AR)

Adrenergic receptors (AR) are a family of G protein-coupled receptors (GPCR). These receptors can initiate multiple signaling cascades and therefore lead to numerous pathological conditions [START_REF] Strosberg | Structure, function, and regulation of adrenergic receptors[END_REF]. There are two classes of adrenergic receptors, the α-adrenergic and the βadrenergic families [START_REF] Wachter | Beta-adrenergic receptors, from their discovery and characterization through their manipulation to beneficial clinical application[END_REF]. It has been demonstrated that β-ARs may regulate different processes of cancer initiation and progression, including the adenylyl cyclase/cAMP/PKA/CREB pathway (Figure 2 (ex Fig. 3)), which transactivates the epidermal growth factor receptor (EGFR) pathway, the SRC/STAT pathway as well as the arachidonic acid (AA) cascade [START_REF] Maki | The beta-adrenergic system in man: physiological and pathophysiological response. Regulation of receptor density and functioning[END_REF]. The physiological agonists for β-ARs are catecholamine stress neurotransmitters noradrenaline and adrenaline [START_REF] Wallukat | The beta-adrenergic receptors[END_REF], and their physiological inhibitor is a neurotransmitter GABA that can block the activation of adenylyl cyclase. NNK structurally resembles classical β-AR agonists [START_REF] Barnes | Beta-adrenergic receptors and their regulation[END_REF], and consequently, it can activate downstream signaling cascades of this receptor. There is a body of evidence suggesting that NNK can induce cancer through β-AR activation, for which there is evidence from lung and pancreatic adenocarcinoma [START_REF] Schuller | Mechanisms of smoking-related lung and pancreatic adenocarcinoma development[END_REF]. As discussed below, β-AR activation can also modulate mitochondrial biology and energy metabolism, which might participate to the bioenergetic reprogramming of lung exposed to cigarette smoke [START_REF] Miller | Gene and metabolite time-course response to cigarette smoking in mouse lung and plasma[END_REF]. The TSNAs-β-AR signaling pathway can be divided in three branches detailed below: (i) TSNAs-β-AR-PLA2, (ii) TSNAs-β-AR-ERK1/2 and (iii) TSNAs-β-AR-EGFR.

At first, NNK can bind to β-AR on pulmonary epithelial cells and initiate proliferation cell signaling pathways, which leads to lung adenocarcinoma [START_REF] Schuller | The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is a beta-adrenergic agonist and stimulates DNA synthesis in lung adenocarcinoma via beta-adrenergic receptor-mediated release of arachidonic acid[END_REF]. After NNK binding to β-AR, GPCR triggers the activation of adenylyl cyclase and cyclic AMP (cAMP). cAMP itself activates protein kinase A (PKA), which subsequently activates phospholipase-A2 (PLA2)

and defines the TSNAs-β-AR-PLA2 branch. PKA can also stimulate RAP-1 to define the TSNAs-β-AR-ERK1/2 branch. Then, cAMP can also activate EGFR which define the TSNAs-β-AR-EGFR branch.

In the TSNAs-β-AR-PLA2 branch, arachidonic acid (AA) acts as a second messenger (Figure 2) to activate mitogen-activated protein kinases and protein kinase C, which leads to increased DNA synthesis and cell proliferation in adenocarcinoma [START_REF] Schuller | The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is a beta-adrenergic agonist and stimulates DNA synthesis in lung adenocarcinoma via beta-adrenergic receptor-mediated release of arachidonic acid[END_REF]. Cyclooxygenase (COX) and lipoxygenase (LOX) are the two significant metabolic enzymes in AA metabolism [START_REF] Li | COX-2/12-LOX dual pathway, a novel strategy for treatment of multiple myeloma?[END_REF] that have been shown to be overexpressed by pulmonary adenocarcinomas and pancreatic adenocarcinoma cells [START_REF] Hida | Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas[END_REF][START_REF] Molina | Increased cyclooxygenase-2 expression in human pancreatic carcinomas and cell lines: growth inhibition by nonsteroidal anti-inflammatory drugs[END_REF][START_REF] Tucker | Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer[END_REF]. In response to NNK, several products downstream of the COX pathway are produced, among which thromboxane A2 (TxA2) has a significant role in cancer development and metastasis [START_REF] Nie | Differential expression of thromboxane synthase in prostate carcinoma: role in tumor cell motility[END_REF][START_REF] Sakai | Upregulation of thromboxane synthase in human colorectal carcinoma and the cancer cell proliferation by thromboxane A2[END_REF]. In lung cancer cells, NNK leads to higher expression of thromboxane synthase TxAS, which facilitates TxA2 synthesis from PGH2 (prostaglandin H2) and leads to TxA2 receptor activation [START_REF] Huang | 4-Methylnitrosamino-1-3-pyridyl-1-butanone (NNK) promotes lung cancer cell survival by stimulating thromboxane A2 and its receptor[END_REF]. It has been demonstrated that overexpression of TxAS and TxA2 receptor has a potential role in tumorigenesis and promotion of the progression of many types of cancers [START_REF] Nie | Differential expression of thromboxane synthase in prostate carcinoma: role in tumor cell motility[END_REF][START_REF] Huang | Cell signalling through thromboxane A2 receptors[END_REF].

Regarding the TSNAs-β-AR-ERK1/2 branch, the NNK-mediated stimulation of β-AR signaling activates the cytoplasmic serine/threonine kinase effector B-RAF, which further activates signal-regulated kinase 1 and 2 (ERK1 and ERK2) (Figure 2) [START_REF] Vossler | cAMP activates MAP kinase and Elk-1 through a B-Raf-and Rap1-dependent pathway[END_REF]. The main genetic effectors of the TSNAs-β-AR signaling pathways include transcription factors as cAMP response element binding protein (CREB), cAMP response element modulator (CREM), activating transcription factor-1 (ATF-1), activator protein 1 (AP1) and the nuclear factor κB (NF-κB) [START_REF] Walker | Inducible cAMP early repressor ICER downregulation of CREB gene expression in Sertoli cells[END_REF]. Those effectors alter cell proliferation, differentiation, survival, energy metabolism (new Figure 3) and consequently the promotion of oncogenesis [START_REF] Sakamoto | CREB in the pathophysiology of cancer: implications for targeting transcription factors for cancer therapy[END_REF].

Lastly, the TSNAs-β-AR-EGFR involves the transactivation of the EGFR pathway and downstream recruitment of the SRC kinase [START_REF] Luttrell | Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes[END_REF][START_REF] Ahn | Src-mediated tyrosine phosphorylation of dynamin is required for beta2-adrenergic receptor internalization and mitogen-activated protein kinase signaling[END_REF][START_REF] Maudsley | The beta(2)adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor[END_REF][START_REF] Biscardi | Characterization of human epidermal growth factor receptor and c-Src interactions in human breast tumor cells[END_REF][START_REF] Maa | Potentiation of epidermal growth factor receptor-mediated oncogenesis by c-Src: implications for the etiology of multiple human cancers[END_REF] that promotes activation of Ras oncogenic proteins. RAS, as a signal transducer, stimulates RAF and consequently mitogen-activated protein kinase kinases (MEKs), ERK1/2 and ribosomal protein S6 kinases (RSKs). These factors activate transcription factors such as AP1 and Myc, which regulate cell proliferation [START_REF] Schuller | Mechanisms of smoking-related lung and pancreatic adenocarcinoma development[END_REF]. Additionally, SRC can activate K + channels, which can stimulate the AA pathway [START_REF] Muyderman | Activation of betaadrenoceptors opens calcium-activated potassium channels in astroglial cells[END_REF]. It has been reported that through this pathway, NNK regulates the growth and development of lung adenocarcinomas [START_REF] Laag | NNK activates ERK1/2 and CREB/ATF-1 via beta-1-AR and EGFR signaling in human lung adenocarcinoma and small airway epithelial cells[END_REF]. In addition to RAF, RAS can also interact with phosphatidylinositol 3-kinase (PI3K), which is important in various cellular processes (Figure 2). Upon activation of PI3K, a secondary messenger phosphatidylinositol 3,4,5-triphosphate (PIP3) is generated, which further phosphorylates and activates serine/threonine kinase Akt or protein kinase B (PKB). The PI3K/Akt pathway is a critical pathway in cancer, as it is a regulator of key cellular processes that contribute to tumorigenesis, tumor growth and therapeutic resistance. As an example, it has been documented that NNK can, in this way, lead to the phosphorylation of the Bad/Bcl2 antagonist, the dissociation of which hampers the pro-apoptotic activity of Bad and consequently promotes cell survival and chemoresistance [START_REF] Jin | Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional cooperation of Bcl2 and c-Myc through phosphorylation in regulating cell survival and proliferation[END_REF]. The TSNAs-β-AR-EGFR branch includes activation of RAS (Figure 2) and involves stimulation of signal transducer and activator of transcription 3 (STAT3), a transcription factor involved in lung cancer cells proliferation, metastasis and bioenergetics [START_REF] Momi | Nicotine/cigarette smoke promotes metastasis of pancreatic cancer through alpha7nAChR-mediated MUC4 upregulation[END_REF][START_REF] Gough | Mitochondrial STAT3 supports Ras-dependent oncogenic transformation[END_REF][START_REF] Demaria | A STAT3-mediated metabolic switch is involved in tumour transformation and STAT3 addiction[END_REF].

Accordingly, the STAT3 target IKBKE, a serine/threonine kinase, is overexpressed in response to NNK and promotes proliferation [START_REF] Momi | Nicotine/cigarette smoke promotes metastasis of pancreatic cancer through alpha7nAChR-mediated MUC4 upregulation[END_REF]. These findings might suggest that activation of beta-adrenergic signaling by NNK could mediate bioenergetic changes through STAT3 and further molecular investigation will be required to verify this hypothesis.

Nicotinic acetylcholine receptors (nAChRs)

nAChRs consist of five subunits with homo-or heteropentamers that form ligand-gated ion channels located in the plasma membrane of mammalian cells (Figure 3). Initially, these receptors were identified in the nervous system, at the neuromuscular junction, and classified into neuronal or muscle nAChRs (108-111). The neuronal nAChRs can be homomeric (composed of five identical α7, α8 or α9 subunits) or heteromeric (composed of combinations of α2-α6 or α10 subunits and β2-β4 subunits), whereas muscle nAChRs may comprise combinations of α1 subunits with β1, γ, δ or ε subunits [START_REF] Portugal | Genetic variability in nicotinic acetylcholine receptors and nicotine addiction: converging evidence from human and animal research[END_REF]. Binding of agonist to nAChRs leads to the activation of these receptors, which is followed by ion influxes or recruitment of β-arrestin to the receptor to stimulate a number of intracellular signaling pathways [START_REF] Schuller | Is cancer triggered by altered signalling of nicotinic acetylcholine receptors?[END_REF][START_REF] Dasgupta | Nicotine induces cell proliferation by beta-arrestin-mediated activation of Src and Rb-Raf-1 pathways[END_REF].

Acetylcholine is the initial agonist for these receptors, but nicotine and TSNAs can serve as an agonist for nAChRs, especially the α7 subtype [START_REF] Lindstrom | Neuronal nicotinic acetylcholine receptors[END_REF]. The affinity of NNK for α7nAChR was found to be 1,300 fold higher than that of nicotine, whereas the affinity of NNN for heteromeric α-βnAChRs was 5,000 fold higher than that of nicotine [START_REF] Arredondo | Nicotinic receptors mediate tumorigenic action of tobacco-derived nitrosamines on immortalized oral epithelial cells[END_REF][START_REF] Schuller | Tobacco-specific carcinogenic nitrosamines. Ligands for nicotinic acetylcholine receptors in human lung cancer cells[END_REF]. This higher affinity implies that NNK and NNN can win in the competition for binding to nAChRs.

Therefore, many cardiovascular, neuropsychological and cancer-stimulating effects currently attributed to nicotine are probably caused by these nitrosamines [START_REF] Schuller | Is cancer triggered by altered signalling of nicotinic acetylcholine receptors?[END_REF]. It has been shown that the binding of NNK to α7nAChR in lung cells lead to activation of voltage-gated calcium (Ca 2+ ) channels and causes an influx of Ca 2+ and consequently membrane depolarization [START_REF] Sheppard | Activation of voltage-operated Ca2+-channels in human small cell lung carcinoma by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone[END_REF].

Following receptor activation and ion flux, three main sub-pathways have been described: i) nAChr-PKC-Erk1/2, ii) nAChr-VEGF and iii) nAChr-EGFR-RAS/PI3k. These sub-pathways are further discussed below. Please note the redundancy with the sub-pathways described above for the beta-adrenergic signaling also activated by TSNas.

Regarding the nAChr-PKC-Erk1/2 branch, PKC activation leads to stimulation of the serine/threonine-protein kinase RAF (Figure 3). This activation initiates the MEK/ERK cascade as well as the downstream activation of number of transcription factors such as FOS, JUN and Myc, leading to proliferation in lung cancer [START_REF] Jin | Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional cooperation of Bcl2 and c-Myc through phosphorylation in regulating cell survival and proliferation[END_REF]. In this sub-pathway, NNK induces the phosphorylation of cellular proteases (μ-and m-calpain) and leads to the migration and invasion of lung cancer cells [START_REF] Xu | Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone induces phosphorylation of mu-and m-calpain in association with increased secretion, cell migration, and invasion[END_REF]. Additionally, contactin 1, which is a neural cell adhesion molecule located in the plasma membrane, can be upregulated by NNK through the ERK pathway. Contactin 1, for which a role has been demonstrated in the development of various types of cancer including lung adenocarcinoma, is able to interact with several other membrane proteins or the extracellular matrix to activate downstream signaling pathways [START_REF] Reid | Identification and characterization of the human cell adhesion molecule contactin[END_REF][START_REF] Berglund | Molecular cloning and in situ localization of the human contactin gene (CNTN1) on chromosome 12q11-q12[END_REF]. The role of this molecule in lung cancer cell invasion and metastasis via the VEGF-C/Flt-4-mediated SRC-p38-C/EBP pathway has been confirmed [START_REF] Liu | VEGF-C promotes the development of esophageal cancer via regulating CNTN-1 expression[END_REF][START_REF] Su | The VEGF-C/Flt-4 axis promotes invasion and metastasis of cancer cells[END_REF][START_REF] Shigetomi | Abstract 2818: Flt-4 expression on tumor cells and its autocrine mechanism with VEGF-C/contactin-1 stimulate progression of head and neck squamous cell carcinoma[END_REF]. In addition, contactin 1 can prevent PHLPP2-mediated Akt dephosphorylation, which leads to Akt pathway activation [START_REF] Yan | Contactin-1 reduces E-cadherin expression via activating AKT in lung cancer[END_REF]. Additionally, nAChRs activation can activate the SRC kinase that leads in turn to RAF kinase stimulation and induction of the Rb/E2F1 axis which regulates proliferative genes as Cdc25A and Cdc6 [START_REF] Dasgupta | Nicotine induces cell proliferation by beta-arrestin-mediated activation of Src and Rb-Raf-1 pathways[END_REF].

The nAChr-VEGF sub-pathway involves HIF-1α activation and plays a role in nicotine-induced tumor cell proliferation [START_REF] Ma | Alpha5 nicotinic acetylcholine receptor mediates nicotine-induced HIF-1α and VEGF expression in non-small cell lung cancer[END_REF]. In vitro studies performed on human lung cancer cell lines demonstrated that nicotine induces Hypoxia-Inducible Factor-1α Expression through the nicotinic acetylcholine receptor with implication for lung cancer cell migration, and invasion [START_REF] Zhang | Nicotine Induces Hypoxia-Inducible Factor-1 Expression in Human Lung Cancer Cells via Nicotinic Acetylcholine Receptor Mediated Signaling Pathways[END_REF].

Lastly, the nAChr-EGFR-RAS/PI3k sub-pathway involves the NNK-mediated stimulation of PI3K and Akt kinases (Figure 3) that leads to the activation of numerous (ASK1) or MAP3K5, a member of the mitogen-activated protein kinase kinase kinase that activates downstream MAPKs, c-Jun N-terminal kinases (JNKs) and p38 MAPKs, (e) 4EBP-1, which is a substrate for the mTOR signaling pathway and (f) S6K1, which leads to mTOR activation [START_REF] Singh | Nicotinic acetylcholine receptor signaling in tumor growth and metastasis[END_REF]. The activation of nAChRs can also occur through the recruitment of a scaffolding protein, β-arrestin-1.

Additionally, nAChRs might functionally network with β-AR and, consequently, the EGFR pathway (Figure 3). The indirect stimulation of β-AR via nAChRs has been reported in NNK-treated small airway epithelial cells [START_REF] Schuller | The tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is a beta-adrenergic agonist and stimulates DNA synthesis in lung adenocarcinoma via beta-adrenergic receptor-mediated release of arachidonic acid[END_REF].

Nitrosamine signaling implication in the metabolic reprogramming of cancer cells

Alterations in cellular metabolism are considered an important hallmark of cancer, as this reprogramming can be observed in most types of cancer cells [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. In a simplified view, one could describe two main types of metabolic reprogramming: glycolytic and oxidative [START_REF] Obre | Emerging concepts in bioenergetics and cancer research: Metabolic flexibility, coupling, symbiosis, switch, oxidative tumors, metabolic remodeling, signaling and bioenergetic therapy[END_REF]. Glycolytic tumors rely more on glycolysis to generate ATP as well as building blocks, with little contribution of the mitochondrion. Several mechanisms and determinants of the glycolytic phenotype have been described and reviewed in the last decade. Briefly, tissue hypoxia or oncogene mediated pseudo-hypoxia promotes the HIF1alpha-dependent expression of specific glycolytic genes and inhibits oxidative metabolism through PDK1 expression [START_REF] Papandreou | HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption[END_REF]. In contrast, oxidative tumors rely on mitochondrial energy transduction from various sources of carbons as carbohydrates, amino-acids or fats. The determinants of the oxidative profile are also numerous and include specific oncogenes activation (as KRAS in lung tumors [START_REF] Padanad | Fatty Acid Oxidation Mediated by Acyl-CoA Synthetase Long Chain 3 Is Required for Mutant KRAS Lung Tumorigenesis[END_REF] or Myc in breast cancer ( 92)) as well as environmental factors as tissue perfusion [START_REF] Hensley | Metabolic Heterogeneity in Human Lung Tumors[END_REF] or oxidative stress [START_REF] Gentric | PML-Regulated Mitochondrial Metabolism Enhances Chemosensitivity in Human Ovarian Cancers[END_REF]. Cancer cells metabolic reprogramming can occur independently of metabolic substrates availability as exemplified by aerobic glycolysis or Warburg effect, where cancer cells use glycolysis even in presence of oxygen. The bioenergetic profile of a given tumor depend on several parameter and inter-patient as well as intra-tumor heterogeneity have been described [START_REF] Hensley | Metabolic Heterogeneity in Human Lung Tumors[END_REF]. A large number of signaling pathways have been involved in the reprogramming of tumor energy metabolism but, surprisingly, little attention has been given to the role of nitrosamine signalling in this hallmark of cancer. As discussed above, several lines of evidence indicate that nitrosamines can activate the PI3K/Akt, Myc, HIF1alpha, KRAS and NRF2 signaling pathways through β-AR, nACHr and ROS-dependent or oncogenic-dependent signaling. These pathways also play a role in the control of energy metabolism, suggesting that nitrosamines could participate to the reprogramming of cancer cells bioenergetics, in concert with changes in other cancer hallmarks such as genomic and epigenomic alteration, deregulated cell proliferation and increased migration (Figure 4).

PI3K/Akt :

The PI3K/Akt pathway, which is located downstream of receptor tyrosine kinase (RTK), is a crucial actor in metabolic reprograming that leads to cell growth (161,163).

Inappropriate activation of PI3K, which leads to activation of the pro-survival kinase Akt, is among the most common perturbations in various human cancers; several well-characterized oncogenes and tumor suppressors are located in this network [START_REF] Yuan | PI3K pathway alterations in cancer: variations on a theme[END_REF]. One of the consequence of activation of this pathway is the promotion of glucose uptake and glycolytic metabolism, directing cellular metabolism toward anabolic growth through Akt-mediated membrane translocation of glucose transporters, and Akt-dependent activation of hexokinase and phosphofructokinase [START_REF] Buzzai | The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid beta-oxidation[END_REF]. By phosphorylation and activation of ACL, Akt can stimulate the conversion of citrate to acetyl-CoA and OAA, which leads to de novo fatty acid synthesis [START_REF] Berwick | The identification of ATPcitrate lyase as a protein kinase B (Akt) substrate in primary adipocytes[END_REF]. A major consequence of the activation of this pathway is the activation of the cell growth regulator mTOR complex 1 (mTORC1). This downstream activation is mediated by the inhibitors of this regulator: TSC2 or proline-rich Akt substrate 40 kDa (PRAS40) [START_REF] Laplante | mTOR signaling in growth control and disease[END_REF].

Activation of mTORC1 enhances protein synthesis by phosphorylation of the translational regulators 4E-binding protein 1 (4E-BP1) and S6 kinase 1 (S6K1) [START_REF] Laplante | mTOR signaling in growth control and disease[END_REF]. Moreover, mTORC1

activation has an effect on the promotion of mitochondrial biogenesis and respiration, gluconeogenesis as well as many other metabolic processes. These effects can be modulated by a nuclear transcriptional coactivator, PGC1α [START_REF] Handschin | Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism[END_REF](100)(101)(102). mTORC1 also has an effect on a number of transcription factors that coordinate metabolic gene expression: HIF1α, Myc, and sterol regulatory element-binding protein 1 (SREBP-1) (103).

The impact of NNK on the PI3K/Akt pathway will also depend on the microenvironment of the cell, in particular the availability to nutrients and to oxygen. As mentioned above, the PI3K/Akt pathway responds to external growth factors and hormonal signals by promoting glucose transport, aerobic glycolysis, and anabolic synthesis of macromolecules.

Subsequently, in the presence of insufficient glucose to feed this pathway, the cell should find another source to supply its ATP demand. Adenosine monophosphate-activated protein kinase (AMPK) is a known sensor of cellular energy status that monitors ATP levels in the cell and is activated under energetic stress, when intracellular ATP decreases and intracellular AMP increases, such as during hypoxia or nutrient deprivation. Activation of this kinase stimulates a program of metabolic adaptation to maintain energy homeostasis (by limiting energy consumption or enhancing energy production) and thus allows adaptation to a given metabolic stress (104,105). In fact, upstream of this kinase is a serine-threonine kinase, liver kinase B1 (LKB1), which is a tumor suppressor and is considered an activator of AMPK.

The LKB1/AMPK pathway, as a metabolic checkpoint in the cell, initiates suppression of the PI3K pathway by inhibiting the mTORC1 pathway and arresting cell growth under conditions of low intracellular ATP levels (106). mTORC1 inhibition is accomplished through phosphorylation of TSC2 and regulatory associated protein of mTOR (raptor) (107,108). By this inhibition, AMPK blocks protein translation and fatty acid synthesis, which consume ATP. Fatty acid synthesis can be blocked by acetyl-CoA carboxylase-1 (ACC1) phosphorylation ( 106). Another consequence of AMPK activation is the induction of autophagy (109). Moreover, fatty acid oxidation will increase to generate acetyl-coenzyme A (CoA) as a substrate for the TCA cycle and oxidative phosphorylation [START_REF] Buzzai | The glucose dependence of Akt-transformed cells can be reversed by pharmacologic activation of fatty acid beta-oxidation[END_REF]. In metabolic adaptation, some of the downstream effects of AMPK are related to phosphorylation and activation of p53 tumor suppressor. This tumor suppressor is one of the most important transcription factors, playing a vital role in the response and regulating metabolic stress to prevent tumor development. p53 activation stimulates transcriptional activity and initiates a metabolic cell cycle checkpoint that restricts cell proliferation (110). As a vital cancer defender, p53 has a diverse range of functions in the promotion of oxidative phosphorylation and downregulation of glycolysis, and its mutation can lead to cancer development. Moreover, as p53 regulates cell growth and autophagy, its role is significant in the cellular response to starvation (111).

HIF1α: the Hypoxia-induced factor 1-alpha (HIF1α) is responsible for metabolic adaptation in hypoxia. At first, during hypoxia the main reason to use glycolysis is the lack of oxygen and HIF-1 further increases glycolysis through the induction of glycolysis related genes (112).

Enhancement of the glycolytic machinery involved the increased expression of glucose transporters, glycolytic enzymes, and of PDK1 that will in turn inhibit pyruvate dehydrogenase and glucose-dependent oxidative phosphorylation [START_REF] Papandreou | HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption[END_REF]. The other transcription factor, Myc, which is activated downstream of mTORC1, also acts in the regulation of cell growth and proliferation (113). Myc targets genes that support the proliferative utilization of glutamine, including glutamine transporters, and those involved in both mitochondrial biogenesis and glutaminolysis. It can also stimulate enhanced expression of many genes involved in glucose uptake, glycolysis, and lactate production (114). Myc increases the expression of many genes that support anabolic growth, including transporters and enzymes involved in glycolysis, fatty acid synthesis, glutaminolysis, serine metabolism, and mitochondrial metabolism (115). Myc-transformed cells undergo apoptosis in the absence of exogenous glutamine (116)(117)(118). Moreover, Myc also induces the expression of enzymes in other anabolic pathways, such as serine hydroxymethyltransferase (SHMT) (serine/glycine metabolism) and fatty acid synthase (FAS)(lipid biosynthesis) (119). The last mentioned transcription factor, SREBP (sterol regulatory element-binding protein), plays a role by inducing the expression of several genes involved in fatty acid biosynthesis in response to growth factor signaling (120). As a downstream effector of mTORC1, SREBP1 can lead to the deregulation of de novo lipid synthesis (121).

KRAS:

Regarding KRAS, NNK can induce activating-KRAS mutations and causes primarily lung adenomas (in susceptible mouse strains) (122). Ras is a type of small GTPbinding protein that functions as a component of several signaling cascades that function in other various signaling cascades, such as RAF/MEK/ERK, PI3K/Akt, and RalEGF/Ral pathways ( 123). RAS has different subtypes, such as H-RAS, N-RAS and K-RAS, and the effect of each of them differs in mediating different signaling pathways. These genes are commonly mutated in human cancer, leading to cellular proliferation [START_REF] Osada | Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer[END_REF]124). KRAS mutation is more frequent in comparison to the other subtypes in most cancer types (including lung adenocarcinoma) (125). Activation of KRAS can increase the glycolytic flux but decreases glucose consumption in the TCA cycle and associated anabolic pathways. Instead, to sustain biosynthetic reactions, this oncogene activation promotes the utilization of glutamine for anabolic pathways (126). Thus, in KRAS mutated cells, glutamine can be used as both carbon and nitrogen sources for biosynthetic reactions to sustain cell growth and quench ROS production metabolism, leading to an efficient utilization of both carbon and nitrogen and in this way playing an essential role in the proliferation and survival of cancer (127-129). Through MEK/ERK kinases activation, KRAS can phosphorylate proteins directly or promote protein phosphorylation and consequently regulate various cellular functions (130). ERK signaling downstream of KRAS mutation can lead to lipogenesis and alter lipid metabolism in cancer ( 131). The proposed target in this pathway inducing lipogenesis is fatty acid synthase (FASN) (126). Additionally, the PI3K/Akt/mTOR pathway is critical for NNKinduced lung tumorigenesis ( 132).

NRF2:

The potential role of NFE2-related factor 2 (or nuclear respiratory factor) (NRF2) in NNK-mediated metabolic reprogramming was suggested by studies showing that NNK can induce oxidative stress in lung tissues in mice and rats by creating a major premutagenic lesion, 8-hydroxy-2'-deoxyguanosine , which is considered a marker of DNA oxidative damage produced by . ROS are intracellular chemical species that contain oxygen and include the superoxide anion (O2 -), hydrogen peroxide (H2O2), and hydroxyl radical (OH•). As these radicals have a single unpaired electron, they are highly reactive (137). The initiator of ROS in aerobic cells is O2 -, which is generated from the incomplete reduction of molecular O2 to H2O in the mitochondrial respiratory chain and peroxisomal oxidation (138) (and cytosolic NADPH oxidases (NOXs) (139)). After O2 - generation, it can be converted to H2O2, which is facilitated in this reaction by superoxide dismutase 1 or 2. H2O2 can be converted either to H2O (catalyzed by peroxiredoxins (PRXs)

and consequently detoxified, or to OH• in the presence of ferrous or cuprous ions. OH• can lead to the oxidation of lipids, proteins, and DNA, which consequently damage cells (140).

Low amounts of ROS can be considered a positive regulator of cell proliferation and cellular adaptation to metabolic stress (particularly H2O2) (141), while higher amounts of ROS can lead to cell death signaling pathways, initiated by H2O2 and followed by OH• generation, which can damage cellular components. In such conditions of oxidative stress the main function of NRF2 is to activate the cellular antioxidant response. Thus, NRF2 is an important regulator of cell survival and is considered a main defense system of the cells (142). The primary regulator of NRF2 is Kelch-like ECH-associated protein 1 (KEAP1). KEAP1 is a substrate adaptor for a Cul3-containing E3 ubiquitin ligase. KEAP1 and the E3 ubiquitin ligase complex degrade NRF2 to maintain its low level. High levels of fumarate can also cause KEAP1 inactivation and consequently stabilize NRF2. Excess NRF2 binds to antioxidant response elements (AREs) and turns on nDNA stress-response genes, including haem oxygenase 1 (HMOX1), which appears to be important in tumorigenesis. NNK can stimulate the generation of mitochondrial ROS and mimic the effects of hypoxia to induce inside the mitochondrion where it could phosphorylate a complex-I subunit, leading to changes in cell respiration (165,166). Similar findings have been reported for STAT3 with a mitochondrial-form of this transcription factor involved in the stimulation of OXPHOS [START_REF] Gough | Mitochondrial STAT3 supports Ras-dependent oncogenic transformation[END_REF]167)

Conclusion

Cigarette smoke, which is the predominant form of tobacco consumption, is the largest preventable cause of cancer, attributed to approximately 30% of all cancer deaths (182-184).

Cigarette smoke has a complex composition, and the exact mechanism by which these components can lead to carcinogenesis is still not well understood. To understand the molecular events related to cigarette smoke, several epigenetic (185-187), genomic (188-190), transcriptomic (191,192), proteomic (193-195) and metabolomic (196,197) studies have been conducted. In particular, metabolomics showed that tobacco smoke immediately ( 2hours) leads to altered level of glycerophospholipids, glutamate and 2-octenoylcarnitine in the blood, suggesting changes in lipid and glutamine metabolism (190). Conversely, long-term effect studies identified specific metabolites that varied in proportion with tobacco-smoke exposure (189). These included nicotine metabolites and xenobiotic metabolites involved in benzoatic or xanthine metabolism as well as specific amino acids, lipids, vitamins or cofactors and one carbohydrate. No metabolite from glycolysis or TCA cycle were identified in these studies. However, the specific changes that occur in lung tissue were not investigated in these studies focused on the systemic changes induced by tobacco-smoke. Therefore, metabolomics studies will be required on lung tissue of animal model expose to TSNAs to evidence potential changes at the level of energy metabolism.

Cancer metabolism, an emerging hallmarks of cancer, is an important aspect of cancer initiation and development, the reprogramming of which is often a consequence of tumorigenic mutations and epigenetic alterations (198). Further dissecting the role of cancer metabolism and tracking its plasticity in addition to other molecular aspects of cancer studies can help us to better understand the pathobiology of cancer, which will in turn lead to the identification of early detection biomarkers and/or therapeutic targets. Cigarette smoke has been reported to enhance lactate production in human bronchial epithelial cells and lead to glucose consumption (199). Moreover, one of the most important effects of cigarette smoke is the induction of oxidative stress. Activation of aryl hydrocarbon receptor (AHR) by cigarette smoke can be associated with the expression of oxidative stress and antioxidant genes (200)(201)(202)(203), as documented in an human airway epithelium cell line (i.e., activation caused increased ROS production ( 204)). AHR is a cytosolic ligand-activated transcription factor that is activated upon encountering multiple foreign ligands, such as cigarette smoke. Activation of AHR can lead to the transcription of genes related to biotransformation enzymes such as CYP1A1. A prominent role of CYP1A1 is related the metabolism of some carcinogens found in cigarette smoke, such as nitrosamines and poly aromatic hydrocarbons (205). Upon chronic exposure of lung cancer cells to cigarette smoke, higher expression of AHR regulatory genes are observed, followed by an alteration in mitochondrial proteins, specifically enzymes involved in TCA and OXPHOS. Moreover, this chronicity leads to higher expression of glutamine synthase, fatty acid degradation and lactate synthesis (195), in addition to reduced expression of many glycolytic enzymes such PKM2 (195), which is a rate-limiting glycolytic enzyme and acts as a key component in tumor metabolism and growth. As a consequence of the lower activity of this enzyme, the upstream intermediates of glycolysis will accumulate and enter anabolic pathways (206). As higher ROS production can lead to inhabitation of PKM2 (207), mitochondria exposed to cigarette smoke are metabolically altered to support their survival and proliferation (195). In general, there is a body of evidence that indicates cigarette smoke plays a potential role in mitochondrial metabolic reprogramming (195). All these reports indicate that cigarette smoke plays a profound role in metabolic alterations.

Several studies have also examined the role of each carcinogenic component of tobacco smoke separately, such as the investigations of TSNAS reviewed in this article. Despite several reports on this subject, little is known about their mechanisms, especially regarding metabolic reprogramming. This suggests that more research in the field is necessary.

  nitrosamines consists of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-4-(3-pyridyl) butanal (NNA), N′-nitrosonornicotine (NNN), N′nitrosoanabasine (NAB), N′-nitrosoanatabine (NAT), 4-(methylnitrosamino)-1-(3-pyridyl)-1butanol (NNAL), 4-(methylnitrosamino)-4-(3-pyridyl)-l-butanol (iso-NNAL), and 4-(methylnitrosamino)-4-(3-pyridyl) butanoic acid (iso-NNAC) (3), among which NNN and NNK, due to sufficient evidence in experimental animals, are classified as human carcinogens

  by nitrosamines, and little attention has been given to the global impact of TSNAs on the emerging cancer hallmarks. The hallmarks of cancer include the sustained proliferative signaling, evasion of growth suppressors, resistance to cell death, enabling of replicative immortality, induction of angiogenesis, activation of invasion and metastasis, reprogramming of energy metabolism, genome instability and mutation, avoidance of immune destruction and promotion of tumor inflammation. In particular, one emergent hallmark of cancer is metabolic reprogramming, and our review will discuss the link between TSNAs and the changes in tumor bioenergetics associated with carcinogenesis. In particular, the main signaling pathways activated by TSNAs, namely β-AR and nAChR signaling, have also been involved in the regulation of energy metabolism but only few studies have considered the direct bioenergetic impact of TSNAs on pre-cancer or cancer cells. The modulation of mitochondrial biology by TSNAs is another point of discussion as changes in organelle biogenesis were described upon activation of Beta-adrenergic signaling. Lastly, TSNAs have been shown to activate different proto-oncogenes that could consecutively modulate energy metabolism. These different aspects are covered in what follows.

  downstream effectors: (a) forkhead transcription factors (FAKHR or FOXO), which have a wide range of roles in physiological processes, including cell cycle arrest, apoptosis, angiogenesis, stress resistance, energy metabolism, and stem cell differentiation (86), (b) serine/threonine kinase glycogen synthase kinase-3 (GSK-3), which can regulate glycogen synthesis, (c) tuberous sclerosis complex 2 (TSC2) or tuberin, which can control mechanistic target of rapamycin (mTOR) signaling pathway, (d) Apoptosis signal-regulating kinase 1

  HIF1α accumulation and activity in lung cancer cell lines (143). In cancer cells, DNA lesions can also lead to excess generation of ROS. Higher ROS levels can stimulate the PI3K and MAPK/ERK signaling pathway and activate transcription factors such as HIF1α and. Many lines of evidence indicate that levels of intracellular ROS were closely linked to HIF1α stabilization, possibly through the modification of prolyl-hydroxylase (PHD) activity (147). Furthermore, mitochondria, which are the main source of ROS production, appear to play a critical role in the regulation of HIF1α stability (148). Intratumoral hypoxia can cause metastasis, which leads to cancer progression (149). Based on the available evidence, most cellular consequences related to hypoxia are mediated by the HIF1, which consists of two proteins -HIF1α and HIF1β. These two proteins can bind to hypoxia response elements (HREs) in the promoter region of numerous metastasis-associated genes and in this way promote metastasis (147,150). HIF1α degradation under normoxia is regulated by PHD enzymes, growth factor stimulation and free radical generation.β-AR-and nAChR-dependent regulation of mitochondrial biology: Biochemicalstudies have shown that mutations in β-AR receptor can increase cell respiration and fatty acid oxidation (151). This increase in cell respiration as a result of activation of β-AR associated with an increased production of ROS (152). It is also demonstrated that β-AR signaling through agonist binding can induce mitochondrial biogenesis (153-156) and in this way modulate oxidative metabolism, energy expenditure, lipolysis, glucose transport, and glucose oxidation (157-159). The regulation of mitochondrial biogenesis by nitrosamine signaling could involve stimulation of the nuclear transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). This transcriptional coactivator interacts with CREB and and nuclear respiratory factors 1 and 2 to (NRF1 and 2) (160). As discussed above, CREB is activated by stimulation of Beta-adrenergic signaling (Figure2).The transcription factor CREB can induce the expression of a series of genes involved in mitochondrial biogenesis and mitochondrial respiration(161)(162)(163). CREB participates to the so-called retrograde signaling involved in the stimulation of mitochondrial function in response to increased energy needs (163). Studies in neurons further showed that CREB can act directly on mitochondrial DNA-encoded gene transcription (164), raising the need to investigate this possibility in the context of cancer. Likewise, the SRC kinase and STAT3, two components of the non-canonical activation of β-AR signaling have also been related to the stimulation of mitochondrial function. For instance, SRC kinase has been discovered

Figure 1 .

 1 Figure 1. Schematic illustration of the NNK and NNN pathways producing DNA adducts. Via P450s, NNK is metabolized to α-hydroxy NNK or can undergo reduction to NNAL, which is converted to NNAL glucuronides (NNAL-Glucs). Like NNK, NNAL is metabolized to α-hydroxy NNALs by P450s, and then both α-hydroxy NNKs and α-hydroxy NNALs are decomposed to diazonium ions and aldehydes. NNN undergoes α-hydroxylation, as well by P450s and is converted to α-hydroxy NNNs. α-hydroxy NNKs, α-hydroxy NNALs and α-hydroxy NNNs are unstable products, and they are rearranged into diazonium ions, which are highly reactive and produce DNA adducts. If DNA adducts are not repaired they can cause miscoding followed by mutational activation of oncogenes and/or inactivation of tumor suppressor genes. As a consequence, these mutations lead to loss of normal cellular growth control functions which results in cellular proliferation and cancer (208). DNA adducts formation happen both in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA)(209). Based on critical role of mitochondria in cellular energy production, apoptosis, and cellular growth and differentiation, its alteration may be involved in the carcinogenic process.These alterations can happen due to accumulation of some carcinogens in mitochondria that can bind to mtDNA and remain because of low-efficiency mtDNA repair mechanisms (210,211). There is growing body of evidence that confirm the role of mtDNA mutations in cancer. Such evidence includes the link between mtDNA mutations and constitutive oxidative stress in cancer cells. Moreover, in malignant cells, mtDNA abnormalities and alterations in respiratory activity seems a general feature. These mutations can changes the cell surface produced by mtDNA mutations in cancer cells. Abnormal expression of mtDNA-encoded proteins due to mutations and deletions in mtDNA, have been observed in various solid tumors (212). It is reported that pyridyloxobutyl (POB)(213) and pyridylhydroxybutyl (PHB) (214) DNA adducts form and accumulate in NNK chronicaly treated rats (209). Based on this study, NNK treatment in rats gives higher levels of POB and PHB adducts in mtDNA than in nDNA of their lungs (209). NNK can form POB-DNA adducts via methyl hydroxylation. It can also convert to NNAL by carbonyl reduction. NNAL can form PHB-DNA adducts via hydroxylation of the methyl group(215,216). Results from this study support the hypothesis that tobacco carcinogens can bind to mtDNA and consequently develop smoking-induced lung cancer (209).

Figure 2 .

 2 Figure 2. β-adrenergic receptor (B-AR)-mediated nitrosamines signaling. Binding of TSNAs as agonists to β-AR on cancer cells activates GTP-binding proteins (G-proteins) coupled to receptor signaling, resulting in the activation of adenylyl cyclase and cyclic AMP

Figure 3 .

 3 Figure 3. Nicotine/Acetylcholine receptor (nAChR)-mediated nitrosamine signaling. Binding of TSNA to nAChRs causes a change in the conformation of the receptor subunits, which opens the receptor gates. Opening the gates, Ca 2+ flows into the cell, which leads to membrane depolarization. This depolarization is enhanced by activation of the gate of voltage-activated Ca 2+ -channels and greater influx of Ca 2+ . The increased intracellular Ca 2+ concentration triggers EGFR/RAF/MAPK, PI3K/Akt signaling pathways. Moreover, this depolarization leads to the release of the autocrine growth factor serotonin and the angiogenic factor VEGF. Serotonin can also activate PKC and its downstream RAF and the MAPK cascade. All these pathways can induce activation of different transcription factors and, consequently, regulate gene and protein expression; this leads to the stimulation of cell proliferation and migration and inhibition of apoptosis. In addition, nAChR can indirectly stimulates β-AR signaling through the release of adrenaline and noradrenaline.

Figure 4 :

 4 Figure 4: Regulation of energy metabolism by tobacco-smoke derived nitrosamines (TSNAs). Two main mechanisms can relate nitrosamine signaling to the regulation of energymetabolism. On the one hand, TSNAs activate the beta-adrenergic and the acetylcholinenicotine receptors to activate a series of transcriptional regulators (pink box) that will in turn trigger the expression of various energy genes involved in glucose metabolism, lipid metabolism, autophagy and mitochondrial function. On the other hand, TSNAs induce oxidative stress and genotoxicity as well as oncogenic activation that also induce a metabolic response. Fine tuning of the metabolic response to TSNAs exposure will depend on the cellular context, the microenvironment and the type and dose of exposure.

  . It is also suggested that β-AR can regulates mitochondrial function directly and indirectly through cAMP production. In this context, cAMP activates various effectors like PKA and Epac that lead to PGC1α activation(168)(169)(170)(171)(172)(173). Moreover, other B-AR signaling could trigger mitochondrial biogenesis through Ca 2+ and CamKKβ or AMPK (174-177).

	Regarding nAChR, cell biology studies have revealed that this receptor is expressed
	on mitochondrial outer membrane of non-neuronal cells (178). It was also demonstrated that
	mt-nAChR is coupled with voltage-dependent anion channel (VDAC) and regulate
	Ca 2+ accumulation and cytochrome c release during apoptosis. The expression of mt-nAChR
	can also regulate mitochondrial permeability transition (179) and it was proposed that
	activation of mt-nAChR by some agonists that can permeate the cells (such as nicotine) may
	inhibit mPTP opening to reduce apoptosis (178-181).

International Agency for Research on Cancer IDH1: isocitrate dehydrogenase 1 iso-NNAC: 4-(methylnitrosamino)-