Lung carcinoma signaling pathways activated by smoking

β-Adrenergic Receptor–Mediated Signaling

β-Adrenergic receptors β-ARs) are members of the G-protein–coupled receptor family. The tobacco-specific nitrosamine, nicotine-derived NNK, is structurally similar to the classical β-AR agonist[7]^,[8] and can bind β-AR on pulmonary epithelial cells. This binding stimulates cell proliferation signaling pathways by triggering β-AR activation[9], suggesting that β-ARs play an important role in NNK-induced lung cancer (Figure 1). Once G-protein–coupled receptor family signaling is activated in NNK-treated lung adenocarcinoma cells, it can trigger the activation of adenylyl cyclase and cyclic AMP (cAMP) and the subsequent activation of protein kinase A (PKA). PKA further activates phospholipase-A2 and causes an NNK concentration-dependent release of arachidonic acid (AA) from cell-membrane phospholipids, increasing DNA synthesis and cell proliferation in adenocarcinoma. This signaling pathway can be completely blocked by β-AR antagonists[7]. In addition, both aspirin, a cyclooxygenase (COX) inhibitor, and ML-886, a lipoxygenase inhibitor, can partially inhibit DNA synthesis in cells after being exposed to NNK, suggesting that the AA-dependent mitogenic signaling transduction cascade' s role in Smoking-related lung Carcinogenesis involves both COX- and lipoxygenase-dependent messengers[7]. High systemic levels of AA-metabolizing enzymes COX-2[10], AA metabolites prostaglandin E2 and leukotriene B4[11] have been identified in lung cancers in NNK-treated mice. Consistently, COX-2–positive tumors are more common in smokers (32%, 29 of 90) than in non-smokers (10%, 1 of 10, P = 0.15)[10].

In addition to AA metabolites, several transcription factors can be stimulated as downstream targets of β-AR/PKA signaling, such as the PKA-dependent overexpression and phosphorylation of cAMP response element binding protein (CREB). Phosphorylation of CREB induced by NNK in human small airway epithelial cells and adenocarcinoma cells[12] and overexpression of p-CREB in NNK-induced adenocarcinomas[13]^,[14] have been reported. CREB regulates the expression of certain genes, including Cyclins, Bcl-2 family members and Egr-1, whose aberrant expression promotes oncogenesis[15]. An inhibitor of the CREB signaling pathway can block CREB activation in lung cancer cells by arresting the cell cycle at the G₂/M phase and by inducing apoptosis through suppression of Bcl-2 and Bcl-X_L expression[16]. However, CREB activation may not be specific in Smoking-related lung cancers. An immunohistochemical analysis of a tissue microarray containing adenocarcinoma, bronchioloalveolar carcinoma, and squamous cell carcinoma specimens collected from 310 patients revealed a significant association between decreased survival and CREB or p-CREB overexpression in non-smokers but not in current or former smokers with lung cancer[17].

β-AR–initiated cAMP signaling may transactivate the epidermal growth factor receptor (EGFR) pathway, including overexpression of EGFR-specific phosphorylated tyrosine kinase, Raf-1, and extracellular signal-regulated kinase 1/2 (ERK1/2) and ERK1/2 phosphorylation (Figure 1). cAMP signaling and the EGFR/Raf-1/ERK1/2 pathway can synergistically regulate the growth and development of NNK-induced lung adenocarcinomas [12]^,[13]. NNK phosphorylates EGFR at tyrosine residues 991, 1068 and 1173 via β-adrenergic stimulation[12]. However, in one study, no mutations were detected in EGFR exons 18–21, which typically occur in non-smokers with lung adenocarcinomas[12]. The phenomenon suggests that the EGFR pathway plays an important role in Carcinogenesis through distinct mechanisms in smoking- and non-smoking–related lung adenocarcinomas.

Another signal transduction pathway, phosphatidylinositol 3-kinase (PI3K)/Akt, is reported to induce Bad phosphorylation downstream of β-AR/PKA signaling (Figure 1). In human lung adenocarcinoma A549 cells, nicotine induces ERK1/2, Akt, and PKA activation through an upstream β-AR to trigger multi-site Bad phosphorylation. Bad phosphorylation blocks apoptosis and subsequently promotes cell survival[18].

Nicotinic Acetylcholine Receptor–Mediated Signaling

Nicotinic acetylcholine receptors (nAChRs) are ion channels located in the plasma membrane of mammalian cells. Initially identified at the neuromuscular junction, nAChRs are classified as neuronal or muscular. Neuronal nAChRs are composed of five identical α7, α8, or α9 subunits or a combination of α2–α6 or α10 subunits and β2–β4 subunits, whereas muscle nAChRs are composed of combinations of α1 subunits with β1, γ, δ, or ε subunits[19]. Binding of agonist to nAChRs results in conformational changes to the receptors, with consequent ion influxes. These can result in the release of neurotransmitters or the stimulation of various intracellular signaling cascades[20]. Nicotine can mimic the actions of acetylcholine as an agonist by binding to the a subunits of nAChRs[20]. Up-regulated expression and activation of nAChRs has been reported in both lung cancer and normal bronchial epithelial cells by exposure to nicotine or NNK[21]^–[24].

Nuclear Factor–кB Signaling

Nuclear factor-κB (NF-κB) is a ubiquitous nuclear transcription factor. The activation of NF-κB by smoke may be mediated by ERK1/2 signaling[60]^,[61] or through a pathway similarto that of tumor necrosis factor[62]. NF-κB is in the inactive state in the cytoplasm as a heterotrimer consisting of p50, p65, and IκBα subunits. Upon activation, IκBα undergoes sequential phosphorylation by IκBα kinase and ubiquitination and degradation; the p50/p60 heterodimer is released and translocated to the nucleus, where it binds to specific sequences in the promoter regions of target genes[63].

In squamous cell lung cancer and lung adenocarcinoma, smoke has been reported to induce NF-κB activation, which regulates the expression of downstream molecules such as COX-2[62], cyclin D1, matrix metalloproteinase-9[64]^,[65], p21, C-IAP2, Bcl-2, and Bad[61]^,[62]^,[66], consequently promotes cancer cell proliferation and survival.

Furthermore, NF-κB directly exerts carcinogenic effects to enhance cancer cell growth. Cigarette smoking induces the production of pro-inflammatory factors, such as COX-2, prostaglandin E2[67], IL-8[68], IL-6, and macrophage inflammatory protein 2[69], via NF-κB activation in normal human lung fibroblasts and alveolar macrophages, creating a pro-inflammatory environment. Long-term smoking induces chronic inflammatory lung diseases. Emerging evidence suggests that chronic inflammation plays a significant role in lung cancer pathogenesis by promoting tumor formation[70] and NF-κB activation may be a link between chronic inflammation and lung cancer.

EGFR Signaling

EGFR may be activated by its autocrine ligands, which are produced by oxygen radicals during cigarette smoking. In lung cancer cells, tobacco smoke-generated oxygen radicals stimulate the tumor necrosis factor-α-converting enzyme, resulting in an increased shedding of EGFR pro-ligands, such as transforming growth factor-α, heparin-binding EGF-like growth factor, and amphiregulin. By binding to EGFR, these ligands activate EGFR phosphorylation and induce the expression of downstream targets such as IL-8 and mucin, resulting in lung cancer cell proliferation[71]^–[73].

Gamma-Aminobutyric Acid Signaling

Gamma-aminobutyric acid (GABA) is the most common inhibitory neurotransmitter in the central nervous system. In a recent study, significant GABA underexpression was reported in NNK-induced hamster lung adenocarcinoma[74]. An in vitro study using the immortalized human small airway epithelial cell line HPL1D and human lung adenocarcinoma cell line NCI-H322 revealed that signaling via the GABA receptor strongly inhibited base-level and isoproterenol-induced cAMP, p-CREB, element-luciferase and pERK1/2, effectively blocking DNA synthesis and cell migration[74]. Therefore, GABA may have a tumor-suppressor function in small airway epithelial cells and lung adenocarcinomas. GABA down-regulation by NNK may contribute to cancer development in smokers.

Conclusions

The number of smokers in the United States is decreasing. However, tobacco smoke is still the major etiologic risk factor for lung cancer, which is more common in previous smokers than in current smokers. A comprehensive understanding of the signals activated by Smoking-related carcinogens may help us develop targeted therapy for lung cancer patients with a history of smoking.