Introduction
Tobacco smoke is strongly linked to the onset of various types of human malignancies. According to epidemiological studies, about 30% of cancer deaths every year in the United States are associated with exposure to tobacco smoke or tobacco products, indicating the importance and urgency for cessation of active and passive cigarette smoke [
1,
2]. Tobacco smoke is known to be the main cause of lung, head and neck tumors [
1,
3‐
5]. Recently, evidence has been emerging for the increasing breast cancer risk associated with tobacco smoke exposure [
6‐
9]. Nicotine, one of the important constituents of tobacco interacts with nicotine acetylcholine receptors (nAChR) and functions in either the motor endplate of muscle or at the central nervous system for the establishment of tobacco addiction [
10‐
13]. Studies also showed that nAChR is expressed in various non-neuronal cells and the ligation of the receptor activates various intracellular signaling pathways in these cells, suggesting that nicotine has the potential to regulate cell proliferation [
14‐
16]. It was reported that nicotine potently induced secretion of different types of calpain from lung cancer cells, which then promoted cleavage of various substrates in the extracellular matrix to facilitate metastasis and tumor progression [
5]. In mammary epithelial or tumor cells, the exposure of nicotine initiated a signaling cascade that involved PKC (protein kinase C) and cdc42, and consequently accelerated cell migration [
7]. Furthermore, the anti-apoptotic property of nicotine in breast cancer cells has been demonstrated to be through upregulation of Bcl-2 family members [
8]. The addition of nicotine desensitized MCF7 cells to doxorubicin-mediated cyctoxicity [
17]. All these data indicate that nicotine plays a positive role in the regulation of cell growth and survival. However, the underlying mechanisms of nicotine in facilitating mitogenic activities remain unclear.
nAChR consists of nine α-subunits (α2 to 10) and two β-subunits (β2 and 4) [
10‐
13]. The subunits of nAChR form heteromeric or homoeric channels in different combinations in neuronal cells, which are highly Ca
++ permeable to allow the penetration of Ca
++ flux [
10‐
13]. Upon the engagement with nAChR in non-neuronal cells, nicotine activates calmodulin-dependent protein kinase II, PKC, phosphodylinositol-3-kinase (PI3K)/Akt and Rac family that are often involved in the regulation of cell growth, adhesion or migration [
7,
18‐
20]. The activation of nicotine receptors was also shown to trigger Ras/Raf/MEK/ERK--Ras/Raf/MEK (mitogen-activated protein kinase)/ERK (extracellular-signal-reguated kinase)-- signaling [
7,
21,
22]. In addition, the involvement of nicotine in the activation of the tyrosine kinase JAK-2 (Janus Kinase-2) and transcription factor STAT-3 (Signal Transducer and Activator of Transcription-3) in oral keratinocytes was also observed [
22].
The epidermal growth factor receptor (EGFR) is a transmembrane protein receptor that possesses an intrinsic tyrosine kinase activity [
23,
24]. The EGFR family consists of several members, including EGFR, ERBB2/HER2/NEU, ERBB3 and ERBB4. The ligation of EFGR activates mitogenic-related signaling pathways, leading to various cellular responses. An increased level of mutation of EGFR has been detected in many human tumors, including breast cancer, which were often accompanied with a poor prognosis [
25,
26]. Upon growth factor stimulation, EGFR undergoes conformational changes and being phosphorylated, followed by being internalizated [
24‐
26]. EGFR signaling subsequently mobilizes multiple signaling cascades, including MAPK (microtubule-associated protein kinase), PI3K (phosphodylinositol-3-kinase) and STAT (signal transducer and activator of transcription) pathways. However, a specific biological outcome, following EGFR activation, is determined by cross-talk or cooperation of its downstream effectors and parallel pathways.
As with EGFR, nAChR subunits appear to be activated through tyrosine phospohrylation [
18,
27]. Using
Xenopus oocytes, neuroblastoma or other types of cells, it was shown that the α7 subunit of nAChRs was regulated by tyrosine phosphorylation and Src family kinases [
18]. The treatment of colon cancer cells with nicotine activated c-Src as well as augmented EGFR expression [
28]. Furthermore, in the colon cancer xenograft model, inhibitors of EGFR and Src dramatically blocked the tumor formation promoted by nicotine injection [
29]. All studies suggest the existence of cooperation between nAChR and EGFR.
During the process of tumor initiation and progression, aberrant growth signaling plays an important role in the perturbation of growth restriction and cell cycle checkpoints. Numerous factors play a role in the regulation of this process, which includes growth factors, kinases, phosphatases as well as extracellular matrix components. Growth receptors, when interacting with corresponding ligands, initiate the process of cell cycle progression and migration in cells. In order to successfully transmit signaling from the membrane to the nucleus, receptors appear to communicate with each other to modulate the magnitude of signaling cascades and further activate transcription factors for the promotion of various biological processes. Nicotine has been demonstrated to induce nAChR phosphorylation, which further stimulated the dissociation of E2F1 from Rb and subsequent binding to cdc6 and cdc25A promoters for cell cycle progression in lung cancer cells [
18]. These events which are induced by nicotine are most likely responsible for the increase of breast cancer risk by active or passive tobacco smoking.
In this study, we demonstrate a novel signaling mechanism whereby nAChR promotes breast cell growth through the sensitization of EGFR-mediated signaling. Upon nicotine-induced EGFR activation, Src, Akt and ERK1/2 were phosphorylated in MCF10 and MDA-MB-231 breast cancer cells, leading to the upregulation of E2F-1, Bcl-2 expression, and long-term cell survival. In this process, Src functioned directly downstream of nAChR to activate EGFR/ERK1/2 as well as Akt pathways, respectively. The identification of the cross-talk between nicotine and EGFR connected through Src provides a new insight into the potential carcinogenic effect of tobacco smoke on the breast.
Materials and methods
Cells, reagents and infection procedure
Human benign MCF10A and malignant MDA-MB-231 breast cancer cells were purchased from ATCC (Manassas, VA, USA). MCF10A cells were cultured in DMEM/F12 medium supplemented with 5% donor horse serum and antibiotics without growth factors. MDA-MB-231 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal calf serum, 4 mM L-glutamine and antibiotics. dn-Src or dn-Akt (dominant-negative Src or Akt) was inserted into MSCV (murine stem cell virus) retroviral vector and subsequently transiently infected into the cells.
Nicotine and the nAChR inhibitor mecamylamine hydrochloride (MCA) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). The Akt inhibitor KP372-1 and the ERK inhibitor PD98059 were obtained from EMD Chemicals Inc. (San Diego, CA, USA). The antibodies were purchased from BD Parmingen (La Jolla, CA, USA).
The procedure for the infection with genes inserted in the MSCV retroviral vector was detailed in the User Manual provided by the company (BD Biosciences Clontech, Heidelberg, Germany). Briefly, after co-transfected expression vector, Gag and Env constructs, PT67 cells were grown for 48 hours. Subsequently, the medium was collected for the infection.
The experiments performed in this study do not require Institute Ethics Board approval, because only commercially available cell lines were used.
Immunoblotting
Following treatment, cell lysates were prepared and proteins were separated by SDS-PAGE gels. Membranes were incubated with the designated primary antibody (1:1000 for all antibodies) overnight in a cold-room at 4°C. Bound primary antibodies were reacted with corresponding second antibodies for 2 hours and detected by chemiluminescence. The anti-phosphor-EGFR (tyr-1068), EGFR, phosphor-E2F, E2F, phosphor-Src, Src and Bcl-2 antibodies were purchased from Santa Cruz, Inc. The anti-phosphor-PDGFRβ, PDGFRβ, phosphor-ERK1/2, ERK1/2, phosphor-Akt and Akt antibodies were from Cell Signaling Technology, Inc, Donvers, MA, USA
GST/Grb2 pull-down assay
GST/Grb2 fusion protein was purchased from Invitrogen. After treatments, cell lysates were incubated with the fusion protein (1 μg) immobilized on glutathione-sepharose beads as indicated in the protocol provided by the company. Bound proteins were washed and subjected to SDS-PAGE.
ChIP assay
After treatments, cells were cross-linked with 1% formaldehyde for 15 minutes at room temperature. The cross-linking was stopped by the addition of glycine. Cells were harvested and sonicated. Lysates were immunoprecipitated with the corresponding antibodies. The different bindings of E2F1, Rb to cdc25A were analyzed by PCR. The sequences of the primers used are: cdc25A promoter size of 209 bp (5'-tctgctgggagttttcattgacctc and 3'-ttggcgccaaacggaatccaccaatc); c-Fos promoter size of 209 bp (5'-tgttggctgcagcccgcgagcagttc and 3'-ggcgcgtgtcctaatctcgtgagcat) [
18]. PCR products were resolved on a gel.
[3H]thymidine incorporation
Cells were grown in Petri dishes until 60% to 70% confluence and five wells were for the control and each treatment. The cells were cultured in medium containing 0.5% serum for 24 hours. Subsequently, the cells were grown in fresh medium containing 0.5% of serum plus 4 μCi/ml of [3H]thymidine (Perkin Elmer Life Sciences, Waltham, MA, USA) with or without various treatments. The cells were labeled for 8 hours at 37°C. After precipitation with cold 10% trichloroacetic acid, the cells were dissolved in 0.5 ml of 0.1 M NaOH overnight at 4°C. The amount of radioactivity in each sample was counted using a scintillation machine.
Cell proliferation assay
Cells (2 × 105) were plated in 12-well plates and cultured in medium containing 0.5% serum, which is designated as day 1. Subsequently, the cells with or without nicotine treatment were grown for another three days. The numbers of viable cells were determined by trypan blue staining and counted daily using a hemocytometer.
Cells (250 cells/plate) were seeded in 100 mm-Petri dishes and cultured in growth medium containing nicotine alone or nicotine plus other inhibitors for ten days. The medium with nicotine or its combination with other inhibitors was changed every four days. After staining, the numbers of colony were counted.
Statistical analysis
Three to five independent repeats were conducted in all experiments. Error bars represent these repeats. A Student's T test was used and a P value of < 0.05 was considered significant.
Discussion
Cigarette smoke contains a variety of genotoxic carcinogens, many of which are derivatives of nicotine that are formed during the curing of tobacco [
1‐
3]. The direct link between cigarette smoke and the onset of lung cancer has long been established. Although the correlation of the smoke with other types of cancer, in particular breast cancer, has been suggested by epidemiological investigations, the underlying molecular mechanisms by which cigarette smoke promotes breast cancer genesis and progression remain unclear. It is known that nAChR is widely expressed in neurons and neuromuscular junctions, but is also present in various non-neuronal organs, tissues or cells, such as epithelial cells from different organs and endothelial cells. Ligation of nAChR has been shown to facilitate cell growth and promote pro-survival activities in lung cancer or other types of malignant cells [
4‐
6]. We previously demonstrated that exposure to nicotine augmented the migration or invasion ability of benign or malignant breast cancer cell lines, in which PKC and cdc42 played a crucial role [
7]. As the continuation of the investigation of the role of nicotine exposure in breast tumorigenesis, we found that the engagement of nicotine with nAChR sensitized EGFR signaling via Src, resulting in the activation of ERK1/2 and upregulation of E2F1 transcriptional activity. We also found that the inhibition of nAChR or Src abrogated the promotion of cell proliferation conferred by nicotine treatment. Furthermore, in response to nicotine treatment, ERK1 and 2 functioned downstream of EGFR and the suppression of these kinases prevented the nicotine-mediated activation of E2F1 and DNA synthesis. We also showed that Akt appeared to be directly activated by Src in nicotine-governed action and responsible for upregulated Bcl-2 expression and increase cell survival activity. Collectively, these findings identified the novel intracellular targets Src/Akt and EGFR/ERK1/2 that are differentially affected by nicotine exposure to facilitate breast cancer progression.
Since there is a lack of understanding about the underlying molecular mechanisms by which tobacco smoke promotes turmorigenesis in other organs of human body, rather than in the lung, nicotine has become a major object of investigation, because it exists in high concentrations in the blood stream of first-, heavy second-hand smokers and nicotine users [
37‐
39]. Although nicotine is not a conventional carcinogen, this tobacco smoke-related compound has been shown to induce the secretion of growth factors (such as bFGF, TGF-α, calpains, and VEGF), resulting in the activation of Raf, Akt or PKC pathways for the growth promotion of lung epithelial or cancer cells and upregulation of Bcl-2 signaling that is responsible for the increase in the resistance to anti-cancer therapies [
35,
36]. The binding of nicotine to nAChR initiated the activation of Src tyrosine kinase that further mediated cell cycle progression of non-small cell lung cancer (NSCLC) [
18]. Our current study demonstrated that exposure of human breast benign or malignant cancer cells to nicotine induced the phosphorylation of Src that augmented cell growth- and survival-related signaling. As a substance, nicotine is able to diffuse rapidly into various organs and tissues. Thus, it is conceivable that this major component of tobacco smoke in the blood stream can efficiently reach the breast and bind to nAChR on the surface of breast epithelial or cancer cells, which provides a growth advantage locally. Indeed, studies have demonstrated that cancer patients who were smokers or nicotine users were more resistant to chemotherapy and had increased metastasis of breast cancer. Furthermore, nicotine was also reported to augment the proliferation of cell lines derived from gastric, colon, bladder or pancreatic tumors [
14‐
16]. Therefore, the interaction of nicotine and nAChR is an un-neglected factor in the regulation of the growth in different tissues or organs.
EGFR belongs to a family of the receptor tyrosine kinases and functions as a mediator to transmit cell signaling initiated by extracellular growth factors to the nucleus. Overexpression of EGFR or other family members is frequently found in human tumors of epithelial origin. Targeting EGFR family members has been attractive for developing new therapeutics with promising clinical results [
23‐
26]. In our current investigation, we demonstrated that EGFR was activated and subsequently internalized in breast cancer cells in response to nicotine treatment, accompanied by the cascade of the phosphorylation of several intracellular effector kinases. Among these kinases, Src acted as a key regulator to link nAChR signaling to EGFR and ERK1/2. In nicotine-treated neuroblastoma or
Xenopus oocytes cells, the α7 subunit of nAChR has been shown to undergo tyrosine phosphorylation and Src was responsible for the activation of this subunit of the receptor [
18]. Using
in vitro and xenograft assays, it was also reported that the levels of Src and EGFR in colon cancer cells were significantly increased following nicotine exposure [
18]. Our experiments showed that Src functions as a key downstream effector of nAChR and links nicotine signals to EGFR and ERK1/2 to promote transient cell growth activities.
By studying the mechanisms of nicotine-mediated cell growth promotion, we revealed that a cross-talk occurred specifically between two important cell surface receptors: nAChR and EGFR. This is the first demonstration of nicotine-induced sensitization of EGFR in benign and malignant breast cancer cells. Intriguingly, we found that in nicotine-mediated action, EGFR activation led to an increase of E2F1 activity, resulting in the promotion of DNA synthesis and cell proliferation. In this process, EGFR appears as a rate limiting factor and ERK1/2 functions as an executor of the cell growth program. Previously, we established that exposure to nicotine activates Raf and PKC pathways in Rat or murine lung epithelial or cancer cells, which facilitate the genesis and development of tumors [
23‐
26]. EGFR has been shown to mediate at least two pathways in cancer cells: the cytosolic and the nuclear pathways. Emerging evidence indicates that upon activation, some of EGFR or its family members in cancer cells relocate to the nucleus, where they participate in the regulation of gene transcription, cell cycle checkpoints and DNA repair. It is still under investigation whether EGFR upon nicotine treatment in our experimental setting translocates to the nucleus or is degraded. The present data suggest that upon nicotine exposure, EGFR appears to play a significant role in breast tumorigenesis.
Tobacco smoke or nicotine can reduce the efficacy of chemo-treatments and increase cancer onset, development or recurrence. Studies showed that in response to nicotine exposure, cancer cells became resistant to cytotoxicity triggered by anti-cancer drugs. Bcl-2 was reported to play an important role in nicotine-induced anti-apoptotic or pro-survival activities [
35,
36]. It was demonstrated that nicotine treatment significantly protected breast cancer cells against the cytotoxicity of doxorubicin [
35,
36]. Here, we determined that Bcl-2 is one of the targets of nicotine exposure. Our study also demonstrated that Akt was involved in the regulation of Bcl-2 expression and responsible for the long-term survival of the breast cancer cells. Together, it seems that nicotine, through activation of Src and Akt, promotes anti-apoptotic or pro-survival activities in breast cancer cells. Thus, Src and Akt pathways might be the intracellular targets for improving the treatment efficacy of breast cancer patients who are active or passive smokers or nicotine users.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TK, HK, LL, YH and JG carried out the experiments and initial analysis and interpretation of the data. CC conceived and designed the studies, made further data interpretations and wrote the manuscript. All authors approved the final version of the manuscript.