Abstract
Background/Aim: The purpose of our study was to test whether EBV infection affects the response of human breast cancer cells to nicotine. In addition, the underlying signaling mechanisms were evaluated. Materials and Methods: EBV-infected MDA-MB-231 and LMP1-transfected MDA-MB-231 breast cancer cells were established. Reverse transcription-polymerase chain reaction and western blotting were performed to evaluate nicotine receptor expression. To verify the functional role and underlying signaling mechanism of nicotine receptor expression induced by EBV infection, morphologic analysis, and proliferation and inhibition assays were performed. Results: Both EBV infection and LMP1 transfection increased cell proliferation and induced the up-regulation of α9-nAChR expression. Additionally, nicotine treatment induced tumorigenic activity in both EBV-infected and LMP1-transfected MDA-MB-231 breast cancer cells. Western blot and inhibitor assays showed that the nicotine-induced signaling was mediated through MAPK/ERK and AKT signaling pathways in EBV-infected and LMP1-transfected breast cancer cells, respectively. Conclusion: These results suggest that EBV infection and EBV-related LMP1 may act as potential molecular targets for breast cancer risk associated with nicotine, and provide a novel insight into the mechanism of nicotine stimulation in EBV-positive breast cancer cells.
- Breast cancer
- Epstein-Barr virus (EBV)
- latent membrane protein-1 (LMP1)
- nicotine receptor
Breast cancer is a malignant tumor in women that originates from the ductal epithelium of the breast or breast lobule (1). According to WHO, in 2008, 1.38 million new cases of breast cancer were reported, which represented 23% of all new cancer cases. Breast cancer is the second most common cancer and the fifth leading cause of all cancer deaths in women (2). The current treatment methods for breast cancer are not sufficient and the prognosis of breast cancer remains poor. Therefore, identifying new targets for the treatment of breast cancer is critical (3).
Human viruses are the second most common risk factor for the occurrence of human cancers, and the most common example is Epstein-Barr virus (EBV). The development of malignant transformation from EBV infection comprises lytic and latent phases. EBV-encoded gene products are important for controlling many critical events such as cell proliferation and development (4). LMP1 and LMP2A are important factors in latency type II infection and also EBV-induced B-cell transformation and oncogenesis (5, 6). LMP1 regulates cell proliferation, migration and motility by binding to oncogenic signaling factors, such as NF-κB, phosphatidylinositol 3-kinase (PI3K), AKT, MAPK/ERK, and JAK/STAT (7-10).
Nicotine is a major chemical compound present in tobacco. It affects the central nervous system and is also responsible for addiction to tobacco (11, 12). Previous studies have demonstrated that nicotine may also increase the likelihood of development of many cancers, including colon, lung, gastric and breast cancers (13-15). Many studies have reported the roles of nicotinic acetylcholine receptors (nAChRs) in angiogenesis, carcinogenesis, proliferation, inhibition of apoptosis and metastasis in breast, colon and lung cancers (16-18). Nicotine interacts with nAChRs, which further activate signaling pathways that result in cancer progression and metastasis. In the development and progression of many cancers in vivo, nicotine mimics acetylcholine, which binds to the α subunit of nAChRs and plays a major role as stimulator (19). Four different subunits (α5, α7, α9, and β4) of nAChRs have been identified, and in breast cancer cells, nicotine exposure mostly increases the expression of α9-nAChR (20).
Our study showed over-expression and activation of α9-nAChR in EBV-infected breast cancer cells, which were more aggressive than those in the control breast cancer cell line. We also demonstrated that nicotine exposure increased EBV-infected breast cancer cell proliferation through activation of the MAPK/ERK and AKT pathways, and pre-treatment with inhibitors blocked nicotine-enhanced EBV-infected breast cancer cell proliferation.
Materials and Methods
Cells and culture conditions. MDA-MB-231 human breast cancer cells and B95-8 cells were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were maintained in keratinocyte serum-free medium (Invitrogen, Carlsbad, CA, USA) with 5 ng/ml epidermal growth factor and 0.05 mg/ml bovine pituitary extract or RPMI medium (Mediatech Inc., Corning Subsidiary, Manassas, VA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Tissue Culture Biologicals, Tulare, CA, USA), 100 IU/ml penicillin, and 100 mg/ml streptomycin. Cells were maintained at 5% CO2 humidified atmosphere in a 37°C incubator.
Establishment of EBV-infected cell lines. The virus titer of B95-8 EBV virions from the B95-8 cell line was used for infecting MDA-MB-231 cells using culture supernatant. After MDA-MB-231 cells were completely attached in 6-well plates, the EBV supernatant was added. Cells were maintained for two weeks to visualize the expression of EBV latent genes. EBV-infected MDA-MB-231 cells were cultured in RPMI-1640 medium (as described above) at 37°C in a 5% CO2 incubator. The cells were washed three or four times with phosphate-buffered saline (PBS) to remove the remaining B95-8 cells.
LMP1 gene interference. MDA-MB-231 cells were seeded into 6-well plate. Lipofectamine 2000 (Invitrogen) was used to transfect LMP1 into cells for 24 h according to the manufacturer’s protocol.
Reverse-transcription polymerase chain reaction (PCR). Cells were harvested and washed three times with PBS (pH=7.4). Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Equal amounts of RNA (2 μg) from each sample were reverse-transcribed into cDNA using the RT PreMix Kit (Bioneer, Daejeon, Republic of Korea). PCR amplification was performed using primer sets (Bioneer) for LMP1 and α9-nAChR mRNA. β-actin mRNA was used as control. PCR products were visualized on 1% agarose gels with ethidium bromide.
Western blotting. Cells were collected and washed with PBS two times and lysed in RIPA lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 0.1% Triton X-100; Elpis Biotech, Daejeon, Republic of Korea) containing protease and phosphatase inhibitors (Sigma-Aldrich). Equal amounts of proteins (10 μg) were separated by electrophoresis on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Amersham, Braunschweig, Germany) by electroblotting. The membranes were incubated with 5% non-fat skim milk. The blots were probed with specific primary antibodies overnight at 4°C and then incubated with diluted enzyme-linked secondary antibody. Bands were visualized by enhanced chemiluminescence as recommended by the manufacturer (Amersham). Protein loading equivalence was assessed by GAPDH expression.
Investigation of morphologic changes. Cells were seeded in 6-well plates and cultured for 24 h. Cell morphology and condition were observed with an inverted microscope digital imaging system (CELENA®S Digital Fluorescence Imaging System; Logos Biosystems, Anyang-si, Gyeonggi-do, Republic of Korea).
Inhibitor treatment. Cells were pre-treated with PD98059 (Selleck Chemicals, Houston, TX, USA) and Akti-1/2 (Selleck Chemicals), which are chemical inhibitors for MAPK/ERK and AKT, respectively. The inhibitor was removed from cell culture before treatment with nicotine.
MTT assay. Cells were seeded into 96-well plates, cultured for 24 h and treated with different concentrations of nicotine. The 3-(4,5-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) growth assay was applied to measure the cell proliferation rate using a Cell Titer 96 nonradioactive cell proliferation assay kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocol. The absorbance at a wavelength of 490 nm was recorded using a 96-well plate reader.
Statistical analysis. Statistical analysis was performed using the GraphPad Prism 8.0.2 software (Graph Pad Inc., La Jolla, CA, USA). Data are expressed as the mean±standard error of the mean. The unpaired t-test was used to evaluate the differences between means, and p-value of 0.05 or less was considered significant (*p<0.05, **p<0.005).
Results
EBV-infected MDA-MB-231 breast cancer cells express the EBV latent gene LMP1. MDA-MB-231 human breast cancer cells, which are EBV negative, were infected with EBV to explore the potential impact of EBV infection in human breast cancer cell lines. We also established LMP1-transfected cell lines using MDA-MB-231 cells. We examined viral gene expression in EBV-infected breast cancer cells by PCR and western blot assays. As shown in Figure 1, we demonstrated for the first time that EBV-infected MDA-MB-231 cells express LMP1, which is an oncoprotein of EBV latent gene products. PCR and western blot results showed LMP1 gene and protein expression in both LMP1-transfected and EBV-infected MDA-MB-231 breast cancer cells.
EBV infection and LMP1 transfection lead to morphologic changes and rapid proliferation compared with control breast cancer cells. We next compared the morphologic changes between EBV-infected and control MDA-MB-231 cell lines as well as EBV-infected and LMP1-transfected cells. Both cells were spindle-shaped in normal culture conditions, but were converted into larger spindle-shaped cells with a different phenotype (Figure 2A and B). MTT assays showed that these cells showed higher proliferation compared with breast cancer cells (Figure 2C and D). Together these data indicate that EBV may increase the tumorigenic activity of breast cancer cells.
Stimulation of cell proliferation by the phosphorylation of MAPK/ERK in both LMP1-transfected and EBV-infected MDA-MB-231 breast cancer cells. Activation of survival signaling plays an important role in tumor development. Many studies have demonstrated that the MAPK/ERK signaling pathway is activated and promotes proliferation, invasion and migration of breast cancer cells. Therefore, we examined whether survival signaling is involved in LMP1-transfected and EBV-infected cell proliferation. To test this hypothesis, we examined AKT, SAPK/JNK and MAPK/ERK in both LMP1-transfected and EBV-infected MDA-MB-231 cells. Western blotting showed that the phosphorylation of MAPK/ERK was drastically increased in both LMP1-transfected and EBV-infected MDA-MB-231 cells (Figure 3). These data suggest that activation of the MAPK/ERK pathway is important for the proliferation of both LMP1-transfected and EBV-infected breast cancer cells.
Both LMP1-transfected and EBV-infected MDA-MB-231 cell proliferation was correlated with the expression of nicotine receptor. Nicotine induces the expression of α9-nAChR in breast cancer. We next examined whether α9-nAChR plays a role in breast cancer cell progression and examined the levels of nicotine receptor using both western blot and PCR. Both LMP1-transfected and EBV-infected MDA-MB-231 cells showed higher α9-nAChR expression. This observation confirmed our finding that α9-nAChR expression in breast tumor tissues is significantly higher, thus indicating that α9-nAChR plays a strong metastatic role during breast tumorigenesis. Expression of higher levels of α9-nAChR in breast cancer cells suggests that nicotine may promote breast cancer development via α9-nAChR expression (Figure 4).
Nicotine induces tumorigenic activity in EBV-infected and LMP1-transfected MDA-MB-231 breast cancer cell lines through MAPK/ERK and AKT signaling pathways, respectively. The pathophysiological effects of nicotine occur through nicotinic acetylcholine receptors. We next examined the proliferative effects of nicotine in the breast cancer cell lines. Treatment of EBV-infected cells (Figure 5A) and LMP1-transfected cells (Figure 5B) with nicotine for 24 h induced significant cell proliferation. Nicotine enhanced proliferation of both EBV-infected and LMP1-transfected cells in a dose-dependent manner; the maximum effect was observed at 1 μM in EBV-infected and 100 μM in LMP1-transfected cells. As EBV-infected MDA-MB-231 cell lines showed higher proliferation, a lower concentration of nicotine was needed compared to LMP1-transfected cell lines.
As mentioned, survival signaling activation is important for tumor development, and survival signaling pathway activation in cancer cells promotes cancer cell proliferation. Therefore, we examined whether survival signaling enhanced cell growth after nicotine treatment in both EBV-infected and LMP1-transfected breast cancer cell lines. Our results clearly show that nicotine altered the proliferative and survival properties of cells through the phosphorylation of MAPK/ERK pathway in EBV-infected cells (Figure 5C) and AKT phosphorylation enhanced the growth of LMP1-transfected cells (Figure 5D).
PD98059 and Akti-1/2 activate cell death by the phosphorylation of MAPK/ERK and AKT in EBV-infected and LMP1-transfected MDA-MB-231 breast cancer cells, respectively, following nicotine treatment. We found that MAPK/ERK regulates the proliferation of EBV-infected MDA-MB-231 breast cancer cell lines. To further confirm these results, EBV-infected MDA-MB-231 breast cancer cells were treated with nicotine in the presence or absence of PD98059, an inhibitor of MAPK/ERK. Nicotine effects were significantly decreased compared with the control in the presence of PD98059 (Figure 6A and B). The AKT pathway also promotes survival and growth of breast cancer cell lines, and nicotine treatment activates this signaling pathway. Our previous results showed that AKT regulates the proliferation of LMP1-transfected MDA-MB-231 breast cancer cell lines. Therefore, we also examined the effects of nicotine in the presence or the absence of Akti-1/2, an inhibitor of AKT. Nicotine effects were decreased compared with the control in the presence of Akti-1/2 (Figure 6C and D). Western data also showed that MAPK/ERK expression in nicotine-treated EBV-infected MDA-MB-231 breast cancer cell was decreased in the presence of PD98059 (Figure 6E). The effect of nicotine on AKT in LMP1-transfected MDA-MB-231 breast cancer cells was reversed in the presence of Akti-1/2 (Figure 6F).
Nicotine-induced cell proliferation of EBV-infected and LMP1-transfected MDA-MB-231 breast cancer cell lines was blocked by selective inhibitors. We showed that nicotine treatment activates various signaling pathways associated with cell proliferation. Upon nicotine treatment, MAPK/ERK signaling pathway mediates EBV-infected MDA-MB-231 breast cancer cell proliferation. In LMP1-transfected MDA-MB-231 breast cancer cells, the AKT pathway promotes cell proliferation. We also examined the proliferation of these cells after nicotine treatment in the presence or absence of selective inhibitors. Nicotine induced higher proliferation of EBV-infected and LMP1-transfected MDA-MB-231 breast cancer cells, and these effects were reversed in the presence of the selective inhibitors (Figure 7).
Discussion
EBV is causally associated with approximately 200,000 malignancies worldwide each year. However, the mechanism has not been identified (21, 22). Our findings indicate that EBV may contribute to the pathogenesis of human breast cancer cell proliferation. To investigate the molecular mechanisms through which EBV upregulates signaling molecules in EBV-infected breast cancer cells, we examined the expression of EBV gene products in EBV-infected cells. Notably, we found increased LMP1 expression and activation of proliferation in EBV-infected MDA-MB-231 breast cancer cells. We also showed that EBV infection may directly enhance breast cancer cell proliferation through specific pathways and to inhibit apoptosis by upregulating EBV gene expression. Previous studies showed that nicotine enhances breast cancer cell proliferation, but the key molecules mediating this effect were not identified. Some studies indicated that smoking correlated with progression and metastasis in breast cancer (14, 17, 23, 24). It has been shown that nicotine treatment stimulates the expression of α9-nAChR and subsequently causes normal breast epithelial cell transformation to cancer cells (25). Our results also showed that nicotine is involved in breast cancer cell proliferation through nicotine receptors. Together, these results indicate that α9-nAChR may be a potential therapeutic target to prevent the development of breast cancer.
The main molecular modulators underlying nicotine enhancement of breast cancer cell proliferation are not clear. Some reports indicate that α9-nAChR is related to the development of breast cancer (22, 26). Consistent with those findings, we observed that nicotine induced up-regulation of α9-nAChR in EBV-infected MDA-MB-231 breast cancer cells. Nicotine-dependent activation of MAPK/ERK and AKT have been reported as important signaling pathways downstream of α9-nAChR (26, 27). So, we considered α9-nAChR mediated MAPK/ERK and AKT activation as a signaling cascade in EBV-infected MDA-MB-231 breast cancer cells exposed to nicotine, which also suggests that MAPK/ERK or AKT signaling pathways regulate EBV-infected breast cancer cell proliferation through nicotine receptors. Both in ER-positive and triple negative breast cancer cells, nicotine significantly induces MAPK/ERK and AKT protein phosphorylation via α9-nAChR activation (25, 26). The AKT and MAPK/ERK pathways are well-known modulators of LMP1-mediated transformation and tumorigenesis (28, 29). These reports and our study show that AKT and/or MAPK/ERK pathways play a significant role in EBV-infected breast cancer cell survival and proliferation. AKT, MAPK/ERK and SAPK/JNK are important transcription regulators, and we investigated whether nicotine treatment caused AKT, MAPK/ERK or SAPK/JNK activation. Indeed, AKT and MAPK/ERK were activated through α9-nAChR in EBV-infected MDA-MB-231 breast cancer cells exposed to nicotine. We found that nicotine treatment enhances the proliferation of EBV-infected MDA-MB-231 breast cancer cells and that pre-treatment with inhibitors of these signaling pathways abolishes these effects. These results are compatible with the previous results that nicotine-enhanced cell proliferation was mediated through the nicotine receptor. Our data demonstrate that both EBV and LMP1 promote cell proliferation and induce the up-regulation of α9-nAChR expression. Furthermore, inhibition of MAPK/ERK and AKT was assessed in both EBV-infected and LMP1-transfected cells where the effect of nicotine was significantly decreased by using MAPK/ERK or AKT inhibitors. Together these findings show that α9-nAChR plays an important role in breast cancer through the activation of AKT or MAPK/ERK and demonstrate the functional significance of nicotine in the proliferation of EBV-infected MDA-MB-231 breast cancer cells. Thus, the role of LMP1 and nicotine in EBV-infected MDA-MB-231 breast cancer cells was verified, and LMP1 could be a potential target for treating EBV-associated breast malignancies.
In summary, our data suggest that LMP1-mediated induction of nicotine receptor expression may enhance the tumorigenic activity of EBV-infected breast cancer cells by activating MAPK/ERK and AKT signaling pathways. and provide insights into the roles of LMP1 in EBV-associated breast cancer malignancies. Although tobacco avoidance is still the best strategy for cancer prevention, the emerging understanding that nAChRs can mediate the carcinogenic mechanism provides a basis for targeting nAChRs for cancer prevention and/or cancer treatment in the clinical setting.
Acknowledgements
This work was supported by the 2019 Inje University research grant.
Footnotes
Authors’ Contributions
Conceptualization, Lata Rajbongshi and Dae Young Hur; Data curation, Lata Rajbongshi and Min Hye Noh; Formal analysis, Lata Rajbongshi and Yeong Seok Kim; Funding acquisition, Dae Young Hur; Investigation, Lata Rajbongshi; Project administration, Lata Rajbongshi and Dae Young Hur; Writing – original draft, Lata Rajbongshi; Writing – review & editing, Yeong Seok Kim and Dae Young Hur.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received May 4, 2021.
- Revision received June 15, 2021.
- Accepted June 16, 2021.
- Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.