Abstract
Background/Aim: Rta, a transactivator of Epstein-Barr virus, is associated with progression of nasopharyngel carcinoma (NPC); however, its mechanism of contribution to the pathogenesis of NPC remains unclear. Interleukin-6 (IL-6), a tumor promoter, is detected in NPC. This in vitro study examined whether and how Rta promotes NPC progression by up-regulating IL-6. Materials and Methods: Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR, ELISA, immunoblotting assays, reporter gene assays, and transwell migration assays were performed. Results: In NPC cells, Rta up-regulated IL-6 expression at the mRNA and protein levels, and the Rta's C-terminus was essential for promoter activation and expression of IL-6. The induction of IL-6 by Rta also required activation of extracellular signal-regulated kinase 1/2 and activator protein-1. Furthermore, IL-6 secreted from Rta-expressing NPC cells promoted migration of Rta-negative NPC cells by activating IL-6 receptor/Janus kinase/signal transducer and activator of transcription 3 pathway. Conclusion: Rta contributes to progression of NPC cells through induction of IL-6 in vitro.
Interleukin-6 (IL-6) is a cytokine that is expressed in multiple tumor tissues, e.g., breast cancer, colorectal cancer and nasopharyngeal carcinoma (NPC) (1-3), suggesting that it exists in the tumor microenvironment. Binding of IL-6 to IL-6 receptor (IL-6R) on tumor cells in a paracrine or an autocrine manner activates the downstream Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway, thus promoting the migration, invasion, or metastasis of tumor cells (4). Patients with NPC exhibit elevated serum IL-6 levels associated with distant metastasis and poor survival (5, 6). IL-6 also promotes the growth, migration and invasion of NPC cells (7). Therefore, IL-6 may contribute to NPC progression.
Rta, an Epstein-Barr virus (EBV) lytic transactivator, participates in the initiation of EBV reactivation (8). The N-terminus of Rta protein mediates DNA binding and dimerization, whereas its C-terminus contains a transactivation domain functions for regulating its target gene expression and protein-protein interaction (9, 10). Additionally, Rta also has a nuclear localization signal (NLS) in its C-terminus, which helps the protein to enter the nucleus (11). Acting as a transactivator, Rta activates viral or cellular genes via direct binding to the Rta-responsive element (RRE) (9, 12, 13). However, Rta can regulate gene expression through several RRE-independent mechanisms, such as activation of some cellular signaling pathways (14, 15) and interaction with other transcription factors (16).
Recently, clues for linking Rta to the pathogenesis of NPC were reported, e.g., the protein was detected in NPC biopsy specimens (17). Moreover, the levels of anti-Rta antibodies were found elevated in the serum of NPC patients, especially those with advanced stages (18). However, how Rta contributes to pathogenesis of NPC remains unclear. Since Rta might induce a senescence-associated secretory phenotype (SASP) in NPC cells (19, 20), which is characterized by the induction of various secreted proteins, including cytokines (21). We hypothesized that Rta may contribute to the progression of NPC by inducing IL-6.
In this study, our results indicate that IL-6 is up-regulated and secreted from Rta-expressing NPC cells and the underlying mechanism involves activation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-activator protein 1 (AP-1) pathway. Furthermore, IL-6 secretion from Rta-expressing NPC cells enhanced the migration of Rta-negative NPC cells by activating the IL-6R/JAK/STAT3 pathway. Our integrated evidence not only indicate that Rta can contribute to NPC cell progression through induction of IL-6, but also suggest that the IL-6/STAT3 axis may serve as a potential therapeutic target to prevent NPC progression.
Materials and Methods
Cell culture and drug treatment. Two NPC cell lines, HONE-1 and NPC-TW01, a breast cancer cell line, MCF-7, and a gastric cancer cell line, AGS, were used for this study and obtained from Dr. Chin-Hwa Tsai (National Taiwan University, Taiwan, ROC). These cell lines, were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum with 5% CO2 at 37°C. In order to investigate whether the signaling pathways, including MEK/ERK, E2F, estrogen receptor α (ERα), c-Jun N-terminal kinase (JNK), AP-1, are involved in Rta-induced IL-6 expression, their specific inhibitors were used respectively based on the following conditions: To block the MEK/ERK pathway, cells were treated with 5 μM U0126 (Calbiochem, CA, USA) or 10 μM PD184352 (Enzo Life Sciences, NY, USA) for 48 h. To block E2F, ERα, JNK, and AP-1 pathways, cells were treated with 0.5 μM HLM006474 (Enzo Life Sciences), 10 μM MPP (Santa Cruz, CA, USA), 5 μM SP600125 (Calbiochem, CA, USA), and 10 μM SR11302 (R&D Systems, MN, USA) for 48 h, respectively. In order to investigate whether IL-6R and JAK/STAT3 pathway contribute to Rta-negative NPC cell migration triggered by Rta-induced IL-6, the specific inhibitors of IL-6R and JAK/STAT3 were used respectively based on the following conditions: To block the JAK/STAT3 pathway, cells were treated with 10 μM pyridone 6 (P6) (Sigma, MO, USA) for 24 h. To inhibit IL-6R, cells were treated with 10 μM LMT-28 (Sigma).
Plasmids, siRNAs, and cell transfection. Plasmids expressing wild-type Rta, an Rta mutant with NLS mutation or an Rta mutant with a C-terminal deletion were kindly provided by Dr. Chin-Hwa Tsai (National Taiwan University, Taiwan, ROC). To construct a reporter plasmid driven by the IL-6 promoter, the IL-6 promoter fragment (−508 to +53) was PCR amplified using a forward primer (5’-GCCAGAACACAGAAGAACTCAG-3’) and a reverse primer (5’-TGAGCCTCAGACATCTCCAG-3’). The PCR fragment was cloned into pGL3-basic vector (Promega, WI, USA) through the 5’ KpnI site and the 3’ XhoI site. The IL-6-targeted siRNA(5’-AAAUCUGUUCUGGAGGUACUCUAGG-3’) and a control siRNA with comparable GC content were purchased from Invitrogen. Lipofectamine 2000 reagent (Invitrogen) was used to transfect cells with plasmid DNA and/or siRNA as described previously (22).
Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Extraction of cellular total RNA and synthesis of cDNA were performed as described previously (22). The IL-6 and GAPDH cDNA samples were PCR amplified using the following conditions: 95°C for 2 min, followed by 25 cycles of 95°C for 20 s, 56°C for 20 s and 72°C for 20 s. The PCR products were electrophoresed on 1.5% agarose gels. The primers for IL-6 and GAPDH were 5’-AGTGAGGAACAAGCCAGAGC-3’ and 5’-GGTGCCCATGCTACATTTG-3’; and 5’-CAACGACCACTTTGTCAAGC-3’ and 5’-AGGGGTCTACATGGCAACTG-3’. Representative results from at least two independent experiments are shown.
Quantitative real-time PCR. The cDNAs of IL-6 and TATA box-binding protein (an internal reference gene for calibration) were quantified by real-time PCR using LightCycler reagents and the compatible detection system (Roche, CT, USA). PCR primers for detecting IL-6 were 5’-GATGAGTCAAAAAGTCCTGATCCA-3’ and 5’-CTGCAGCCACTGGTTCTGT-3’, and the locked nucleic acid-based probe was 5’-GGATGGAG-3’. Primers for detecting the TATA box-binding protein were 5’-GCTGGCCCATAGTGATCTTT-3’ and 5’-TCCTTGGGTTATCTTCACACG-3’, and the probe was 5’-CCCAGCAG-3’. Relative IL-6 mRNA levels were calculated using the LightCycler software (Roche). Each assay was carried out in duplicate, and the whole set of the experiments was performed at least twice independently.
Enzyme-linked immunosorbent assay (ELISA). IL-6 protein concentrations in the cell culture supernatants were quantified using the DuoSet ELISA kit (R&D Systems, MN, USA) according to the manufacturer's instruction. Each assay was carried out in duplicate, and the whole set of experiments was performed at least twice independently.
Immunoblotting assay. Extraction of cellular proteins and the immunoblotting assay were performed as described previously (23). The anti-Rta primary antibody (467) was kindly provided by Dr. Ching-Hwa Tsai. Antibodies for detecting ERK1/2 phosphorylated at Thr202/Tyr204, total ERK1/2, STAT3 phosphorylated at Tyr705, and total STAT3 were from Cell Signaling Technology (Cell Signaling Technology, MA, USA). The anti-β-actin antibody was from Chemicon (Chemicon, MA, USA). The anti-IL-6R antibody was from Abcam (Abcam, Cambridge, UK). Representative results from at least two independent experiments are shown.
Transwell migration assay. Transwells (6.5-mm diameter and 8-μm pore size; Corning, MA, USA) were used for studying cell motility. Cells were suspended in serum-free medium and plated on the upper chamber, and the cell culture supernatants were added to the lower chamber. After 24 h of incubation, non-migratory cells on the upper chamber were removed, and the migratory cells at the bottom side of the membrane were fixed with methanol and stained with Giemsa dye. The numbers of stained cells were counted from five randomly selected microscopic fields and shown are the average cell numbers per field. Experiments were performed three times.
Promoter activity assay. Cells were co-transfected with 2 μg of reporter plasmids (with a firefly luciferase gene driven by IL-6 promoter) and 2 μg of effector plasmids according to our previous protocol (22). At 48 h post-transfection, the cells were harvested and the firefly luciferase activity was measured by using a Bright-Glo assay kit (Promega) according to the manufacturer's instructions. Each assay was carried out in duplicate, and the whole set of the experiments was performed at least twice independently.
Dual luciferase reporter assay for detecting AP-1 activity. Cells were co-transfected with reporter plasmids (a mixture of an AP-1-responsive firefly luciferase plasmid and a control Renilla luciferase plasmid, purchased from Qiagen, Hilden, Germany) and effector plasmids. At 48 h post-transfection, the cells were harvested and subjected to the luciferase assay by using a dual luciferase kit according to the manufacturer's protocol (Promega). Each assay was carried out in duplicate, and the whole set of experiments was performed at least twice independently.
Statistical analysis. The SPSS (version 17.0) software was used to perform statistical analysis. Statistically significant differences between groups were determined by one-way ANOVA and followed by least significance difference (LSD) test. Data were expressed as mean±SEM. Statistical significance was set at p<0.05.
Results
Rta induces IL-6 expression in NPC cells. First, we assessed whether Rta could induce IL-6 expression in two NPC cell lines (HONE-1 and NPC-TW01) transfected with a vector plasmid or an Rta-expressing plasmid. The mRNA and protein levels of IL-6 were examined by using semiquantitative RT-PCR and ELISA, respectively. Figure 1A shows that both mRNA and protein levels of IL-6 were increased by ectopic expression of Rta, suggesting that Rta up-regulates IL-6 at the transcriptional level. We also demonstrated that Rta was unable to induce IL-6 production in two non-NPC epithelial cell lines, MCF-7 and AGS, (Figure 1B), suggesting that the Rta-mediated induction of IL-6 is cell-type dependent.
The C-terminus of Rta is required, whereas its nuclear localization signal is not essential, for the induction of IL-6. Rta's NLS is required for the protein to enter the nucleus (11), and Rta's C-terminus is known to be involved in signaling transduction (10). Both of them play an important role in target gene transactivation (11, 20). One of the two Rta mutants has an NLS mutation, designated RNLSm, and the other lacking the C-terminus, designated RtaΔC, were used to evaluate their importance in IL-6 expression. The expression of Rta and its mutants was confirmed by using an immunoblotting assay (Figure 2A). The promoter activity assay, quantitative RT-PCR, and ELISA (Figure 2B, C and D), show that wild-type Rta activated the IL-6 promoter and up-regulated IL-6 at the mRNA and protein levels. Moreover, RNLSm enhanced the promoter activity and expression of IL-6 (Figure 2B, C and D), whereas RtaΔC failed to activate IL-6 promoter and induce IL-6 expression (Figure 2B, C and D), indicating that the C-terminus of Rta, but not the its NLS, is required for IL-6 induction.
Rta activates the MEK/ERK-AP-1 pathway, which is required for Rta-induced IL-6 expression. The NLS of Rta is not required for IL-6 induction (Figure 2B, C and D), and we did not find any typical RRE sequence in the IL-6 promoter, suggesting that Rta may not enter the nucleus to transactivate the IL-6 gene. We hypothesized that Rta up-regulates IL-6 via activation of cellular signaling pathways. Therefore, several signaling pathways associated with IL-6 induction, i.e., MEK/ERK, JNK, E2F, and ERα (24-27), were screened in relation to Rta-induced IL-6 expression. Inhibitors of these signaling pathways, i.e., U0126, SP600125, HLM006474, and MPP, respectively, were used. Figure 3A shows that among the inhibitors, U0126 significantly inhibited Rta-triggered IL-6 expression, suggesting that MEK/ERK pathway is involved in Rta-induced IL-6 expression. We also treated cells with PD184352, another specific inhibitor of the MEK/ERK pathway, which significantly inhibited Rta-induced ERK1/2 activation and IL-6 expression (Figure 3B), indicating that the MEK/ERK pathway is required for Rta-induced IL-6 expression. AP-1, the downstream transcription factor of MEK/ERK pathway, is activated by Rta (20) and is required for IL-6 expression (28). To test whether AP-1 plays a role in Rta-induced IL-6 expression, we treated cells with an AP-1 activation inhibitor, SR11302. Figure 3C shows that treatment with SR11302 reduced Rta-induced IL-6 expression (Figure 3C) and inhibited Rta-induced AP-1 activation (Figure 3D), suggesting that activation of AP-1 is involved in Rta-induced IL-6 expression. Since MEK/ERK is an upstream signaling pathway of AP-1, we also treated cells with the MEK/ERK inhibitor U0126 or PD184352, which significantly reduced Rta-induced AP-1 activation (Figure 3E and F, respectively), indicating that Rta induces AP-1 activation through the MEK/ERK pathway. Our results suggest that Rta-activated MEK/ERK pathway contributes to AP-1 activation, leading to induction of IL-6.
IL-6 secreted from Rta-expressing NPC cells enhances migration of Rta-negative NPC cells through activating IL-6R/JAK/STAT3 pathway. IL-6 is associated with cell migration (29); therefore, we tested the migratory ability of Rta-expressing NPC cells by using a transwell migration assay. Rta-expressing NPC cells had a lower migratory activity compared to vector control cells (data not shown), probably because of Rta-induced cellular senescence. Despite having reduced migratory or invasive ability (30, 31), senescent cells can still promote bystander tumor cells migration or invasion through paracrine of SASP proteins (32, 33). Therefore, we tested whether the IL-6 secreted from Rta-expressing NPC cells could exert a pro-migratory effect on Rta-negative NPC cells. We found that when Rta-induced IL-6 production was significantly suppressed by the siRNA (Figure 4A), the conditioned medium of Rta-expressing NPC cells (designated “Rta-conditioned medium”) had a reduced pro-migratory effect on Rta-negative NPC cells (Figure 4B), supporting that Rta-expressing NPC cells can promote migration of bystander tumor cells through paracrine of IL-6. Binding of IL-6 to IL-6R leads to activation of JAK/STAT3 pathway that is associated with cell migration (29). Therefore, we evaluated whether the Rta-induced IL-6 promotes migration of bystander tumor cells through activating IL-6R/JAK/STAT3 pathway. First, we checked whether the bystander tumor cells have IL-6R. The immunoblotting data show that bystander tumor cells expressed the IL-6R protein (Figure 4C). Treatment with an IL-6R specific inhibitor not only blocked STAT3 activation (Figure 4C) but also reduced the cell migration (Figure 4D) induced by Rta-conditioned medium. In addition, treatment with a JAK/STAT3 inhibitor also abolished Rta-conditioned medium-induced STAT3 activation (Figure 4E), and cell migration (Figure 4F). According to the above results, we suggest that IL-6 secreted from Rta-expressing NPC cells promotes Rta-negative NPC cell migration by activating IL-6R/JAK/STAT3 pathway.
Discussion
Although Rta is associated with progression of NPC, how it promotes NPC progression is still unknown. Rta has been reported to induce senescence in NPC cells. Senescent cells can promote cancer progression by secreting various proteins, such as growth factors or cytokines (21). IL-6, one of the cytokines, is associated with NPC progression (5, 6). Therefore, in this study, we first evaluated whether Rta-expressing NPC cells could produce IL-6. Our results showed that both mRNA and protein levels of IL-6 were increased by ectopic expression of Rta in NPC cells (Figure 1A), suggesting that Rta induces transcription of the IL-6 gene. Notably, Rta was unable to induce IL-6 in non-NPC cell lines (Figure 1B). We also found that the MEK/ERK pathway required for IL-6 induction in NPC cells was not activated by Rta in the non-NPC cell lines (data not shown), suggesting that cellular backgrounds may be involved in determining Rta-induced IL-6 expression.
The cytoplasm-confined Rta mutant with disruption in its NLS could still promote IL-6 promoter activation (Figure 2B) and induce IL-6 expression (Figure 2C and D), and we also did not find any typical RRE on the IL-6 promoter, suggesting that Rta itself does not regulate the transcription of the IL-6 gene directly in the nucleus. Rta has been shown to up-regulate its target genes through activation of several cellular signaling pathways (14, 15). Herein, we found that MEK/ERK pathway is required for Rta-induced IL-6 expression (Figure 3A and B). Additionally, Rta could activate IL-6 promoter (Figure 2B), indicating that some cellular transcription factors participating in IL-6 expression may be activated by the Rta-activated MEK/ERK pathway. According to our results, AP-1 is the transcription factor that was found involved in IL-6 expression (Figure 3C), suggesting that Rta-activated MEK/ERK pathway may drive AP-1 transcription factor to up-regulate IL-6 gene expression in NPC cells.
How Rta activates MEK/ERK is still unclear. Kinase suppressor of Ras 1(KSR1) is a scaffold protein that can promote MEK/ERK activation (34). It is known that BRCA1-associated protein 2 (BRAP2) can interact with KSR1, resulting in the inhibition of MEK/ERK activation (34). Recently, a study showed that the interaction of Rta's C-terminus and BRAP2 helps release KSR1 from BRAP2 (10). Acting as a signaling scaffold protein, the released KSR1 can promote MEK/ERK activation (34). Because the Rta's C-terminus is important for activation of ERK1/2 (data not shown), we speculate that the BRAP2-KSR1 pathway may mediate the Rta-induced activation of ERK1/2.
It has been reported that IL-6 and activated STAT3 can be detected in NPC biopsies at the protein level (3, 35), and the IL-6/STAT3 pathway plays an important role in promoting the malignant process of NPC (6, 36). Although Rta is associated with NPC progression (17, 18), whether it can regulate the IL-6/STAT3 pathway to promote NPC progression remains unknown. Knockdown of IL-6 or inhibition of STAT3 activation could block the Rta-induced pro-migratory effect (Figure 4B and F), suggesting that Rta may promote NPC progression by activating the IL-6/STAT3 pathway. Previous studies show that blocking the IL-6/STAT3 pathway can decrease tumor growth and metastasis in vivo (37, 38), and our study also found that inhibition of IL-6/STAT3 pathway by using IL-6-targeted siRNA, IL-6R inhibitor, or JAK/STAT3 inhibitor significantly inhibits Rta-negative NPC cell migration (Figure 4B, D and F). Therefore, IL-6/STAT3 pathway-targeted therapies may also prevent the progression of NPC.
Fascin, an actin-bundling protein, is implicated in filopodia formation (39). The fascin is up-regulated in NPC (40), and is also involved in migration and invasion of NPC cells (41). It has been reported that IL-6/JAK/STAT3 pathway contributes to up-regulate fascin, leading to migration of cancer cells (42, 43). In addition, E-cadherin, a molecule that mediates cell-cell adhesion, is commonly down-regulated in NPC (44). The loss of E-cadherin expression is involved in epithelial-mesenchymal transition process, which promotes migration, invasion, and metastasis of tumor cells (45-47). The JAK/STAT3 pathway is also reported to down-regulate E-cadherin expression through regulating a transcriptional repressor, ZEB1 (48). According to the clues above, we postulate that IL-6 secreted from Rta-expressing NPC cells may up-regulate fascin and/or down-regulate E-cadherin via the JAK/STAT3 pathway to promote the migration of Rta-negative NPC cells. To further confirm the in vitro findings in this study, in vivo studies will be performed in the future.
Acknowledgements
This study was supported by the Ministry of Science and Technology, Taiwan, R.O.C. (MOST-107-2320-B-471-001).
Footnotes
↵* These Authors contributed equally to this study.
Authors' Contributions
Kuo-Lung Tung, Yen-Ting Wu and Yu-Yan Lan designed the study and analyzed the data and results. Kuo-Lung Tung, Yen-Ting Wu, Cheng Liu, Sheng-Chieh Lin, Chin-Han Wu, Shih-Yi Wu, and Yao Chang performed all the experiments in this study. Kuo-Lung Tung, Yen-Ting Wu, and Yu-Yan Lan wrote the manuscript. Yu-Yan Lan revised the manuscript.
Conflicts of Interest
The Authors declare that they have no conflicts of interest.
- Received April 1, 2020.
- Revision received April 12, 2020.
- Accepted April 21, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved