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Research Center for Emerging Viral Infections and the Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan
Address reprint request to Ching-Hwa Tsai, Ph.D., Graduate Institute of Microbiology, College of Medicine, Jen-Ai Rd., Room 719, No. 1, 1st Section, National Taiwan University, Taipei 10051, Taiwan
Nasopharyngeal carcinoma (NPC) is characteristic for its strong association with Epstein-Barr virus (EBV) and high metastatic rate. Recently, overexpressed recepteur d'origine nantais (RON) (MST1R), receptor tyrosine kinase has been reported in human cancers and tumor metastasis. Therefore, the role of RON in EBV-associated NPC and its metastasis was investigated. Here we show that RON was found in NPC but not in control tissues. A significant correlation of latent membrane protein 1 (LMP1) and RON expression was found in NPC (Pearson's χ2 test; P = 0.0023). At the molecular level, LMP1 stimulates nuclear factor-κB binding to the RON promoter through its carboxyl-terminal activation region 1 to induce expression of RON. Knockdown of RON in cells expressing LMP1 significantly reverses LMP1-induced epithelial-mesenchymal transition and suppresses LMP1-induced cell migration and invasion. These results suggest an important role of RON in the tumorigenesis and metastasis of NPC and RON may be a novel therapeutic target for EBV-associated NPC.
Among the head and neck cancers, nasopharyngeal carcinoma (NPC) is notorious for its highly metastatic character. Epstein-Barr virus (EBV), the first human oncogenic virus described, is detected in most biopsies of NPC and is highly associated with NPC tumorigenesis in endemic regions.
In addition, EBV is associated with several other human malignancies, including Burkitt's lymphoma, Hodgkin's lymphoma, and post-transplantation lymphoproliferative disorder.
The close association between EBV and malignancies has raised the question as to how this tumor virus contributes to the invasive character of the tumor.
NPC has been shown to display a type II latency that expresses Epstein-Barr nuclear antigen 1, latent membrane protein 1 (LMP1), LMP2A, LMP2B, and EBV encoded RNAs.
Induction of c-Met proto-oncogene by Epstein-Barr virus latent membrane protein-1 and the correlation with cervical lymph node metastasis of nasopharyngeal carcinoma.
LMP1, the key EBV oncoprotein, is essential for EBV immortalization of primary B lymphocytes. LMP1 is capable of transforming rodent fibroblasts and inducing lymphomas in transgenic mice.
The oncogenic activity of LMP1 depends on its ability to affect cellular biological processes, which includes increasing cell cycle progression, anti-apoptosis, transformation, enhancement of cytokine expression, and promoting metastatic activities.
Structurally, LMP1 is a transmembrane protein that acts as a constitutively active CD40 receptor. Without requiring a ligand, LMP1 engages a number of signaling pathways, including phosphatidylinositol 3-kinase/Akt, nuclear factor (NF)-κB, mitogen-activated protein kinases via its carboxyl-terminal activation region (CTAR) 1 and 2.
Epstein-Barr virus latent membrane protein 1 induces the matrix metalloproteinase-1 promoter via an Ets binding site formed by a single nucleotide polymorphism: enhanced susceptibility to nasopharyngeal carcinoma.
MUC1 induced by Epstein-Barr virus latent membrane protein 1 causes dissociation of the cell-matrix interaction and cellular invasiveness via STAT signaling.
Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells.
RON is first synthesized as a 190 kDa single-chain precursor (pro-RON) and mature RON is composed of 40 kDa extracellular α-chain (RON-α) and 150 kDa transmembrane β-chain (RON-β) linked by one disulfide bond.
The product of the RON gene is a 180 kDa heterodimeric protein composed of a 40 kDa extracellular α-chain and a 150 kDa transmembrane β-chain with intrinsic tyrosine kinase activity.
Activation of RON triggers downstream signaling cascades, including Ras, phosphatidylinositol 3-kinase, and mitogen-activated protein kinases to induce cell scattering, migration, survival, and invasion, all of which are seen commonly in tumor formation and progression.
Thus, in vitro and in vivo studies hinted that RON is capable of regulating various oncogenic activities and is highly associated with tumor metastasis.
However, the role of RON in NPC and its metastasis has not been investigated yet. Here we show that RON is overexpressed in NPC biopsies, but not in control tissues, and there is a strong correlation between the expression of RON and LMP1 in the biopsies of NPC by immunohistochemical staining. At the molecular level, LMP1 induced RON expression via NF-κB binding to the RON promoter. LMP1 induction of epithelial-mesenchymal transition (EMT) to promote cell migration and invasion is through the RON receptor tyrosine kinase. These results suggest that LMP1-induced RON expression may play an important role in the pathogenesis and metastasis of EBV-associated NPC. This is the first study demonstrating the expression of RON in NPC and suggesting the possibility of targeting RON in the treatment for NPC.
Materials and Methods
Immunohistochemistry Assay
The control lymphoid hyperplasia of nasopharynx and NPC specimens were obtained from the National Taiwan University Hospital. Experiments involving human samples were approved by the Institutional Review Boards of the National Taiwan University Hospital (Taipei, Taiwan). Immunohistochemistry (IHC) assays were performed using the Super Sensitive Link-Label IHC Detection System (BioGenex, Fremont, CA) as previously described.
The human epidermal keratinocyte cells (RHEK) were a nonmalignant cell line that was established by infecting it with a hybrid virus (adenovirus-12-simian virus-40) of normal foreskin keratinocytes.
Epstein-Barr virus latent membrane protein 1 induces micronucleus formation, represses DNA repair and enhances sensitivity to DNA-damaging agents in human epithelial cells.
All cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS) plus 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C with 5% CO2. C666-1 cells are an EBV-positive NPC cell line.
C666-1 cells were maintained in RPMI 1640 Medium (Hyclone), supplemented with 10% FBS plus 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C with 5% CO2.
RON-expressing and vector control RHEK cell lines were established using a Neon kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, and cells were selected with 400 μg/mL neomycin for 4 weeks.
Preparation and Infection of LMP1 Expressing Lentiviruses
The pSIN LMP1 and its deletion mutants were constructed as previously described.
The control and cytomegalovirus-based RON expression plasmids were gifted from Dr. Hsing-Jien Kung (UC Davis Cancer Center, Sacramento, CA). The following were used: antibodies against GAPDH (6C5; Biodesign, Saco, ME), phospho-IκB-α Ser32/36 (9246; Cell Signaling, Danvers, MA), LMP1 (CS1-4; Dako, Glostrup, Denmark; S12
), p65 (C20; Santa Cruz, Santa Cruz, CA), phosphotyrosine (4G10; Millipore, Billerica, MA), E-cadherin (610182; BD Bioscience, San Jose, CA), γ-catenin (610254; BD Bioscience), vimentin (V6389; Sigma-Aldrich, St Louis, MO), Snail (H130; Santa Cruz), phospho Y1238/Y1239 RON (ab125264; Abcam; Cambridge, MA), and RON-β (C20; Santa Cruz).
RNA Extraction and RT-qPCR
Total RNA was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's protocol, and the method of RT-qPCR has been previously described using specific Taqman Gene Expression Assays (Applied Biosystems, Carlsbad, CA).
E-cadherin mRNA was detected using Roche universal probe (no. 77) and forward primer 5′-CCACCAAAGTCACGCTGAA-3′ and reverse primer 5′-TGCTTGGATTCCAGAAACG-3′. γ-catenin mRNA was detected using Roche universal probe (no. 77) and forward primer 5′-CAGCATCCTGCACAACCTC-3′ and reverse primer 5′-ACTCCACAGGGGAGCTGA-3′. Vimentin mRNA was detected using Roche universal probe (no. 13) and forward primer 5′-TACAGGAAGCTGCTGGAAGG-3′ and reverse primer 5′-ACCAGAGGGAGTGAATCCAG-3′. Snail mRNA was detected using Roche universal probe (no. 66) and forward primer 5′-GCTGCAGGACTCTAATCCAGA-3′ and reverse primer 5′-ATCTCCGGAGGTGGGATG-3′.
Immunoprecipitation and Western Blot
For immunoprecipitation, cell lysates were incubated with 3 μg antibody against RON overnight at 4°C on a rotating rocker and incubated with protein A beads for 2 hours. After washing with PBS, immunoprecipitated complexes were added to 25 μL 2× sample buffer at 95°C for 5 minutes and analyzed by using Western blot as previously described.
Cells were electroporated using a Neon kit (Invitrogen) according to the manufacturer's instructions. After 1100 V, 20 milliseconds, and 2 pulse analyses, cells were incubated for 5 days before being subjecting to functional assays.
Chromatin Immunoprecipitation Assay
The chromatin immunoprecipitation assay of DNA and p65 complex were performed as previously described.
Observation of Morphological Changes and Immunofluorescence Staining
RHEK cells were electroporated and seeded at 1 × 105 cells per well in a 6-well plate. The cell morphology was observed after 5 days using an inverted microscope (Axiovert 10; Zeiss, Göttingen, Germany). For immunofluorescence assays, electroporated cells were cultured for 5 days and re-seeded on glass coverslips. After 24 hours, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and then blocked with 1% bovine serum albumin. The cells were incubated with primary antibodies against LMP1 (S12) and RON (1:30 dilution), and were then incubated with Rhodamine-conjugated anti-mouse IgG (Millipore) and fluorescein isothiocyanate-conjugated anti-rabbit IgG (Millipore), respectively. The DNA content was stained with Hoechst 33258. The florescent signals were observed by fluorescence microscopy (Axioskop 40 FL; Zeiss).
Wound-Healing Assay
The confluent monolayer cells were cultured in a medium containing 0.5% FBS DMEM, scratched with a micropipette tip, and the migrating cells were monitored for 24 hours by microscope (Axiovert 10; Zeiss).
Transwell Migration and Matrigel Invasion Assay
For migration assay, 1 × 105 electroporated cells in 0.2 mL medium containing 0.5% FBS were loaded into the upper chamber of a 24-well transwell apparatus (Corning Costar Corp., Cambridge, MA) and incubated for 48 hours with 10% FBS DMEM medium in the lower chamber. For matrigel invasion assay, the transwell membrane in the upper chamber was coated with 50 μL Matrigel (BD Bioscience) at room temperature for 1 hour. The 1 × 105 electroporated cells in 0.2 mL medium containing 0.5% FBS were loaded into the upper chamber of the transwell apparatus and incubated with 10% FBS DMEM medium in the lower chamber at 37°C for 72 hours. The migrating and invading cells were fixed with 3% formaldehyde and stained with 0.5% crystal violet. For quantification, cells were quantitated by dissolving the stained cells in a solution of 500 μL 1% SDS solution, and a colorimetric reading was taken at an optical density at 595 nm using an enzyme-linked immunosorbent assay reader (uQuant, BioTek, Winooski, VT).
Statistical Analysis
Pearson's χ2 test was used to analyze the correlation of LMP1 and RON expression in the NPC biopsies. The differences of cell migration and invasion assays were analyzed by the Student's t-test.
Results
Expression of LMP1 and RON Is Correlated in NPC Tissues
The expression of LMP1 and RON was detected by IHC assay in 14 control lymphoid hyperplasia of nasopharynx and in 69 NPC. In NPC, IHC staining showed that LMP1 and RON were located in the cytoplasm and cell membrane. RON could not be detected in all 14 cases of lymphoid hyperplasia control (Figure 1A). LMP1 and RON were expressed in 22 of 69 (31.9%) and 26 of 69 (37.7%) NPC biopsies (Table 1). Moreover, the phosphorylated RON was observed in 16 of 26 (61.5%) RON-positive NPC biopsies (Figure 1B) (Table 1). Of note, we found a significant correlation between LMP1 and RON expression in NPC (Pearson's χ2 test; P = 0.0023) (Table 2).
Figure 1RON is expressed in LMP1 expressing NPC biopsies. A: IHC assays of control lymphoid hyperplasia of the nasopharynx and NPC sections were performed. Positive diaminobenzidine signals of LMP1 and RON expression were observed as a light brown color in LMP1-positive NPC and counterstained with hematoxylin which indicated the localization of nucleus. Negative immunoreactivity against LMP1 and RON were characterized by a blue background in control lymphoid hyperplasia of the nasopharynx and LMP1-negative NPC. Original magnification, ×200. Scale bar = 50 μm. B: The total and phosphorylated RON were detected by IHC assays. Positive diaminobenzidine signals of total RON and phosphorylated RON were observed as brown color signals and counterstained with hematoxylin which indicated the localization of nucleus. Original magnification, ×200. Scale bar = 50 μm.
To examine whether EBV affects the RON expression in epithelial models, we investigated the expression of RON in RHEK cells, two EBV-negative NPC cell lines (TW01 and TW03), and an EBV-positive C666-1 cell line. As shown in Figure 2A, the expression of RON in EBV harboring C666-1 cells was higher than that in the EBV-negative NPC cell lines and RHEK cells. To determine whether LMP1 affects RON expression, we investigated the expression of RON in RHEK and two NPC cell lines (TW01 and TW03) infected with LMP1 expressing lentiviruses. The Western blot results indicated that the expression of RON was increased in the LMP1-transfectants (Figure 2B). To determine which domains of LMP1 are essential for the expression of RON, cells were infected with lentivirus expressing pSIN vector control, pSIN-LMP1 full length, pSIN-LMP1ΔCTAR1, pSIN-LMP1ΔCTAR2, or pSIN-LMP1ΔCTAR1/2 for 5 days. LMP1 and LMP1ΔCTAR2 induced the expression of RON, whereas LMP1ΔCTAR1 and LMP1ΔCTAR1/2 could not induce the expression of RON. This result suggested that LMP1 increased RON expression through its CTAR1 domain (Figure 2C) that is well-known for NF-κB activation. Another study has reported that p65 is the main regulator of RON expression.
Therefore, we observed whether LMP1 regulates RON expression through p65 by using LMP1 expressing cells treated with NF-κB inhibitor (BAY11-7082). The result indicated that the expression of RON decreased in the presence of the NF-κB inhibitor (Figure 2D). Furthermore, chromatin immunoprecipitation assays were performed to investigate whether p65 binds directly to the RON promoter. It showed that binding of p65 to the RON promoter was detectable in LMP1 and LMP1ΔCTAR2 expressing cells, but not in pSIN vector control, LMP1ΔCTAR1, or LMP1ΔCTAR1/2 expressing cells (Figure 2E). These results suggested that LMP1 induces RON expression through NF-κB binding to the RON promoter.
Figure 2LMP1 increases RON expression through activation of nuclear factor-κB and constitutively phosphorylated RON is observed in LMP1-expressing RHEK cells. A: RHEK, TW01, TW03, and C666-1 cells were harvested and total protein was extracted. Expression of RON and GAPDH was detected by using Western blot. The pro-RON (190 kDa) and RON-β (150 kDa) were detected by anti-RON antibody. GAPDH served as an internal control. B: Vector and LMP1 expressing RHEK cells were harvested and total protein was extracted. TW01 and TW03 cells were infected with pSIN vector control or pSIN-LMP1 expressing lentiviruses at a MOI of 1 for 5 days. Expression of RON, LMP1, and GAPDH were detected by using Western blot. GAPDH served as an internal control. C: TW01 cells were infected with pSIN vector control, and pSIN-LMP1, pSIN-LMP1ΔCTAR1, pSIN-LMP1ΔCTAR2, or pSIN-LMP1ΔCTAR1/2 expressing lentiviruses at an MOI of 1 for 5 days and total protein was harvested. The expression of RON, full-length LMP1, LMP1ΔCTAR1, LMP1ΔCTAR2, LMP1ΔCTAR1/2, and GAPDH was detected by using Western blot. D: Vector and LMP1 expressing RHEK cells were seeded at a density of 4 × 105 cells per well in 6-well plates for 24 hours and were treated with DMSO or 10 μmol/L of BAY11-7082 for 6 hours. The transcripts of RON were detected by RT-qPCR (upper panel). The expression of RON, LMP1, phospho-IκB-α, and β-actin were measured by using Western blot (lower panel). E: DNA was harvested from TW01 cells infected with pSIN vector control, LMP1, LMP1ΔCTAR1, LMP1ΔCTAR2, or LMP1ΔCTAR1/2 expressing lentiviruses. Complexes of DNA and p65 were immunoprecipitated by using anti-p65 antibody or rabbit IgG. RON promoter DNA and the control GAPDH promoter DNA were detected by PCR. Total DNA was harvested and used as the input control. F: Vector and LMP1 expressing RHEK cells were harvested and total protein was extracted. Both pro-RON (190 kDa) and RON-β (150 kDa) were immunoprecipitated with anti-RON antibody. Phosphorylated RON-β (p-RON-β) was detected with anti-phosphotyrosine antibody by using Western blot. G: The vector, LMP1, LMP1ΔCTAR1, LMP1ΔCTAR2, or LMP1ΔCTAR1/2 lentiviruses infected TW01 cells were harvested and total protein was extracted. Both pro-RON (190 kDa) and RON-β (150 kDa) were immunoprecipitated with anti-RON antibody. Phosphorylated RON-β (p-RON-β) was detected with anti-phosphotyrosine antibody by using Western blot. IP, immunoprecipitation; WB, Western blot.
The Phosphorylation Status of RON Is Increased in LMP1-Expressing Cells
RON is a tyrosine kinase, and therefore we wondered whether LMP1 induction of RON expression affects the phosphorylation status of RON. Indeed, the expression of LMP1 increased the tyrosine phosphorylation status of RON in cells constitutively expressing LMP1 (Figure 2F). Moreover, we tested which domains of LMP1 are required for RON activation. According to our data, LMP1 and LMP1ΔCTAR2 induced the activation of RON, whereas LMP1ΔCTAR1 and LMP1ΔCTAR1/2 could not induce RON activation (Figure 2G). This result suggested that LMP1 increases RON activation through its CTAR1 domain. These experiments showed that LMP1 increases not only the amount of RON expression, but also the tyrosine phosphorylation of RON, suggesting that LMP1 facilitates the expression and kinase activity of RON in this epithelial model.
RON Plays a Pivotal Role in LMP1-Mediated Cell Morphological Changes and EMT
Alteration of the cell morphology after LMP1 expression was examined as shown in Figure 3A. RHEK vector control cells exhibited cobblestone morphology, but the LMP1 expressing cells displayed long spindle-shaped morphology. Furthermore, knockdown of RON changed the morphology of LMP1-expressing cells from a long spindle-shape to cobblestone morphology. To confirm this, immunofluorescence staining for LMP1 and RON was performed and the results showed similar morphological changes (Figure 3B). To determine the molecular changes of EMT, we detected the epithelial markers, mesenchymal markers, and Snail transcription factor after knockdown of RON. As shown in Figure 3C, LMP1 down-regulated the expression of the epithelial markers, E-cadherin, and γ-catenin. In the meantime, LMP1 increased the expression of the mesenchymal markers, vimentin. When RON was knockdown in LMP1-expressing cells, down-regulation of E-cadherin, and γ-catenin expression was restored, and the upregulation of vimentin expression was diminished. We also checked the expression of Snail, which is the key E-cadherin repressor that is essential for EMT regulation in embryogenesis.
After depleting RON expression, the expression of Snail, which was originally induced in LMP1 transfectants, was undetectable (Figure 3C). We analyzed the expression of epithelial and mesenchymal markers, and the Snail transcription factor at the transcriptional level by RT-qPCR, and their expression patterns were similar to their protein expressions (Figure 3, D–F). Hence, these results demonstrated that the morphological and molecular changes of EMT induced by LMP1 are mediated via the presence of RON. These evidences indicate that RON is required in the process of LMP1-induced EMT.
Figure 3LMP1 induces epithelial-mesenchymal transition (EMT) via RON. A: Vector and LMP1-expressing RHEK cells were electroporated with green fluorescent protein small interfering (si)RNA and RON siRNA plasmids for 5 days and both cell lines were re-seeded in 6-well plates and incubated for 24 hours. The morphology was observed microscopically. Original magnification, ×100. Scale bar = 100 μm. B: The expression of RON after siGFP and siRON transfection was observed by immunofluorescence assay. Red fluorescence indicated LMP1 expression and green fluorescence indicated RON expression in vector and LMP1-expressing RHEK cells. Blue fluorescence indicated the cell nuclei were stained with Hoechst. Original magnification, ×400. Scale bar = 25 μm. C: The expression of epithelial markers (E-cadherin and γ-catenin), mesenchymal marker (vimentin), and EMT transcription factor (Snail) was detected by using Western blot in siGFP or siRON RHEK cells, which were stably expressed vector or LMP1. The transcripts of epithelial markers (E-cadherin and γ-catenin) (D), mesenchymal maker (vimentin) (E), and transcription factor (Snail) (F) were detected by RT-qPCR.
To determine whether LMP1 induced migration and invasion through RON, a cell migration and invasion assay was performed. Compared to the vector control, LMP1-expressing cells were shown to have significantly higher migration activity in a wound healing assay. However, this migration activity was reduced when LMP1-expressing cells were transfected with RON small interfering RNA (Figure 4A). In addition, data from transwell migration and matrigel invasion assays also showed that LMP1 enhanced the cell mobility and invasiveness, whereas depletion of RON expression suppressed LMP1-induced migration and invasion (Figure 4, B and C). To further examine whether overexpression of RON affected cell migration, we performed cell migration assay in RON overexpressing RHEK cells. As showed in Figure 5, A and B, overexpression of RON induced the cell migration. Therefore, these data suggested that RON is the key mediator of LMP1-induced cell migration and invasion.
Figure 4LMP1 induces cell migration and invasion through RON. A: Vector and LMP1 expressing RHEK cells were electroporated with green fluorescent protein small interfering (si)RNA and RON siRNA plasmids after 5 days and then re-seeded in 24-well plates. The migration of cells was observed 24 hours after scratching monolayer cultures and being photographed. Original magnification, ×50. Scale bar = 200 μm. B: The siRNA electroporated vector and LMP1 expressing RHEK cells were plated in the upper chamber of a transwell. After 48 hours, cells were fixed and stained with crystal violet. The migrating cells were photographed (left panel), quantitated by dissolving the stained cells in 500 μL 1% SDS and the optical density was measured at 595 nm (OD 595) using an ELISA reader (right panel). Original magnification, ×100. Scale bar = 100 μm. C: After 72 hours, the invading cells were fixed, stained with crystal violet, and photographed (left panel), quantitated by dissolving the stained cells in 500 μL 1% SDS and the optical density was measured at 595 nm (OD 595) using an ELISA reader (right panel). Original magnification, ×100. Scale bar = 100 μm. **P < 0.01 by Student's t-test.
Figure 5Overexpression of RON induces cell migration. A: Vector and RON expressing RHEK cells were established and observed 24 hours after scratching monolayer cultures and being photographed. Original magnification, ×50. Scale bar = 200 μm. B: Vector and RON expressing RHEK cells were harvested and total protein was extracted. Expressions of RON and GAPDH were detected by using Western blot. GAPDH served as an internal control.
One of the characteristics distinguishing NPC from other head and neck cancers is its EBV association and high metastatic rate, and this is also the most critical issue in clinical treatment. Recently, EMT was considered as a new marker of cancer metastasis.
Collaborative activities of macrophage-stimulating protein and transforming growth factor-beta1 in induction of epithelial to mesenchymal transition: roles of the RON receptor tyrosine kinase.
We would, therefore, like to explore the involvement of RON in the process of EMT in NPC. Several studies have demonstrated the down-regulation of E-cadherin, the hallmark of EMT, in NPC, and the low expression of E-cadherin correlates with metastasis of NPC.
In vitro, LMP1 has been reported to decrease E-cadherin expression by inducing and activating DNA methyltransferase, resulting in the loss of cell-cell contact and enhancement of cell motile activity.
The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases.
Here, a novel mechanism of down-regulation of E-cadherin by LMP1 in NPC may be suggested. According to our data, the expression of E-cadherin was decreased in cells expressing LMP1 as reported elsewhere.
The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases.
Importantly, the down-regulation of E-cadherin could be reversed by the knockdown of RON (Figure 3C). These data suggested that RON is intermediated for LMP1-induced repression of E-cadherin. Furthermore, Snail is a zinc finger transcription factor that directly binds to the E-box in the E-cadherin promoter and represses expression of E-cadherin; the presence of Snail in LMP1-expressing cells was prohibited by the RON small interfering RNA (Figure 3C). Consistent with our finding, the expression of LMP1 and Snail is highly associated in NPC biopsies.
At the molecular level, we hypothesize that RON-mediated upregulation of Snail may be responsible for the LMP1-triggered down-regulation of E-cadherin. In addition, we found that knockdown of RON restored the γ-catenin expression in cells expressing LMP1 (Figure 3C). According to other reports, LMP1 decreased the expression of γ-catenin (also called plakoglobin) to enhance cell migration.
In summary, RON is the key mediator of LMP1-triggered EMT. The mechanism(s) that allow RON to regulate the expression of γ-catenin and Snail are worthy of further study.
At the molecular level, we showed that the CTAR1 domain of LMP1 is responsible for the upregulation of RON and stimulates p65 of NF-κB components binding to NF-κB binding sites on the RON promoter (Figure 2). Previous studies already demonstrated a sophisticated regulatory model of LMP1-induced epidermal growth factor receptor expression in that LMP1 CTAR1 activates p50/p50 homodimers and binding of the Bcl3 complex to the epidermal growth factor receptor promoter.
LMP1, apparently, can regulate diverse cellular genes by activating different NF-κB subunits. In addition to the NF-κB binding site, Sp1 is the potential transcription factor regulating RON expression.
We, therefore, tested the involvement of Sp1 in LMP1-induced RON expression by addition of Sp1 inhibitor, mithramycin. Obviously, the expression of LMP1-induced RON expression was not affected in the presence of effective Sp1 inhibitor (data not shown). Data from the JNK inhibitor (SP600125) and PI3K inhibitor (LY294002) assays also indicated that both LMP1 downstream JNK and PI3K signaling pathways were not participants in the RON expression (data not shown). However, we cannot completely exclude the involvement of other factors in the LMP1-induced RON expression.
RON has been demonstrated to play a pivotal role in promoting human cancers to invasive and metastatic phenotypes. To our knowledge, however, this is the first evidence presented that indicated RON was overexpressed in NPC tissues; and the expression of LMP1 and RON is significantly correlated (Figure 1) (Table 1, Table 2). Vigorous research on the expression of RON has been reported in several human carcinomas, although the regulation of RON is still poorly understood. We demonstrated that RON is upregulated by the EBV oncogene LMP1 in NPC, an EBV-associated malignancy. The presence of the EBV oncoprotein LMP1 was assumed to be one of the major features that promote NPC metastasis by triggering RON expression. Recently, for the clinical application, a dual RON/hepatocyte growth factor receptor (MET) inhibitor, compound I, has been shown to inhibit cell migration and suppress tumor growth in xenograft models.
In addition, a phase I clinical trail was already conducted using Foretinib, a multi-target inhibitor, which inhibits RON, MET, Axl tyrosine kinase, and vascular endothelial growth factor receptor in ovarian cancer, breast cancer, and colorectal cancer.
We showed that RON is overexpressed in 39.3% of NPC (Figure 1) (Table 1); these results suggest that RON could be a novel therapeutic target in certain NPC patients.
In this study, we showed that LMP1 induced EMT through RON and reported a strong correlation of LMP1 and RON expression in NPC. This result is not only important for explaining the metastatic character of NPC, but it also provides a new mechanism for the induction of EMT in human cancers.
Acknowledgments
We thank Dr. Tim J. Harrison (UCL Medical School, London, UK) for critically reviewing the manuscript.
Induction of c-Met proto-oncogene by Epstein-Barr virus latent membrane protein-1 and the correlation with cervical lymph node metastasis of nasopharyngeal carcinoma.
Epstein-Barr virus latent membrane protein 1 induces the matrix metalloproteinase-1 promoter via an Ets binding site formed by a single nucleotide polymorphism: enhanced susceptibility to nasopharyngeal carcinoma.
MUC1 induced by Epstein-Barr virus latent membrane protein 1 causes dissociation of the cell-matrix interaction and cellular invasiveness via STAT signaling.
Induction of cyclooxygenase-2 by Epstein-Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells.
Epstein-Barr virus latent membrane protein 1 induces micronucleus formation, represses DNA repair and enhances sensitivity to DNA-damaging agents in human epithelial cells.
Collaborative activities of macrophage-stimulating protein and transforming growth factor-beta1 in induction of epithelial to mesenchymal transition: roles of the RON receptor tyrosine kinase.
The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases.
Supported by grants from the National Science Council (NSC 100-2320-B-002-100-MY3) and the National Health Research Institute (NHRI-EX101-10031BI to C.-H.T.), and grants from the National Science Council (NSC98-2320-B-400-005-MY3 and NSC101-2325-B-182-002 to S.-J.L).
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