Ka-Po Tse1, Chih-Kai Chien2, Jau-Song Yu2,3, Yu-Sun Chang2

附件（一）

The Epstein-Barr virus oncogene product, latent membrane protein 1, regulates Nm23-H1 at transcriptional level

1Institute of Microbiology and Immunology, National Yang-Ming University, Bei-tou, Taipei 112, Taiwan Republic of China, 2Graduate Institute of Basic Medical Sciences and 3Proteomic core Laboratory, Chang-Gung University, Kwei-shan, Taoyuan 333, Taiwan Republic of China

Grant support:

Reprint requests: Yu-Sun Chang, Graduate Institute of Basic Medical Sciences, Chang- Gung University, 259 Wen-Hwa 1st Road, Kwei-shan, Taoyuan 333, Taiwan, Republic of China. Phone: 011-886-3-2118683; Fax:011-886-3-2118683; E-mail: [email protected]

Keywords: Metastasis suppressor, Nm23-H1,

Abbrevations: NPC, Nasopharyngeal carcinoma; EBV, Epstein-Barr Virus; LMP1, Latent membrane protein 1; Nm23, Nonmetastatic protein 23 Abstract: Epstein-Barr Virus (EBV) is closely related with many human malignancies like Burkitts’s lymphoma, Hodgkin’s lymphoma and Nasopharyngeal carcinoma (NPC). Among them, NPC is prevalent in southern China and is notoriously metastatic. Latent membrane protein 1 (LMP1), one of the viral proteins expressed in NPC biopsies, has been reported to induce morphological changes and enhanced invasiveness of epithelial cell when over-expressed. However, little is known about the underlying mechanisms or target proteins regulated by LMP1. To get a global view of protein expression modulated by LMP1, proteomic strategies were used. From NPC cell lines that stably express LMP1 or the control vector, whole cell extracts were separated by two-dimensional electrophoresis (2-DE), stained and analyzed. Protein differentially expressed in two cell lines were identified by matrix-associated laser desorption ionization mass spectrophotometry-time of flight (MALDI-TOF). Comparative analysis of the respective spot patterns on two-dimensional electrophoresis showed that Nm23-H1, which is a metastasis-suppressor, was downregulated in the LMP1-expressing cell line. Reduction of Nm23-H1 protein and mRNA levels in LMP1- stable cell line were further confirmed by Western blot and quantitative real-time PCR, respectively. Data from Nm23-H1 promoter studies demonstrated that LMP1 represses NM23-H1 expression at the transcriptional level. Furthermore, the mechanism underlying the repression is studied. Since down-regulation of Nm23-H1 in NPC has been correlated with lymph node metastasis, our preliminary data further suggests that LMP1 may be the critical viral protein mediates Nm23-H1 down-regulation in NPC and which can explain the role of LMP1 in enhancing tumor metastatic ability. Introduction: Epstein-Barr virus (EBV), a ubiquitious human gammaherpesvirus, is implicated in the etiology of human malignancies, such as Burkitt’s lymphoma (BL), Hodgkins’s disease and Nasopharyngeal carcinoma (NPC) [1], gastric carcinoma [2] and invasive breast cancer. Nasopharyngeal carcinoma (NPC) is one of the common malignancies in the south of China. This cancer comprises～40% of the head and neck cancers and is notorious for its highly metastatic nature. In NPC, EBV infection is predominantly latent and viral gene expression is restricted, similar to other EBV-associated malignant tumors. One of the viral genes, latent membrane protein 1 (LMP1), is expressed in～70% of NPC. This protein has the ability to transform rodent cells [3] and render cell growth in soft agar [4]. Recent studies showed that human epithelial cells expressing LMP1 display significant higher invasive capacity, correlating with decreased E-cadherin and RECK expression [4, 5] or increased MMP-9 activity. LMP1 is a 63-kDa integral membrane protein comprising a short N-terminal domain, six transmembrane domains and a 200-aa C-terminal domain. The C-terminal domain can be subdivided into two major activating regions, CTAR1 and CTAR2. CTAR1 associates with tumor necrosis factor receptor-associated proteins (TRAFs), whereas CTAR2 interacts with tumor necrosis factor receptor-associated death domain protein (TRADD), which may mediates nuclear factorB activity [6] and induces expression of the epidermal growth factor receptor [7]. Although several studies indicated that LMP1 is involved in multiple cellular functions, the mechanism used by the protein in mediating cell migration activity is still unknown. The first nm23 gene (nm23-M1) was originally identified by subtractive cloning in murine melanoma cell lines as a putative tumor metastasis suppressor [8] . The metastasis suppressor function of nm23 has been demonstrated by both in vivo and in vitro experiments that show reduced incidence in metastatic potential on transfection of nm23- M1 and nm23-H1 cDNA into highly metastatic murine melanoma cells [9] and human breast cancer cells [10], respectively. In addition, nm23 transfection inhibits motility of human and murine tumor cells in response to different factors [11]. Nm23 possesses several enzymatic activities, including a nucleoside diphosphate kinase activity [12], a histindine kinase activity [13], a serine protein kinase activity [14], and a GTPase- activating protein of the Ras-related GTPase Rad [15]. A number of previous studies have reported a correlation between nm23-H1 expression and poor prognosis for various human tumors (breast carcinoma, colon carcinoma, gastric carcinoma, and hepatocellular carcinoma [16-18] In this study, we tried to get a global screening of target proteins modulated by LMP1, and proteomic approaches were used. Protein expression profiles of LMP1- positive and negative cell lines was compared after 2-DE. We found that Nm23-H1, one of the most representative metastasis suppressor, was downregulated in the LMP1- expressing cell lines. Western blotting and quantitative real-time RT-PCR further confirmed the result. Studies from nm23-H1 promoter activity assay indicated that LMP1 represses nm23-H1 gene expression at the transcriptional level in a dose-dependent manner and C-terminal of LMP1 is responsible for the repression. Ectopic expression of HA-tagged nm23-H1 with LMP1 can inhibit LMP1-induced cell migration, which suggested that LMP1 may enhance cell migration ability, one of the criteria essential for tumor metastasis, through down-regulation of nm23-H1. Materials and Methods: Cell culture MCF-7 cells (ATCC no.HTB22) was obtained from the American Type Culture Collection. The NPC076 cell line was kindly provided by C. T. Lin (National Taiwan University, Taiwan). LMP1 expressing cell clones were established as described previously [19]. All cell were cultured in DMEM (Gibco, UK) supplemented with 10% FBS and penicillin/streptomycin.

Plasmid construction The Flag-NLMP plasmid was constructed as described previously [19], and LMP1 mCTAR1 (PXQXT→AXAXT) constructed was designed based on previous studies[6]. HA-tagged Nm23-H1 construct pGL2-Nm23-H1 plasmid [20] was kindly provided by Dr. Kwang-Huei Lin.

Sample preparation and 2D-gel Electrophoresis Cells were washed with ice-cold phosphate-buffered saline (PBS) for three times, scrapped by rubber policeman and centrifuged at 1,000×g for 2min. The cell pellet were then solubilized in lysis buffer containing 8M urea, 4% CHAPS and 2% IPG (v/v) buffer (pH4-7NL), mixed with rehydration buffer (9M urea, 4% CHAPS, 20mM dithiothreitol (DTT), 0.05% IPG buffer and 0.002% Bromophenol Blue) respectively and separated in the first-dimension for isoelectric focusing by using commercial Immobiline DryStrip (nonlinear pH gradient 3-10, 13cm length, Amersham Biosciences) on an IPGphor isoelectric focusing system (Amersham Biosciences) at 20℃ under the following condition: 50V for 12h, 100V for 0.5h, 150V for 0.5h, 250V for 0.5h, 500V for 0.5h, 1000V for 0.5h, 4000V for 0.5h, then 4000-8000V for about 12h with a total of 45,000V- h. After IEF, the IPG strips were immediately equilibrated for 2×15 min with gentle shaking in 10ml equilibration solutions (50mM Tris-HCl pH8.8, 6M urea, 30% glycerol, 2% SDS, 1% DTT and 0.01% Bromophenol Blue). The strips were then subjected to second-dimension separation in 8-15%, 16×16 cm2 gradient SDS-PAGE.

Silver staining After 2-DE, the gels were fixed in 50% methanol and 25% acetic acid for at least 2h, washed with 30% methanol for 15min followed by washing with deionized water for 5min×3. The gels were treated with 0.8mM sodium thiosulphate for 2min and washed with deionized water for 30sec×3. Prechilled 0.2% silver nitrate was used for staining for 25min, the gels were then washed with deionized water twice before developing with 0.0185% formaldehyde and 0.016mM sodium thiosulfate in 2% sodium carbonate solution. The developing process was terminated by washing with 0.042M ethylenediaminetetraacetic acid disodium salt dihydrate for 10min, and finally replaced with deionized water for storage.

In-gel digestion and MALDI-TOF analysis Protein bands are excised, cut into 1mm3 pieces, place into 0.65ml siliconized tubes (PGC Scientific), and washed three times with 40% acetonitrile/50mM ammonium bicarbonate for 15 min with shaking and destained with 6mM potassium ferricyanide in 1mM sodium thiosulphate. After destaining, gel spots were washed with 25mM ammonium bicarbonate until it was colorless and then washed with acetonitrile. Remove the acetonitrile after the gel spots were shrunk and followed by in-gel digestion with freshly prepared enzyme solution (20g/l of trypsin (Promega) in 25mM ammonium bicarbonate ) at 37℃ overnight. The digested peptides were extracted by 100% acetonitrile containing 0.05% trifluoroacetic acid and analyzed by UltraFlex MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Germany).-cyano-4-hydroxy cinnamic acid was used as matrix. The time-of-flight was measured using the following parameters: 21kV accelerating voltage and 150ns delay extraction time. Laser shots at 500 per spectrum were used to acquire the spectra with mass range from 500 to 3000Da. External calibration was performed using Angiotansion II (M+H+, 1046.54) and ACTH (M+H+, 2465.20) in the same series as the samples to be measured. Internal calibration was also performed using autolytic fragment peaks of procine trypsin (M+H+, 2211.1 and 842.56). The peptide mass profiles produced by MALDI-TOF were analyzed by Biotools. Protein identification of peptide fragments was performed by using Mascot search engine based on the entire MSDB database, on the assunption that peptides were monoisotopic, oxidized at methionine residues and carbamidomethylated at cysteine residues. Up to 1 missed trypsin cleavage and mass error tolerance of 50ppm for matching the peptide mass value was allowed. Score greater than 62 (95th percentile) were considered significant (p<0.05).

Western Blot The cells were collected and the total protein concentration was determined as mentioned above. Five g of protein lysate were dissolved in 4× sampling buffer (0.25M Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 20% 2-mercaptoethanol and 0.4% Bromophenol Blue) and separated by SDS-polyacrylamide gel electrophoresis (SDS- PAGE). The gel was then electro-transferred to a nitrocellulose membrane (HybondTM ECLTM, Amersham pharmacia biotech) in transfer buffer (2.9g Glycine, 5.8g Tris-base,

200ml methanol, add ddH2O to final volume of 1L, pH 8.3) at 250mA for 2 hr. After electro-transfer, the nitrocellulose paper was stained with Ponceau S (Merck) to check if protein was transfered competely and then was blocked with 5% non-fat milk in the Tris- buffered saline-with 1 %Tween 20 (TBS-T) solution for 2 hr at RT. The membrane was incubated with rabbit polyclonal anti-Nm23-H1 (C-20, Santa Cruz, USA), anti-LMP-1 (S12) [21], and anti-tubulin (NeoMarkers, USA, was used as the internal control ) antibodies. After incubation at RT for 3 hr, the membrane was washed with TBS-T for 20 min, 3 times. After that, the membrane was incubated with HRP-conjugated anti-mouse IgG (1:10000, Amersham Biosciences) that was diluted in 5% non-fat milk in the TBS-T for 1 hr at RT and then was washed for 15 min, 3 times with TBS-T solution. The blots were developed by ECL system (Amersham Biosciences, USA) and exposed to an X-ray film (Kodak BioMax MR film).

Quantitative real-time Reverse Transcription (RT)-PCR Total RNA of the LMP1-expressing and control cells was isolated with the TRIzol reagent (GIBCO/BRL). The mRNA (1g) was used for the synthesis of first-strand cDNA with an oligo-(dT) primer and Taqman Reverse transcription kit (Applied Biosystems). Primers used for the detection of Nm23-H1 and the internal control glyceraldehydes-3- phosphate dehydrogenase (GAPDH) are described in the literature[22]. The primers, Nm23-H1 Forward (5’—3’) that generate a 200bp product were used to detect the expression level of the Nm23-H1. Quantitative RT-PCR was performed according to the manufacturer’s instructions on a Light-Cycler instrument (Roche Diagnostics) with Faststart DNA Master SYBR Green I (Roche Diagnostics), which fluoresces on binding to double-stranded DNA. Results were normalized to GAPDH.

Transient transfection NPC076 (2×105) cells were transfected with 0.5g Flag-tagged LMP1, 3g pGL2-Nm23-H1 constructs, and 0.5g pSV2-gal (Promega) using CaPO4-precipitation method. MCF-7 (2×105) cells were transfected with 0.5g Flag-tagged LMP1, 1g pGL2-Nm23-H1 constructs, and 0.2g pSV2-gal using lipofectamine (GIBCO/BRL), according to manufacturer’s instructions. Cells were collected 24 hours post transfection.

Luciferase Reporter assay Cells were washed twice with PBS and lysed in 50l luciferase lysis buffer (25mM Tris-phosphate, pH 7.8, 2mM 1,2- diaminocyclohexane-N,N,N’, N’-tetraacetic acid, 10% glycerol, 10%Triton X-100 and 2M DTT) (Promega, USA) to mix well by vortexing and then kept on ice for 10 min. After centrifugation at 12,000 r.p.m. at 4℃ for 15 min, the supernatant was transferred to a new Eppendorff tube and the protein concentration was estimated by measuring in a spectrophotometer (SHIMADZU UV-

160A, Japan) at OD595nm using Bradford’s method. Fifty g of total protein from each cell extract was automatically mixed with 100ul of luciferase assay buffer (20mM Tricine,

1.07mM (MgCO3)4Mg(OH)2 ． 5H2O, 2.67mM MgSO4, 0.1mM EDTA, 33.3mM DTT, 270uM coenzyme A, 470uM luciferin and 530uM ATP) (Promega, USA) and the relative light unit (R.L.U.) was measured by Autolumat LB953 (Berthold, Germany) within 10 sec.

Transwell migration assay Cell migration ability was evaluated by using the chemotaxis chamber (Neuroprobe, Cabin John, MD). 48 hours post transfection, cells were washed, trypsinized and counted. (1×104) cells in 50l of culture medium were applied to the upper chamber of the device, and 30l of medium containing 10g/ml collagen type IV was added to the lower chamber. A polycarbonate membrane with a pore size of 8m was placed in between the two chambers. After 6 hours of incubation at 37℃, the membrane was fixed in methanol for 10 min and stained in 10:1 water/Giemsa solution for 1 hr. Migrated cells on the membrane were counted under a microscope. Results: Proteomic analysis of differentially expressed proteins between LMP1-expressing and control cell lines by 2-DE To study the proteins regulated by LMP1 and may involved in LMP1-induced cell transformation or cell invasiveness, we extracted the total proteins of NPC cell line- derived LMP1-expressing and control cells and used large-format, high-resolution 2-DE to monitor changes in the abundance of proteins. The pH range for the first dimension is pH4-7NL. The second dimension runs in an 8-15% gradient SDS-polyacrylamide gel. We obtained high resolution (over 1000 spots/gel) and high reproducibility (>95%) 2-D gels (Figure 1). For each group, we integrated three 2-DE silver-stained gels and used Image Master 2D Elite Version 3.01 software to analyze the 2-DE images. Through image analysis, we found that 15 protein spots showed significantly different expression patterns between two cell lines.

Identification of proteins by MALDI-TOF/MS Based on the comparative analysis of protein expression patterns between LMP1- expressing and control cell lines, the 15 differentially expressed proteins were picked from the silver-stained gel. All of them were digested by trypsin and got peptide masses maps from MALDI-TOF analysis. Table 1 showed the proteins differentially expressed and identified by MALDI-TOF as shown in figure 2. In the identified protein candidates, transformation upregulated nuclear protein, type II cytoskeletal 8, protein disulfide- isomerase ER60 precursor, Adenylate kinase 6, stratifin and tropomyosin were all lowly expressed in the control (LMP1-negative) cell and significantly up-regulated in LMP1- expressing cells, while dnaK-type molecular chaperone HSPA5 precursor, ER endoplasmic reticulum lumenal Ca2+ binding protein grp78, Bip protein, Keratin type II cytoskeletal 1, Annexin II, FUSE binding protein, -actin, -actin 1 and nonmetastatic protein 1 (NME1/Nm23-H1) have a higher expression in control cells and a low expression in LMP1-expressing cells.

LMP1 downregulates the expression of Nm23-H1 Among the identified proteins, nm23-H1 is one of the best representative metastasis-suppressor, which may play a role in LMP1-induced tumor metastasis. The protein spot (M.W./ pI =17/5.83) identified as nm23-H1 was further confirmed by MS/MS analysis (Figure 3A and 3B) and Western blotting using nm23-H1 specific antibody (C-20) (Figure 4B). To check whether expression of nm23-H1 is modulated by LMP1, whole cell extracts of 1) LMP1-expressing cell lines and control cell lines; 2) 293T cells transiently transfected with Flag-tagged LMP1 expression vector or empty vector were examined by western blotting (Figure 5A). We found that nm23-H1 protein was moderately downregulated in LMP1-stably expressed NPC076 cells, which correlates with the result from proteomic studies. However, nm23-H1 is significantly repressed by LMP1 in 293T cells, which suggested that LMP1 down-regulates nm23-H1 in epithelial cells and the fold of repression is cell-type specific. To evaluate if this reduction of protein level is the result of transcriptional down- regulation. Total RNA of both cell lines was isolated and reverse transcripted using Nm23-H1-specific primers. Nm23-H1 mRNA level was determined by quantitative real- time PCR using specific primers and GAPDH was used as internal control. Data from quantitative PCR indicated that Nm23-H1 mRNA level was also downregulated (about 50%) in LMP1-expressing cell line (Figure 5b), which is consistent with the result of Western blotting. Our results showed that expression of nm23-H1 at the protein and mRNA levels was down-regulated in the presence of LMP1.

LMP1 downregulates nm23-H1 at transcriptional level Since Nm23-H1 mRNA level was downregulated in LMP1-expressing cell lines, Nm23-H1 promoter activity was studied in the presence of LMP1. Luciferase-reporter gene driven by Nm23-H1 promoter (pGL2B-Nm23-H1, ranging from –500 to +95) was transiently cotransfected with LMP1 expression plasmid (Flag-LMP1) or control plasmid (Flag-CMV) into NPC076 and MCF-7 cells, transfectants were collected and luciferase activity was assayed according to the manufacturer’s recommendation. Nm23-H1 promoter activity was repressed (about 60% in NPC076 and 85% in MCF-7) in the presence of LMP1 (Figure 7A). In addition, luciferase-reporter plasmid containing nm23- H1 promoter was cotransfected with increasing amount of Flag-tagged LMP1 expression plasmid into MCF-7, nm23-H1 promoter activity reduced as the LMP1 protein increased (Figure 7B). Taken together, data indicated that LMP1 represses Nm23-H1 promoter activity in NPC and MCF-7 cells in a dose-dependent manner.

C-terminal of LMP1 is essential for the repression of nm23-H1 promoter activity CTAR1 and CTAR2 of LMP1 are two critical regions responsible for most of the LMP1-mediated functions. Mutations or deletions of these regions may abolishes LMP1- mediated signaling. We used this criterion to clarify the clarify the functional motif on LMP1 responsible for the repression. Flag-tagged LMP1 and its mutants (Figure 8.) (LMP1YYD, LMP1295, LMP1260, LMP1186, LMP1N-mut and LMP1mCTAR1) were co-transfected with nm23-H1 promoter-reporter constructs into NPC076 cells, and effects were measured by analyzing luciferase activity. Figure 9A indicated that nm23-H1 promoter activity could only be restored when C-terminal of LMP1 is completely deleted. Neither deletion of CTAR2, 3 nor mutated SH3 motif on N-terminal cannot restored the nm23-H1 promoter activity. However, residues between amino acid 186-260 containing CTAR1 motif seemed to be required for the repression. Although mCTAR1 construct used in the studies cannot restored the nm23-H1 promoter activity, but it could not exclude the possibility of signaling activated from CTAR1 downregulates nm23-H1 promoter activity because the mutated PXQXT motif is only required for the interaction between LMP1 and TRAF molecules. The involved signaling pathway will be further examined by the use of specific signaling inhibitors.

Ectopic expression of Nm23-H1 in epithelial cells can blocks LMP1-induced cell migration Since expression of LMP1 in epithelial cell has been reported to increase cell migration ability, expression level of NM23-H1 correlates with cell metastatic ability and re-introduction of Nm23-H1 into highly metastatic tumor cell lines can block cell invasiveness. We performed transwell migration assay to answer whether restoration of Nm23-H1 expression in LMP1- expressing cell lines can block LMP1-mediated cell migration. We generated a HA-tagged Nm23-H1 expression plasmid and co-transfected with Flag-tagged LMP1 expression plasmid into MCF-7, a breast cancer cell line with low migration ability and was usually used as a model in cell migration studies. Overexpression of LMP1 alone in MCF-7 increase cell increased cell migration ability as reported before. Re-introduced Nm23-H1 in MCF-7 can reduced the migrated cell even in the presence of LMP1. All these results suggested that LMP1 may enhance cell migration ability through down-regulation of Nm23-H1 in epithelial cells. Discussion:  Our data demonstrated that LMP1 induced downregulation of nm23-H1 via transcriptional repression in epithelial cell lines. In addition, we found that C- teriminal of LMP1 is responsible for the repression.  The nm23 gene is known as the best representative of metastasis-suppressor genes, and the nature of its matastatic inhibition was confirmed by a transfected-cell-line experiment [10]  In 1998, Guo et al. reported that the absence of nm23-H1 protein expression was significantly associated with lymph-node metastasis, recurrence and distinct metastasis in NPC patients.  The function of the nm23 gene product in relation to regional lymph-node metastasis and distant metastasis of NPC remains undetermined. reference

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