Role of a novel EGF-like domain-containing gene NGX6 in cell adhesion modulation in nasopharyngeal carcinoma cells
Jian Ma1,
Jie Zhou1,
Songqing Fan1,
Lili Wang1,
Xiaoling Li1,
Qun Yan2,
Ming Zhou1,
Huaying Liu1,
Qiuhong Zhang1,
Houde Zhou1,
Kai Gan1,
Zheng Li1,
Cong Peng1,
Wei Xiong1,
Chen Tan1,
Shourong Shen3,
Jianbo Yang1,
Jiang Li1 and
Guiyuan Li1,4
1 Cancer Research Institute, 2 Xiang-Ya Hospital and 3 The Third Xiang-Ya Hospital of Central South University Xiang-Ya School of Medicine, 110 Xiang-Ya Road, Changsha, Hunan, 410078, China
4 To whom correspondence should be addressed Email: ligy{at}xysm.net
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Abstract
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The epidermal growth factor (EGF)-like domain is involved in receptorligand interactions, extracellular matrix formation, cell adhesion and chemotaxis. Nasopharyngeal carcinoma associated gene 6 (NGX6) is a novel EGF-like domain-containing gene located at the high frequent loss of heterozygosity (LOH) region 9p21-22 associated with nasopharyngeal carcinoma (NPC). It is down-regulated in NPC and its over-expression can delay the cell cycle G0G1 progression in NPC cells. In the present study, in situ hybridization analysis, using NPC tissue microarrays, showed that loss of NGX6 expression was associated with NPC lymph node metastasis. The Tet-on gene expression system and cDNA array techniques were used to profile the potential targets of NGX6. We found that NGX6 can influence the expression of some cell adhesion molecules in NPC cells. NGX6 can associate with ezrin, a linkage between the cell membrane and cytoskeleton. The NGX6 protein was expressed on the cell surface as a glycoprotein. Ectopic induction of NGX6 can impair NPC cell migration and invasive ability as well as improve cell adhesion and gap junctional intercellular communication, and can suppress tumor formation in vivo. The data revealed that NGX6 plays a role in cell adhesion modulation in NPC cells.
Abbreviations: Dox, doxycyclin; EGF, epidermal growth factor; EGFP, enhanced green fluorescent protein; EST, expressed sequence tag; GJIC, gap junctional intercellular communication; H&E, hematoxylin and eosin; HNE, human nasopharyngeal carcinoma epithelial cell line; LOH, loss of heterozygosity; NGX6, nasopharyngeal carcinoma associated gene 6; NPC, nasopharyngeal carcinoma; PBS, phosphate-buffered saline; SLDT, scrape-loading and dye transfer; TMA, tissue microarray
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Introduction
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The short arm of chromosome 9 is thought to include multiple tumor suppressor genes, because the loss of heterozygosity (LOH) on 9p is one of the most frequent genetic alterations in many common cancers (15). In particular, LOH at 9p21-22 has been observed in up to 6187% of nasopharyngeal carcinomas (NPC) (69). NPC incidence shows striking geographical and ethnic distribution. Similar to other human cancers, the development of NPC likely involves multiple genetic events including activation of oncogenes and inactivation of tumor suppressor genes (10).
In order to isolate a putative tumor suppressor gene, we previously delineated a novel minimal LOH region between markers D9S161D9S1853 at 9p21-22 using 11 high-density microsatellite polymorphic DNA probes and 25 primary NPC samples. We subsequently studied the expression patterns of 25 novel expression sequence tags (ESTs) in NPC cell lines, primary culture of nasopharyngeal epithelial cells, and NPC biopsies to identify novel genes in this region (11). One of these ESTs (Genbank entry R38763) was found to be down-regulated in NPC. We obtained the full-length cDNA corresponding to the human EST R38763 from the IMAGE Consortium and named it nasopharyngeal carcinoma associated gene 6 (NGX6) (Genbank accession AF188239) (12).
The NGX6 cDNA is 2134 bp in length and encodes a putative protein of 338 amino acids with a predicted molecular weight of 37 kDa. The NGX6 protein includes two transmembrane regions. The extracellular region contains one EGF (epidermal growth factor)-like domain and three potential N-glycosylation sites, and the short cytoplasm contains a tyrosine residue that is a potential phsophorylation site by tyrosine kinases. Searching protein database for the NGX6-related protein revealed a high similarity of amino acid sequence between the NGX6 and the human M83 proteins (Genbank accession NM_021259). The M83 protein is a five-span transmembrane molecule and contains a tyrosine residue in the cytoplasmic domain (13). NGX6 and M83 may define a new subfamily of the multi-span transmembrane protein superfamily. N-Glycosylation sites are signature of some cell adhesion molecules. The EGF-like domain is involved in the interaction between receptors and ligands in cell adhesion and signal transductions, some cell adhesion-related molecules and extracellular matrix components contain this domain (14).
Our previous study showed that NGX6 was expressed at high levels in normal nasopharyngeal epithelial tissues, but at low levels in nasopharyngeal carcinoma biopsies and cell lines (12). NGX6 was also down-regulated in colorectal carcinomas, and the frequency of down-regulation of NGX6 in colorectal carcinoma tissues with lymph node or distance metastasis (15/16) was significantly greater than in patients without metastasis (25/34) (P < 0.05) (15). Ectopic expression of NGX6 can induce the reversion of some of the malignant phenotypes of NPC cells (data obtained from cell-cycle analysis, cell growth rate curves, soft agar colony formation and nude mice injection analysis) (16).
In the present study, we characterized NGX6 as a glycosylated membrane protein. We also performed in situ hybridization for NGX6 using an NPC tissue microarray (TMA) and found that the NGX6 mRNA expression level was correlated with NPC lymph node metastasis status. Then, we employed the Tet-on gene expression system (17) to make the expression of NGX6 inducible in the presence/absence of doxycyclin (Dox) in NPC cells, and identified several differentially expressed genes, induced by NGX6 over-expression, using a cDNA array. Since the differentially expressed genes included a number of cell adhesion molecules, we determined the role of NGX6 in cell adhesion events, and found that NGX6 can influence NPC cell adhesion properties.
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Material and methods
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Cell culture, transfection, plasmid construction and antibodies
The human NPC cell line, human nasopharyngeal carcinoma epithelial cell line (HNE)2, was maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) (18), and the mammary COS7 cells (from American Type Culture Collection, Rockville, MD) were grown in DMEM (Invitrogen), supplemented with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated at 37°C in a 5% CO2, humidified environment. Transfections were performed using Lipofectamine (Invitrogen) according to the manufacturer's instructions. For these experiments four vectors were constructed. The pTREhyg-NGX6 expression vector was used to create the Tet-on-NGX6-HNE2 system. The pCMV-Myc-NGX6 expression vectors, used for transient transfection of the NGX6 ORF (open reading frame), were tagged with a Myc tag at the N-terminus. The enhanced green fluorescent protein (EGFP)-C2-NGX6 expression vector was used to determine NGX6 cellular localization. The EGFP-C2-NGX6 (1183 aa) encoded a mutant protein deleted of the EGF-like domain and two-span transmembrane regions. All vectors were confirmed by DNA sequencing. Antibodies were purchased from the indicated companies: mouse anti-Myc (Becton Dickinson Bioscience, Palo Alto, CA), rabbit anti-ezrin, anti-integrin
2, anti-integrin ß1 (Upstate, Lake Placid, NY), anti-
tublin, anti-nm23 H1, anti-VE cadherin, anti-catenin
2, protein-A (or G) agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-mouse IgG, goat anti-rabbit IgG (US Biological, Swampscott, MA), fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG, Cy3-conjugated anti-rabbit IgG (Sigma, St Louis, MO).
Immunofluorescence staining, GFP detection and confocal microscopy
COS7 cells were plated on coverslips and transfected as indicated in the figure legend. Cells were fixed in containing 3.7% paraformaldehyde for 1 h. The cells were permeabilized using 0.2% Triton X-100 and blocked using normal goat serum as recommended (Molecular Probes, Eugene, OR). The primary antibodies, mouse anti-Myc and rabbit anti-ezrin, were added and incubated at room temperature for 2 h followed by washing with phosphate-buffered saline (PBS). The secondary antibodies, FITC-conjugated sheep anti-mouse IgG and Cy3-conjugated sheep anti-rabbit IgG, were then added and incubated for 1 h. The coverslips were washed four times with PBS and mounted using Gel/Mount mounting media (Biomeda, Foster City, CA). Control experiments were carried out without primary or secondary antibodies. The staining was examined under a 60x oil immersion objective using a Bio-Rad laser confocal microscope. For detection of GFP-fusion proteins in live cells a 60x water immersion objective was used.
Immunoprecipitation, western blotting and glycosylation detection
Immunoprecipitation was performed as described (19). Briefly, the cells were lysed in modified radioimmune precipitation buffer and insoluble material was removed by centrifugation. Antibodies were then added to lysates, incubated for 16 h at 4°C, collected with protein A or Gagarose beads, and the immunocomplexes were washed three times with lysis buffer at 4°C. The immunoprecipitates were resolved on SDSPAGE followed by western blot analysis. The bound primary antibody was detected with a HRP-conjugated secondary antibody and visualized by Super Signal West Pico Chemiluminescent Substrate Reagent (Pierce, Rockford, IL) according to the manufacturer's instruction and exposed to Kodax film (Rochester, NY). For the detection of NGX6 glycosylation, COS7 cells were transiently transfected with the pCMV-Myc (negative control) or pCMV-Myc-NGX6 expression vectors. The NGX6 protein was immunoprecipitated with a monoclonal mouse anti-Myc antibody. Hydrolysis of N-linked oligosaccharides from the immunoprecipitated NGX6 protein was performed using 0.5 U of N-glycosidase F (Roche, Penzberg, Germany), according to the manufacturer's instructions. Then, the treated protein was resolved on a 12% gel by SDSPAGE followed by western blotting using anti-Myc as the primary antibody.
In situ hybridization for NGX6 using NPC TMA
Tissue samples. Nasopharyngeal biopsy specimens were obtained from 231 patients including 100 NPC patients with lymph node metastasis, 48 NPC patients without metastasis and 83 non-NPC patients (chronic inflammation of nasopharyngeal mucosa, CINM) at Xiang-Ya Hospital (Changsha, Hunan, China) during 2002 and 2003. The main characteristics of the patients were: age (45 ± 9.7, mean ± SD) and gender (male 159, female 72). Written informed consent was obtained from all of the patients participating in the study. The biopsied nasopharyngeal tissues were immediately fixed in 10% buffered paraformaldehyde, routinely processed, and embedded with paraffin. The entire paraformaldehyde-fixed nasopharyngeal sample of each patient was cut into 5-µm-thick sections. Microscopic examination of the biopsy tissues was performed following hematoxylin and eosin (H&E) staining. The diagnosis and lymph node metastasis status of the NPC were based on the International Union Against Cancer's TNM Classification of Malignant Tumors.
TMA construction. Representative areas of NPC and nasopharyngeal epithelium were selected and marked on the H&E slide and the corresponding tissue block was sampled for the TMA. The TMA was assembled with a tissue-arraying instrument (Beecher Instruments, Silver Springs, MD). Briefly, the instrument was used to create holes in a recipient block with defined array coordinates. A solid stylet was used to transfer the tissue cores into the recipient block. Two composite high-density TMA receptive blocks, including all spots, were designed. The 0.6-mm diameter tissue cores were taken from each NPC and nasopharyngeal epithelium sample. Serial 5-µm-thick sections were cut with a Leica microtome (Wetzlar, Germany) and transferred to adhesive-coated slides with the help of a Paraffin Tape-Transfer System (Instrumedics, Hackensack, NJ). One section from each tissue array block was stained with H&E. The remaining sections were covered with thin paraffin and stored at 4°C before the in situ hybridization assay.
In situ hybridization for NGX6. We used three 30-base nucleotide probes from different regions of the NGX6 cDNA corresponding to bp 380409, 16441673 and 18251854 of the NGX6 cDNA. The sequences were 5'-GAC CGG AAA TAC ACA GTC ACA CTT TGG TCT-3', 5'-GTA CAG ATA ATT GTG TGT GCG CAG CAG CTT-3' and 5'-AGG TGT AGA CTG CAG CTT CCA GCA CAT ATC-3' (Bioasia Biotech., Shanghai, China). The probes were labeled with 11-DIG-dUTP at the 3' end (DIG Olignucleotide 3'-tailing labeling Kit, 2nd Generation, Roche) and mixed together. Five-micrometer-thick sections of TMA were deparaffinized, re-hydrated and post-fixed in diethyl pyrocarbonate (DEPC)-treated PBS containing 4% paraformaldehyde for 5 min at 4°C. After washing with PBS, the sections were digested with 2 µg/ml proteinase K at 37°C for 15 min and again incubated with DEPC-treated PBS containing 4% paraformaldehyde for 10 min. Sections were subsequently incubated with 0.2 M HCl for 20 min, 0.25% (v/v) acetic anhydride with 0.1 M triethanolaminine buffer for 2x 5 min. Sections were incubated with pre-hybridization solution at 37°C for 2 h and then hybridized with sheared salmon sperm DNA (Invitrogen) and yeast tRNA along with the appropriate probes. The mixed three probes were used at a concentration of 500 ng/ml and hybridized overnight. After washing at 37°C in a graded series of SSC (2x SSC, 2x 15 min, 1x SSC, 2x 15 min, 0.25x SSC, 2x 15 min), immunological detection of digoxigenin was performed using a DIG nucleic acid detection kit (Roche). Sections were incubated in a solution of anti-digoxigenin horseradish peroxidase, Fab fragments (anti-DIG-POD), and the colorimetric substrate 3,3'-diaminobenzidine tetrahydrochloride. The slide was counterstained using Myers hematoxylin. A poly d(T) probe was used as a control for total RNA preservation. The expression of NGX6 was observed by microscopy. Correlations between different clinical status and NGX6 positive expression were analyzed using a
2 test.
Tet-on-NGX6-HNE2 system construction
The Tet-on-HNE2 cell line was generously provided by Dr Ya Cao (Central South University Xiang-Ya School of Medicine) (20). The cells were grown in RPMI 1640 medium supplemented with 100 µg/ml G418 (Sigma). The procedure for stable transfection is described below. Tet-on-HNE2 cells were allowed to grow to 60% confluency in a six-well plate and washed with 2 ml serum-free RPMI1640 media. A total of 2 µg of plasmid DNA and 6 µl of Lipofectamine were gently mixed in a total of 400 µl RPMI1640 and incubated at room temperature for 30 min. One milliliter of serum-free medium RPMI1640 was added to each tube containing the DNALipofectamine complex, mixed gently, and added to the cells. After incubation at 37°C for 12 h, the complex-containing medium was replaced with normal growth medium and cultured for 24 h. The transfected cells were selected in culture media containing 150 µg/ml Hygromycin B (Calbiochem, La Jolla, CA). About three weeks later, colonies were picked using the cloning cyclinder technique and expanded into cell lines: Tet-on-NGX6-HNE2.
Assay of the Tet-on-NGX6-HNE2 system by RTPCR, northern and southern blotting
RTPCR. Total RNAs of Tet-on-NGX6-HNE2 cells were extracted using the Trizol reagent (Invitrogen) and then treated with DNase (Roche) to eliminate contaminated DNA. Reverse transcription of the RNA was performed according to the instructions of Promega (Madison, WI) followed by PCR amplification. The PCR primers for NGX6 were: 5'-TGA CCT GTT CCA AAG AGT CCC TG-3' and 5'-GCA GCT TCC AGC ACA TAT CGA CT-3', the primers for GAPDH were: 5'-CAA CAG CCT CAA GAT CAT CAG CA-3' and 5'-GAG GAG GGG AGA TTC AGT GTG GT-3'. The GAPDH primers were added to the PCR reaction at the end of the fifth cycle as a control.
Northern blotting. Total RNA was resolved on a 1.2% agarose gel containing 2 mM formaldehyde and transblotted onto a nylon membrane. The membrane was then pre-hybridized at 68°C for 3 h with solution containing 5 ml ExpressHyb (BD) and 100 µg/ml salmon sperm DNA. The PCR-amplified probes were 32P-labeled by the random prime labeling system (Promega). The hybridization was carried out at 68°C for 24 h. The membrane was washed twice in 2·SSC/0.1% SDS at room temperature for 15 min, followed by a 30-min wash in 0.1·SSC/0.1% SDS, and then exposed to Kodak film for 4872 h at 70°C. The relative abundance of individual mRNA in each sample was normalized with a GAPDH mRNA band detected by hybridization to the 32P-labeled GAPDH probe.
Southern blotting. Genomic DNA from M22, K10 or Tet-on-HNE2 cells was digested with EcoRI and NdeI endonucleases (Takara, Otsu, Japan). Following electrophoretic separation in a 0.8% agarose gel, DNA fragments were transblotted onto a nylon membrane. The DNA blot was then subjected to Southern blot hybridization using a 32P-labeled DNA probe corresponding to a fragment of vector pTRE2hyg.
cDNA array analysis
Total RNA was isolated from Tet-on-NGX6-HNE2 cells using the Trizol reagent and treated with DNase I to eliminate possible genomic DNA contamination and confirmed by PCR for 35 cycles with primers of GAPDH. The concentration and quantity of total RNAs were assessed by absorbency at 260/280 nm and then labeled according to the manufacturer's instructions. Radioactivity of synthesized probes was
5 x 106 c.p.m./µg. Prior to hybridization, the Atlas human cancer cDNA expression array (BD) membranes were pre-hybridized with ExpressHyb and 100 µg/ml salmon sperm DNA at 68°C for 30 min. Subsequently, membranes were hybridized with the probes in ExpressHyb hybridization solution overnight at 68°C. The membranes were washed and then exposed to Kodax X-ray film at 70°C. The results of the arrays were scanned using a confocal laser scanner and the digitalized image data processed using AtlasImage software (BD). We used the mean of nine housekeeping genes (ubiquitin C, tyrosine 3-monooxygenase, hypoxanthine phosphoribosyltransferase 1, G3PDH,
-tubulin, MHC-I, ß-actin, ribosomal protein L13a and ribosomal protein S9) to normalize the intensity of the signals. A ratio of down/up regulation
2 was considered to be significant. To eliminate transcriptional profiling changes caused by Dox, we performed parallel experiments with the vector-control cell line (Tet-on-vector-HNE2, K10). The array experiments were performed twice, and selected genes that showed the same changes in the two independent experiments.
Adhesion assay
Cell adhesion was measured according to the method described by Busk et al. (21). In brief, the wells of a 96-well culture plate were coated with 0.1 ml fibronectin or laminin (Sigma) at different concentrations. In addition, 1 mg/ml of poly-D-lysine and 1% bovine serum albumin (BSA) in PBS were each coated into two wells as maximal- and minimal-adhesion controls, respectively. The 96-well plate was incubated at 37°C for 1 h and blocked with 1% BSA at 37°C for 0.5 h. Tet-on-NGX6-HNE2 cells (105 cells) were added to each coated well and incubated for 2 h at 37°C; wells were then washed twice and stained with crystal violet and the absorbance at 595 nm was measured. The cells adherent to the coated wells were calculated as follows (the data were expressed as the means of triplicate wells):
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Cell invasion and migration assay
Cell invasion was measured by a Matrigel invasion chamber assay, which was performed using 6.5-mm, 8-µm pore size Transwell chambers (Corning, Corning, NY). Matrigel (BD) was diluted in cold distilled water (200 µg/ml), 0.1 ml added to the upper well of the Transwell chamber, and dried in a sterile hood. The Matrigel was reconstituted with medium for 1 h at 37°C before the addition of cells. Tet-on-NGX6-HNE2 cells were starved overnight in serum-free medium and re-suspended at a concentration of 2.5 x 105 cells/ml in serum-free medium containing 0.1% bovine serum albumin. A sample (0.2 ml) of the cell suspension was added to the top of each well, and a 10 µg/ml fibronectin solution was added to the bottom well of the chamber as a chemo-attractant. After 36 h, the cells that had not invaded were removed from the upper surface of the filters using a cotton swab. The cells that had invaded to the lower surface of the filter were fixed with methanol, stained with H&E and eight high-power fields (200x) were counted. The data were expressed as the mean value of cells per high-power field in triplicate in two independent experiments. Cell motility was also determined by the invasion chamber assay in the absence of a Matrigel barrier and a decrease in incubation time to 10 h. To determine the mechanisms underlying the NGX6-dependent changes in the invasion and migration assay, the assay was performed in the presence of anti-integrin
2 and ß1 antibodies. Before the assay, the Tet-on-NGX6-HNE2 cell suspension (2.5 x 105 cells/ml) was incubated with anti-integrin
2 (10 µg/ml) and anti-integrin ß1 (10 µg/ml) antibodies for 20 min to block the integrin
2ß1, then was added to each transwell. Control mouse IgG (10 µg/ml) was used as the negative control.
Gap junctional intercellular communication (GJIC)
We used a rapid and simple technique, scrape-loading and dye transfer (SLDT), to study GJIC in NPC cell monolayers. Tet-on-NGX6-HNE2 cells were plated and grown overnight to confluence in a six-well plate. Monolayers of cells were rinsed with PBS, and immersed in the 0.05% lucifer yellow (Mr 457, Sigma) in PBS. Scrape-loading was performed using a sharp knife. Cells were incubated in dye solution for an additional 3 min at room temperature, rinsed with PBS and observed. Lucifer yellow does not diffuse through intact cell membranes, but its relative molecular mass permits diffusion through patient gap junctions in knife-injured cells. Cells competent in GJIC showed transfer of lucifer yellow from the injured border to interior cells, while cells incompetent in GJIC did not show dye transfer. Transfer of the fluorescent dye was observed using a phase-contrast inverted microscope equipped with epifluorescence. Quantification of cell communication was performed by counting the number of fluorescently labeled cells surrounding and including the injured cells.
Tumor formation assay in nude mice
To assess the effect of NGX6 on tumorigenicity in vivo, Tet-on-NGX6-HNE2 cells were washed once with PBS and then injected s.c. in the flank region at a concentration of 2 x 106 cells/mouse (6-week-old male nude mice). The mice were divided into two groups of 10 mice, and were administered drinking water supplemented with either 4% sucrose as control or 4% sucrose plus 1 µg/ml Dox to induce NGX6 expression. The animal experiments were performed in accordance with institutional guidelines.
Statistical analysis
All the data except for the TMA analysis were analyzed by the Student's t-test using the SPSS program. Differences were considered significant whenP < 0.05.
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Results
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Clinicopathological significance of NGX6 in NPC with or without lymph node metastasis
Since a previous study showed that the loss of NGX6 expression was associated with lymph node or distance metastasis in colorectal carcinomas (15), we investigated the association between NGX6 mRNA expression and the clinicopathological factors (lymph node metastasis) of NPC patients using in situ hybridization with a NPC TMA containing 231 tissue samples. The positive expression rate of NGX6 was 44% (44/100) in the group of 100 NPC patients with lymph node metastasis, while was 67% (32/48) in the group of 48 NPC patients without lymph node metastasis, and 82% (68/83) in the group of 83 non-NPC patients (see Figure 1 and Table I). Statistical analysis using a
2 test revealed that non-metastasis group had a higher frequency of NGX6 expression than the metastasis group, and the non-NPC group had a higher frequency of NGX6 expression than the two NPC groups.

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Fig. 1. In situ hybridization detection of NGX6 from an NPC-related tissue microarray. (A) Overview of tissue microarray (H&E). (B) H&E staining of tissue microarray of higher magnification. (C) NGX6 mRNA in situ hybridization showing positive (left) and negative (right) reactions.
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Inducible expression of NGX6 in HNE2 cells
To facilitate research to determine the function of the NGX6 gene, we employed a Tet-on system to make the expression of NGX6 inducible in the presence of Dox. We transfected Tet-on-HNE2 cells with the NGX6-expressing plasmid, pTRE2hyg-NGX6. After hygromycine selection for 3 weeks, we obtained 32 hygromycine-resistant colonies, RTPCR was performed to detect the expression of NGX6 in the presence or absence of Dox. One clone, named M22, was confirmed by northern blotting and RTPCR (Figure 2A and B), to show low background and high inducibility of NGX6 expression. The expression of NGX6 in M22 cells peaked in the presence of 1 µg/ml Dox at 2448 h post-induction. Southern blot analysis was used to confirm the vector DNA had been introduced into the M22 genome (Figure 2C). We also transfected the mock vector, pTRE2hyg, into Tet-on-HNE2 cells, and selected a cell clone as the negative control and named it K10. The NGX6 transcript was not detected in K10 cells in the presence or absence of Dox.

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Fig. 2. The Dox-dependent expression of NGX6. (A) Northern blot analysis of M22 (Tet-on-NGX6-HNE2) and K10 control cells (Tet-on-vector-HNE2) in the presence or absence of 1 µg/ml Dox. M22 cells showed low background and high inducibility, peak expression of NGX6 was observed at 2448 h post-induction. The NGX6 transcript was not detected in K10 under the conditions used. Part of the NGX6 cDNA, 1.1 kb, was used as a probe. The housekeeping gene GAPDH was used as an internal control. (B) RTPCR analysis of M22 cells. (C) Southern blot analysis was used to confirm that the vector DNA has been integrated into the M22 and K10 cells but not the Tet-on-HNE2 cell genome. Part of the vector pTREhyg vector was used as a probe.
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Ectopic expression of NGX6 influenced some cell adhesion molecules expression
To explore the consequences of the expression of the novel gene, NGX6, the Atlas human cancer cDNA expression array that contains 588 genes related to carcinogenesis was employed to screen for potential targets of NGX6. We established the expression profiles of the 588 genes in Tet-on-NGX6-HNE2 cells (clone M22) in the presence and absence of Dox, as well as in vector control cells (clone K10). No signals were visible in the blank spots and negative control spots, indicating that the hybridization was specific. The intensity of hybridization to housekeeping genes was very similar among the cell lines as expected. We used the mean intensities of nine housekeeping genes to normalize the intensity of signals. A ratio of differential expression
2 was considered to be significant. Five differentially expressed genes (data not shown) between non-induced and induced control K10 cells were considered to be caused by Dox itself. Only these genes differentially expressed by the M22 cells but not by the K10 cells were considered significant. Sixteen genes were found to be differentially regulated by the induction of NGX6, among which 11 genes were related to cell adhesion (Table II) such as ezrin, catenin
2 and VE-cadherin. The results of the cDNA array analysis were confirmed by western blot analysis of some molecules including ezrin, catenin
2, VE-cadherin and nm23-H1. The changes in the expression levels of ezrin, nm23-H1 and catenin
2 were similar to their mRNA level, while the levels of the VE-cadherin protein level did not change (Figure 3).
Characterization of NGX6 as a glycosylated membrane protein
Full-length NGX6 ORF was fused with GFP and over-expressed in COS7 cells. As shown in Figure 4A, NGX6 was primarily localized to the cell membrane and could also be found in the cytoplasm. To define the sequence element responsible for the membrane targeting of NGX6, we generated GFPNGX6 (1183 aa, a mutant vector lacking the EGF-like domain and two-span transmembrane regions), and found that the mutant protein localized only in the cytoplasm. Therefore, the EGF-like domain and the two-span transmembrane regions determine targeting of NGX6 to the membrane. To investigate the modification of NGX6 protein by glycosylation, immunoprecipitated NGX6 protein was treated with N-glycanase. As shown in Figure 4B, a clear deglycosylation was observed following N-glycanase treatment, resulting in the reduction of NGX6 protein molecular mass from 37 into 30 kDa.

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Fig. 4. Characterization of NGX6 as a glycosylated membrane protein. (A) Localization of NGX6 and its mutant. COS7 cells were transiently transfected with EGFP-C2-NGX6 or EGFP-C2-NGX6 (1183 aa, deleted of the EGF-like domain and two-span transmembrane regions) for 24 h. Confocal images of the transfected live cells were obtained using a water immersion objective. (B) Glycosylation of the NGX6 protein. Myc-NGX6 protein was transiently expressed in COS7 cells (lane 2) and immunoprecipitated with anti-Myc antibody and incubated in the presence or absence of 0.5 U of N-glycosidase F (N-Gly). Immunoprecipitates were then separated on a 12% SDSPAGE gel, transferred, and probed with anti-Myc antibody. Lane 1 stands for the negative control COS7 cells transfected with an empty vector.
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Association of NGX6 with ezrin
NGX6 can influence the expression levels of some cell adhesion molecules. Therefore, the association between NGX6 and these cell adhesion molecules was examined. In this experiment, we determined whether NGX6 could associate with ezrin, catenin
2, VE-cadherin or nm23-H1, respectively. COS7 cells were transiently transfected with Myc-NGX6 or Myc (as the negative control). Cell lysates were immunoprecipitated with anti-Myc or anti-ezrin (or catenin
2, VE-cadherin and nm23-H1) antibodies, and then western blot analysis was performed using anti-ezrin (or VE-cadherin, nm23-H1, catenin
2) or anti-Myc primary antibodies. We determined that NGX6 cannot associate with VE-cadherin, nm23-H1 or catenin
2 (data not show), but can associate with ezrin (Figure 5A). To verify the association of NGX6 with ezrin as observed in the co-immunoprecipitation experiment, we used the immunofluorescence staining and found that NGX6 and ezrin were co-localized as shown in Figure 5B. This is the first report that shows that NGX6 can associate with ezrin.

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Fig. 5. (A) Co-immunoprecipitation of ezrin with NGX6 in COS7 cells. Myc-NGX6 protein transiently expressed in COS7 cells, were immunoprecipitated with an anti-Myc or an anti-ezrin antibody. Immunoprecipitates were then separated on 8% SDSPAGE gels, transferred and probed with an anti-ezrin or an anti-Myc antibody. (B) Immunofluorescence co-localization of ezrin with NGX6 in COS7 cells. COS7 cells, transiently expressing Myc-NGX6 or Myc (negative control) proteins were incubated with rabbit anti-ezrin and mouse anti-Myc antibodies followed by staining with secondary FITC-conjugated anti mouse IgG and Cy3-conjugated anti rabbit IgG antibodies. Images were obtained using a confocal microscope. The yellow color in the merged image represents the extent of the co-localization of NGX6 (green) with ezrin (red).
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Ectopic expression of NGX6 influenced cell adhesion behavior of NPC cells
As shown above, a loss of NGX6 expression was associated with NPC lymph node metastasis, and NGX6 can influence the expression of some cell adhesion molecules. Therefore, we performed cell migration and invasion assays using the Tet-on-NGX6-HNE2 cell system to examine the changes in motility and invasive capacity induced by NGX6. As shown in Figure 6A, the migration assay indicated that cell motility was significantly suppressed, by 54%, in Tet-on-NGX6-HNE2 cells that over-expressed NGX6 (in the presence of 1 µg/ml Dox for 24 h) compared with HNE2 cells that did not express NGX6 (P < 0.01). As shown in Figure 6B, the cell invasion assay revealed that cell invasion was also significantly suppressed, by 42%, in HNE2 cells that over-expressed NGX6 (in the presence of 1 µg/ml Dox for 24 h) compared with HNE2 cells that did not express NGX6 (P < 0.01). We also determined whether the
2ß1 integrin was a fundamental requirement for the NGX6-dependent changes in cell migration and invasion. The addition of neutralizing antibodies against-integrin
2 and ß1 to the Tet-on-NGX6-HNE2 cells significantly inhibited their ability to migrate and invade Matrigel-coated substrata. Addition of neutralizing antibodies to the
2ß1 integrin reduced the rate of migration and invasion of Tet-on-NGX6-HNE2 cells by 60 and 53%, respectively. Control antibody isotype mouse IgG did not affect the migration and invasive ability of HNE2 cells. We found that NGX6 can still affect the migration and invasion ability of HNE2 cells after the
2ß1 integrin was blocked, P < 0.05 (Figure 6). Thus, these experiments indicated that the
2ß1 integrin is important for HNE2 cells invasion and migration ability, but that the effect of NGX6 on these abilities may not completely dependent on
2ß1 integrin.
The adhesion of HNE2 cells to fibronectin- or laminin-coated wells increased with the concentrations of fibronectin or laminin. The adhesion of cells to fibronectin or laminin was higher in Tet-on-NGX6-HNE2 cells that over-expressed NGX6 (in the presence of 1 µg/ml Dox for 24 h) than in HNE2 cells that did not express NGX6 (P < 0.01 at 1030 µg/ml of fibronectin or laminin) (Figure 7).

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Fig. 7. Increase in cell adhesion after induction of NGX6 (1 µg/ml Dox induction for 24 h) in Tet-on-NGX6-HNE2 cells. (Left) Adhesion of cells to fibronectin. (Right) Adhesion of cells to laminin. *P < 0.01 compared with the control cells (absence of NGX6 expression).
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Loss of GJIC is a characteristic of cancer cells. A coordinated interaction of epithelial tumor cells with stromal cells is a prerequisite for tumor invasion and metastasis. In the present study, we detected the GJIC of the Tet-on-NGX6-HNE2 cells with or without Dox induction. After NGX6 induction, HNE2 cell GJIC increased. Table III summarizes the results of three independent experiments (at least 100200 cells were examined for each individual experiment) using lucifer yellow dye transfer to assess GJIC between the HNE2 cells (Figure 8 and Table III).

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Fig. 8. Increase in GJIC after induction of NGX6 (1 µg/ml Dox induction for 24 h) in Tet-on-NGX6-HNE2 cells. The GJIC assay was performed by SLDT. Three minutes after scrape-loading, the NGX6 over-expressing cells allowed lucifer yellow transfer into neighboring contiguous cells while in control cells (absence of NGX6 expression) very little dye transfer occurred.
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Ectopic expression of NGX6 inhibited NPC cells tumorigenicity in vivo
To directly evaluate the role of NGX6 in tumor formation in vivo, Tet-on-NGX6-HNE2 cells were xenotransplanted into nude mice (each group of 10 mice). After 4 weeks, a significant reduction in the size of the tumor was observed in the group of mice, which over-expressed NGX6 because they were administered 1 µg/ml Dox in their drinking water. The average weight of these tumors was significantly lower than that of tumors arising from the control group (Figure 9). To determine whether tumors obtained from the group of Dox induction mice expressed NGX6, RNA isolated from these tumors was analyzed by RTPCR. This analysis revealed that all of the tumors from the group of Dox induction mice expressed high levels of NGX6 while tumors from control group mice expressed low levels of NGX6 (data not shown). We concluded that activation of NGX6 caused a reversal of HNE2 cell tumorigenicity.

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Fig. 9. Induction of NGX6 expression inhibited in vivo HNE2 tumor cells growth in nude mice. (A) s.c. tumors in the NGX6 over-expression group (doxcyclin treatment) and in the control group (absence of NGX6 expression) after injection of 2 x 106 cells at 4 weeks. (B) The average weight of 10 tumors was measured four weeks after injection. Each group included 10 mice. *P < 0.01 compared with the control group.
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Discussion
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In the present study, we examined the role of a novel EGF-like domain-containing gene, NGX6, in cell adhesion in NPC cells.
In situ hybridization analysis using NPC TMA suggested that loss of NGX6 expression was associated with NPC lymph node metastasis. It is noteworthy that Zhang et al. (15) reported similar results in colorectal carcinomas. We also found that NGX6 is located at the high frequent LOH region 9p21-22 of NPC. These findings suggest a role of NGX6 in NPC carcinogenesis and metastasis.
We employed the tetracycline-inducible expression system and the cDNA array technique to screen for potential targets of NGX6 in NPC cells. From the list of genes that were induced by NGX6 over-expression, we found that NGX6 could influence the expression of some important cell adhesion-related molecules such as RhoA, vitronectin, KRT1, integrin
2, integrin ß7, PSCD2L, CD9, ezrin, nm23-H1, VE-cadherin and catenin
2. The changes in ezrin, nm23-H1, VE-cadherin and catenin
2 mRNA expression were confirmed and western blot analysis found that ezrin, nm23-H1 and catenin
2 can be confirmed in protein level, but VE-cadherin's protein expression level had no change. This could be due to the observation that mRNA and protein expression levels are not always identical.
In this study, the expression of RhoA and vitronectin were both decreased following the induction of NGX6. RhoA is implicated in the invasion of human microvascular endothelial cells (HMEC-1). Ectopic expression of active-RhoA GTPase induced the expression of the MMP-9 metalloproteinase (22). Vitronectin can promote cell adhesion and spreading. In normal tissue, vitronectin has a homogeneous periductal occurrence, with local accumulations much lower than in carcinoma tissues (23). Their down-regulation reflected a possible role of NGX6 in tumor invasion and metastasis. We also found that induction of NGX6 up-regulated the expression of KRT1, integrin
2, integrin ß7, PSCD2L, CD9, ezrin, nm23-H1, VE-cadherin and catenin
2. KRT1 is a member of the keratin gene family associated with differentiation (24). Integrins are a family of transmembrane glycoproteins that participate in a wide range of cellular events including cell adhesion, proliferation, apoptosis, differentiation and cell-surface-mediated signaling (25). PSCD2L (Cytohesin-1) can specifically interact with CD18 (integrin ß2) and can promote cell adhesion to ICAM1 (26). CD9 is a cell-surface glycoprotein, that can modulate cell adhesion and migration and that can also trigger platelet activation and aggregation (27). Nm23-H1 is a tumor metastasis inhibitor in many tumors (28). VE-cadherin is a calcium-dependent cellcell adhesion glycoprotein (29). The function of cadherin cell adhesion molecules is thought to be regulated by a group of cytoplasmic proteins including catenin
. Catenin
is a linkage between cellcell adhesion molecules and the cytoskeleton (30). Ezrin will be discussed below. It attracted our attention that NGX6 can influence the expression of these cell adhesion related proteins. At present, we do not know whether induction of NGX6 directly accounts for these genes expression changes, but it provides a possibility that NGX6 participates in cell adhesion related proceedings.
To investigate whether NGX6 can associate with the differentially expressed molecules, we performed co-immunoprecipitation assays and found that NGX6 can physically associate with ezrin but not with VE-cadherin, nm23-H1 or catenin
2. Catenin
2 and VE-cadherin are important cell adhesion molecules and are located on or near the membrane while nm23-H1 is located in the cytoplasm or nucleus. Our results showed that these molecules do not associate with NGX6 so more research is required to investigate their relationship with NGX6. The association of ezrin with NGX6 was confirmed by immunofluorescence microscopy. Immunofluorescence staining showed NGX6 co-localized with ezrin on or near the cell membrane of COS7 cells. Ezrin belongs to a group of intracellular proteins that includes moesin and radixin, collectively known as the ezrin/radixin/moesin (ERM) family. ERM family members provide a linkage between the cell membrane and the actin-based cytoskeleton and may play a key role in the control of cell morphology and motility (31). The N-terminal residues of ezrin bind directly, or indirectly, to the plasma membrane, with the C-terminal residues binding laterally to actin (31). So it is possible that ezrin binds to the membrane protein, NGX6, with its N-terminal domain. Ezrin binds to cell adhesion molecules such as CD44, CD43, CD46, ICAM-1, ICAM-2 and ICAM-3, all of which are implicated in cell migration and metastasis (33). Ezrin has been suggested to be a mediator of cell motility, is essential for the maintenance of cellcell adhesion, and is inhibitory towards cellmatrix adhesion in human colonic epithelial cells. Ezrin can also be co-precipitated with E-cadhein and ß-catenin, two key proteins involving in cellcell adhesion (34). The association of NGX6 with ezrin provides the possibility that NGX6 participates in the function of ezrin.
Detection of the GFPNGX6 fusion protein showed that NGX6 mainly localized to the cell membrane and partially in the cytoplasm. NGX6 was also detected as a glycosylated protein. As expected from its three potential N-glycosylation sites, N-glycanase treatment resulted in a 7-kDa reduction in NGX6 molecular mass, indicating that the 30 and 37 kDa proteins represent the core and N-glycosylated proteins, respectively. As shown in Figure 4B, there was no apparent proteolytic modification of NGX6. Thus, there is a significant discrepancy between the predicted molecular mass of 37 kDa and the bioinformatical findings. However, the tendency to migrate faster than predicted on the basis of molecular mass is a well-known feature of many multi-span proteins (35). This also seems to be true of NGX6. The transmembrane protein superfamily includes receptors that bind to biologically active factors and adhesion molecules (36). The EGF-like domain is a sequence of
40 aa residues long, which has a significant homology to EGF. The EGF-like domain has been found in many different proteins, including receptors, receptor-like molecules, growth factors or cell adhesion molecules. Functional analysis has shown that the EGF-like domains play important roles in the interaction between a receptor and a ligand or in cell adhesion events (37).
In vivo tumor formation was significantly suppressed by NGX6 over-expression NPC cells. This result indicated that NGX6 expression suppressed multiple phenotypes related to malignancy of the HNE2 NPC cells.
Cancer metastasis is a very complicated biological process involving many sequential steps. Cellcell and cellextracellular matrix (fibronectin, laminin, collagen, etc.) interactions are involved in the metastatic process (38). We investigated whether cell adhesion properties can be altered by ectopic expression of NGX6 in NPC cells in vitro. In a cell adhesion assay, NGX6 over-expression increased NPC cells adhesion to fibronectin or laminin. In cell migration and invasion assays, NGX6 over-expression reduced the migration and invasive ability of NPC cells. We investigated whether the NGX6-dependent alterations in cell migration and invasion were associated with integrin
2ß1 because
2ß1 is an important receptor for collagen and laminin. In vivo,
2ß1 has been shown to function in cell migration and is implicated in tumor invasion and metastasis (39). We found that
2ß1 can affect the migration and invasion ability of HNE2 cells, but only partially reverse the NGX6-dependent alterations in cell migration and invasion. Therefore, additional unknown mechanisms are involved. In addition, we found that NGX6 can improve NPC cell GJIC. Cellular interactions with tissue stroma and extracellular matrix and interactions among cells and with different cells are important in controlling cell growth and development. The invasive and metastatic phenotypes could be explained, in part, by the loss of regulatory signals that are passed between adjacent epithelial cells through intercellular junctions. These findings suggest that NGX6 plays a role in cell adhesion modulation in NPC cells. Its molecular mechanism can be partly explained by our experimental data: NGX6 is a novel EGF-like domain containing, glycosylated, membrane protein, that can associate with the cell adhesion molecule ezrin, loss of NGX6 expression is associated with NPC metastasis status, cDNA array analysis showed that NGX6 can increase the expression level of some tumor metastasis suppressor genes such as nm23-H1, and NGX6 can influence NPC cell adhesion-related properties, and suppress NPC growth in a nude mouse assay.
In conclusion, our present results provide important clues for NGX6 function in cell adhesion modulation, but the function of NGX6 remains to be fully elucidated especially its correlation with cell adhesion molecules.
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Acknowledgments
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We thank Professor Ya Cao for kindly providing Tet-on-HNE2 cell line. Research was supported by grants from the National Natural Scientific Foundation of China (30330560, 30271403, 30200160, 30300064), the Special Funds for Major State Basic Research of China (2002BA711A03, 2002BA711A08).
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Received April 13, 2004;
revised October 6, 2004;
accepted October 10, 2004.