Molecular Mechanism Of Ezrin Facilitating Degradation Of NGX6a Protein Promoting Invasion And Metastasis In Nasopharyngeal Carcinoma Cells

Recently, nonrandom loss of heterozygosity at 3p,9p, 11q,13q and 14q in NPC has been detected by comparative genomic hybridization (CGH) and genome-wide analysis with microsatellite allelotyping. By using positional candidate cloning strategy, Dr. Jian-Bo Yang isolated a novel NPC-related gene which was down-regulated in NPC. This gene was designated human nasopharyngeal carcinoma associated gene6 (NGX6) (GenBank accession number:AF188239).Bioinformatics reveals that the full-length of NGX6 cDNA is 2134 bp, which encodes a protein of 338 amino acids with a predicated molecular weight of 37 kDa. NGX6 protein contains two transmemberane regions, an EGF-like domain and three potential N-glycosylation sites in the extracellular domain. The short cytoplasmic region contains a tyrosine residue that is a potential phsophorylation site of tyrosine kinase.Previous studies showed that NGX6 was expressed in high level in normal nasopharyngeal epithelial tissues, while expressed in very low or undetectable level in nasopharyngeal carcinoma biopsies and cell lines. It was also found that NGX6 was down-regulated in colorectal carcinomas, and that the down-regulation rate of NGX6 in colorectal carcinoma tissues with lymph-node or distance metastasis (15/16) was significantly higher than that without metastasis (25/34) (P<0.05). Transfection of NGX6 into nasopharyngeal and colorectal carcinoma cells could induce the reversion of some malignant phenotypies, and down-regulate the expression of key molecules of the EGFR pathway. It was found that NGX6 could interact with Ezrin by immunoprecipitation assay in COS7 cells. NGX6 could decrease the invasive ability, increase the adhesive rate and improve communication ability of 5-8F cells. There was a delay in tumor formation and metastasis in lung when cells with NGX6 overexpression were injected into scid mice. The EGF-like domain and CYTO region are important to modulate cell adhesion. The role ofâ–³EGF orâ–³CYTO to increase the adhesion ability decreased compared with NGX6, indicating that EGF and CYTO were involved in the modulation of the adhesion ability of 5-8F cells; the CYTO domain of NGX6 also is critical for in the modulation of cell invasion, migration, proliferation and growth. Epidermal growth factor could induce the invasion and mobility of 5-8F cells. The expression of EGFR, PI3K and RhoA were increased as the concentration and duration time of EGF induction increased, and RhoA was translocated from cytosol to membrane. While NGX6 could partly inhibit the EGF-induced invasion and migration of 5-8F cells by EGFR-PI3K-IKK-NF-κB pathway.These previous studies illuminated that NGX6 gene may be a suppressor in the invasion and migration of NPC. To better understand the cellular role of the NGX6 gene, in this study, His-NGX6â–³TM2 recombinant protein were expressed in E.coli and Polyclonal anti-NGX6 antibody was produced by immunizing New Zealand white rabbits with the purified His-NGX6â–³TM2 recombinant protein. The specificity of the antibody was identified by Western blot and immunofluorescence. With the prepared NGX6 antibody, we detected the distribution of NGX6 in the human fetus. The result of western blot showed the protein of NGX6 had two type of isoforms, isoform a (NGX6a) and isoform b (NGX6b). Searched for the NCBI nucleinic acid and protein database, there are three mRNA transcript variants of NGX6. Transcript variant 1 (NM₀01042590) and variant 2 (NM₀01042589) encode the same NGX6 protein isoform a (NP₀01036055 or NP₀01036054) which is composed with 472 amino acids with a calculated molecule weight of 52 kDa, and transcript variant 3(NM₀16446)encodes isoform b. The isoform a is composed with 472 amino acids with a calculated molecule weight of 52 kDa, while the isoform b is composed with 338 amino acids with a calculated molecule weight of 37 kDa. It is predicated that there is an EGF (epidermal growth factor) domain in N-terminal of both a and b isoforms, and seven transmembrane domains in NGX6a, but only two transmembrane domains in NGX6b. The expression level of NGX6a was higher than NGX6b in human fetus tissues. Obvious high expression of NGX6a protein presents in human fetus nervous system and epithelial tissues, but the NGX6b protein (37 kDa) is mainly expressed in the nervous system. We further analyzed tissue microarray (TMA) contained 308 nasopharyngeal carcinoma biopsies and 140 non-NPC biopsies and found that NGX6a was significantly down-regulated in the nasopharyngeal carcinoma and associated with tumor metastasis.We analyzed the expression of NGX6a in several human nasopharyngeal carcinoma cell lines using Western blot. A high expression of NGX6a at the protein level was detected in normal nasopharyngeal epithelial cell line NP69 and normal nasopharyngeal epithelial tissue. However, all of the nasopharyngeal carcinoma cell lines showed downexpression of NGX6a compared with NP69. To investigate whether over-expression of NGX6a in cancer cells leads to suppression of cancer cells growth, NGX6a was stablely transfected into 5-8F,6-10B, HNE1, cell line. In these cells, the mRNA levels of NGX6a were increased more than 20 folds, but it can't be detected any signals in the protein levels. After treated with proteasomes inhibitor MG132, NGX6a protein could be detected to increase as the concentration a of MG132 induction increased by using Western Blot and immunofluorescence. Transfect pCMV-myc-NGXa vector into 5-8F cell and transient expressed NGX6a protein, then isolate and purify NGX6a protein by immunoprecipitation and used anti-ubiqutin antibody to procee to Western Blot. The result showed NGX6a protein didn't be ubiqutinated. These results demonstrated that NGX6a protein was degraded in NPC cell through proteasomes but not ubiqutin pathway.Previous studies illuminated that NGX6b protein interacted with ezrin. The present study showed that NGX6a protein also could interact with Ezrin by immunoprecipitation assay and immunofluorescence test. Ezrin, a linker between membrane protein and cytoskeleton, played important role in the cell morphology, cytoskeleton reorganization, adhesion, invasion and metastasis. Detected by tissue-microarray (TMA) and western blot, the overexpression of Ezrin in NPC and in NPC cell lines contrast to NGX6a suggested that Ezrin was involved in the progression and invasion of NPC. Another interesting fact is that the distribution of Ezrin is contrary to NGX6a in the human fetus tissues. For example, in the nerve system ezrin was detected predominantly in astrocytes, while NGX6a was expressed in neurons, no ezrin was detected in neurons in most tissues. In human fetus cerebellum, very strong ezrin staining was detected in the top of the molecular layer. However, Purkinje cells were ezrin-negative, while NGX6a was expressed in the purkinje cells and nerve fibers in the molecular layer. Further, using over-express and RNAi technology, we demonstrated that Ezrin Facilitates degradation of NGX6a protein in cell. We also demonstrated that seven-transmemberane domain was critical region for the degradation of NGX6a in cell by using mutant technology.NGX6a could inhibit the invasion and mobility of NPC cells detected by matrigel migration assay, scraping test. Further invesgation showed that expression silence of Ezrin by RNAi could reduce the invasive ability of the 5-8F cells by matrigel migration assay. Simultaneously knocking-down Ezrin expression level and over-expressing NGX6a could make this inhibitory effect more significant. Using mithramycin which is SP1 inhibitor to treat 5-8F cells could down-regulated the expresson of SP1 and Ezrin and up-regulated NGX6a, which reduced the invasion and mobility of 5-8F cells. Combined use of mithramycin and MG132 to treat 5-8F cells could further inhibit the degradation of NGX6a protein, and is more effective to reduce the invasion and metastasis of 5-8F cells than single use of mithramycin or MG132 respectively.Taken together, we generated a specific NGX6 antibody and investigated the distribution of NGX6 protein in human fetus. We found a novel subtype protein of NGX6:NGX6a; Obvious high expression of NGX6a protein presents in human fetus nervous system and epithelial tissues. NGX6a was significantly down-regulated in the nasopharyngeal carcinoma and associated with tumor metastasis; NGX6a was degraded in NPC cells. Ezrin was over-expressed in the nasopharyngeal carcinoma and associated with tumor metastasis. The distribution pattern of Ezrin was contrary to NGX6a in tissues and cells; Ezrin could interact with NGX6a and facilitates degradation of NGX6a protein; The seven-transmemberane domain of NGX6a was critical region for the degradation of NGX6a; Decreasing the expression level of Ezrin and increasing the expression level of NGX6a can reduce the ability of invasion and migration of 5-8F cells significantly.