Molecular Mechanism On NAG7 Promote Migration And Invasion Of Nasopharyngeal Carcinoma Cells By Estrogen Receptor

【Background of NAG7】By using positional candidate cloning strategy, Dr. Yi Xie isolated anovel nasopharyngeal carcinoma (NPC)-related gene located in3p25.3-26.3, named NAG7. Its GenBank accession number wasAF086709.Bioinformatics revealed that the full-length of NAG7 cDNA is 1677bp, which encodes a protein of 94 amino acids with a predicatedmolecular weight of 11 kDa. NAG7 protein contains a transmemberaneregion which N-terminal side is inside membrane, a Protein kinase Cphsophorylation site and an N-myristoylation site.Previous studies showed that NAG7 was expressed in very low orundetectable level in NPC biopsies and NPC cell line HNE1, whileexpressed in high level in normal nasopharyngeal epithelial tissues.Subcellular localization of NAG7 protein is mostly in cytoplasm by usinga GFP-NAG7 fusion protein in HNE1 cells. NAG7 re-expression inducedthe changes of protein expression profile and gene expression profile,reduced the proliferation rate of HNE1 cells, and inhibited growth of xenografts after the transfected cell was injected into nude mice in vivo.It also down-regulated the expression of cyclins, increased the G0/G1phase cells arrest and decreased the S phase cells, and induced theapoptotic cells increase.【Expression of NAG7 in malignant cell lines】We found that the expression of NAG7 is very low in six malignantcell lines (HNE1, CNE1, 6-10B, SW480, Bel7402 and U251) by RT-PCRassay, but is high in 5-8F cell line. Sequencing of PCR products generatedfrom gDNA of HNE1, CNE1, 6-10B, SW480, Bel7402 and U251 celllines showed that they all contained point mutation at the same position(ORF of NAG7: T89G), which is a termination codon mutation.Sequence of PCR products generated from gDNA of 5-8F is precise. Thedownexpression of NAG7 in these six cell lines may be caused by genemutation.NAG7 gene suppresses the proliferation of HNE1 cells in vitro andin vivo obviously, and has the differential expression and point mutationin the various potential of invasion and metastasis of cell lines, whichdecreased expression and point mutation in low/none potential cells andincreased expression and right sequence in high potential cell. For NAG7expression varys with different invasive potential tumor cells, especiallyin 5-8F and 6-10B cells, so we propose the hypothesis: does NAG7 participate in the invasion and metastasis of NPC cells besides thesuppressive function on cell growth?【NAG7 promotes migration and invasion of NPC】In order to investigate the effect of NAG7 gene on invasion andmetastasis of NPC, we planned to use three cell lines with differentinvasion and metastasis potential: high potential cell line 5-8F, lowpotential cell line HNE1 and none potential cell line 6-10B. As a result ofabsent appropriate RNAi sequences and lacking of effective RNAi effectin 5-8F cells, NAG7 gene was introduced into HNE1 and 6-10B cells byliposome transfection and the stable cell lines pEGFP-C2/NAG7-HNE1and pEGFP-C2/NAG7-6-10B were established. Our data demonstrate thatNAG7 significantly enhances cell-matrix adhesion, cell-endothelial celladhesion, cell invasion and cell migration in HNE1 cells, but has triflingeffect on 6-10B cells. NAG7 could not restore the Gap JunctionalIntercellar Communication (GJIC) ability of HNE1 and 6-10B cells.Moreover, NAG7 also promote angiogenesis of human umbilical veinendothelial cells (HUVEC). The metastasis tumor of the xenograft ofNAG7/HNE1 and HNE1 cells implanted to the subrenal capsule ofBABL/c mice are undiscovered, while the invading depth and area ofNAG7/HNE1 cell in mice's kidney are lager than that of HNE1 cells.NAG7 gene can promote the migration and invasion potential of HNE1 cells rather than 6-10B cells, which means NAG7 do not have the abilityto launch the migration and invasion potential of cancer, but only boostthe potential.【Molecular mechanism on NAG7 promote migration and invasionof NPC cells by estrogen receptor】The different gene expression profile of NAG7 regulated HNE1cells were tested using the human cancer oligonucleotide array. Amongthe total 2747 tested genes, the expression of 34 genes were up-regulatedand 67 genes were down-regulated in NAG7/HNE1 cells as comparedwith HNE1 cells. The expression of CAV1 and MMP1 gene inNAG7/HNE1 and HNE1 cells determined by qRT-PCR were consistentwith the expression in array. The result of array accurately reflects thechanges of genes.The main groups of different expression gene functionalclassification based on Gene Ontology as follows:①Regulate cell cycle,②Cellular framework,③Cellular metabolism,④Catalytic activity,⑤Signal transducer,⑥Response to stimulus,⑦Cellular localization andCell adhesion. The pathway analysis of different expression genes usedKEGG and BIOCARTA database. There are 42 genes participated in 54KEGG pathways, especially 9 genes in MAPK signaling pathway and 6genes in cytokine-cytokine receptor interaction, and 31 genes in 78 BIOCARTA pathways. The relativity of different expression genes andthe key word of migration and invasion of cancer were found byPubmatrix, which searches the abstract of articles in PubMed. The resultshowed a great significant association of the different expression genesand invasion and metastasis of cancer.ERαprotein interacts with NAG7, and has paradoxical actions asboth anti-invasive and proliferative actions in human cancer. Proteinexpression of ERαin the tissue microarray of nasopharyngeal tissues wasscreened by Immunohistochemistry (ICH). It was found that theexpression of ERαin the NPC, adjacent-cancer and benign disease fromthe nasopharynx was similar, and was no relation of gender of patientsand clinical stages of NPC. The strong positive expression ratio of ERαinnon-metastasis NPC group was obviously higher than that in metastasis.It was suggested that the high expression of ERαmight take part in NPCcell proliferation, and the expression of ERαdecreased in the metastasisprocess of NPC.NAG7 protein interacts with ERα, and decreases17beta-estrodial-induced activation of ERαtranscriptional activity inmammalian cells. NAG7 down-regulated the expression of ERαin HNE1,and the faint increase expression of ERαin NAG7/HNE1 and HNE1 cellstreated by 10nM 17beta-estrodial detected by Western blot andimmunohistochemistry. There is no change of cell-matrix adhesion, cell-endothelial cell adhesion and cell migration in NAG7/HNE1 andHNE1 cells treated by 17beta-estrodial. We suggest that the dualfunctions of NAG7, anti-proliferation and promote invasion, actualizedby interact with ERα.In this study, we showed that NAG7 up-regulates JNK2 and c-Junexpression, inhibits c-Fos expression, and invalidates ERK, p-ERK, P53and NF-κB. We also found that 17beta-estrodial treat the cells does notchange the protein expression. NAG7 increases H-ras expression andactivate p-c-Raf, while does not regulate K-ras,N-ras and c-Raf. NAG7regulates H-ras/p-c-Raf and JNK/AP-1 signaling pathway, and increasesthe promoter activity of AP-1. Moreover, NAG7 activates AKT/p70s6Ksignaling pathway and increases MMP1 and Ezrin expression.In a word, NAG7 inhibits ERα, activates H-ras/p-c-Raf, JNK/AP-1and AKT/p70s6K signaling pathway, and upregulates CAV1, MMP1,Ezrin expression, then promotes the migration and invasion of HNE1cells.