| BACKGROUND & OBJECTIVENasopharyngeal carcinoma(NPC) is a type of cancer common in Southern China and Southeast Asia,especially those of Cantonese origin.Although the levels of diagnosis and the treatment on NPC have being evidently improved in recent years, the survival rate of 5 years for patients with NPC is still very low,which the main cause of higher mortality is early lymph node and distant organ metastases.Recently, many molecular markers relating invasion and metastasis of NPC have been found such as VEGF,CXCR4,CD44,Cadherin,MMP,nm-23,Wt-p53,etc.They play an important role in NPC's invasion and metastasis.However,the function and mechanism of Tiam1(T lymphoma invasion and metastasis inducing factor 1)in NPC's invasion and metastasis are still unclear.Tiam1 was identified in 1994 by proviral tagging in combination with in vitro selection for invasiveness from murine lymphoma cells.It had been reported that Tiam1 was extensively expressed in a variety of cancers,such as breast cancer,lung cancer,colorectal cancer,and so on,especially in adenocarcinoma and squamous cell cancer with local infiltration and/or distant metastasis.Moreover,examination on more than 40 rodent and human tumor cell lines demonstrated that Tiam1 expression in virtually all lines.Furthermore,the level of Tiam1 was positively correlated with metastatic potential of tumor cell lines.Another research found that Tiam1 overexpression promoted progression of colorectal cancer cell lines,while downregulating Tiam1 protein inhibited their bionomics such as the invasion and metastasis.Tiam1-/- mice were resistant to the development of Ras-induced skin tumors.Considerable work had demonstrated that the molecular mechanisms of Tiam1-induced invasion and metastasis were closely correlated to cystoskeleton rearrangement,cellular adhesion,cell cycle progression,gene transcription,formation of cell membrane ruffles,etc.Several recent studies had demonstrated that Tiaml binded to the cytoskeletal protein ankyrin,causing Tiam1-mediated Rac1 activation as well as an increase in breast tumor cell migration and invasion.Similarly,the cellular adhesion molecule CD44 binded to Tiam1 at the PHn-CC-Ex region in SP1 murine breast carcinoma cells.Furthermore,the binding of HA to CD44v3 causes an increase in Tiam1-mediated Rac activation and promoted cytoskeleton-mediated tumor cell migration.Invasion and metastasis are the main bionomics and directly cause of invalid treatment or death of NPC.Up to now,few reports was found about function and mechanism of Tiam1 in NPC.In this study,we mainly focus on the functional alterations of Tiam1 in NPC tissues and cell lines by gene transfection,RNAi,etc.The aim is insighting into the role of Tiam1 in invasion and metastasis,and providing a molecular marker for NPC's treatment and prognosis.METHODS1.Expression of Tiam1 and it's relationship with biological behaviour of NPC.Immunohistochemistry(SP-9000) was performed to examine the expressions of Tiam1 protein in 71 specimens of NPC tissue,20 specimens of chronic nasopharyngitis tissue,and 6 specimens of tumor tissues from nude mice inoculated with metastatic human NPC cells.The expression of Tiam1 in 6 NPC cell lines was detected in protein level by immunofluorescence staining and in mRNA level by RT-PCR respectively.2.Establishment and functional verification of Tiam1-overexpressed NPC Cells. We firstly established stably Tiam1-overexpressed CNE2 cells by transfecting Tiam1/c1199 cDNA with lipofectamin2000.The Tiaml-overexpressed CNE2 clones was screened with G418 and verified by RT-PCR and Western blot.The functional alterations of the resulting transfectants were performed by MTT assay,plate colony formation assay,soft agar assay,cell adhesion assay,cell wounding heal assay, invasion assays in vitro and metastasis assay in vivo through the lateral tail-vein injection.3.Establishment of Tiam1-knockdown NPC cells and functional alterations in vitro and in vivo.Lentiviral expression vectors containing enhanced green fluorescence protein (GFP) and Tiam1 small interfering RNA(Lenti-Tiam1si),or the control siRNA (Lenti-NC) gene were constructed.A human monocyte-derived cell line CNE2 was transfected with a different multiplicity of infection(MOI) of Lenti-Tiam1-si-A,B,C, D or Lenti-NC,and cultured to obtain stably-transfected CNE2-KD and CNE2-NC cells.The expression of Tiam1 mRNA was determined by real-time PCR,and Tiam1 protein was detected by western-blot.Analysis of cell cycles by flow cytometry,soft agar assay,cell adhesion assay,invasion assays,cytoskeleton staining(coomassie brilliant blue R250),morphological changes viewed by scanning electron microscope (SEM) and transmission electron microscope(TEM),metastasis assay through the lateral tail-vein injection,respectively.Metastasis assay in vivo by whole-body visualizing animal imaging system and H&E staining were used to evaluate the functional e alteration s of Tiam1-knockdown NPC cells in vivo.4.Screening and identification of Tiam1-interacting proteinsTo explore the Tiam1-mediated possible molecular mechanism of relating to invasion and metastasis in NPC cells,we firstly screened Tiam1-interacting protein in (http://www.genome.jp/kegg/) and(http://visant.bu.edu/),then we chose ankyrin1, rac1 and CD44 proteins which closely correlated to invasion and metastasis for co-localization analysis by double immunofluorescence staining and co-immunoprecipitation(CO-IP). RESULTS1.The expression of Tiam1 in nasopharyngeal carcinoma tissues and cell lines.The results of IHC for Tiam1 were summarized as follow:The localization of Tiam1 protein is in cytoplasm and cytomembrane.The average scores for Tiam1 expression were significantly higher in NPC tissues than those in chronic nasopharyngitis tissues(t=7.224,P＜0.01);The level of Tiam1 protein in 6 tumor tissues from nude mice inoculated with metastatic human NPC cells was also higher. Comparison with the TNM stages of NPC patients,no difference was found in Tiam1 expression among NPC patients in different T stages(F=1.670,P=0.182),while the expressions differed significantly between the patients with lymph node metastasis and those without metastasis,and also between patients with organ metastasis and those without(t=9.005,P＜0.01;t=5.069,P＜0.01).The expression of Tiam1 in 6 NPC cell lines was detected in protein level by immunofluorescence staining and in mRNA level by RT-PCR respectively.The results were shown that higher-level Tiam1 was found in 5-8F cell line with higher metastasis property(0.560±0.020);In 6-10B cell line without metastasis ability,fewer Tiam1 was detected(0.110±0.010) and in others cell lines such as CNE1,CNE2,HONE1 and C666-1,moderate-expressed Tiam1 mRNA was detected.2.Generation and functional verification of Tiam1-overexpressed CNE2 Cells.We firstly generated CNE2 cells stably transfected with Tiam1/c1199 cDNA and empty vector(named as CNE2/Tiam1 or C/T,CNE2/Mock or T/M).RT-PCR and western-blot methods were used to analysis the expression of Tiam1 in four chosen Tiam1-transfected clones of CNE2.The highest level of Tiam1 expression was seen in the Tiam1 transfected clone3.We chose T3 clone as function experiments in vitro or vivo,and the results shew that Tiam1 overexpression promoted proliferation(MTT assay:F=323.158,P＜0.01;plate colony formation assay:t=25.065,P＜0.01;soft agar assay:F=318.960,P＜0.01),adhesive(F=86.689,P＜0.05) and invasive capabilities (F=323.158,P＜0.05) in CNE2/Tiam1 cells compared with CNE2 or CNE2/Mock in vitro.To evaluate the role of Tiam1 overexpression on metastatic ability of NPC,we performed in vivo metastasis assay by tail-vein injection.On the fifteenth day after injection,one of the mice in CNE2/Tiam1 group was died from multiple abscesses. By the nineth week after injection,when nude mice had not died but some appeared to be moribund,all mice were sacrificed.Then their lungs,livers and other organs were removed and fixed in 10%formalin for H&E staining.We found that all 8 nude mice in CNE2/Tiam1 group emerged lung metastatic nodes,while in CNE2/Mock group,4 of 9 nude mice shew lung metastatic nodes under dissecting microscope.The average number of lung metastatic nodes in CNE2/Tiam1 group in each nude mice (7.75±2.315) was higher than that in CNE2/Mock group(3.78±4.549)(t'=2.305, P＜0.05).All the suspected metastatic nodes were confirmed by histopathological examination.In addition,scattered NPC cells infiltration in liver tissues was found in one of CNE2/Tiam1 group under microscope.No metastatic focus was found in the other organs.3.Establishment and identification of lentiviral-mediated Tiam1-knockdown CNE2 cellsTo require prolonged suppression of Tiaml protein in CNE2 cells,we constructed 4 shRNA(pGC-LV recombination vector) containing Tiam1 interfere sequence A,B,C and D.Real-time PCR analysis showed that the mRNA levels of Tiaml in four Tiam1-si cells were all suppressed,especially in Tiam1-si-B cells (0.124±0.055),next to si-C(0.156±0.046).Besides,we reinfected CNE2 cells with si-B,si-C lentivirus for further identifycation.On thirth days after transfection, fluorescence microscope photographs were firstly demonstrated the efficient transduction of CNE2 cells.The results in mRNA or protein levels by Real-time PCR and western-blot were consistent with above preliminary identification.We named Lenti-si-B,si-C infected CNE2 cells as KD1 and KD2 respectively.Furthermore,we chose KD1 cells as functional experiments in vitro or in vivo.4.Functional alterations of lentiviral-mediated Tiam1-knockdown NPC cells.To further detect the role of Tiam1 in NPC,we established Tiam1-knockdown CNE2 cells,and a series of assays were performed to evaluate the function alterations of Tiam1-knockdown cells in invasion and metastasis.Flow cytometry was used to detect the alteration of cell cycle in CNE2,CNE2/NC and CNE2/KD cells.The results showed that their percentages in S stage were 51.3%,52.7%and 21% respectively,which indicated that the ability of proliferation on CNE2 cell was declined after Tiam1 depletion.Soft agar assay was used to detect the abilitiy of colony formation and obtained declined the ability of proliferation of single Tiam1-knockdown CNE2 cells(F=645.863,P＜0.05).In order to further determine whether Tiam1 protein has a role in enhancing cell adhesion to fibronectin,we conducted cell adhesion assay and found that compared to CNE2 and CNE2/NC,the adhesive capability to fibronectin of CNE2/KD cells was decreased(F=55.388,P＜0.05). Results from the invasion assay showed the reduced ability of CNE2/KD cells to migrate through the matrigel membrane,and it demonstrated that suppression of Tiaml effectively reduced the invasive capability of CNE2 cells in vitro(F=181.727, P＜0.05).Besides,coomassie brilliant blue staining was used to compare cytoskeleton changes in the content and distribution of NPC cells.The results showed that the cytoskeleton in CON,NC cells were large,intensive and fascicularis arranged along with cell morphology;In contrast,in KD cells small and sporadic distribution of cytoskeleton were seen.Scanning electron microscopy(SEM) showed CON,NC cells closely attached to the slide,alive with in microvilli each other;Adversely, surface microvilli of KD cells were sparse,short,and observed disjunction phenomenon.Ultrastructural changes of cells were observed by transmission electron microscope(TEM).In KD cells,nuclear heterochromatin increased margination. Vesicular or lacunar endoplasmic reticulum and Golgi complex,and apoptotic bodies were viewed in comparision with CON and NC cells.In addition,to evaluate the effects of Tiaml depletion on metastasis in vivo,we injected NC cells(n=9,groupl) and Tiam1-KD CNE2 cells(n=9,group2) into 6-8-week-old athymic mice through the lateral tail vein and detected.By the nineth week,all mice were executed and their metastatic nodules in the lung or other organs were firstly observed under a GFP imaging system,and then their lungs,livers and other organs were fixed in 10%formalin for further analysis of histopathology.We found that 5 of 9 mice in groups1 formed metastatic nodules in the lung of mice analysed(5/9,all together 36 foci).The average number in every mouse was(4.00±4.093).In contrast,only one lung micro metastatic focus was found in group2 under a GFP imaging system.The discrepancy between group1 and group2 was significant by statistical analysis(t'=2.841,P＜0.05);No other organ but lung was found metastatic nodules,indicating that Tiam1 depletion reverses cancer metastasis in vivo.5.Co-localization analysis of Tiam1-interacting proteinsDouble immunofluorescence staining was firstly used to confirm whether heterogenous Tiam1-Ankyrinl,Tiam1-Rac1 or Tiam1-CD44 were colocalized in CNE2 cells.Cells were simultaneously stained with rabbit-anti-Tiam1 antibodies and mouse-anti-Ankyrin1,Rac1,CD44 antibodies followed by TRITC and FITC conjugated antibodies;The results were observed under confocal microscopy: Tiaml protein presenting red fluorescence and Ankyrin protein showing green fluorescence were punctiformly colocalized in cytoplasm of CNE2;Tiam1 and Rac1 proteins were extensively colocalized in cytoplasm while Tiam1 and CD44 proteins were only filiformly colocalized in medial surface of cell membrane.In addition, co-immunoprecipitation(CO-IP) was also applied to further detect the co-localization of Tiam1-Ankyrin1,Tiam1-Rac1 or Tiam1-CD44.By following western-blot ananlysis,we only observed the co-localization of Tiam1-Ankyrin1 and Tiam1-Rac1, no co-localization was found from Tiam1-CD44 proteins.CONCLUSION1.Tiam1 gene is closely correlated to NPC progression,and may be a useful gene of NPC invasion and metastasis.2.Tiam1 protein can directly interact with Ankyrinl protein,Rac1 protein in NPC cells,indicating that it is a key protein in signal pathways related to invasion and metastasis of NPC.
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