The Role Of Tight Junction Protein Claudin-1 In Metastatic Behavior In Colon Cancer

Background and objectivesColorectal adenocarcinoma(CRC)is the third most common type of cancer and the fourth most frequent cause of death due to cancer worldwide.In developed countries it is the second most common type of tumor,with a lifetime risk of 5%.Surgery is the cornerstone of therapy when the disease is confined to the bowel wall.This results in 70%-80%of patients who have tumors that,when diagnosed,can be resected with curative intent.After curative surgery the five-year survival rate for patients with localised disease is 90%,decreasing to 65%in the case of metastasized disease to the lymph nodes.Therefore early detection of CRC will increase survival the most.It is well recognized that CRC arises from a multistep sequence of genetic alterations that result in the transformation of normal mucosa to a precursor adenoma and ultimately to carcinoma.In cancer research,biomarkers are molecules that indicate the presence of cancer in the body.Most biomarkers are based on mutations in genes or abnormal changes in RNA,proteins and metabolites,can potentially be used to detect early colorectal cancer.Furthermore,they might be used to predict prognosis,monitor disease progression and therapeutic response.A new family of membrane proteins Claudins are recently identified members of the tetraspanin family of proteins,which are integral to the structure and function of tight junctions,consisting of at least 24 members.Claudins are the most apical cell-cell contacts and are important for barrier function in epithelial and endothelial cells.Claudins encode proteins with 4 transmembrane domains,and their N- and C-terminal ends are located in the cytoplasm.In normal epithelia,the primary functions of the tight junction are to seal the apical intercellular spaces of glandular epithelia creating a barrier against the paracellular passage of macromolecules and to separate the plasma membrane into the apical and basolateral domains.Tight junction is key molecular components governing cellular adhesion,polarity and glandular differentiation.It's one of the key molecular components governing cellular adhesion and polarity.It also play a critical role in cellular proliferation and neoplastic pathways via their functions as couplers of the extracellular milieu to intracellular signaling pathways and the cytoskeleton.More and more study certificated that Claudins proteins play an important role in migration of malignant tumor.The role of Claudins in cancer has not been clearly defined.Neoplastic cells, particularly in those cancers that manifest high metastatic potential,frequently exhibit structural and functional deficiencies in the tight junction,while the levels of Claudin-I(CLD1)was frequently elevated in CRC.CLD1 had been reported to recruit and promote the activation of pro-MMP-2, which suggests potential involvement in invasion and metastasis.CID1 was also recently identified as a probable target ofβ-catenin/Tcf signaling,which supports a potential role for CLD1 regulation in colorectal carcinogenesis.Based on these studies,we hypothesized that C1D1 have a causal role in the process of cellular invasion and metastasis. The histological grade of colorectal carcinomas is an important prognostic variable.There is no report about the study of CLD 1 in human colon carcinogenesis by organizational classification at the present.Therefore,we detected the expression of CLD1 among colorectal peritumor normal-adenoma-carcinoma(different Dukes stage)tissues by immunohistochemical,in order to investigate the relationship between the expression of CLD 1 and colorectal cancer.In this experiment,three colorectal tumor cells of different pathological source and biological feature:SW620,SW480,SWlll6 are chosen to identify their differential expression of CLD1 in various ways.After verifying that CLD1 is one of related proteins of colorectal tumor,the research on its expression and regulation are carried out to further prove its effect on biological activity of colorectal tumor cell. Using genetically manipulated colorectal cancer cells as tools for in vitro and xenograft tumor models,we demonstrate a role for C1D1 in the regulation of cellular metastasis.The involvement of CLD1 with progression of colorectal cancer may bring an insight for the treatment of colorectal carcinoma patients.Material and methods1.The detection of CLD1 among colorectal tissues by immunohistochemistryTo explore the roles of CLD1 on the progression of CRCs systemically and directly,we observed its distributing and expressing differences among colorectal peritumor normal-adenima-carcinoma tissues.The exprssions of CLD1 in 87 colorectal peritumor nomal,15 cases of colorectal polypi(Non-cancerous polypi),16 adenomas(Neoplastic polypi),and 87 CRCs with different Dukes stages were detected using immunohistochemistry.2.The selection of cellsThree different sources of colorectal cancer cells:SW1116(Dukes A),SW480 (Dukes B)and SW620(Dukes C)were chosen for studied.Immunocytochemistry and Laser scanning confocal microscope were used to detect the expression site of CLD1,Real-time RT-PCR(FQ RT-PCR)and Western Blot(WB)confirmed CLD1 expressions at mRNA and protein expression levels.We manipulated the expression levels of Claudin-1 expression in SW480 and SW620 cell lines.Since SW480 cells express lower levels of Claudin-1 than do SW620 cells,we overexpressed Claudin-1 in SW480 cells to achieve Claudin-1 levels comparable to the level in SW620 cells.These cell lines were chosen because they are derived from primary(SW480)and metastatic lesions(SW620)of a single colon cancer patient.3.The influences on SW480 cell metastasis behavior by CLD1 gene transfectionRecombinant CLD1 eukaryotic expression vector CLD1/pEGFP-C1 was constructed at first.SW480 cell line were transfected with the corresponding native pEGFP-C 1 and recombinant plasmids CLD 1/PEGFP-C 1 using Lipofectamine 2000TM (Invitrogen).The positive cell comes were selected by G418 and subsequently proliferated respectively.The expression changes of CLD1 were detected using FQ RT-PCR.Laser confocal microscope detected the expression site of CLD1. Homotypic adhesion assay detected the adhesion between cells,cell-matrix adhesion test detected the adhesion between cells and typeâ…£collagen,chemotactic motion assay detected cell invasion,wound-healing assay detected cell migration.4.The influences on SW620 cell metastasis behavior by RNAiCLD1 overexpression in SW620 was downregulated by RNAi(RNA interference), and its effect was obserzed by FQ RT-PCR,Western Blot and laser confocal microscope in screened cell lines.To justify the influences of CLD1 on cellular biological activity,the cell adhesion,invasion and migration of SW620 were detected by homotypic adhesion assay,cell-matrix adhesion test,chemotactic motion assay and wound-healing assay in vitro.In vivo experiment,xenografled tumor cells models were performed in athymic mice,that were mice inoculated tumor cells subcutaneously into flanks(local neoplasm formed models),mice inoculated tumor cells subcutaneously into the nail pad(lymphatic metastasis model),mice inoculated tumor cells intrasplenically(blood metastasis models).5.Statistical analysisSPSS13.0 Statistics soft was employed to analysis statistically and P＜0.05 was significantly.Results1.CLD1 immunolocalization in colonic tissues by immunohistoehemistry(1)CLD1 localized in colonic tissues CLD1 localized in colorectal epithelia mass mainly,while almost no CLD1 signals was detected in the peritumor extracellular matrix.(2)CLD1 immunolocalization in the cell membrane Kruskal-Wallis Test showed thatχ²=2.469,P=0.872,no significant correlation was detected between the cell membrane expression of any of the proteins studied with normal,colorectal polypi,adenomas and CRCs.(3)CLD1 expression in colon cells cytoplasm compare between normal mucosa-polypi -adenoma- colorectal cancer Kruskal-Wallis Test showed thatχ²=106.83,P=0.000,significant correlation was detected in the cell cytoplasmic expression.The positive rates in normal mucosa-polypi - adenoma- carcinoma tissues were increased.Most of normal colonic mucosa and polypi,where the staining was restricted to the cell membrane less in cells cytoplasm,while adenoma 62.5%showed cytoplasmic expression,CRCs were 84.6%.Normal colonic mucosa and polypi only negative and weak,while adenoma showed positive and strong positive,and CRCs positive and strong were 43.7%. (4)CLD1 expression in colon cells cytoplasm compare between normal mucosa-polypi-adenoma-colorectal cancer stage A-B-C-D Kruskal-Wallis Test showed thatχ²= 95.71,P=0.000,significant correlation was detected.the highest positive rate of cell cytoplasmic expression was colorectal cancer A(87.5%),the sum of positive and strong positive also in colorectal cancer stage A(56.3%).(5)CLD1 expression in colon cell nucleus Only four cases were found in adenomas,colorectal stage A,B,C.Kruskal-Wallis Test showed thatχ²=4.46,P= 0.108,no significant correlation was detected.(6)CLD1 expression in colon cells membrane,cytoplasm,nucleus compare between normal mueosa-polypi - adenoma- eoloreetal cancer Kruskal-Wallis Test showed thatχ²= 23.42,P=0.000,significant correlation was detected.The positive rates in normal mucosa-polypi - adenoma- carcinoma tissues were increased.The expression of normal mucosa and polypi were lesser,adenoma and colorectal cancer strong positive(Above +3)gradually increase(P=0.000). (7)CLD1 expression in cells membrane,cytoplasm,nucleus compare between normal mueosa-polypi-adenoma-eoloreetai cancer A-B-C-D Kruskal- Wallis Test showed thatχ²= 42.52,P=0.000,significant correlation was detected.The positive rates in normal mucosa-polypi - adenoma- carcinoma tissues were increased. The highest positive rate were adenoma and colorectal cancer A(100%),while stage A strong positive rate(62.4%)was higher than adenoma(37.5%).The change of CLD1 expression imply the early colorectal cancer,that is CLD1 expression were increased significantly since adenoma,and stronger in stage A.(8)Relation between the expression of CLD1 with mueinous differentiation No significant correlation was detected between the expression positive rates of CLD1 with mucinous differentiation.2.The differential expression of CLD1 in SW620,SW480 and SWlll6 cell (1)Immunocytochemistry and confocal microscopy detected the distribution of CLD1 in cells CLD1 was mainly expressed in the cytomembrane and cytoplasma in SW1116,and in the cytoplasma in SW480,also in the nucleus and cytoplasma in SW620 cell.With the develop of CRCs,the expression of CLD1 was mislocalized from cytomembrane to cytoplasm and nucleus.(2)The mRNA expression of CLD1 was detected by FQ RT-PCR The Ct values of SW620 was smallest,SW480 was in the middle,the Ct values of SW1116 was largest.There was a significant difference between SW620,SW480 and SW1116 (P=0.000).The sequence was SW1116＜SW480＜SW620.(3)The protein expression of CLD1 was detected by Western Blot The analysis of Western Blot showed that the protein expression of CLD 1 in SW620 was the most manifest,the inferior was SW480,and the minimum was SWlll6.Their relative expression level(CLD1/β-actin)were SW1116＜SW480＜SW620.3.The influences on metastatic behaveior of SW480 cell by CLDI transfection (1)Vector constructed Recombinant CLD1 eukaryotic expression vector pEGFP-C 1/CLD1 was constructed successfully.(2)Cell clones cultured Two cell clones,SW480^CLD1and SW480^control,transfected with pEGFP-C1/CLD1 and pEGFP-C1 correspondingly,were cultured successfully.(3)Detection of the expression of CLD1 The expression of CLD1 in SW480^CLD1,SW480^controland SW480 cells were detected by FQ RT-PCR,showed that the expression of SW480^CLD1was higher than SW480^controland SW480.(4)Laser confocal microscopy showed localized of vectors The result indicated that CLD 1 expressed localized in the cytomembrane of SW480 cells.(5)The result of homotypic adhesive The SW480^CLD1cell morphology appeared spindly and fibroblastoid in monolayer culture,whereas the SW480^*control)cells showed round or round-like,similar to that of parental cells SW480.The number of homotypic adhesive SW480^CLDIcells were less than those of SW480^control(P=0.000) and SW480 cells(P=0.000).No significant correlation was detected between SW480^controland SW480 cells(P=0.376).The sequence was SW480^CLD1＜SW480^control and SW480.A inverse trend was detected between CLD1 and the cells homotypic adhesive,helping tumor cells leave primary tumor and metastasis.Alterations in tight junction permeability may also allow increased diffusion of nutrients and other factors critical for tumor growth and survival.In addition,loss of tight junction integrity may be an important step in the development of the metastatic phenotype.(6)The result of cell-matrix adhesive tests There were more SW480^CLD1adhesive cells than those of SW480^control(P=0.000)and SW480 cells(P=0.000),significant correlation was detected.No significant correlation was detected between SW480^controland SW480 cells(P=0.334).The sequence of number of adhesive cell were SW480^CLD1＞SW480^controland SW480.The adhesion was increase between SW480 cells and collagen IVmediated by CLDI gene transfection.(7)The result of cell invasion tests Tests showed the numbers of SW480^CLD1 membane-filtered cell were significantly more than that of SW480^control (P=0.000)and SW480(P=0.000).No significant correlation was detected between SW480^controland SW480 cells(P=0.408).The sequence of number of cell were SW480^CLD1＞SW480^controland SW480.(8)The result of cell migration tests A monolayer wound-healing assay revealed that the scratched areas of SW480^CLD1cell were significantly smaller than those of SW480^controland SW480 cell after 48h.Migrated cells in scratched areas of SW480^CLD1were more than those of SW480^controland SW480 cell.4.The influences on metastatic behaveior of SW620 cell by RNAi target to CLD1(1)Constructed RNAi recombinant plasmids and transfected into SW620 cells shRNA eukaryotic expression vectors targeting to CLD1 and a control vector were constructed successfully.Two Cell clones,SW620^siRNAand SW620^control,were cultured successfully after the recombinant plasmids were transfected into SW620 cells,and G418-resistant cell lines were screened,mRNA and protein expression of CLD1 in SW620^siRNA,SW620^controland SW620 cells were detected by FQ RT-PCR and Western Blot,showed that the expression of SW620^siRNAwas lower than SW620^controland SW620.Immunofluorescence indicated a decrease in the nucleus expression of SW620^siRNAclones compared with SW620^controland SW620 cells.All these improve that expression of CLD1 in SW620 was interfered successfully.(2)The result of homotypie adhesive The SW620^siRNAcells exhibited a spreading morphology and tended to form multicellular aggregates in monolayer culture, whereas SW620 and SW620^controlcells exhibited round,round-like,and dispersed. The number of homotypic adhesive SW620^siRNAcells were more than those of SW620^control(P=0.000)and SW620 cells(P=0.000).No significant correlation was detected between SW480^controland SW480 cells(P=0.213).The sequence of number ofhomotypic adhesive cells was SW620(control)＞SW620^controland SW620.(3)The result of cell-matrix adhesive tests There were less SW620^siRNAadhesive cells than those of SW620^control(P=0.000)and SW620 cells(P=0.000).No significant correlation was detected between SW620 and SW620^controlcells(P=0.269).The sequence of number of adhesive cell were SW620^siRNA＜SW620^controland SW620. The adhesion was decrease between SW620 cells and collagen IVmediated by RNAi.(4)The result of cell invasion tests The numbers of SW620^siRNAmembane-filtered cell were significantly more than that of SW620^control(P=0.000)and SW620 (P=0.000).No significant correlation was detected between SW620^controland SW620 cells(P=0.514).The sequence of number of invasion cell were SW620^siRNA＜SW620^controland SW620.(5)The result of cell migration tests A monolayer wound-healing assay revealed that almost no migration in SW620^siRNAcells compared with SW620^controland SW620 cells after 48h,whereas SW620 and SW620^controlcells migrated and created faster. Migrated cells in scratched areas of SW620^siRNAcells were less than those of SW620^controland SW620.(6)The result of inoculated tumor cells subcutaneously into mice flanks Based on our cell culture data,we postulated that CLD1 expression would affect tumor formation and metastasis in vivo.In mice injected with the SW620(control)and SW620 cells,measurable tumors appeared within 2 weeks,while mice injected with the SW620^siRNAbecame detectable only after 2 weeks of growth.All of the mice were appeared tumors when euthanized and subjected to autopsy after 8 weeks. Tumorigenicity assay showed the tumor sizes of SW620^siRNAwere less than thoes of SW620^controland SW620 cells significantly,respectively(P=0.000).The sequence of average tumor volume were SW620^siRNA＜SW620^controland SW620.Histological examination HE of all tumors belonged to poorly differentiated adenocarcinomas.(7)The result of inoculated tumor cells subcutaneously into the nail pad In mice injected with the SW620^siRNAhad no claw subcutaneous neoplasm formed while SW620^controland SW620 all had subcutaneous neoplasms formed within 3-4 weeks. All of the mice were appeared tumors when euthanized and subjected to autopsy after 8 weeks.There were 75%(3/4)nude mice lymph nodes metastasis in mice injected with the SW620^controland SW620,and the average number of metastatic lymph nodes were 2.25 and 2 each mice.No lymph node metastasis was appeared in nude mice injected with the SW620^siRNA.Histological examination HE stain of lymph nodes belonged to poorly differentiated adenocarcinomas.(8)The result of inoculated tumor cells into mice intrasplenically All of the mice were appeared intrahepatic metastatic tumors in mice injected with the SW620^control and SW620(4/4,100%),whereas only a mice had intrahepatic metastatic tumors in mice injected with the SW620^siRNA(1/4,25%)when autopsy after 8 weeks.The average number of livers surface tumor of mice injected with SW620,SW620^control, SW620^siRNAcells were 3.75,3.50 and 0.25 each mice.Histological examination HE stain of lymph nodes belonged to poorly differentiated adenocarcinomas.Conclutions1.Immunohistochemistry detected CLD1 localization in colonic tissues.CLD1 localized in colorectal epithelia mass mainly,while almost no CLD1 signals was detected in the peritumor extracellular matrix.Most of normal colonic mucosa and polypi,where the staining of CLD1 was restricted to the cell membrane less in cells cytoplasm,and only negative and weak positive.The positive ratio of CLD1 cytoplasmic expression and intensity increase since adenomas,the most notable period of mislocalization from the cell membrane to the cell cytoplasm was the early stage A of darcinogenesis.The sum of CLD1 positive ratio of cells membrane, cytoplasm,nucleus were increased significantly since adenoma,and more stronger in the early colorectal cancer stage A.The change of CLD1 positive expression and mislocalization suggested the growth early stage of colorectal cancer.We should alert colorectal carcinogenesis when the change of CLD1 positive expression and mislocalization from the cell membrane to the cell cytoplasm.This is especially critical for determining which stage A colon cancer patients should receive therapy.2.Using immunocytochemistry and laser confocal microscopy detected the distribution of CLD1 in three different sources of colorectal cancer cell: SW1116,SW480å’ŒSW620.The result showed that with the develop of CRCs,the expression of CLD 1 was mislocalized from cytomembrane to cytoplasm and nucleus. FQ RT-PCR and Western Blot confirmed CLD1 expressions at mRNA and protein expression levels.There was a significant difference between three cells.The sequence was SW1116＜SW480＜SW620. 3.CLD1 eukaryotic expression vector enhance the expression of SW480 CLD1 effectively,inhibited its homotypic adhesion,helping tumor cells leave primary tumor and metastasis;accelerated its cell-matrix adhesion,enhance cells invasion ability, increase cells migration ability in vitro.4.Constructed CLD1 hairpin siRNA eukaryotic expression vectors significantly downregulated SW620 CLD1 expression,enhance its homotypic adhesion,inhibit its cell-matrix adhesion,decrease cells invasion ability,reduce cells migration ability in vitro,decreased the tumorigenicity,lymphatic metastasis ability and blood metastic ability in vivo,which indicates CLD1 expression can regulates the invasion and metastasis procession of CRC.5.CLD1 might plays an important role in the development and progression of colorectal carcinogenesis,and it may be used as a new prognostic and therapeutic maker in patients with the colorectal tumor.