| [BACKGROUND]:EWI2(also called CD316,IgSF8 or PGRL in mouse)is a newly identified IgSF(immunoglobulin superfamily)member.EWI-2 protein has an extracellular domain consisting of 4 and 6 Ig domains,it also has a short cytoplasmic tails containing 10 and 27 amino acids which allow it physically associates with some tetraspanins like CD81,CD9,besides,EWI-2 was previously found as a partner of tetraspanins[1-4].The intracellular structure of EWI-2 can link tetraspanins and cytoskeleton via its binding to ERM proteins.[14]Cell Adhesion Molecules(CAMs)are proteins located on the cell surface.They are involved in binding with the extracellular matrix(ECM)and with other adjacent cells in the process called cell adhesion.Basically,these proteins are transmembrane proteins and are composed of three domains:1,an extracellular domain that interacts either with other cell adhesion molecule;2,an intracellular domain which can interact with cytoskeleton;3,a transmembrane domain.[91].IgSF family is one member of cell adhesion molecule which is calcium independent in cell adhesion process,these proteins are soluble proteins and share immunoglobulin structural features.One subfamily in this IgSF family which called EWI family was composed by some noval proteins such like EWI-2,EWI-F,FPRP/CD9P-1,IgSF3,CD101 and so on.EWI2 relatively shares a highly homologyical amino acid sequence with other IgSF proteins and thus forms a novel Ig subfamily called the EWI Ig subfamily[1-4].Among the subfamily members,FPRP/CD9P-1 was also associated with tetraspanins[5,6].However,whether the rest of the members of the subfamily,such as IgSF3 and CD101,bind to tetraspanins remains unclear.EWI-2 also associates with tetraspanin KAI1/CD82(Tspan27),KAI1/CD82 has close relationship with cancer metastasis and has been defined as a cancer metastasis suppressor.The association between EWI-2 and CD82 is inversely correlates with the cancer metastatic potential[7,8].Although the biological functions of EWI proteins remains unclear,the EWI-2-tetraspanin associations suggest that EWI proteins might affect the tetraspanin functions such as cell adhesion,cell migration,cell invasion and fusion[9-12].Because tetraspanins has a prominent feature which can regulate cell cancer cell migration,EWI2 probablly regulate cancer cell motility through them via direct or indirect associations.For exsample,EWI2 overexpression can inhibit cancer cell migration onto collagen and fibronectin,by contrast,silencing of EWI2 can promote cancer cell migration[3,14,15].However,as an IgSF protein,EWI2 has location on microprotrusion which means it may function as a cell-cell adhesion molecule and be involved in the interaction between cells in different organs such as the prostate,testis,brain and kidney,where EWI2 is highly expressed[3].As a matter of fact,previous study has demonstrated that at the surface of dendritic cells,heat-shock protein A8 can be expressed in a cell-bound mode,and thus serves as a EWI2 ligand[16].In previous study,enforced overexpression of EWI2 can prohibite brain tumour growth size and growth rate in U87 glioma animal model[17].Furthermore,in malignant transformation in the brain the expression of EWI2 is down-regulated,besides,its overexpression also associated with better prognosis for brain tumours patients[17].CHAPTER 1 EWI-2 regulates cancer cell surface protein expression and cell motility[Objectives]To investigate whether EWI-2 can regulate cell surface protein expression,organization and further regulate cell motility.[Method]We choose PC3-human prostate cancer cell line and U87-human glioma cell line as medels to investigate into the flow cytometry analysis.In PC3 group,cells were silenced by EWI-2 SiRNA and named as PC3-KD,cells without knocking down procedure was named as PC3-NEG.U87 glioma cells were transfected with U87-MOCK as negative control,EWI-2 over-expression WT as well as U87-M3 palmitoylation mutant.Both of the two groups were stained with anti-tetraspanin primary antibodies and anti-integrin primary antibodies,followed by the staining of FITC conjugated secondary antibody.Flow cytometry analysis was proceeded after staining.We use Flowjo 7.6.1 and SPSS 13.0 for data analysis,and then followed by data quantitative statistics of comparison of mean fluorescent intensity by(1)two independent t test for the difference between PC3-NEG and PC3-KD under each antiboday staining or(2)One Way ANOVA for U87 group under each antiboday staining,respectively.P<0.05 was considered as statistically significant.Cancer cell motility anaylsis was proceeded by wound healing,transwell migration and invasion assay.For wound healing assay,PC3 cells were cultured in 6-well plates and then proceeded EWI-2 RNAi.After the cells became confluence,cells were treated with mitomycin-C,wounds were created with pipette tips by scratching the monolayer.During 16-24 hour healing,Wound closure was analyzed using an inverted light microscope every four hours.Wound closure was imaged and analysed by Image J software.PC3 cells were silenced by SiRNA and trans-well migration assay and invasion assay were proceeded in Trans-well insert,for trans-well migration assay the inserts was pre-coated with either fibronectin or laminin 1 and then blocked with heat inactivated BSA.(Group 1:to compare the difference between PC3-NEG and PC3-KD when coated the membrane with fibronectin;Group 2:to compare the difference between PC3-NEG and PC3-KD when coated the membrane with laminin 1).Six thousand cells were suspended in DMEM/BSA and then plated onto insert and incubated the chamber in 37℃,5%C02 for 3-6 hours.Invasion assay was proceeded on insert which was pre-coated with Matrigel,105 cells were added on the gel and incubated the chamber in 37℃,5%C02 overnight.Cells which can invade or migrate onto the insert were stained using Diff-Quick solution.Cells were imaged and the number was counted using Image J software.Data quantitative statistics was proceeded in SPSS 13.0 software and three individual experiments were quantified for the number of cells that migrated or invaded onto the membrane using two independent t test,P<0.05 was considered as statistically significant.[Result]In our study,after knocking down EWI-2,the expression level of CD9 was remarkably decreased for homo-clustered CD9(PC3-NEG=1919.67±23.674,n=3;PC3-KD=1349.00±75.408,n=3;P=0.002).The ratio C9BB staining/MAB7 staining is also decreased which means CD9 was transforming towards hetro-clustered form and this phenomenon was also observed in U87 EWI-2 over-expression group.However,the EWI-2 directly partner CD81 was not changed according to EWI-2 knocking down(P>0.05).Besides,knocking down EWI-2 can remarkably down-regulate the expression of a6 integrin which is laminin receptor(PC3-NEG=1498±22.51666,n=3;PC3-KD=1016±52.20153,n=3;P=0.001),however,the fibronectin receptor α5 integrin is up-regulated(PC3-NEG=9.5667±0.74923,n=3;PC3-KD=15.2433±0.09171,n=3;P=0.002).The following cell-matrix adhesion experiment demonstrated this further.In EWI-2 over-expression group,EWI-2 WT can slightly increase the expression level of homo-clustered CD9(U87-MOCK=1164.6667±240.00162,n=3;U87-WT=2704±488.42024,n=3;P=0.017),nevertheless,homo-clustered CD9 was decreased remarkably in EWI-2 M3 mutant(U87-MOCK=1164.6667±240.00162,n=3;U87-M3=1068.6667±183.43603,n=3;P=0.013)compared with WT.CD82/KAI-1 is reported to associate with EWI-2 and regulates cancer progression and metastasis,in our study,EWI-2 M3 mutant robust increased CD82 expression compared to MOCK(U87-MOCK=6.5733±0.56972,n=3;U87-M3=29.19±4.67339,n=3;P=0.004)and WT(U87-WT=14.8067±1.33834,n=3;U87-M3=29.19±4.67339,n=3;P=0.034).Coherence with in PC3 SiRNA group,CD151 was down-regulated in EWI-2 WT compared to MOCK(U87-MOCK=3.46333±0.14051,n=3;U87-WT=2.12±0.42548,n=3;P=0.044)and M3 mutant(U87-WT=2.12±0.42548,n=3;U87-M3=4.31±0.46608,n=3;P=0.006).The change of integrin a6 is different from PC3 SiRNA group which means over-expression of EWI-2 can decrease the a6 integrin and further affect the cell-matrix adhesion on laminin(U87-MOCK=3.46±0.51481,n=3;U87-WT=1.4333±0.23681,n=3;P=0.030).By contrast,the a6 integrin in M3 mutant was evaluated compared with-MOCK(U87-MOCK=3.46±0.51481,n=3;U87-M3=3,5422±0.67097,n=3;P=0.019)and-WT(U87-WT=1.4333±0.23681,n=3;U87-M3=3.5422±0.67097,n=3;P=0.001).Compared with PC3 SiRNA group,the WT and MOCK transfectants express equivalent amounts of a5 integrin,we only observed relatively higher level of a5 integrin in U87-M3 mutant(U87-MOCK=12.22±2.75308,n=3;U87-M3=21.2133±2.03850,n=3;P=0.045).a2 integrin expression level was up-regulated in U87-WT compared with the MOCK(U87-MOCK=14.6567±0.92726,n=3;U87-WT=19.8033±2.04452,n=3;P=0.036)and M3(U87-MOCK=14.6567±0.92726,n=3;U87-M3=11.7133±0.66519,n=3;P=0.045).Besides,the expression level of CD44 was consistant with PC3 group(P>0.05).The motility-suppressive function of EWI-2 does not reflect in collective cell migration-wound healing.Cell migration onto fibronectin was evaluated in PC3-KD compared to negative cells(PC3-NEG=278.3333±34.16789,n=3;PC3-KD=546.3333±48.75904,n=3;P=0.011),but decreased if we coated the insert with laminin 1(PC3-NEG=626.6667±32.31271,n=3;PC3-KD=522.6667±8.64741,n=3;P=0.036).Interestingly,this result was consistant with flow cytometry result.The result from the invasion assay revealed that the invasive potential of the PC3-KD had been significantly evaluated compared to the PC3-NEG(PC3-NEG=186±46.92902;n=3;PC3-KD=970.6667±87.15785,n=3;P=0.001).It has been reported that EWI-2 inhibits the invasive potential of cells by KAII1.Interestingly,the expression level of CD82/KAI1 in PC3 cell line is pretty low which means the effect may not come from CD82/KAI1 but other mechanism.[Conclusion](1)EWI-2 can regulate cancer cell surface protein level.(2)Alter the molecular organization of CD9.(3)Futher affect the cancer cell motility.CHAPTER 2 The role of EWI-2 on cancer cell morphology,microprotrusion formation and cell adhesion[Objectives]Cancer cell migration is typically regulated by growth factors,chemokines,matrix-degrading enzymes,and cell-cell and cell-matrix adhesion molecules.EWI-2 located at the cell-cell contact micro-protrusion and functioned as cancer suppressor protein.In thin study,we over-expressed EWI-2 in human glioma cell line and meanwhile silenced EWI-2 in human prostate cell line in order to demonstrate the function of EWI-2 on the protrusion formation and its effect on cell-cell adhesion.[Method]We choose PC3-human prostate cancer cell line and U87-human glioma cell line as medels to investigate into the flow cytometry analysis.In PC3 group,cells were silenced by EWI-2 SiRNA and named as PC3-KD,cells without knocking down procedure was named as PC3-NEG U87 glioma cells were transfected with U87-MOCK as negative control,EWI-2 over-expression WT as well as U87-M3 palmitoylation mutant.Both of the two groups were stained with different cell-cell adhesion related antibody such as CD9,CD81,CD44.Immnofluorescent microscopey imaging was proceed after staining.We used Image J and Carl Zeiss ZEN lite software for the images analysis,we counted the number of cell-cell zipper formation,measured micro-protrusion number and length as well as cell morphology change.All of this procedure was followed by data quantitative statistics in SPSS 13.0 software,three individual experiments were quantified(1)The difference of percentage of cell zipper formation(number of zipper/total cell number(%))between PC3-NEG and PC3-KD were quantified using Independent-Samples t test;(2)The difference of number of microprotrusion between PC3-NEG and PC3-KD were quantified using Independent-Samples t test;(3)The difference of cell morphology(number of cells having spherical morphology,number of cells having fusiform morphology)were quantified using X2 test;(4)The multiple comparison of the number or length of microprotrusion formation was by One Way Anova in U87 group,respectively).P<0.05 was considered as statistically significant.Cell-cell adhesion was analyzed by hanging drop aggregation assay.The PC3 cells were EWI-2 silenced and then allowed to aggregate over night in the hang-drop suspension.The experiment was designed into two group:No drug treatment group and Y27632 treatment group.For each group,we treated cells in two conditions:calcium-dependent condition or calcium independent condition.(Group 1:compare the difference of aggregation size between PC3-NEG and PC3-KD with no drug treatment,calcium-dependent condition;Group2:compare the difference of aggregation size between PC3-NEG and PC3-KD with no drug treatment,calcium-independent condition;Group3:compare the difference of aggregation size between PC3-NEG and PC3-KD with Y27632 treatment,calcium-dependent condition;Group4:compare the difference of aggregation size between PC3-NEG and PC3-KD with Y27632 treatment,calcium-independent condition).After aggregation,the cells went through mechanical pressure and then photographed under a light microscope,followed by quantification of the area of aggregates using Image J software.Results are displayed as the area of aggregate.The area of aggregates was measured by image J software.Data quantitative statistics was proceed by two independent t test based on the grouping situations above using two independent t test.P<0.05 was considered as statistically significant.We then use DU 145 cells to analyze the β-catenin and E-cadherin staining in cell-cell contact.Western blot and immnofluorescent staining were described as above and imaging were proceeded on Leica SP2 MP cofocal microscope,63x Plan APO 1.4 NA oil immersion,the image were then analysed using Image J software,then did data quantitative statistics in SPSS 13.0 software,the difference between the percentage of β-catenin staining in cytoplasm/total cell number were quantified using two independent t test.P<0.05 was considered as statistically significant.Cell-Matrix adhesion was also analyzed,PC3 cells were silenced by EWI-2 SiRNA and then followed by the cell-matrix adhesion assay proceeded on fibronectin or laminin1 coated plastic.For each group,we treated cells in two conditions:calcium-dependent condition or calcium independent condition.(Group 1:compare the difference of cell number attached on the plastic between PC3-NEG and PC3-KD with fibronectin coated,calcium-dependent condition;Group2:compare the difference of cell number attached on the plastic between PC3-NEG and PC3-KD with fibronectin coated,calcium-independent condition;Group3:compare the difference of cell number attached on the plastic between PC3-NEG and PC3-KD with lamininl coated,calcium-dependent condition;Group4:compare the difference of cell number attached on the plastic between PC3-NEG and PC3-KD with lamininl coated,calcium-independent condition).After 1 hour incubation,cells were rinsed with DMEM 3 times to get rid of the cells which were not adhere on the extracellular matrix and then stained with Diff-Quick solution.We counted the cell number on the plastic using Image J software and then did data quantitative statistics in SPSS 13.0 software based on the grouping situations above using two independent t test.P<0.05 was considered as statistically significant.[Result]EWI-2 silencing in PC3 cell line can transform the cell morphology from spherical,polygonal-shape to fusiform,spindle-shape(P<0.05).Besides,the protrusion formation in PC3-NEG is more regular,smooth,by contrast,the protrusion formation in PC3-KD is shorter and easy to break down.Statistic results showed that the percentage of fusiform,the number of microprotrusion formation is decreased in PC3-KD compared to PC3-NEG(PC3-NEG=117.2±7.23080,n=10;PC3-KD=52±5.43446,n=10;P=0.000).The spindle-shaped cells in PC3-KD group is 82.9%,this percentage is much higher compared to negative group(17.2%)which means the silencing of EWI-2 can induce PC3 cells changing their morphology towards spindle shape(PC3-NEG=82.8%,n=233;PC3-KD=17.1%,n=175;N=3;P=0.000).The zipper structure between cells in PC3-KD group(10%)was remarkably decreased compared to PC3-NEG(52.5%,P<0.05).we figured out that no matter what antibody we used to stain the cells,over-expression of U87-M3 mutant always showed higher number of protrusions formation compared with-MOCK(P<0.05),U87-WT transfectant showed up the trend to have more protrusion formation compared to MOCK but unfortunately quantitative statistics showed there was no significant difference compared with another two transfectants(P>0.05).We then analyzed the length of the protrusion formation with CD9 and CD81 staining,we observed that in U87-WT transfectant,as shown in statistic result,in CD9 staining,U87-WT has relative longer protrusion compared with-MOCK(U87-MOCK=7.3267±0.58015,U87-WT=10.3061±0.77284;P=0.002)and M3(U87-WT=10.3061±0.77284,U87-M3=8.0238±0.60308;P=0.024),nevertheless,in CD81 staining,there was significant difference between MOCK and WT(P<0.05)with no significant difference between WT and M3(U87-WT=15.7525±8.97587,U87-M3=13.2460±6.82862;P>0.05).For phalloidin staining,we observed that no matter we measure the length or count the number,-MOCK group always had the shortest and smallest number of protrusion formation and M3 group had the longest and largest number of protrusion formation(all of the P values were less than 0.010).We then quantified the difference of cell-cell adhesion under different condition.We found that there is statistically significant decrease in terms of the area of aggregate.In cell-cell adhesion assay,it was observed that,after shear stress,the area of aggregates from the calcium-dependent and-independent group were smaller in PC3-KD group when compared to PC3-KD(Calcium dependent group:PC3-NEG=115.2859±7.37516,n=25;PC3-KD=89.7471±5.85023,n=23;P=0.003;Calcium independent group:PC3-NEG=133.4103±8.42085,n=27;PC3-KD=86.8984±3.33575,n=23).Moreover,after shear stress,the PC3-KD cells exhibited much more single cells compared to the PC3-NEG.Then we use Y27632 to treat the cells and interestingly,the difference of the area of aggregate in calcium-dependent group was totally diminished(PC3-NEG=136.3948±7.01654,n=15;PC3-KD=126.0439±11.71461,n=15,P>0.05)but still existed in calcium-independent group(PC3-NEG=157.9326±9.427704,n=16;PC3-KD=116.6084±7.96081,n=15,P=0.002).After knocking down EWI-2 in DU145 cells,from western-blot result we can see the obvious up-regulation of total β-catenin expression.And then we did immune-staining using anti-β-catenin and anti-E-cadherin antibody,we observed that after knocking down EWI-2,cytoskeleton showed more bundle and more β-catenin and E-cadhrin turn over back to cytoplasm or nuclear.We then counted the cells which had β-catenin staining in cytoplasm,the number of cells which have cytoplasmβ-catenin staining was doubled in DU145-KD compared with negative control(DU145-NEG=0.11412±0.023446,n=6;DU145-KD=0.29950±0.038099,n=6;P=0.002).Besides,the staining of E-cadherin in cell-cell junction was also decreased after silencing EWI-2.In cell-matrix adhesion assays,in PC3-KD group,cell number attached on fibronectin coated surface was higher compared to PC3-NEG group(PC3-NEG=253.4444±54.91815,n=9;PC3-KD=446.8889±52.32390,n=9;P=0.021),by contrast,the attached cell number on laminin.1 coated surface was much lower in PC3-KD(PC3-NEG=712±123.08913,n=6;PC3-KD=315.16679±38.82990,n=6;P=0.012).[Conclusion](1)EWI-2 contributes to the cancer cell motility regulation at least partly via micro-protrusion formation and cell morphology change.(2)As a member of IgSF,EWI-2 can regulate cell-cell adhesion in calcium-dependent and-independent manner.Rock inhibitor can’t alter the cell-cell adhesion in calcium independent condition.Besides,the cell-matrix adhesion can also be altered and is ECM and integrin dependent.CHAPTER 3 EWI-2 affects the mesenchymal to amoeboid transition[Objectives]To demonstrate whether EWI-2 can affect the mesenchymal to amoeboid transition.[method]We choose PC3 cell as model for transmission electron microscopic analysis,PC3 cells were silenced by EWI-2 SiRNA and named as PC3-KD,cells without knocking down procedure was named as PC3-NEG After SiRNA silencing,cells were detached,rinse with PBS and then resuspended in DMEM complete medium on ice for the micro-protrution reformation.After four hours,cells were washed with cacodylate buffer twice to get rid of the serum and then fixed with 2.5%gluteraldehyde in sodium cacodylate buffer.The cells together with insert membrane was then cut into thin sections and stained with 0.3%potassium ferrocynide in 2.0%osmium tetroxinde and 4.0%uranyl acetate.The cells were then examined under a transmission electron microscope,and the images were acquired with a digital camera.The cell membrane blebs and protrusions formation were analyzed by Image J software and then did data quantitative statistics in SPSS 13.0 software.The morphology change(number of cells with microprotrusion formation,number of cells with blebs formation)was quantified by X2 test.P<0.05 was considered as statistically significant.Western blot was used to investigate the signaling pathway during the mesenchymal to amoeboid transition.Cells from PC3 SiRNA group and U87 EWI-2 over-expression group were lysed with RIPA buffer and separated by SDS/PAGE(12%)gel and then electrically transferred to nitrocellulose membranes.The nitrocellulose membranes were blocked and then blotted with anti-ERK,P-ERK,PAN-ERK,VIMENTIN,FAK,PY319-FAK,EGFR,P-EGFR,tyrasinated-α-tubulin,,MEK1,MEK2,mAb and horseradish peroxidase-conjugated IgG and sequentially detected with chemiluminescence.DU 145 prostate cells were then silenced by EWI-2 SiRNA and then placed on different 3D culture gel under different condition.After the incubation,cells in the gel or on the gel were imaged under inverted light microscope and the cell morphology formation was analyzed using Image J software.After morphology imaging,cells were stained with certain antibody.After staining,gel and cell together were removed from the 96 wells plate,plated on glass slides and then mounted in hard-set fluo-save mounting medium.Imaging were proceeded on Leica SP2 MP confocal microscope,63x Plan APO 1.4 NA oil immersion,the image were then analyzed using Image J software.Followed by data quantitative statistics in SPSS 13.0 software.The percentage of number of cells with membrane blebs formation was quantified by two independent t test.P<0.05 was considered as statistically significant.The method of immune-staining for tyrosinated-α-tubulin was the same as described above in chapter 2.[Result]In transmission electron microscopy study,after 4 hours incubation in suspension condition,the PC3 EWI-2 silencing cells displayed more abundant,reversible eruption of blebs compared to the negative control(X2 VALUE=17.385b,df-1,P=0.000).During the 3D culture,we figured out the cell membrane blebbing in DU145 cell lines after the EWI-2 silencing.This observation was also consistant with TEM result above.Conversely,we noticed in negative group,cells had more micro-protrusion formation(On gel culture:DU145-NEG=11.157±0.5982,n=3;DU145-KD=71.5±4.5369,n=3;P=0.005;In gel culture:DU145-NEG=16.180±6.7757,n=3;DU145-KD=80.233±2.3849,n=3;P=0.001),We then demonstrated that the EGFR/MAPK/ERK signaling pathway contributed for EWI-2 signaling transduction.As shown in western blot result,after knocking down EWI-2 in PC3 cells,the activation of ERK and EGFR were up-regulated with a higher expression level of MEK1,MEK2 and vimentin.Focal adhesion kinase was downregulated not only on the level of expression but also the autophosphorylation.Besides,we also demonstrated the EWI-2 silencing was not Src dependent for the phosporylation of Src was not altered after EWI-2 silencing.Detyrasinated α-tubulin is relative to the progression of cancer.In this study,western-blot result showed up the decreased level of tyrasinated α-tubulin,and in immunofluorescent staining,we also observed the disassembly of tyrasinated-α-tubulin after EWI-2 silencing in PC3 cells.[Conclusion](1)3D culture is essential for the amoeboid phenotype and the EWI-2 silencing can promote MAT.(2)ERK and FAK signaling pathway contribute to the EWI-2 related mesenchymal to amoeboid transition.(3)Microtubule lost its stability during MAT. |
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