| Objective Vasculogenic mimicry(VM)is a nonangiogenesis-dependent pathway that promotes tumor metastasis and disease progression.The previous experimental study of our research group showed that the presence of VM in breast cancer was associated with metastasis and poor prognosis,however the underlying mechanism is still not very clear.On the other hand,recent studies have shown that breast cancer stem cells(BCSCs)were associated with metastatic and therapeutic resistance in breast cancer and were considered to be responsible for forming metastases to tumor cells at the distal site.The Nodal signal plays several vital roles in the embryonic developmental phase,and recent studies have found that it can be re-expressed in malignant tumor tissue.In view of the role of Nodal in embryos and tumors,this study aims to investigate whether Nodal signaling promotes vasculogenic mimicry in breast cancer and its role in breast cancer stem cells,meanwhile further explore its mechanism and signaling pathways.Hope to find a therapeutic target for future applications to inhibit breast cancer recurrence and metastasis,thereby improving the prognosis in breast cancer patients.Methods1.Immunohistochemical staining was used to analyze the expression of Nodal in100 cases of breast cancer.The expression of Nodal protein in breast cancer samples and paired adjacent normal tissues from patients were detected by Western blot.And we further analyzed the relationship between Nodal expression and node metastasis,TNM clinical stages,differentiation grade and overall survival of breast cancer cases.2.CD31/PAS double staining was used to identify VM structure in breast cancer tumors slices,and Kaplan–Meier survival analysis were performed.In addition,the relationship between Nodal expression and VE-cadherin,CD44,Slug expression was determined by immunohistochemical staining.3.The expression of Nodal protein in breast cancer cell lines MCF-7 and MDA-MB-231 was analyzed by Western blot.Use lentiviral plasmids to knock down or up regulating Nodal expression from gene levels.And the cells were selected with puromycin to establish stable Nodal knockdown or Nodal-overexpressing cell lines.Western blot was used to detect the transfection efficiency of four Nodal sh RNA lentiviruses.The Nodal expression of stable transfection cell lines was confirmed by Western blot and RT-PCR.4.In order to exclude the off target effect,the rescue experiments had been performed by overexpressing Nodal in Nodal knockdown sh Nodal4 cells.The western bolts were performed to examine the reexpression of Nodal.The functional experiments were also performed in 231-sh Nodal3 and231-sh Nodal4 and Nodal reexpression group.5.The relationship between Nodal and VE-cadherin expression was detected by Western blot and RT-PCR.The ability of forming VM structures of breast cancer cells in vitro was observed by on matrigel and in matrigel three-dimensional culture.Immunofluorescence Nodal/VE-cadherin staining was performed on VM structures and analyzed by laser confocal microscopy.6.Western blot was used to detect the level of p-Smad2/3 in breast cancer cells.The expression levels of Nodal,p-Smad2/3,VE-cadherin,MMP2 and MMP9 were detected by Western blot in MCF-7-Con,MCF-Nodal,MDA-MB-231-sh Con cells and SB431542 treated group.And the results were verified by immunofluorescence.The activities of MMP2 and MMP9 were detected by gelatin zymography assays.In addition,SB431542 was used in the three-dimensional culture to observe the role of Smad2/3 pathway in VM formation.7.The effects of Nodal signaling and Smad2/3 pathway on the migration and invasion of breast cancer were analyzed by wound-healing assays,transwell assays and invasion assays.The expression of Vimentin,N-cadherin and E-cadherin and the effects of Nodal on the expression of transcription factors Slug,Snail and c-Myc were detected by Western blot.The immunofluorescence staining of breast cancer cells were performed to verify these results.8.The use of colony formation assays to assess the breast cancer cell self-renewal and proliferation capacity,and the sphere formation assays were performed to determine the effects of Nodal on BCSCs.At the same time,the effect of SB431542 application on the breast cancer cells was observed.9.The expression of BCSC related markers ALDH1,CD44 and CD133,and the expression of related transcription factors Sox2,Oct4 and Nanog were investigated by Western blot.The immunofluorescence staining of breast cancer cells were performed to further verify above results.10.56 nude mice were injected subcutaneously with stable transfected cells of each group to confirm the effect of Nodal signaling on tumor formation and proliferation in breast cancer xenograft model.Tumor volume was monitored weekly using vernier calipers for portraying the growth curve.In addition,SB431542 solution(10mg/kg/mouse)was intraperitoneal injected every other day to verify the feasibility of SB431542 in vivo.11.Endomucin / PAS immunohistochemical double staining was performed on each group of xenografts slices,and the number of VM formation was counted under microscope.In addition,Nodal,p-smad2/3,VE-cadherin,E-cadherin,Vimentin,MMP2,Slug and CD44 were immunohistochemically stained to detect the expression of VM and CSC-related proteins in xenografts.Results1.The level of Nodal protein in breast cancer tissues was significantly up-regulated compared with the adjacent normal tissues(P<0.05).In the immunohistochemical results,Nodal positive staining was predominantly localized in the cytoplasm of cancer cells.Among the 100 cases of breast cancer immunohistochemical staining,62 cases were positive for Nodal staining.High expression of Nodal was significantly correlated with tumor metastasis(P = 0.000),differentiation grade(P = 0.003)and low survival rate(P = 0.013).2.The results show that the expression of Nodal and the presence of VM was positively correlated in breast cancer(P = 0.005).Nodal was also associated with the expression of the endothelial-specific marker VE-cadherin(P = 0.000)and the Slug staining(P = 0.001).At the same time,the expression of BCSC associated marker protein CD44 resulted in a decrease in survival(P = 0.022)and a significant correlation with Nodal expression(P = 0.000).3.MDA-MB-231 cells were infected with four lentiviral vectors expressing Nodal sh RNA.Among the four sh RNAs,sh Nodal4 most efficiently knocked down Nodal expression by more than 80%.Meanwhile MCF-7 cells transfected with Nodal overexpression lentivirus plasmids.Western blot and PCR results showed that the expression of Nodal protein and m RNA transcription in MDA-MB-231 were significantly inhibited.And the expression of Nodal protein in MCF-7 Nodal cells was significantly enhanced.As a result,MCF-7-Nodal cells changed from epithelial phenotype to fibroblast-like morphology,but EMT phenotype in MDA-MB-231-sh Nodal cells was not significant reversal(P <0.05).4.Western blot analysis showed that the interference efficiency of sh Nodal4 was about 80% and that of sh Nodal3 was 70%.In addition,the level of Nodal expression in 231-sh Nodal4 rescue group had got recover.5.The results showed that the down-regulation of Nodal expression in MDA-MB-231 cells reduced the level of VE-cadherin protein and m RNA expression.231-sh Control cells formed typical channel-like structures on Matrigel and in Matrigel 3D cultures,while there were no integrated VM-like channels in 231-sh Nodal cells.Nodal overexpression in MCF-7-Nodal up-regulated VE-cadherin protein and m RNA levels,and promoted the formation of channel-like networks both on Matrigel and in Matrigel.The immunofluorescence staining of pipe-like structures showed that Nodal was concentrated in the cells that formed the VM networks and in the outer edges of these structures.At same time,these VM channel walls overexpressed VE-cadherin.6.The knockdown of Nodal in MDA-MB-231 cells decreased the phosphorylation level of Smad2/3,while the overexpression of Nodal in MCF-7 cells significantly increased Smad2/3 phosphorylation.After treatment with SB431542,Western blot showed that the phosphorylation level of Smad2/3was significantly inhibited and the expression of VE-cadherin was down-regulated.Western blot and gelatin zymography showed that Nodal could promote the expressions and activities of MMP2 and MMP9.At the same time,SB431542 inhibited the expressions and activities of MMP2 and MMP9 to a certain extent,and affected the formation of VM in three-dimensional culture in vitro.7.Nodal signaling can promote the migratory ability and invasive ability of breast cancer in vitro.And in the experimental group treatment with SB431542 neutralized the migration and invasion.Western blot and immunofluorescence showed that Nodal could up-regulate the expression of N-cadherin,Vimentin,Snail,Slug and c-Myc,meanwhile down-regulated the expression of E-cadherin.8.The knockdown of Nodal expression in MDA-MB-231 cells apparently reduced colony formation of breast cancer cell.It was also observed that the formation rate and volume of tumor stem spheres were significantly decreased.Moreover,up-regulated Nodal expression promoted the formation of spheres and cell clone in MCF-7 cells.And application of SB431542 inhibited the effects of Nodal.9.The results showed that the Nodal signaling promoted the expression of CSC-related markers and factors ALDH1,CD44,CD133,Sox2,Oct4 and Nanog,and the expression of these markers was inhibited to different degrees when blocking the Smad2 / 3 pathway.10.Compared with the 231-sh Con group,the tumor growth was significantly slower in the 231-sh Nodal group(P <0.01),and the volume of the tumor was also reduced.Overexpression of Nodal significantly promoted tumor growth in the MCF7-Nodal group(P <0.01).In the SB431542 group,the tumor growth rate was inhibited and the tumorigenicity was impaired in vivo.11.The results showed that Nodal signaling promoted the expression of VE-cadherin,Vimentin,MMP2,Slug and CD44,and decreased the expression of E-cadherin in breast cancer xenografts.At the same time,Nodal promoted the formation of VM in vivo.And the feasibility of SB431542 application in vivo was verified by reducing the formation of VM in xenografts.Conclusions1.High expression of Nodal signaling in human breast cancer is associated with breast cancer metastasis and poor prognosis.2.In vitro experiments,Nodal signaling up-regulates the expression of VM-associated protein and promotes the formation of VM on matrigel and in matrigel of breast cancer cells.In addition,the expression of Nodal and VE-cadherin in VM tubules is correlated.3.Nodal promotes Slug,Snail and C-myc expression,and increase the migration,invasion and plastic ability of breast cancer cells.Result in inducing EMT process thereby leading to VM formation.4.On the other hand,Nodal regulates breast cancer stem cell phenotype,promoting ALDH1,CD44 and CD133 expression.Result in increasing the breast cancer cell tumorigenicity and multi-differentiation potential.5.In breast cancer cells,Smad2/3 pathway plays a crucial role in Nodal regulating VM formation and stem cell phenotype.6.Nodal signaling significantly promote breast cancer cell tumorigenicity and VM formation in vivo.At the same time,SB431542 can inhibit Smad2/3phosphorylation and thereby blocking Nodal signal transduction in vivo and in vitro.Therefore,Nodal pathway might serve as a therapeutic target for breast cancer treatment,thereby improving the patients’ prognosis. |
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