The Mechanism Of S1PR1 On The Regulation Of Microangiogenic Mode In Breast Cancer

Objective: Breast cancer is the most commonly diagnosed tumor among women worldwide and its mortality rate is also very high which is inseparable from the abundant angiogenesis in breast cancer.The angiogenic patterns of tumors can be roughly divided into two types: endothelium-dependent vessels and tumor angiogenic mimicry.Tumor angiogenesis mimicry refers to the way in which tumor cells provide energy for their own channels.These tumor cells also have markers of tumor cells and endothelial cells.It has been confirmed in many tumors that a poor prognosis is caused by the appearance of an angiogenic mimicry.Sphingosine 1 receptor 1(S1PR1)is a protein of 382 amino acids and plays a very important role in angiogenesis and lymphatic metastasis.However,its research in tumor angiogenesis is rare,especially in the role of tumor angiogenesis mimicry(VM).This study investigated the role of S1PR1 in angiogenesis in breast cancer and further elucidated the mechanism of S1PR1 on the transition of two angiogenic modes in human breast cancer.Provide new ideas for anti-angiogenic therapy for breast cancer.Methods: 1.Resected specimens of 100 breast cancer patients confirmed by two senior pathologists and their tissue specimens were provided by Tianjin Medical University General Hospital.The expression of S1PR1 in human tissues was detected by immunohistochemical staining and the relationship between the expression of S1PR1 and pathological staging,grading and lymphatic metastasis in clinical case data was further analyzed.The CD31/PAS double staining method was used to correlate the expression of S1PR1 with VM and endothelium-dependent blood vessels.Survival analysis was performed using the Kaplan-Meier method.2.The expressions of VE-cadherin and β-catenin in 100 breast cancer specimens were detected by immunohistochemistry.The expression and correlation of S1PR1,VE-cadherin and β-catenin were analyzed by Pearson statistical method.3.The expression of S1PR1 in a series of breast cancer cell lines(MDA-MB-231,HS-578 T,BT-474,MCF-7,T-47D)was analyzed by Western Blot.The selected cell lines with low(MDA-MB-231)or high(MCF-7)expression of S1PR1 were stably transfected with S1PR1 and sh-S1PR1 plasmids,respectively.The expression of S1PR1 in these cells were confirmed by Western Blot.4.The three-dimensional culture was used to observe the tube formation ability of endothelial cells co-cultured with breast cancer cells and the tube formation ability of breast cancer cells.The proliferation activity of endothelial cells cocultured with breast cancer cells was detected by MTT assay.The effects of S1PR1 on invasion,migration in breast cancer cells were observed by invasion,migration and wound healing assays.5.Western blot and immunofluorescence experiments were performed on breast cancer cells transfected with S1PR1 to observe the expression of VE-cadherin,β-catenin and Eph A2.6.The phosphorylation of VE-cadherin(Y731)was detected by Western blot.The expression of VEGF was detected by enzyme-linked immunosorbent assay(Elisa).7.Rho A activity in S1PR1 transfected breast cancer cells was detected by Glisa.After adding Rho A inhibitor the expression of VE-cadherin(Y731),VE-cadherin and β-catenin was detected by Western blot and immunofluorescence.VEGF secretion was detected by Elisa after inhibition of Rho A activity.After adding the S1PR1 inhibitor,the Rho A activity and the expression of downstream factors were detected by Western blot,immunofluorescence and enzyme-linked immunosorbent assay(Elisa).8.S1PR1 stably transfected human breast cancer cells were injected subcutaneously into nude mice and the tumor volume changes were measured and the tumor volume growth curve was drawn.The expression of S1PR1,VE-cadherin and β-catenin in tumors and the number of endothelium-dependent blood vessels and VM were detected by immunohistochemical staining and Endomucin /PAS double staining.Results:1.Among the 100 cases of human breast cancer tissues,S1PR1 was highly expressed in 63 cases(63/100)which was associated with tumor grade(p =0.027),metastasis(p=0.019)and VM(p=0.004).Kaplan-Meier analysis showed that the high expression of S1PR1 in non-tri-negative breast cancer was associated with poor prognosis but there was no significant difference in triplenegative breast cancer.2.Pearson chi-square test showed that S1PR1 was correlated with the expression of VE-cadherin and β-Catenin.Pearson correlation analysis showed that S1PR1 was negatively correlated with VE-cadherin expression and S1PR1 was positively correlated with β-catenin expression.(p< 0.05).3.Western blot analysis was performed in five breast cancer cell lines(MDA-MB-231,HS-578 T,BT-474,MCF-7,T-47D).The MCF-7 with high expression of S1PR1 and the MDA-MB-231 with low expression of S1PR1 were selected.The selected cell lines with low(MDA-MB-231)or high(MCF-7)expression of S1PR1 were stably transfected with S1PR1 and sh-S1PR1 plasmids,respectively.The expression of S1PR1 in these cells were confirmed by Western Blot.4.After MCF-7 was down-regulated S1PR1,the ability of invasion,migration and tube formation was enhanced(p<0.05).While breast cancer cell line MDA-MB-231 with up-regulated S1PR1,the ability of invasion,migration and tube formation was attenuated(p<0.05).The activity and tube formation ability in endothelial cell co-cultured by S1PR1 low expression were decreased(p<0.05).While the activity and tube formation ability in endothelial cell co-cultured by S1PR1 high expression were enhanced(p<0.05).5.The protein level of breast cancer cell line MCF-7 transfected with S1PR1 downregulated plasmid showed that the expression of VE-cadherin and Eph A2 was increased and the expression of β-catenin was decreased(p <0.05).The protein level of breast cancer cell line MDA-MB-231 transfected with S1PR1 upregulated plasmid showed that VE-cadherin,Eph A2 expression decreased,and β-catenin expression increased(p<0.05).6.After breast cancer cell line MCF-7 down-regulated S1PR1,VE-cadherin(Y731)phosphorylation decreased and the secretion of VEGF was decreased(p<0.05).After breast cancer cell line MDA-MB-231 up-regulated S1PR1,VE-cadherin(Y731)increased phosphorylation,and the secretion of VEGF was increased(p<0.05).7.In the S1PR1 high expression cells(MCF-7 cells and MDA-MB-231-S1PR1)the activity of Rho A was higher than that of the control group.After the addition of an inhibitor of Rho A to MCF-7 cells,the expression of VE-cadherin was increased,while the expression of VE-cadherin(Y731)and β-catenin was decreased.After adding an inhibitor of Rho A in the MCF-7 cells the tube formation ability was enhanced and the secretion of VEGF was reduced.The same results could be achieved by adding an inhibitor of S1PR1 to breast cancer cells and transfection the plasmid with down-regulating S1PR1 in breast cancer cells.8.Compared with the control group,the MCF-7-sh-S1PR1 group had fast growth,large tumor volume and more VM channels.The expression of VE-cadherin was increased and the expression of β-Catenin was decreased in tumor tissues.Compared with the control group,the MDA-MB-231-S1PR1 group had slow growth,small tumor volume and fewer VM channels.The expression of VEcadherin was decreased and the expression of β-Catenin was increased in tumor tissues.Conclusions: 1.In human breast cancer tissues S1PR1 was associated with breast cancer staging,lymphatic metastasis and VM.In non-three-negative breast cancer,it was associated with poor prognosis of patients.2.In vivo experiments found that S1PR1 promoted endothelium-dependent angiogenesis in breast cancer and inhibited the formation of VM.The expression of S1PR1 relatively slowed the invasion and migration ability of breast cancer cells.3.S1PR1 induced VEGF secretion by inducing VE-cadherin(Y731)phosphorylation and induced endothelial cells more active.The phosphorylation of VE-cadherin lead to the detachment of β-Catenin(linked to VE-cadherin Y731 site).4.After inhibition of Rho A,S1PR1 attenuated the induction of VE-cadherin(Y731)phosphorylation and initially confirmed that Rho A is a key factor in the regulation of VE-cadherin(Y731)phosphorylation by S1PR1.5.In vitro,the high expression of S1PR1 was positively correlated with the number of tumor endothelium-dependent vessels and negatively correlated with VM,tumor size.