Study On The Scutellarin-Provided Inhibition On Triple-Negative Breast Cancer Metastasis And Its Engaged Mechanism

Backgound: Triple-negative breast cancer(TNBC)is a high aggressive sub-type of breast cancer with frequent metastasis and recurrence,and it is still incurable at present.It is well-known that abundant abnormal vasculature and inflammatory micro-environment will accelerate tumor metastasis during the development of TNBC.Scutellarin(SC),with excellent cardiovascular protective function,is one of the main effective compounds from Chinese medical herb Ban-Zhi-Lian(Scutellaria barbata D.Don)that has great effects of clearing away heat and detoxifying.Previous studies have reported that scutellarin inhibited tumor metastasis in a variety of tumors including hepatocellular carcinoma,colorectal cancer and renal cancer.However,it is still not clear whether scutellarin can also inhibit TNBC metastasis.Objective: This study aims to investigate the inhibitory effect of scutellarin on TNBC metastasis and its engaged mechanism.The results will supply scientific evidences for the potential application of scutellarin in TNBC treatment and provide new therapeutic strategy for clinical TNBC treatment.Methods:1.In fluorescence labeled MDA-MB-231-luc-GFP and 4T1-luc-GFP orthotopic tumor bearing mice model,optical imaging and node counting were performed to evaluate the inhibitory effect of scutellarin on TNBC metastasis in vivo.2.Circulating tumor cells(CTCs)counting,tumor vessels isolation,Western-blot and immunofluorescence assay,combined with the analysis of The Cancer Genome Atlas(TCGA)data were used to reveal the influence of scutellarin on the expression of some endothelial junctional proteins and the function of endothelial barrier.Enzyme-linked immunosorbent assay(ELISA)was performed to detect the content of tumor necrosis factor α(TNFα)in tumor tissues.3.Transendothelial electrical resistance(TEER)and fluorescein isothiocyante(FITC)conjugated dextran cell permeability assay were used to investigate the injury of endothelial barrier induced by TNFα in human mammary microvascular endothelial cell(HMMEC)and human umbilical vein endothelial cell(HUVEC),and the protective function of scutellarin.Western-blot,real-time polymerase chain reaction(Real-time PCR),3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide(MTT)assay,Hoechst staining,combined with using inhibitors and si RNA were applied to reveal the important role of TNFα receptor 2(TNFR2)-mediated the phosphorylation of extracellular regulated protein kinase 1/2(ERK1/2)and downstream nuclear translocation of enhancer of zeste homolog-2(EZH2)in the TNFα-induced endothelial barrier injury and TNBC transendothelial invasion in vitro.Next,whether scutellarin can inhibit this signal axis was investigated both in vivo and in vitro.Furthermore,molecular docking,cellular thermal shift assay(CETSA),drug affinity responsive target stability(DARTS)and Co-immunoprecipitation(Co-IP)were used to explore the potential interaction between scutellarin and TNFR2.4.Cell migration assay and immunofluorescence assay were used to evaluate the promotion of supernatants from TNFα-stimulated HMMEC on TNBC cell migration.Protein array and ELISA were used to analyze the expression of human growth factors including granulocyte colony-stimulating factor(G-CSF)from TNFα-stimulated endothelial cells.Furthermore,cell migration assay,immunofluorescence assay in vitro and the injection of G-CSF neutralizing antibody in vivo were performed to confirm the crucial role of G-CSF involved in TNBC metastasis.5.Cell migration assay and immunofluorescence assay were used to evaluate the effect of scutellarin on the expression of G-CSF and G-CSF-induced migration of TNBC cells.Western-blot,ELISA,chromatin immunoprecipitation(CHIP)and Real-time PCR,combined with using si RNA and inhibitors were used to reveal the important role of TNFR2-Runt-Related Transcription Factor 1(RUNX1)signal axis in TNFα-induced G-CSF expression in endothelial cells,and to elucidate the engaged mechanism in the scutellarin-provided inhibition on endothelial G-CSF expression induced by TNFα.Results:1.The results of fluorescence labeled MDA-MB-231-luc-GFP and 4T1-luc-GFP orthotopic tumor bearing mice model showed that scutellarin had no obvious inhibitory effect on the growth of TNBC,but obviously reduced the distant metastasis of TNBC.2.Scutellarin reduced the enhanced number of CTC in TNBC orthotopic bearing mice.Analysis of TCGA data showed that the expression of endothelial junctional proteins in breast tumor tissues including vascular endothelial cadherin(VE-cadherin),and platelet/endothelial cell adhesion molecule-1(PECAM-1/CD31)was lower than in normal tissues.Results of Western-blot and immunofluorescence assay showed that scutellarin significantly restored the decreased expression of VE-cadherin,CD31 and tight-junction protein claudin-1 in TNBC-related vessels in vivo.Result of ELISA showed that the content of TNFα in tumor tissues was significantly higher than that in normal tissues,but scutellarin had no inhibitory effect on this increase in vivo.3.Results of Western-blot showed that TNFα reduced the expression of VE-cadherin,occludin and CD31 in both HMMEC and HUVEC.Results of TEER,FITC-dextran assay and transendothelial invasion showed that TNFα decreased the TEER value,increased the permeability of HMMEC and HUVEC monolayer barrier,and promoted the transendothelial invasion of TNBC through endothelial monolayer.Scutellarin reversed the decrease of TEER value and increase of permeability in TNFα-stimulated HMMEC and HUVEC.Scutellarin also reduced the increased transendothelial invasion of TNBC induced by TNFα.4.In vitro,the results of Western-blot,Real-time PCR and FITC-dextran permeability assay showed that TNFα stimulation increased the nuclear translocation of EZH2 and the phosphorylation of ERK1/2 in both HMMECs and HUVECs.Both GSK126(an inhibitor of EZH2)and blocking ERK1/2 phosphorylation could restore the decrease of junction proteins expression and reverse the damage of barrier function in TNFα-stimulated endothelial cells,and also reduced the enhanced transendothelial invasion of TNBC induced by TNFα.Inhibition of ERK1/2 also reduced the increase of EZH2 nuclear translocation.Results of MTT assay and Hoechst staining showed that TNFα didn’t cause cytotoxicity in HMMEC and HUVEC when cells were incubated with TNFα for 24 h.R-7050(TNFR1 inhibitor)had no obvious effect on the increased transendothelial invasion of TNBC induced by TNFα.Further results showed that TNFR2 si RNA restored the decreased expression of endothelial junction proteins,reduced the enhanced ERK1/2 activation and EZH2 nuclear translocation induced by TNFα.Knockdown of TNFR2 also reduced the increased FITC-dextran leakage from HMMEC and HUVEC monolayer and transendothelial invasion of TNBC induced by TNFα.Results of Western-blot showed that scutellarin obviously inhibited the activation of ERK1/2 and nuclear accumulation of EZH2 in both TNBC-associated vessels and TNFα-stimulated HMMEC and HUVEC.Results of molecular docking imply the potential interaction between scutellarin with TNFR2.Furthermore,results of CETSA,DARTS and Co-IP showed that scutellarin couldn’t change the thermal stability and protease susceptibility of TNFR2,but could reduce the binding between TNFR2 and TNFα.5.Results of cell migration assay and immunofluorescence assay showed that the supernatant from TNFα-stimulated HMMEC obviously increased the migration of TNBC cells.Results of the human growth factor array analysis,Real-time PCR and ELISA showed that TNFα enhanced the expression of granulocyte colony-stimulating Factor(G-CSF)in endothelial cells.Results of cell migration assay and immunofluorescence assay showed that G-CSF accelerated the migration of TNBCs in vitro.G-CSF neutralizing antibody could significantly inhibit the TNBC metastasis in 4T1-luc-GFP orthotopic mice in vivo.6.Results of cell migration,ELISA and Western-blot showed that scutellarin had no effect on TNBC migration induced by G-CSF,but could decrease the enhanced expression of G-CSF in endothelial cells.Besides,TNFR2 si RNA decreased G-CSF expression in HMMEC and HUVEC induced by TNFα,while GSK126 or U0126 had no this effect.TNFα induced nuclear accumulation of RUNX1 in endothelial cells.RUNX1 could bind to the promoter regions of G-CSF gene,and Ro5-3335(RUNX1inhibitor)significantly decreased the enhanced expression of G-CSF in TNFα-stimulated endothelial cells.Scutellarin suppressed the enhanced nuclear translocation of RUNX1 in both TNBC-associated vessels and TNFα-stimulated HMMEC and HUVEC.Conclusions: Scutellarin inhibited TNBC metastasis in vivo.The mechanism may be that scutellarin alleviates the injury of TNBC-related vascular endothelial barrier by inhibiting TNFα-initiated TNFR2-ERK1/2-EZH2 signal axis,and also reduces the G-CSF-induced TNBC migration via deceasing the TNFα-induced expression and release of G-CSF in endothelial cells.