Effect And Mechanism Of Luteolin On Suppressing Triple Negative Breast Cancer

Purposes:Triple negative breast cancer(TNBC)is the most malignant subtype of breast cancer.Due to the lack of endocrine therapy and targeted therapy,it is of paramount importance to develop new therapeutic targets and drugs for TNBC.Aberrant activation of HippoYAP/TAZ signaling pathways is closely related to the tumorigenesis and metastasis of TNBC.We screened a natural compound library and discovered that luteolin inhibited the transcriptional activity of YAP/TAZ effectively.Therefore,our study aims to clarify the mechanism between luteolin and YAP/TAZ,to determine the inhibitory effect of luteolin against TNBC,and thus to provide an evidence supporting the efficacy of.natural drugs against TNBC via Hippo-YAP/TAZ signaling pathways.Methods:(1)Screening the potential inhibitors on regulating YAP/TAZ activity in TNBC:Using the MDA-MB-231 cell as a cellular model,we screened a natural compound library by the luciferase activity test,and discovered the potential inhibitors of YAP/TAZ.qRT-PCR assay was used to investigate the mRNA levels of CTGF and CYR61 which are the target genes of YAP and TAZ in luteolin-treated TNBC cells.(2)Identification of luteolin as an inhibitor by inhibiting proliferation of TNBC cells:MDA-MB-231,4T1,and MCF10A cells were treated with the concentrations of luteolin(0-80μM)for 24h,48h,and 72 h.SRB assay was performed to determine the cell viability.Colony formation assay:MDA-MB-231 and 4T1 cells were treated with the indicated concentration of luteolin for 14 days,and calculated the number of colonies in TNBC cells.(3)Molecular mechanism of luteolin on inhibiting proliferation of TNBC cells:We used immunofluorescence and confocal microscopy to assess the subcellular localization of YAP/TAZ in luteolin-treated TNBC cells.qRT-PCR assay and western blot were used to investigate the mRNA levels and protein levels of the core contents of Hippo-YAP/TAZ signaling pathways.We verified the sensitivities to luteolin by over-expressing and decreasing the expression of TAZ.Proteasome inhibitor MG132 were utilized to detect luteolin promoting the degradations of YAP/TAZ via the ubiquitin-proteasome pathway.(4)Molecular mechanism of Luteolin on suppressing epithelial-mesenchymal transition(EMT)and migration of TNBC cells:Wound healing assay and transwell migration assay were used to detect luteolin suppressing the migration of TNBC cells.We used RT-qPCR and western blot to test the mRNA levels and protein levels of EMT markers in luteolin-treated TNBC cells and MCF10A over-expressed TAZS89A cells.(5)Validation of luteolin inhibition on xenograft tumor growth via YAP/TAZ:We injected mouse TNBC 4T1 cells into the right front axilla of mice and randomly divided xenografted mice.Mice were treated in traperitoneally with the vehicle and luteolin.Body weights of 4T1 tumor-bearing mice and Tumor volumes were recorded.Tumors were embedded in paraffin and sectioned for IHC to stain with YAP/TAZ.Results(1)Luteolin regulated the Hippo-YAP/TAZ signaling pathway in TNBC cells:Luteolin was screened as a potential inhibitor of YAP/TAZ.qRT-qPCR assay showed that luteolin could effectively decrease the mRNA levels of CTGF and CYR61 in luteolin-treated TNBC cells.(2)Luteolin inhibited proliferation and colony formation of TNBC cells ability:We performed SRB assay to examine the cell viability.The results demonstrated that luteolin inhibited the growths of MDA-MB-231 and 4T1 cells in a dose-and-time dependent manner.MCF10A cells were less sensitive to the treatment of luteolin than the other two cells.Colony formation assay examined that luteolin could significantly decrease the number of colonies in TNBC cells.(3)YAP/TAZ mediated the inhibitory effect of luteolin on TNBC cell growth:Luteolin decreased YAP/TAZ transcriptional activity and nuclear localization in TNBC cells.Luteolin decreased the mRNA levels and protein levels of the core contents of Hippo-YAP/TAZ signaling pathways.TAZS89A-overexpressed MDA-MB-231 cells were more resistant to luteolin treatment.Knockdown of TAZ sensitized MDA-MB-231 cells to luteolin treatment.Luteolin induced proteasome-dependent degradation of YAP/TAZ in TNBC cells.(4)Luteolin Suppressed EMT and metastasis of TNBC cells:Luteolin suppressed cell migration in TNBC cells.Luteolin inhibited EMT in TNBC cells and TAZS 8 9A-overexpressed MCF10A cells by decreasing the expressions of mesenchymal markers and increasing the expressions of epithelial markers.(5)Luteolin inhibited xenograft-tumor growth in vivo by inhibiting YAP/TAZ activity:Luteolin significantly decreased the final tumor volume and weight of xenograft.Moreover,treatment of luteolin decreased the expressions of YAP/TAZ in xenograft tissues.Conclusion:Luteolin can inhibit the transcriptional activity of YAP/TAZ and suppress the abilities of proliferation,EMT and migration in TNBC cells.Luteolin can induce the phosphorylation of YAP/TAZ and promote their degradations via the ubiquitin-proteasome pathway.Luteolin can regulate the growth of xenograft tumors and decrease the activity of YAP/TAZ.This study revealed that luteolin suppresses proliferation and migration of triple-negative breast cancer cells by inhibiting YAP/TAZ activity,and provided an evidence for potential clinical application of luteolin to treat TNBC.