Effects of osteopontin-activated Notch signaling pathway on stemness and angiogenic capacity in triple-negative breast cancer
GUO Man
WANG Ruoyan
WANG Cao
NIE Mandi
NIU Shurun
LI Weihan
ZHANG Hao
Abstract：Objective To investigate the regulatory role of osteopontin(OPN)on stemness and angiogenic capacity in triple-negative breast cancer(TNBC)and the involvement of the Notch signaling pathway.Methods Based on in vitro cultures of human normal mammary epithelial cells(MCF-10A),luminal breast cancer cells(MCF-7),and TNBC cells(MDA-MB-436),an OPN-overexpressing(OPN-OE)vector was transfected into MDA-MB-436 cells to establish a stable OPN-overexpressing cell line(OPN-OE group).Control groups included untreated cells(Control group)and empty vector-transfected cells(Vector group).To investigate the role of the Notch signaling pathway,an additional OPN-OE+DAPT group was set up,in which cells were pretreated with the γ-secretase inhibitor DAPT prior to transfection.Subsequently,stemness characteristics were assessed using a tumor sphere formation assay,and pro-angiogenic capacity was evaluated through a tube formation assay with human umbilical vein endothelial cells(HUVECs).Meanwhile,the expression levels of stemness markers,including cluster of differentiation(CD)44,CD24,and aldehyde dehydrogenase 1,as well as vascular endothelial markers such as von Willebrand factor and CD31,were determined by Western blot analysis.Results MDA-MB-436 cells exhibited the highest tumor sphere-forming capacity and expression levels of stemness markers.Their conditioned medium also induced the highest tube formation in HUVECs and expression of vascular endothelial markers.Moreover,the protein levels of OPN and Notch1 in these cells were significantly higher than those in MCF-7 and MCF-10A cells(P<0.05).Compared with the Control and Vector groups,the OPN-OE group showed enhanced tumor sphere formation,upregulation of stemness markers,and significantly increased pro-angiogenic capacity along with related marker expression(P<0.05).However,these OPN overexpression-enhanced stemness properties and pro-angiogenic effects were significantly inhibited by treatment with the Notch pathway inhibitor DAPT(P<0.05).Conclusion OPN enhances stemness and pro-angiogenic capacity in TNBC cells by activating the Notch signaling pathway.Inhibition of the Notch pathway reverses these effects,suggesting that the OPN-Notch axis may represent a potential therapeutic target for TNBC.
Keywords：Triple-negative breast cancerOsteopontinStemnessAngiogenesisNotch signaling pathway
Publication Date：2025-10-28
Online Publishing Date：2025-12-03(First online date of this platform, not the publication date of the document)
Pages：6( 944-949 )