Hispidulin Inhibits Metastasis of Triple Negative Breast Cancer by Promoting MDM2-mediated Apelin Degradation

Background: and Purpose : This study aimed to examine hispidulin (HIS)’s inhibitory and anti-metastatic effects on triple-negative breast cancer (TNBC), determine its capacity to impede TNBC metastasis by inhibiting angiogenesis through regulation of the Apelin/APJ system, and clarify its mechanism of action in regulating Apelin. Experimental Approach : Using in vitro cell trials and an orthotopic TNBC mouse model, we evaluated HIS’s efficacy through cell counting kit-8, migration and invasion assays, hematoxylin-eosin staining, and immunofluorescence assay. The mechanism of action was investigated through integrated approaches, including network pharmacology, molecular docking, microscale thermophoresis (MST) assay, mouse serum enzyme-linked immunosorbent assay, tissue and cell Western blot, quantitative reverse transcription polymerase chain reaction, and immunofluorescence assay. Key Results : HIS exhibited a more potent inhibitory effect on MDA-MB-231 cell migration in scratch assays and inhibited tumor cell proliferation. In orthotopic TNBC mouse models, HIS effectively inhibited tumor proliferation and lung metastasis. Network pharmacology and molecular docking experiments were conducted to investigate the Apelin target protein, whereas the MST assay demonstrated that HIS exhibits specific binding capability with Apelin (affinity: 49.4 μM). HIS can reduce Apelin protein levels both in vivo and in vitro , and inhibit the expression of angiogenesis and metastasis-related proteins. Experiments confirmed that HIS promotes Apelin degradation through the ubiquitin-proteasome pathway, rather than inhibiting its transcription or altering the processing enzyme Furin. Subsequent experiments revealed that HIS promotes the ubiquitination and degradation of Apelin by activating the MDM2-mediated E3 ligase pathway, thereby inhibiting tubule formation and TNBC metastasis. Conclusion: and Implications : We initially discovered that HIS inhibits TNBC metastasis by downregulating the Apelin/APJ system and inhibiting angiogenesis. This mechanism involved MDM2-mediated E3 ligase activation, resulting in Apelin degradation via the ubiquitin-proteasome pathway.