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  • br in vivo This is further supported by the VEGF


    in vivo. This is further supported by the VEGF mAb experiment and the expression of p-VEGFR2 in HUVECs cocultured with TNBC cells, suggesting that VEGF plays a key role in wogono-side’s reduction of angiogenesis in TNBC, consistent with network analysis (Figure 2A). We also tested wogonin (Data S1), a wogonoside analog, in TNBC cell lines. We found that wogonin cannot consistently inhibit VEGF secretion of MDA-MB-231 APTSTAT3-9R at 60 mM (Figures S4A and S4B) and had no effect on the migration of HUVECs cocultured by MDA-MB-468 CM compared with wogonoside (Figures S4C–S4F). In summary, we have identified wogonoside as an effective angiogenesis inhibitor for treating TNBC in vitro and in vivo.
    Emerging data suggest the involvement of Hedgehog signaling in tumor-associated angiogenesis: (1) increased blood vessel den-sity in breast cancer was observed upon Hedgehog signaling acti-vation (Harris et al., 2012); (2) Hedgehog promotes neovasculariza-tion by regulating Ang-1 in bone marrow-derived proangiogenic cells (Nakamura et al., 2010); and (3) canonical Hedgehog signaling augments tumor angiogenesis by induction of VEGF (Chen et al., 2011). tGli1, an alternative splicing form of Gli1, enhances the hVEGF gene promoter resulting in upregulation of VEGF in breast cancer cells (Cao et al., 2012; Carpenter and Lo, 2012). To explore the potential mechanism of wogonoside’s impact on VEGF and angiogenesis in TNBC further, we examined Hedgehog/Gli signaling by the Gli luciferase reporter assay and observed that micromolar wogonoside inhibits the transcriptional activity. In addition, Gli1 nuclear translocation was decreased by wogonoside in a concentration-dependent manner. Importantly, we demon-strated that wogonoside directly inhibited SMO (a key upstream activator involving in the nuclear translocation and transcriptional activity of Gli1 [Cannonier and Sterling, 2015]) protein expression by promoting its proteasome degradation, which contributes to its pharmacologic inhibition of TNBC growth in vitro and in vivo.
    Ubiquitination of SMO regulates its trafficking and cell-surface expression (Li et al., 2012). We investigated whether Cul4-DDB1 is involved in human SMO degradation triggered by wogonoside. We observed that siRNA-mediated downregulation of Cul4A effectively impairs the wogonoside-induced SMO ubiquitination in MDA-MB-231 cells, indicating that Cullin4 is potential E3 ligases for human SMO in wogonoside-triggered SMO ubiquiti-nation. In the light of known mechanism-of-action of existing small-molecule inhibitors on the Hedgehog signaling pathway (Hui et al., 2013; Wang et al., 2013), we tested the direct interac-tion of wogonoside with SMO or Gli1. Via SMO binding and the
    Figure 5. A Physical Interaction between Wogonoside and SMO
    (A) The relative expression of mRNA of Cul4A in MDA-MB-231 cells following specific siRNA treatment.
    (B) SMO ubiquitination was determined by protein immunoprecipitation assay. Cul4A was silenced in MDA-MB-231 cells by a specific siRNA. MDA-MB-231 cells were then treated with wogonoside (100 mM) for 24 h and SMO protein was immunoprecipitated and detected by ubiquitin antibody using western blotting. Relative expression of SMO ubiquitin (% control) was shown in right. The comparisons were made relative to wogonoside treated group with siRNA control and the significance of the difference is indicated as *p < 0.05 and **p < 0.01.
    (C) The binding mode of wogonoside with SMO by molecular docking simulation (see the STAR Methods).
    (E) A physical interaction of wogonoside and SMO was detected by BODIPY-cyclopamine by SMO binding assay. The quantitative data of BODIPY-cyclopamine bingeing was shown on the right.
    (F) MDA-MB-231 cells were treated with DMSO or wogonoside (100 mM) for 12 h and then subjected to cellular thermal shift assay (CETSA). CETSA shows that wogonoside has no effect on stabilizing Gli protein in MDA-MB-231 cells.