A Systems Pharmacology Approach Uncovers Wogonoside as an Angiogenesis Inhibitor of Triple-Negative Breast Cancer by Targeting Hedgehog Signaling

Abstract

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous disease that lacks clinically actionable genetic alterations that limit targeted therapies. Here we explore a systems pharmacology approach that integrates drug-target networks and large-scale genomic profiles of TNBC and identify wogonoside, one of the major active flavonoids, as a potent angiogenesis inhibitor. We validate that wogonoside attenuates cell migration, tube formation, and rat aorta microvessel outgrowth, and reduces formation of blood vessels in chicken chorioallantoic membrane and TNBC cell-induced Matrigel plugs. In addition, wogonoside inhibits growth and angiogenesis in TNBC cell xenograft models. This network-based approach predicts, and we empirically validate, wogonoside's antiangiogenic effects resulting from vascular endothelial growth factor secretion. Mechanistically, wogonoside inhibits Gli1 nuclear translocation and transcriptional activities associated with Hedgehog signaling, by promoting Smoothened degradation in a proteasome-dependent mechanism. This study offers a powerful, integrated, systems pharmacology-based strategy for oncological drug discovery and identifies wogonoside as a potential TNBC angiogenesis inhibitor.

Keywords: Smoothened; angiogenesis; hedgehog signaling; systems pharmacology; triple-negative breast cancer; wogonoside.

Figure 1.. A diagram illustrating systems pharmacology-based prediction of anti-tumor effects of wogonoside in triple-negative breast cancer (TNBC).

(A) An in silico model for predicting anti-TNBC indications for natural products by integrating the drug-target network and experimentally validated functional genes in TNBC. The performance of the in silico model was evaluated using Receiver Operating Characteristic (ROC) curve. The area under ROC curve (AUC) was shown. (B) The chemical structure clustering analysis for 148 natural products having the significantly predicted anti-TNBC indications (q<10⁻⁵). (C) Circos plot (Circo v0.69) representing the predicted anti-TNBC indications (q<10⁻⁵) for 24 steroids and 39 flavonoids. The predicted q values with corresponding Z-score are exhibited as connected lines (edges). Natural products with previously published experimental data in TNBC are highlighted in bold font. Wogonside was selected for experimental validation using subject matter expertise based on a combination of factors (see Data S1 for references). (D) Effect of wogonoside on MDA-MB-231 tumor growth in nude mice. (E) The tumor weight of control and wogonoside treatment group (n=7) was measured after 15 days treatment. (F) The tumor volume of control and wogonoside treatment group (n=7) was measured every three days. The comparisons were made relative to the control group and the significance of the difference is indicated as *P value < 0.05 and **P value < 0.01.

Figure 2.. Effects of wogonoside on VEGF secretion, protein and mRNA expression and transcriptional activity in MDA-MB-231 and MDA-MB-468 cell lines.

(A) Network analysis highlighting the inferred mechanism-of-action for wogonoside in TNBC. The potential molecular mechanisms of wogonoside against TNBC were investigated via integration of known drug targets and experimentally validated TNBC genes into tissue-specific co-expressed protein-protein interactome network (see Methods). Node size indicates the protein-coding gene expression level in breast comparing to other 31 tissues from GTEx database (Consortium, 2015). Larger size highlighting the high expression level in breast comparing to other tissues. Co-expression denotes the co-expressed gene pairs (p-value < 0.05, F-statistics) encoding protein-protein interactions in TNBC RNA sequencing data from The Cancer Genome Atlas database (Cancer Genome Atlas, 2012). (B) Effect of wogonoside on VEGF expression in xenograft model of MDA-MB-231 cells in nude mice (n=5) was detected by Western blot analysis. (C) The expression of VEGF in xenograft model was detected by immunochemistry using specific antibody. (D) The concentration of VEGF in MDA-MB-231 and MDA-MB-468 CM were measured by ELISA kits. (E) VEGF expression was detected by western blot analysis using specific antibodies. (F) The mRNA level of VEGF was investigated by RT-PCR. (G) VEGF transcriptional activity was tested by Dual-Luciferase reporter assay. The comparisons were made relative to the control group and the significance of the difference is indicated as *P value < 0.05 and **P value < 0.01. See also Figures S1 and S4.

Figure 3.. Effects of wogonoside on Hedgehog signaling pathway.

(A) Effect of wogonoside on Gli1 expression in xenograft tissue of MDA-MB-231 cells in nude mice was detected by immunochemistry. (B) Gli1 expression in nucleoplasm of MDA-MB-231 cell xenograft tissue was detected by Western blot analysis using specific antibodies and Lamin A was used as nuclear marker. (C) The mRNA level of Cyclin D2, HIP and GAS1 was detected by RT-PCR. (D) Western blot analysis of Gli1 expression in cytosolic and nuclear lysates. Lamin A and β-tublin were used as nuclear and cytoplasmic markers, respectively. (E) Gli1 nuclear tanslocation was analyzed by immunofluorescence confocal microscopy. The comparisons were made relative to the control group and the significance of the difference is indicated as *P value < 0.05 and **P value < 0.01. See also Figure S2.

Figure 4.. Effects of wogonoside on the expression of SMO.

(A) Effect of wogonoside on SMO expression in xenograft tissue of MDA-MB-231 cells in nude mice was detected by immunochemistry. (B) The total SMO expression in the whole lysate of MDA-MB-231 cell xenograft tissue was tested by Western blot. (C) The total Gli1 and SMO expression in MDA-MB-231 and MDA-MB-468 cell lines was tested by Western blot. (D) The expression of SMO in MDA-MB-231 and MDA-MB-468 cells treated with MG132 (10 μM) or NH₄Cl (25 mM) for different time points (0, 2, 6, 12 and 24 h) by Western blot. (E) The expression of SMO in cells treated with MG132 (0 or 10 μM) or NH₄Cl (0 or 25 mM) and wogonoside (0, 25, 50 and 100 μM) as indicated was investigated by western blot. (F) SMO ubiquitination was tested by protein immunoprecipitation assay. The comparisons were made relative to the control group and the significance of the difference is indicated as *P value < 0.05 and **P value < 0.01.

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 μM) 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. (C) The binding mode of wogonoside with SMO by molecular docking simulation (see Methods). (D) The aromatic ring branched chain of wogonoside stretched into the hydrophobic pocket consisted of Asp384, Ser385, Val386, Ser387, Gly388, Ile389, Cys390, Phe391, Val392, Gly393, Tyr394 and Arg477. (E) A physical interaction of wogonoside and SMO was detected by BODIPY-cyclopamine by SMO binding assay. The quantitative data of BODIPY-cyclopamine binging was shown in right of panel E. (F) MDA-MB-231 cells were treated with DMSO or wogonoside (100 μM) 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. The comparisons were made relative to the control group and the significance of the difference is indicated as **P value < 0.01 and ***P value < 0.001. See also Figure S2.

Figure 6.. Effects of wogonoside on TNBC cell-induced angiogenesis in vitro.

(A) The tube formation ability of HUVECs cultured by CM collected from MDA-MB-231 or MDA-MB-468 pretreated with wogonoside (0, 25, 50 and 100 μM) for 24 h and 1% FBS medium in control group as indicated was tested by the endothelial cell tube formation assay. (B) The rat aortic ring microvessel sprouting induced by CM collected from MDA-MB-231 or MDA-MB-468 pretreated with wogonoside (0, 25, 50 and 100 μM) for 24 h and 1% FBS M199 medium in the control group as indicated was tested by rat aortic ring assay. (C) Effect of wogonoside on the angiogenesis of chicken chorioallantoic membrane. Data are presented as mean±SD. The comparisons were made relative to MDA-MB-231 or MDA-MB-468 CM group and significance of difference is indicated as *P value < 0.05 and **P value < 0.01. See also Figure S3.

Figure 7.. Effects of wogonoside on angiogenesis in vivo.

Matrigel containing saline injection (control group) or MDA-MB-231 cells and MDA-MB-468 cells was injected subcutaneously to assess angiogenesis in vivo. (A) Macroscopic appearance of matrigel plugs isolated from each group of mice (n=5). (B) The whole-mount of CD31 staining was viewed by laser scanning confocal microscope. (C) The hemoglobin content in matrigel plugs was determined. (D) The expression of CD31 in MDA-MB-231 cells xenograft in nude mice was tested by immunochemistry. (E) Effect of VEGF neutralizing antibody (10 μg/ml) on the tube formation of HUVECs induced by CM from MDA-MB-231 or MDA-MB-468 cells was tested by endothelial cell tube formation assay. (F) Effect of VEGF neutralizing antibody (10 μg/ml) on the enhanced angiogenesis induced by MDA-MB-231 cells or MDA-MB-468 cells (1×10⁶ cells/CAM) was tested by chicken chorioallantoic membrane (CAM) assay. The comparisons were made relative to MDA-MB-231 or MDA-MB-468 cells group and the significance of the difference is indicated as *P value < 0.05 and **P value < 0.01.