Syndecan-1 Promotes Angiogenesis in Triple-Negative Breast Cancer through the Prognostically Relevant Tissue Factor Pathway and Additional Angiogenic Routes

Abstract

Triple-negative breast cancer (TNBC) is characterized by increased angiogenesis, metastasis, and poor survival. Dysregulation of the cell surface heparan sulfate proteoglycan and signaling co-receptor Syndecan-1 is linked to poor prognosis. To study its role in angiogenesis, we silenced Syndecan-1 in TNBC cell lines using a 3D human umbilical vein endothelial cell (HUVEC) co-culture system. Syndecan-1 siRNA depletion in SUM-149, MDA-MB-468, and MDA-MB-231 cells decreased HUVEC tubule network formation. Angiogenesis array revealed reduced VEGF-A and tissue factor (TF) in the Syndecan-1-silenced secretome. qPCR independently confirmed altered expression of F3, F7, F2R/PAR1, F2RL1/PAR2, VEGF-A, EDN1, IGFBP1, and IGFBP2 in SUM-149, MDA-MB-231, and MDA-MB-468 cells. ELISA revealed reduced secreted endothelin-1 (SUM-149, MDA-MB-468) and TF (all cell lines) upon Syndecan-1 depletion, while TF pathway inhibitor treatment impaired angiogenesis. Survival analysis of 3951 patients demonstrated that high expression of F3 and F7 are associated with better relapse-free survival, whereas poor survival was observed in TNBC and p53 mutant basal breast cancer (F3) and in ER-negative and HER2-positive breast cancer (F2R, F2RL1). STRING protein network analysis revealed associations of Syndecan-1 with VEGF-A and IGFBP1, further associated with the TF and ET-1 pathways. Our study suggests that TNBC Syndecan-1 regulates angiogenesis via the TF and additional angiogenic pathways and marks its constituents as novel prognostic markers and therapeutic targets.

Keywords: 3D co-culture; CD138; PAR1; PAR2; Syndecan-1; angiogenesis; endothelin-1; prognosis; tissue factor; triple-negative breast cancer.

Figure 1

Sdc-1 depletion in TNBC cells restrains angiogenesis network formation of HUVECs. (A) Sdc-1 knockdown was confirmed by qPCR in the triple-negative cell lines SUM-149, MDA-MB-231, and MDA-MB-468. (B–E) 3D co-culture models of HUVECs and the control siRNA and Sdc-1 siRNA transfected SUM-149, MDA-MB-468, and MDA-MB-231 cells. (B) Phase-contrast images for HUVECs were either grown in 3D alone (as negative or positive controls) or co-cultured with control and Sdc-1-suppressed SUM-149 and MDA-MB-468 cells for 24 h. (C) Confocal immunofluorescence microscopy shows tubule formation by only HUVECs (green fluorescent staining) and not by MDA-MB-231 cells (red fluorescent staining) in a co-culture 3D system. Notably, MDA-MB-231 cells formed tubules when cultured alone in 3D on Matrigel. (D) Quantitative analysis of HUVEC tubulogenesis, namely the total length of HUVEC tubules. (E) Quantitative analysis of nodes and meshes formed by HUVECs as analyzed by angiogenesis analyzer software. Panels (A,D,E): Data represent the mean ± SEM, n = 3. *** p < 0.001, ** p < 0.01, * p < 0.05, and # p = 0.09 as determined by Student’s t-test.

Figure 2

Sdc-1 expression affects the expression and secretion of angiogenesis-related factors. Profiling of angiogenesis-regulating molecules secreted by control and Sdc-1 siRNA transfected cells in a 3D co-culture model with HUVECs as analyzed by proteome profiler™ human angiogenesis antibody array. Media conditioned by the secretome of control or the indicated Sdc-1 siRNA-treated TNBC cells co-cultured in 3D with HUVECs were collected and subjected to profiling. Colored boxes indicate common and cell type-specific dysregulated factors. See Supplementary Figure S2 for densitometric quantification.

Figure 3

Impact of Sdc-1 on the gene expression of angiogenic factors and functional impact of tissue factor on angiogenesis. (A–C) Sdc-1 silencing differentially regulates expression of the TF signaling pathway and angiogenic factors in SUM-149, MDA-MB-231, and MDA-MB-468 cells as assessed by qPCR. (D) Sdc-1 knockdown decreases EDN1 secretion in SUM-149 and MDA-MB-468 cells. EDN1 secretion was quantified by ELISA in the cell culture supernatants of control and Sdc-1 knockdown SUM-149, MDA-MB-231, and MDA-MB-468 cells collected 48 h post-transfection. (E) Sdc-1 knockdown decreases coagulation factor III/TF (F3) secretion in TNBC cells. Coagulation factor III/TF secretion was quantified by ELISA in the cell culture supernatants of control and Sdc-1 knockdown SUM-149, MDA-MB-468, and MDA-MB-231 cells collected 48 h post-transfection. (F) Representative images of the in vitro 3D co-culture model of HUVECs with TNBC cells treated with 50 ng/mL tissue factor pathway inhibitor (TFPI). TFPI treatment blunts the ability of tubule formation of HUVECs in co-culture with TNBC cells. (G) Quantitative analysis of nodes and meshes formed by HUVECs in coculture with TNBC cells as analyzed by angiogenesis analyzer software. Data represent the mean ± SEM, n ≥ 3. *** p < 0.001, ** p < 0.01, and * p < 0.05 as determined by Student’s t-test.

Figure 4

The prognostic value of the expression of F3 in patients with breast cancer. Kaplan–Meier curves are plotted based on the expression of F3 in (A) all patients (n = 3951), (B) p53 mutated (n = 1149), (C) patients with the intrinsic molecular classification Basal and p53 mutated (n = 901), and (D) Triple-negative tumors of the basal subtype (n = 186). Curves were analyzed using the log-rank test. Log-rank p values and hazard ratios are shown. Log-rank p values and hazard ratios (HRs; 95% confidence interval in parentheses) are shown. The corresponding Affymetrix ID is 204363_at.

Figure 5

The prognostic value of the expression of F7 (factor VII), F2R (PAR1), and F2RL1 (PAR2) in patients with breast cancer. Kaplan–Meier curves are plotted based on expression of F7 in (A) all patients showing F7 expression (n = 1734), and (B) patients with luminal A classification (n = 841). Expression of F2R in (C) ER-classified patients (n = 801) and (D) HER2 + classified patients (n = 252). Expression of F2RL1 in (E) all patients (n = 3951), (F) ER- classified patients (n = 801), (G) HER2 + classified patients (n = 252), and (H) patients with tumors of the basal subtype (n = 618). Curves were analyzed using the log-rank test. Log-rank p-values and hazard ratios (HRs; 95% confidence interval in parentheses) are shown. The corresponding Affymetrix IDs are: F7: 204363_at; F2R: 203989_x_at; F2RL1: 213506_at.

Figure 6

The protein–protein interaction network of Sdc-1 with F3, F7, VEGF-A, EDN1, F2R, F2RL1, IGFBP1, and IGFBP2. (A) Interaction analysis of Sdc-1 with F3, F7, VEGF-A, EDN1, F2R, F2RL1, IGFBP1, and IGFBP2 was performed using the STRING database (http://string-db.org/) (accessed on 29 January 2021). The proteins of interest are highlighted in dark red boxes. (B) Gene ontology (GO) analysis of Sdc-1, EDN1, F3, F7, VEGF-A, F2R, and F2RL1. The most significant GO terms (p < 0.05) in the cellular component (orange), molecular functions (green), and biological processes (blue) branches are shown. (C) KEGG pathway analysis. All the selected significant values are presented in (−log10) transformation.