Supplementary Fig. S1: Tissue microarray VN:IGFBP-3 PLA immunoreactivity and patient information; Supplementary Fig. S2: VN peptide array amino acid sequence information; Supplementary Fig. S3: IGFBP-3 peptide array amino acid sequence information; Supplementary Fig. S4: VN and IGFBP-3 immunoreactivity of HMLER-FOXC2 primary tumour xenografts; Supplementary Fig. S5: Single channel images of in situ PLA; Supplementary Fig. S6: IGF-II-induced cell signalling in MCF-7 breast cancer cells; Supplementary Fig. S7: Breast cell migration measured using the xCELLigence assay; Supplementary Fig. S8: Optimisation of ligand binding to VN and its subsequent immunodetection; Supplementary Fig. S9: Identification of ligand binding sites on VN using information from VN peptide arrays; Supplementary Fig. S10: Binding sites on IGFBP-3 for VN using the IGFBP-3 peptide arrays
ARTICLE ABSTRACT
We provide proof-of-concept evidence for a new class of therapeutics that target growth factor:extracellular matrix (GF:ECM) interactions for the management of breast cancer. Insulin-like growth factor-I (IGF-I) forms multiprotein complexes with IGF-binding proteins (IGFBP) and the ECM protein vitronectin (VN), and stimulates the survival, migration and invasion of breast cancer cells. For the first time we provide physical evidence for IGFBP-3:VN interactions in breast cancer patient tissues; these interactions were predominantly localized to tumor cell clusters and in stroma surrounding tumor cells. We show that disruption of IGF-I:IGFBP:VN complexes with L27-IGF-II inhibits IGF-I:IGFBP:VN-stimulated breast cancer cell migration and proliferation in two- and three-dimensional assay systems. Peptide arrays screened to identify regions critical for the IGFBP-3/-5:VN and IGF-II:VN interactions demonstrated IGFBP-3/-5 and IGF-II binds VN through the hemopexin-2 domain, and VN binds IGFBP-3 at residues not involved in the binding of IGF-I to IGFBP-3. IGFBP-interacting VN peptides identified from these peptide arrays disrupted the IGF-I:IGFBP:VN complex, impeded the growth of primary tumor-like spheroids and, more importantly, inhibited the invasion of metastatic breast cancer cells in 3D assay systems. These studies provide first-in-field evidence for the utility of small peptides in antagonizing GF:ECM-mediated biologic functions and present data demonstrating the potential of these peptide antagonists as novel therapeutics. Mol Cancer Ther; 15(7); 1602–13. ©2016 AACR.
