Antibody-drug conjugate aEGFR-E-P125A reduces triple-negative breast cancer vasculogenic mimicry, motility, and metastasis through inhibition of EGFR, integrin, and FAK/STAT3 signaling
Primary tumor growth and metastasis in triple-negative breast cancer (TNBC) require supporting vasculature, which develop through a combination of endothelial angiogenesis and vasculogenic mimicry (VM), a process associated with aggressive metastatic behavior in which vascular-like structures are lined by tumor cells. We developed aEGFR-E-P125A, an antibody-endostatin fusion protein that delivers a dimeric, mutant endostatin (E-P125A) payload that inhibits TNBC angiogenesis and VM in vitro and in vivo. To characterize the mechanisms associated with induction and inhibition of VM, RNA-seq of MDA-MB-231-4175 TNBC cells grown in a monolayer (2D) was compared to cells plated on Matrigel undergoing VM (3D). We then compared RNA-seq between TNBC cells in 3D and cells in 3D with VM inhibited by aEGFR-E-P125A (aEGFR-E-P125A). Gene set enrichment analysis (GSEA) demonstrated that VM induction activated the IL6-JAK-STAT3 and angiogenesis pathways, which were downregulated by aEGFR-E-P125A treatment. Correlative analysis of the phospho-proteome demonstrated decreased EGFR phosphorylation at Y1069, along with decreased phosphorylation of focal adhesion kinase (FAK) Y397 and STAT3 Y705 sites downstream of a5ß1 integrin. Suppression of phosphorylation events downstream of EGFR and a5ß1 integrin demonstrated that aEGFR-E-P125A interferes with ligand-receptor activation, inhibits VM, and overcomes oncogenic signaling associated with EGFR and a5ß1 integrin crosstalk. In vivo, aEGFR-E-P125A treatment decreased primary tumor growth and VM, reduced lung metastasis, and confirmed the inhibition of signaling events observed in vitro. Simultaneous inhibition of EGFR and a5ß1 integrin signaling by aEGFR-E-P125A is a promising strategy for the inhibition of VM, tumor growth, motility, and metastasis in TNBC and other EGFR-overexpressing tumors. Overall design: To investigate the mechanisms associated with the induction of VM, RNA-seq of MDA-MB-231-4175 TNBC cells grown in a monolayer (2D) was compared to cells plated on Matrigel and undergoing VM (3D). To investigate the mechanisms associated with the inhibition of VM by aEGFR-E-P125A, RNA-seq of TNBC cells in 3D was compared to cells in 3D with VM inhibited by aEGFR-E-P125A. We then performed differential gene expression analysis and gene set enrichment analysis using RNA-seq data to identify genes and pathways which were inversely dysregulated upon the induction of VM from 2D to 3D and the inhibition of VM from 3D to aEGFR-E-P125A treatment. RNA was isolated from 2D, 3D, and aEGFR-E-P125A-treatment conditions in four biological replicates per condition.