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posted on 2024-03-11, 14:20 authored by Ankita P. Sankar, Hyun-Mi Cho, Seung-Uon Shin, Tal Sneh, Sundaram Ramakrishnan, Christian Elledge, Yu Zhang, Rathin Das, Hava Gil-Henn, Joseph D. Rosenblatt Inhibition of EGFR signaling is not critical for inhibition of VM by αEGFR-E-P125A. A, αEGFR-E-P125A treatment inhibits VM tube formation in a dose-dependent manner compared with cetuximab and E-P125A controls. B, Quantification of meshes by Image J Angiogenesis Analyzer plugin demonstrates that compared with controls, αEGFR-E-P125A treatment significantly reduced mesh (tube) formation in MDA-MB-231-4175 cell plated on matrigel. C, Increasing doses of cetuximab treatment does not inhibit VM tube formation. D, Western blot analysis demonstrating reduction of EGFR Y1069 phosphorylation upon increasing doses of cetuximab treatment. E, Quantification of meshes by Image J Angiogenesis Analyzer plugin demonstrates that increasing doses of cetuximab do not inhibit VM tube formation. F, siRNA knockdown of EGFR was conducted on MDA-MB-231-4175 cells and a VM tube formation assay was conducted to determine whether knockdown of EGFR inhibited VM tube formation. G, Quantification of meshes demonstrates that number of meshes remained the same upon EGFR knockdown. H, Western blot analysis validating efficiency of EGFR siRNA knockdown. I, Increasing doses of E-P125A results in the formation of fewer VM tubes. J, Quantification of meshes demonstrates that increasing concentrations of E-P125A reduces number of complete meshes. K, Western blot analysis demonstrating reduction of FAK Y397 phosphorylation upon increasing concentrations of E-P125A treatment. VM tube formation assays were imaged at 5x magnification. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Funding
DOD | Department of Defense Education Activity (DoDEA)
Bankhead-Coley Foundation
Small Business Innovation Research (SBIR)
Israel Cancer Research Fund (ICRF)
Israel Cancer Association (ICA)
Israel Science Foundation (ISF)
History
ARTICLE ABSTRACT
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 αEGFR-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 sequencing (RNA-seq) of MDA-MB-231-4175 TNBC cells grown in a monolayer (two-dimensional) was compared with cells plated on Matrigel undergoing VM [three-dimensional (3D)]. We then compared RNA-seq between TNBC cells in 3D and cells in 3D with VM inhibited by αEGFR-E-P125A (EGFR-E-P125A). Gene set enrichment analysis demonstrated that VM induction activated the IL6-JAK-STAT3 and angiogenesis pathways, which were downregulated by αEGFR-E-P125A treatment.Correlative analysis of the phosphoproteome demonstrated decreased EGFR phosphorylation at Y1069, along with decreased phosphorylation of focal adhesion kinase Y397 and STAT3 Y705 sites downstream of α5β1 integrin. Suppression of phosphorylation events downstream of EGFR and α5β1 integrin demonstrated that αEGFR-E-P125A interferes with ligand-receptor activation, inhibits VM, and overcomes oncogenic signaling associated with EGFR and α5β1 integrin cross-talk.In vivo, αEGFR-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 α5β1 integrin signaling by αEGFR-E-P125A is a promising strategy for the inhibition of VM, tumor growth, motility, and metastasis in TNBC and other EGFR-overexpressing tumors.
αEGFR-E-P125A reduces VM, angiogenesis, tumor growth, and metastasis by inhibiting EGFR and α5β1 integrin signaling, and is a promising therapeutic agent for TNBC treatment, used alone or in combination with chemotherapy.
