American Association for Cancer Research
crc-23-0278_fig3.png (1.09 MB)

FIGURE 3 from Antibody–Drug Conjugate αEGFR-E-P125A Reduces Triple-negative Breast Cancer Vasculogenic Mimicry, Motility, and Metastasis through Inhibition of EGFR, Integrin, and FAK/STAT3 Signaling

Download (1.09 MB)
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

αEGFR-E-P125A inhibits EGFR, FAK, and STAT3 phosphorylation. A, Phospho-antibody array depicts changes in phosphorylation from MDA-MB-231-4175 cells plated on matrigel and undergoing the tube formation process to those plated on matrigel and treated with αEGFR-E-P125A for 16 hours in which tube formation was inhibited (left). Zoomed panel depicts decreased EGFR Y1069, FAK Y397, and STAT3 Y705 phosphorylation upon αEGFR-E-P125A treatment (right). B, Bubble plot depicts top downregulated phospho-sites as seen in the phospho-antibody array. Downregulated phospho-sites of interest which correlate to transcriptomic findings include EGFR Y1069, FAK Y397, and STAT3 Y705 sites. C–E, Quantification of EGFR Y1069, FAK Y397, and STAT3 Y705 downregulation from phospho-array. F–H, Western blots demonstrating downregulation of EGFR Y1069, FAK Y397, and STAT3 Y705 upon αEGFR-E-P125A treatment compared with controls. I–K, Immunofluorescence staining at 20x magnification demonstrating inhibition of tube formation and downregulation of EGFR Y1069, FAK Y397, and STAT3 Y705 upon αEGFR-E-P125A treatment. Staining was quantified by percent fluorescent area. **, P < 0.01; ***, P < 0.001.


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)



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.