FIGURE 4 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

Abstract

<p>αEGFR-E-P125A suppresses α5 integrin and EGFR activation, and consequently downregulates FAK/STAT3 signaling. <b>A,</b> Immunofluorescence staining at the 20x magnification demonstrates that expression of α5 integrin is disrupted upon treatment with αEGFR-E-P125A. Staining was quantified by percent fluorescent area. <b>B,</b> Western blot analysis of MDA-MB-231-4175 cells plated on matrigel and treatment with either controls or αEGFR-E-P125A. Western blot analysis demonstrates downregulation of α5 integrin upon αEGFR-E-P125A treatment compared with controls. <b>C,</b> IP of α5 integrin was conducted on samples treated with an IgG control, media, E-P125A, cetuximab, or αEGFR-E-P125A. IP demonstrated that cells treated with αEGFR-E-P125A had decreased levels of α5 integrin after pulldown, resulting in decreased levels of β1 integrin bound to α5 integrin compared with the controls. <b>D,</b> Schematic demonstrating αEGFR-E-P125A binding to EGFR via the α-EGFR antibody end and preventing EGF-induced EGFR phosphorylation at EGFR Y1069. <b>E,</b> EGFR competition experiment between αEGFR-E-P125A and EGF ligands. MDA-MB-231-4175 cells were either stimulated with EGF alone, treated with αEGFR-E-P125A, or pretreated with αEGFR-E-P125A and then stimulated with EGF. <b>F,</b> Reverse EGFR competition experiment between αEGFR-E-P125A and EGF ligands. MDA-MB-231-4175 cells were pretreated with either stimulated with EGF alone, treated with αEGFR-E-P125A, or prestimulated with EGF and then treated with αEGFR-E-P125A. <b>G,</b> Schematic demonstrating αEGFR-E-P125A binding to α5β1 integrin via the E-P125A end and preventing fibronectin-induced FAK Y397 phosphorylation. <b>H,</b> α5β1 integrin competition experiment between αEGFR-E-P125A and fibronectin. MDA-MB-231-4175 cells were either stimulated with fibronectin alone, treated with αEGFR-E-P125A, or pretreated with αEGFR-E-P125A and then stimulated with fibronectin. <b>I,</b> Reverse α5β1 integrin competition experiment between αEGFR-E-P125A and fibronectin. MDA-MB-231-4175 cells were either stimulated with fibronectin alone, treated with αEGFR-E-P125A, or prestimulated with fibronectin and then treated with αEGFR-E-P125A. <b>J,</b> Treatment of MDA-MB-231-4175 cells with increasing concentrations of FAK inhibitor, PF-573,228 demonstrated decrease in FAK Y397 and downstream STAT3 Y705 phosphorylation, and a subsequent increase in EGFR Y1069 phosphorylation. <b>K,</b> Treatment of MDA-MB-231-4175 cells with increasing concentrations of cetuximab demonstrated decrease in EGFR Y1069 and total EGFR levels, and a subsequent increase in FAK Y397 and STAT3 Y705 phosphorylation. <b>L,</b> Treatment of MDA-MB-231-4175 cells with either media, cetuximab, E-P125A, the untargeted FcE dimer, a combination of cetuximab and E-P125A, or αEGFR-E-P125A for 16 hours in 3D demonstrated enhanced inhibition of downstream EGFR and α5β1 integrin signaling upon treatment with αEGFR-E-P125A.</p>