Abstract P2-24-06: αEGFR-E-P125A ANTIBODY-ENDOSTATIN FUSION PROTEIN REDUCES VASCULOGENIC MIMICRY AND TUMOR CELL MOTILITY BY INHIBITION OF EGFR, INTEGRIN AND FAK SIGNALING
Authors: Sankar Ankita P. , Henn Hava Gil , Cho Hyun Mi , Nassar Dania , Ramakrishnan Sundaram , Das Rathin , Zhang Yu , Elledge Christian , Shin Seung-Uon , Rosenblatt Joseph 
Published: March 2023

Abstract
Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer with high metastatic potential and increased morbidity and mortality. Although expression of, and signaling by the epidermal growth factor receptor (EGFR) is commonly seen in TNBC, anti-EGFR antibodies such as Cetuximab have had limited therapeutic efficacy, used alone or in combination with chemotherapy. Primary TNBC tumor growth and metastases require supporting vasculature, which develops through a combination of endothelial angiogenesis and vasculogenic mimicry (VM). VM is more frequently seen in TNBC than other breast cancer subtypes, and is associated with aggressive metastatic behavior. We previously developed αEGFR-E-P125A, an antibody-endostatin fusion protein, linking an anti-EGFR antibody targeting domain to a mutated version of the anti-angiogenic protein endostatin (E-P125A). αEGFR-E-P125A delivers a dimeric E-P125A payload which inhibits TNBC angiogenesis and VM in vitro and in vivo, and markedly decreases metastasis in both the MDA-MB-231-4175 and MDA-MB-468 TNBC xenograft models. To determine the mechanism of αEGFR-E-P125A action on inhibition of VM, we studied the effects of αEGFR-E-P125A on both the transcriptome and proteome. RNA-seq was conducted on MDA-MB-231-4175 TNBC cells plated on matrigel and undergoing VM, and on TNBC cells plated in matrigel and treated with αEGFR-E-P125A. Gene set enrichment analysis demonstrated that αEGFR-E-P125A treatment downregulated genes on the KRAS, JAK-STAT, and angiogenesis signaling pathways, including EGF, VEGFA, STAT3, PTK2, ITGB1, ITGB3, ITGAV, and ITGA5. Phospho-array analysis was used to interrogate the proteome and demonstrated downregulation of phosphorylation on multiple sites downstream of the EGFR and the α5β1 integrin receptors, such as the EGFR Y1069 site, the FAK Y397 site, implicated in the regulation of endothelial and TNBC motility, and the STAT3 Y705 and S727 sites, known for their role in promoting the transcription of angiogenic genes. Since inhibition of EGFR signaling alone does not inhibit VM, we interrogated the mechanistic relationship between αEGFR-E-P125A and VM inhibition through the α5β1 integrin/FAK signaling pathway. Specific inhibition of FAK at the Y397 site using the small molecule inhibitor, PF-573228 inhibited TNBC VM in vitro. Transient siRNA knockdown of FAK in MDA-MB-231-4175 cells and shRNA-mediated knockdown of FAK in MDA-MB-231 TNBC cells confirmed that downregulation of FAK inhibited VM in vitro. To understand the effect of αEGFR-E-P125A on the EGFR and α5β1 integrin receptors, competition assays were conducted. αEGFR-E-P125A competed with EGF for the EGFR receptor and with the binding of fibronectin to α5β1 integrin. Treatment of TNBC cells with αEGFR-E-P125A reduced total α5 integrin protein levels and decreased co-localization of EGFR and α5β1 integrin receptors. These results indicate that αEGFR-E-P125A is bound to both EGFR and α5β1 integrin, simultaneously suppressing downstream EGFR and integrin signaling. Simultaneous inhibition of EGFR and α5β1integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases.
Citation Format: Ankita P. Sankar, Hava Gil Henn, Hyun Mi Cho, Dania Nassar, Sundaram Ramakrishnan, Rathin Das, Yu Zhang, Christian Elledge, Seung-Uon Shin, Joseph Rosenblatt. αEGFR-E-P125A ANTIBODY-ENDOSTATIN FUSION PROTEIN REDUCES VASCULOGENIC MIMICRY AND TUMOR CELL MOTILITY BY INHIBITION OF EGFR, INTEGRIN AND FAK SIGNALING [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-06.