Abstract 2413: αEGFR-E-P125A antibody-endostatin fusion protein reduces vasculogenic mimicry and metastasis by inhibiting FAK, integrin, and EGFR signaling
Authors: Sankar Ankita P. , Cho Hyun Mi , Gil-Henn Hava , Ramakrishnan Sundaram , Das Rathin , Elledge Christian , Zhang Yu , Shin Seung-Uon , Rosenblatt Joseph D. 
Published: June 2022

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 either 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). Our laboratory previously developed a novel targeted therapy, αEGFR-E-P125A, an antibody-endostatin fusion protein, by 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 decreases metastatic tumor growth (lung metastasis) in both the MDA-MB-231-4175 and MDA-MB 468 TNBC xenograft models. Preliminary studies of the mechanism of αEGFR-E-P125A action indicate that αEGFR-E-P125A downregulates VM through combined inhibition of EGFR and integrin signaling. Phospho-array analysis of αEGFR-E-P125A treated MDA-MB-231-4175 cells demonstrated downregulation of phosphorylation of EGFR at Y1069, a site of aberrant EGFR signaling which promotes TNBC motility. The phospho-array also demonstrated inhibition of focal adhesion kinase (FAK) phosphorylation at Y397. FAK signaling occurs downstream of β1 and β3 integrins, and the Y397 site is implicated in the regulation of endothelial and TNBC motility, VM, and metastatic behavior. We specifically inhibited FAK at the Y397 site using a small molecule inhibitor, PF-573228, and demonstrated that FAK inhibition at Y397 alone inhibited the TNBC VM in vitro. shRNA-mediated knockdown of FAK in MDA-MB-231 TNBC cells confirmed that downregulation of FAK inhibited VM formation in vitro. Immunoprecipitation (IP) of EGFR detected the presence of β1 integrin, and IP of β1 integrin detected EGFR in both MDA-MB-231-4175 and MDA-MB-468 TNBC cells treated with αEGFR-E-P125A. αEGFR-E-P125A treatment increased levels of bound β1 integrin following IP of EGFR relative to untreated or Cetuximab treated TNBC cells. These results indicate that αEGFR-E-P125A is binding to both EGFR and β1 integrin simultaneously, suppressing downstream EGFR and integrin signaling. Simultaneous inhibition of EGFR and β1 integrin signaling by αEGFR-E-P125A fusion is a promising approach to inhibition of TNBC growth and metastases.

Citation Format: Ankita P. Sankar, Hyun Mi Cho, Hava Gil-Henn, Sundaram Ramakrishnan, Rathin Das, Christian Elledge, Yu Zhang, Seung-Uon Shin, Joseph D. Rosenblatt. αEGFR-E-P125A antibody-endostatin fusion protein reduces vasculogenic mimicry and metastasis by inhibiting FAK, integrin, and EGFR signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2413.