三阴性乳腺癌(TNBC)的原发性肿瘤生长和转移需要支持的脉管系统,通过内皮血管生成和血管生成拟态(VM)的组合发展,与侵袭性转移行为相关的过程,其中血管样结构被肿瘤细胞排列。我们开发了αEGFR-E-P125A,提供二聚体的抗体-内皮抑素融合蛋白,在体外和体内抑制TNBC血管生成和VM的突变内皮抑素(E-P125A)有效负载。为了表征与VM的诱导和抑制相关的机制,将在单层(2D)中生长的MDA-MB-231-4175TNBC细胞的RNA-seq与接种在经受VM(3D)的Matrigel上的细胞进行比较。然后,我们比较了3D中的TNBC细胞和具有被αEGFR-E-P125A(EGFR-E-P125A)抑制的VM的3D中的细胞之间的RNA-seq。基因组富集分析(GSEA)表明VM诱导激活了IL6-JAK-STAT3和血管生成途径,通过αEGFR-E-P125A治疗下调。磷酸化蛋白质组的相关分析表明,Y1069的EGFR磷酸化减少,α5β1整联蛋白下游的粘着斑激酶(FAK)Y397和STAT3Y705位点的磷酸化减少。EGFR和α5β1整联蛋白下游磷酸化事件的抑制表明αEGFR-E-P125A干扰配体受体激活,抑制VM,并克服了与EGFR和α5β1整合素串扰相关的致癌信号传导。在体内,αEGFR-E-P125A治疗降低原发性肿瘤生长和VM,减少肺转移,并证实了体外观察到的信号事件的抑制。通过αEGFR-E-P125A同时抑制EGFR和α5β1整合素信号是抑制VM的有希望的策略,肿瘤生长,运动性,以及在TNBC和其他EGFR过表达肿瘤中的转移。
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.
UNASSIGNED: 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.