RESUMEN
The EphB4-ephrinB2 signaling axis has been heavily implicated in metastasis across numerous cancer types. Our emerging understanding of the dichotomous roles that EphB4 and ephrinB2 play in head and neck squamous cell carcinoma (HNSCC) poses a significant challenge to rational drug design. We find that EphB4 knockdown in cancer cells enhances metastasis in preclinical HNSCC models by augmenting immunosuppressive cells like T regulatory cells (Tregs) within the tumor microenvironment. EphB4 inhibition in cancer cells also amplifies their ability to metastasize through increased expression of genes associated with epithelial mesenchymal transition and hallmark pathways of metastasis. In contrast, vascular ephrinB2 knockout coupled with radiation therapy (RT) enhances anti-tumor immunity, reduces Treg accumulation into the tumor, and decreases metastasis. Notably, targeting the EphB4-ephrinB2 signaling axis with the engineered EphB4 ligands EFNB2-Fc-His and Fc-TNYL-RAW-GS reduces local tumor growth and distant metastasis in a preclinical model of HNSCC. Our data suggest that targeted inhibition of vascular ephrinB2 while avoiding inhibition of EphB4 in cancer cells could be a promising strategy to mitigate HNSCC metastasis.
RESUMEN
The EphA2 receptor tyrosine kinase activates signaling pathways with different, and sometimes opposite, effects in cancer and other pathologies. Thus, highly specific and potent biased ligands that differentially control EphA2 signaling responses could be therapeutically valuable. Here, we use EphA2-specific monomeric peptides to engineer dimeric ligands with three different geometric configurations to combine a potential ability to differentially modulate EphA2 signaling responses with the high potency and prolonged receptor residence time characteristic of dimeric ligands. The different dimeric peptides readily induce EphA2 clustering, autophosphorylation and signaling, the best with sub-nanomolar potency. Yet, there are differences in two EphA2 signaling responses induced by peptides with different configurations, which exhibit distinct potency and efficacy. The peptides bias signaling when compared with the ephrinA1-Fc ligand and do so via different mechanisms. These findings provide insights into Eph receptor signaling, and proof-of-principle that different Eph signaling responses can be distinctly modulated.
RESUMEN
Eph receptor tyrosine kinases play a key role in cell-cell communication. Lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered understanding of their signaling mechanisms. Here, we use integrative structural biology to investigate the structure and dynamics of the EphA2 intracellular region. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling involving serine/threonine phosphorylation of the linker connecting its kinase and SAM domains. We show that accumulation of multiple linker negative charges, mimicking phosphorylation, induces cooperative changes in the EphA2 intracellular region from more closed to more extended conformations and perturbs the EphA2 juxtamembrane segment and kinase domain. In cells, linker negative charges promote EphA2 oligomerization. We also identify multiple kinases catalyzing linker phosphorylation. Our findings suggest multiple effects of linker phosphorylation on EphA2 signaling and imply that coordination of different kinases is necessary to promote EphA2 non-canonical signaling.
Asunto(s)
Receptor EphA2/química , Serina/química , Motivo alfa Estéril/genética , Treonina/química , Células A549 , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Expresión Génica , Células HEK293 , Humanos , Modelos Moleculares , Imitación Molecular , Células PC-3 , Fosforilación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Receptor EphA2/genética , Receptor EphA2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina/genética , Serina/metabolismo , Electricidad Estática , Especificidad por Sustrato , Treonina/genética , Treonina/metabolismoRESUMEN
Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes via distinct mechanotransduction pathways. In breast cancer, increased ECM stiffness promotes epithelial-to-mesenchymal transition (EMT), cell invasion, and metastasis. Here, we identify a mechanosensitive EPHA2/LYN protein complex regulating EMT and metastasis in response to increasing ECM stiffness during tumor progression. High ECM stiffness leads to ligand-independent phosphorylation of ephrin receptor EPHA2, which recruits and activates the LYN kinase. LYN phosphorylates the EMT transcription factor TWIST1 to release TWIST1 from its cytoplasmic anchor G3BP2 to enter the nucleus, thus triggering EMT and invasion. Genetic and pharmacological inhibition of this pathway prevents breast tumor invasion and metastasis in vivo. In human breast cancer samples, activation of this pathway correlates with collagen fiber alignment, a marker of increasing ECM stiffness. Our findings reveal an EPHA2/LYN/TWIST1 mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis.