Division of Cancer Biology and Genetics, Cancer Research Institute and Department of Pathology & Molecular Medicine, Queen's University, Kingston, ON, K7L 3N6, Canada.
Division of Cancer Biology and Genetics, Cancer Research Institute and Department of Pathology & Molecular Medicine, Queen's University, Kingston, ON, K7L 3N6, Canada.
Lung Cancer. 2020 Apr;142:123-131. doi: 10.1016/j.lungcan.2020.02.004. Epub 2020 Feb 22.
Increased expression of REarranged during Transfection (RET) kinase is reported in 10-20 % of lung adenocarcinomas (LUAD) and is associated with metastasis and reduced survival. Ezrin is a scaffold protein that promotes protein interactions with the actin cytoskeleton to regulate cell migration and is also associated with invasion and metastasis in cancers. RET isoforms interact with unique combinations of scaffold proteins to promote distinct signaling pathways. We hypothesized that RET isoforms associate distinctly with Ezrin for cytoskeletal reorganization and LUAD cell migration processes.
HCC1833 and A549 LUAD, SH-SY5Y neuroblastoma or HEK-293 cells expressing RET and Ezrin were stimulated with the RET ligand glial cell line-derived neurotrophic factor (GDNF) and treated with RET, Ezrin or Src inhibitors. Co-immunoprecipitation or pull-down assays coupled to immunoblotting were used to investigate protein activation and interactions. Immunofluorescence confocal microscopy assessed LUAD cytoskeletal reorganization and colocalization of RET and Ezrin. Live-cell fluorescence imaging was used to measure cell migration and chemotaxis.
GDNF promoted activation, interaction and colocalization of RET51 isoform and Ezrin. Inhibition of RET or Src impaired Ezrin interactions with RET and Src. GDNF stimulation enhanced the formation of actin-rich filopodia, in which both RET and Ezrin were enriched, and promoted chemotaxis in LUAD cells. However, inhibition of RET, Src or Ezrin suppressed filopodia formation, reduced colocalization of Ezrin with RET, and impaired cell migration and/ or chemotaxis. We further showed that GDNF-mediated activation of RET and Ezrin promoted RhoA-GTPase activity and signaling of ROCK1 and ROCK2 in LUAD cells.
Expression and activation of RET51 mediates unique protein interactions with Ezrin to promote LUAD cell chemotaxis for cancer cell dissemination, which may have implications in LUAD metastatic progression.
据报道，在 10-20%的肺腺癌（LUAD）中，REarranged during Transfection（RET）激酶的表达增加，与转移和生存降低有关。Ezrin 是一种支架蛋白，可促进与肌动蛋白细胞骨架的蛋白质相互作用，从而调节细胞迁移，并且还与癌症的侵袭和转移有关。RET 同工型与独特的支架蛋白组合相互作用，以促进不同的信号通路。我们假设 RET 同工型与 Ezrin 独特地结合，以进行细胞骨架重排和 LUAD 细胞迁移过程。
用 RET 配体胶质细胞源性神经营养因子（GDNF）刺激 HCC1833 和 A549 LUAD、SH-SY5Y 神经母细胞瘤或表达 RET 和 Ezrin 的 HEK-293 细胞，并使用 RET、Ezrin 或 Src 抑制剂进行处理。共免疫沉淀或下拉测定与免疫印迹结合使用，以研究蛋白质激活和相互作用。免疫荧光共聚焦显微镜评估 LUAD 细胞骨架重排和 RET 和 Ezrin 的共定位。使用活细胞荧光成像测量细胞迁移和趋化性。
GDNF 促进了 RET51 同工型和 Ezrin 的激活、相互作用和共定位。RET 或 Src 的抑制会损害 Ezrin 与 RET 和 Src 的相互作用。GDNF 刺激增强了富含 RET 和 Ezrin 的富含肌动蛋白的丝状伪足的形成，并促进了 LUAD 细胞的趋化性。然而，RET、Src 或 Ezrin 的抑制抑制了丝状伪足的形成，减少了 Ezrin 与 RET 的共定位，并损害了细胞迁移和/或趋化性。我们进一步表明，GDNF 介导的 RET 和 Ezrin 的激活促进了 RhoA-GTPase 活性以及 LUAD 细胞中 ROCK1 和 ROCK2 的信号转导。
RET51 的表达和激活介导了与 Ezrin 的独特蛋白质相互作用，以促进 LUAD 细胞趋化性，从而促进癌细胞扩散，这可能对 LUAD 转移进展具有重要意义。
