Objectives

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.

Methods

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.

Results

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.

Conclusions

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.

中文翻译：

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与支架蛋白Ezrin的RET亚型特异性相互作用促进了肺腺癌中的细胞迁移和趋化性。

目标

据报道，在10-20％的肺腺癌（LUAD）中，转染过程中（RE）激酶重新排列的表达增加，并且与转移和生存率降低有关。Ezrin是一种支架蛋白，可促进蛋白与肌动蛋白细胞骨架的相互作用，从而调节细胞迁移，并且还与癌症的侵袭和转移有关。RET同工型与支架蛋白的独特组合相互作用，以促进独特的信号通路。我们假设RET亚型与Ezrin明显相关，用于细胞骨架重组和LUAD细胞迁移过程。

方法

用RET配体神经胶质细胞系衍生的神经营养因子（GDNF）刺激表达RET和Ezrin的HCC1833和A549 LUAD，SH-SY5Y神经母细胞瘤或HEK-293细胞，并用RET，Ezrin或Src抑制剂处理。使用共免疫沉淀法或下拉法结合免疫印迹法研究蛋白质的活化和相互作用。免疫荧光共聚焦显微镜评估了RET和Ezrin的LUAD细胞骨架重组和共定位。活细胞荧光成像用于测量细胞迁移和趋化性。

结果

GDNF促进RET51亚型和Ezrin的激活，相互作用和共定位。抑制RET或Src会削弱Ezrin与RET和Src的相互作用。GDNF刺激增强了富含肌动蛋白的丝状伪足的形成，其中RET和Ezrin均富集，并促进了LUAD细胞的趋化性。但是，抑制RET，Src或Ezrin抑制丝状伪足的形成，减少Ezrin与RET的共定位，并损害细胞迁移和/或趋化性。我们进一步表明，GDNF介导的RET和Ezrin激活促进LUAD细胞中RhoA-GTPase活性和ROCK1和ROCK2的信号传导。

结论

RET51的表达和激活介导与Ezrin的独特蛋白质相互作用，以促进LUAD细胞趋化性促进癌细胞的扩散，这可能对LUAD转移进程有影响。