E3 泛素连接酶 NEDD4 介导肺癌细胞中 EGFR 的细胞迁移信号转导。
The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells.
机构信息
School of Medicine, Jiangsu University, Zhenjiang, Jiangsu, 212013, China.
出版信息
Mol Cancer. 2018 Feb 19;17(1):24. doi: 10.1186/s12943-018-0784-2.
BACKGROUND
EGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. However, how NEDD4 promotes the EGFR-dependent lung cancer cell migration is unknown. This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling.
METHODS
Lentiviral vector-loaded NEDD4 shRNA was used to deplete endogenous NEDD4 in lung cancer cell lines. Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound-healing and transwell assays. Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation. Co-expression of NEDD4 with EGFR or PTEN was determined by immunohistochemical (IHC) staining in 63 lung adenocarcinoma tissue samples. Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulation, AKT activation and lysosomal secretion were examined using the GST-Uba pulldown assay, immunoblotting, immunofluorescent staining and a human cathepsin B ELISA assay respectively. The specific cathepsin B inhibitor CA-074Me was used for assessing the role of cathepsin B in lung cancer cell migration.
RESULTS
Knockdown of NEDD4 significantly reduced EGF-stimulated cell migration in non-small cell lung carcinoma (NSCLC) cells. Co-immunoprecipitation assay found that NEDD4 is associated with EGFR complex upon EGF stimulation, and IHC staining indicates that NEDD4 is co-expressed with EGFR in lung adenocarcinoma tumor tissues, suggesting that NEDD4 might mediate lung cancer cell migration by interaction with the EGFR signaling complex. Interestingly, NEDD4 promotes the EGF-induced cathepsin B secretion, possibly through lysosomal exocytosis, as overexpression of the ligase-dead mutant of NEDD4 impedes lysosomal secretion, and knockdown of NEDD4 significantly reduced extracellular amount of cathepsin B induced by EGF. Consistent with the role of NEDD4, cathepsin B is pivotal for both basal and the EGF-stimulated lung cancer cell migration. Our studies propose a novel mechanism underlying the EGFR-promoted lung cancer cell migration that is mediated by NEDD4 through regulation of cathepsin B secretion.
CONCLUSION
NEDD4 mediates the EGFR lung cancer cell migration signaling through promoting lysosomal secretion of cathepsin B.
背景
EGFR 依赖性细胞迁移在肺癌进展中起着重要作用。我们之前的研究观察到，HECT E3 泛素连接酶 NEDD4 与肿瘤转移显著相关，并且是胃癌细胞中 EGFR 迁移和侵袭信号所必需的。然而，NEDD4 如何促进 EGFR 依赖性肺癌细胞迁移尚不清楚。本研究旨在阐明 NEDD4 介导 EGFR 肺癌细胞迁移信号的机制。
方法
使用负载慢病毒载体的 NEDD4 shRNA 耗尽肺癌细胞系中的内源性 NEDD4。通过划痕愈合和 Transwell 测定确定 NEDD4 敲低对 EGFR 依赖性或非依赖性肺癌细胞迁移的影响。通过共免疫沉淀测定 NEDD4 与激活的 EGFR 的结合。通过免疫组化（IHC）染色在 63 例肺腺癌组织样本中检测 NEDD4 与 EGFR 或 PTEN 的共表达。使用 GST-Uba 下拉测定、免疫印迹、免疫荧光染色和人组织蛋白酶 B ELISA 测定分别检测 NEDD4 异位表达或敲低对 PTEN 泛素化和下调、AKT 激活和溶酶体分泌的影响。使用特定的组织蛋白酶 B 抑制剂 CA-074Me 评估组织蛋白酶 B 在肺癌细胞迁移中的作用。
结果
NEDD4 敲低显著降低了非小细胞肺癌（NSCLC）细胞中 EGF 刺激的细胞迁移。共免疫沉淀实验发现，NEDD4 在 EGF 刺激后与 EGFR 复合物结合，IHC 染色表明 NEDD4 在肺腺癌肿瘤组织中与 EGFR 共表达，提示 NEDD4 可能通过与 EGFR 信号复合物相互作用来介导肺癌细胞迁移。有趣的是，NEDD4 促进了 EGF 诱导的组织蛋白酶 B 分泌，可能通过溶酶体胞吐作用实现，因为 ligase-dead 突变体 NEDD4 的过表达阻碍了溶酶体分泌，而 EGF 诱导的细胞外组织蛋白酶 B 量的减少显著降低。与 NEDD4 的作用一致，组织蛋白酶 B 对基础和 EGF 刺激的肺癌细胞迁移都是至关重要的。我们的研究提出了一个新的机制，即在 EGFR 促进的肺癌细胞迁移中，NEDD4 通过调节组织蛋白酶 B 的分泌来介导。
结论
NEDD4 通过促进溶酶体分泌组织蛋白酶 B 来介导 EGFR 肺癌细胞迁移信号。
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