Evidence that depletion of the sorting nexin 1 by siRNA promotes HGF-induced MET endocytosis and MET phosphorylation in a gefitinib-resistant human lung cancer cell line
Evidence that depletion of the sorting nexin 1 by siRNA promotes HGF-induced MET endocytosis and MET phosphorylation in a gefitinib-resistant human lung cancer cell line
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DOI：
10.3892/ijo.2013.2194
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发表时间：
2014-02-01
影响因子：
5.2
通讯作者：
Itoh, Kazuyuki
中科院分区：
文献类型：
--
作者：
Nishimura, Yukio;Takiguchi, Soichi;Itoh, Kazuyuki
已知受体酪氨酸激酶MET及其配体HGF在恶性肿瘤细胞中过表达，它们与肺癌细胞对吉非替尼的耐药有关。我们最近发现，在对吉非替尼耐药的非小细胞肺癌细胞系中，与EGFR相互作用的蛋白--分选连接素1(SNX1)对EGFR从早期到晚期的内体转运具有负性调节作用。为了研究SNX1在HGF刺激的MET内吞作用中的作用及其通过早期/晚期内吞途径下调的作用，我们检测了siRNA对NSCLC细胞SNX1表达的抑制作用。利用免疫荧光技术，我们发现siRNA沉默SNX1导致质膜相关MET在细胞内的分布发生了显著变化，由此产生的MET染色遍及细胞质，并与内吞的德克萨斯红标转铁蛋白在siRNA-SNX1细胞中很好地共定位。我们还发现，在siRNA-SNX1转基因细胞中，高效的MET磷酸化和快速的内吞递送将磷酸化的MET从早期的内体传递到晚期的内体。相反，siRNA对照的细胞显示从早期的内体到晚期的内体的磷酸化MET的内吞送效率低下。此外，即使在HGF刺激60分钟后，仍可观察到大量的磷酸化MET在晚期内体中积累，这表明磷酸化MET的降解是在晚期内体/溶酶体途径中进行的。Western印迹分析显示，siRNA耗尽SNX1后，细胞内磷酸化MET的表达在60min达到最大值，随后HGF刺激后MET的降解速度加快。综上所述，我们认为SNX1在调节HGF刺激的MET/磷酸化MET内吞和下调Gefitinib耐药NSCLC细胞的早期/晚期内吞途径中起抑制作用。
The receptor tyrosine kinase MET and its ligand HGF are known to be overexpressed in malignant tumor cells, and they have been implicated in gefitinib resistance in lung cancer cells. We recently found that sorting nexin 1 (SNX1), a protein that interacts with EGFR, exhibited negative regulation of EGFR trafficking out of early to late endosomes in gefitinib-resistant NSCLC cell lines. To investigate the role of SNX1 on HGF-stimulated MET endocytosis and its downregulation via the early/late endocytic pathway, we examined the effect of depletion of SNX1 expression by siRNA in NSCLC cells. Using immunofluorescence, we found that the silencing of SNX1 by siRNA caused a dramatic change in the intracellular distribution of plasma membrane-associated MET and that the resultant MET staining was spread throughout the cytoplasm, and it co-localized well with the endocytosed Texas red-labeled transferrin in the siRNA-SNX1-transfected cells. We also found efficient MET phosphorylation and rapid endocytic delivery of phosphorylated MET from early endosomes to late endosomes in the siRNA-SNX1-transfected cells. By contrast, the siRNA-control transfected cells showed inefficient endocytic delivery of phosphorylated MET from early endosomes to late endosomes. Furthermore, large amounts of phosphorylated MET that had accumulated in late endosomes were seen even after 60 min of HGF-stimulation in the presence of bafilomycin A1, indicating that degradation of phosphorylated MET proceeds in a late endosome/lysosome pathway. Western blot analysis revealed that depletion of SNX1 by siRNA induced a maximal and dramatic increase in phosphorylated MET at 60 min, followed by an accelerated degradation of phosphorylated MET after HGF stimulation in the cells. Taken together, we suggest that SNX1 plays a suppressive role in the regulation of HGF-stimulated MET/phosphorylated MET endocytosis and downregulation via the early/late endocytic pathway in the gefitinib-resistant NSCLC cells.