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

Yukio Nishimura, Soichi Takiguchi, Shigeru Ito, Kazuyuki Itoh

研究成果: ジャーナルへの寄稿記事

8 引用 (Scopus)

抄録

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.

元の言語英語
ページ(範囲)412-426
ページ数15
ジャーナルInternational journal of oncology
44
発行部数2
DOI
出版物ステータス出版済み - 2 1 2014

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Sorting Nexins
Endocytosis
Endosomes
Small Interfering RNA
Lung Neoplasms
Phosphorylation
Cell Line
Down-Regulation
Proto-Oncogene Proteins c-met
gefitinib
Transferrin
Lysosomes
Fluorescent Antibody Technique
Cytoplasm
Western Blotting
Cell Membrane
Staining and Labeling
Ligands

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

これを引用

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. / Nishimura, Yukio; Takiguchi, Soichi; Ito, Shigeru; Itoh, Kazuyuki.

:: International journal of oncology, 巻 44, 番号 2, 01.02.2014, p. 412-426.

研究成果: ジャーナルへの寄稿記事

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title = "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",
abstract = "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.",
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T1 - 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

AU - Nishimura, Yukio

AU - Takiguchi, Soichi

AU - Ito, Shigeru

AU - Itoh, Kazuyuki

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N2 - 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.

AB - 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.

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