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. 2008 May 21:7:42.
doi: 10.1186/1476-4598-7-42.

Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line

Yukio Nishimura  1 , Kiyoko Yoshioka, Biborka Bereczky, Kazuyuki Itoh
Affiliations

Evidence for efficient phosphorylation of EGFR and rapid endocytosis of phosphorylated EGFR via the early/late endocytic pathway in a gefitinib-sensitive non-small cell lung cancer cell line

Yukio Nishimura et al. Mol Cancer. .

Abstract

Gefitinib (Iressa)-a specific inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase-has been shown to suppress the activation of EGFR signaling required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We recently provided novel evidence that gefitinib-sensitive PC9 cells show normal endocytosis of EGFR: internalized EGF-EGFR complexes were transported to late endosomes/lysosomes 15 min after EGF stimulation, and then degraded within the lysosomes. However, gefitinib-resistant QG56 cells showed internalized EGFR accumulation in early endosomes after 60 min of internalization, instead of its trafficking to lysosomes, indicating an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes. Therefore, we postulate that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines. To further substantiate the detailed internalization mechanism of gefitinib-sensitive and gefitinib-resistant cells, using confocal immunofluorescence microscopy, we examined the endocytic trafficking of phosphorylated EGFR (pEGFR) in the absence or presence of gefitinib. In PC9 and QG56 cells without EGF stimulation, a large number of pEGFR-positive small vesicular structures not colocalized with late endosomes/lysosomes were spread throughout the cytoplasm, and some pEGFR staining was distributed in the nucleus. This implies a novel intracellular trafficking pathway for pEGFR from cytoplasmic vesicles to the nucleus. Furthermore, an aggregated vesicular structure of early endosomes was observed in the perinuclear region of QG56 cells; it was revealed to be associated with SNX1, originally identified as a protein that interacts with EGFR. Therefore, we confirmed our previous data that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to late endosomes/lysosomes occurs in QG56 cells. Furthermore, in PC9 cells, efficient phosphorylation of EGFR and rapid internalization of pEGFR was observed at 3 min after EGF stimulation; these internalized pEGFR-positive vesicles were trafficked to late endosomes at 15 min, indicating rapid trafficking of EGF-pEGFR complexes from early to late endosomes in PC9 cells. Gefitinib treatment strongly reduced the phosphorylation level of EGFR, and subsequent endocytosis of EGFR was significantly suppressed in PC9 cells. In contrast, in QG56 cells, EGFR trafficking via the early endocytic pathway was basically impaired; therefore, gefitinib appeared to slightly suppress the internalization of pEGFR. Collectively, our data provide novel evidence that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib-resistance in QG56 cells.

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Figures

Figure 1
Figure 1
Intracellular distribution of pEGFR in the NSCLC cell lines. The gefitinib-sensitive NSCLC cell line, PC9 (A), or the gefitinib-resistant cell line, QG56 (B) was fixed and double-stained for LIMPII (red in b, h) or cathepsin D (red in e, k) and pEGFR (green) as described in the Materials section. Superimposed images of cathepsin D or LIMPII and pEGFR are shown in c, f, i, l. In both PC9 and QG56 cells, it is notable that pEGFR-positive small punctate vesicles are spreading in the cytoplasm and these vesicles are not colocalized with the LIMPII-, or cathepsin D-positive vesicular structures, and also pEGFR-positive punctate stainings are clearly seen in the nucleus. Bar, 10 μm.
Figure 2
Figure 2
Endocytosed Texas red-transferrin is downloaded into the SNX1-positive aggregated vesicular structure of early endosomes in the perinuclear region of gefitinib-resistant QG56 cells. The PC9 cells (A), or the QG56 cells (B) were fixed and double-stained for SNX1 (green in b, j) and LIMPII (red in c, k) as described in the Materials section. Superimposed images of SNX1 and LIMPII are shown in d, l. Each cell line was stained with DAPI (blue) to reveal nuclei. The merged confocal images as yellow color were quantified and presented as the percentage of total amounts of SNX1-positive vesicles per cell in C. The error bar denotes SD. In PC9 cells (A), SNX1-positive small vesicles are not colocalized with LIMPII-positive vesicles (d), however, in QG56 cells (B), part of LIMPII-positive vesicles colocalized with SNX1-positive early endosomes is seen in the perinuclear region (l). Furthermore, superimposed images of SNX1 and the internalized Texas red-transferrin in the PC9 cells and the QG56 cells are shown in h and p, respectively. The merged confocal images as yellow color as indicated by white arrowheads (h) or white arrows (p) were quantified and presented as the percentage of total amounts of SNX1-positive vesicles per cell in D. Note that SNX1-positive early endosomes form large aggregated vesicular structures in the perinuclear region (p) in QG56 cells, and that these aggregated structures are overlapped with Texas red-transferrin; however no aggregated vesicles are seen in PC9 cells (h).
Figure 3
Figure 3
Evidence for a rapid endocytosis of ligand-induced pEGFR in the gefitinib-sensitive PC9 cells, but for an inefficient endocytic traffic of EGF-pEGFR in the gefitinib-resisitant QG56 cells. The PC9 (A, B) or QG56 (C, D) cells were incubated in the absence (A, C) or presence (B, D) of gefitinib at 37°C with Texas red-EGF for 5, 10, or 15 min, and cells were fixed and double-stained for pEGFR (green) as described in the Materials section. Superimposed images of pEGFR and Texas red-EGF are shown. The white arrowheads indicate the colocalization of the pEGFR-positive vesicles and Texas red-EGF-positive vesicular structures. It is notable that rapid endocytosis of EGF-EGFR occurs in PC9 cells, since large amounts of pEGFR-positive small vesicles co-stained with Texas red-EGF appear in the cytoplasm after 5 min incubation (a) and these co-stained vesicles are increased at 15 min (b, c). By contrast in QG 56 cells, pEGFR stainings are mostly associated with plasma membrane even after 15 min incubation (i). Further, gefitinib significantly suppresses phosphorylation of EGFR in NSCLC cell lines and amount of pEGFR stainings are considerably reduced during the incubation (d, e, f, j, k, l). Bar, 10 μm.
Figure 4
Figure 4
Evidence for an efficient phosphorylation of EGFR and rapid delivery of pEGFR into the early endosomes after EGF stimulation in PC9 cells. The PC9 (A) or QG56 (B) cells stimulated with EGF for 15 min on ice were further incubated at 37°C with Texas red-transferrin (red) for 3, 6, or 15 min, and cells were fixed and double-stained for pEGFR (green) as described in the Materials section. Superimposed images of pEGFR and Texas red-transferrin are shown. Each cell line was stained with DAPI (blue) to reveal nuclei. The white arrows indicate the colocalized early endosomal vesicular structures positive for pEGFR and Texas red-transferrin. The merged confocal images as yellow color as indicated by white arrows (a, d) at 3 min incubation were quantified and presented as the percentage of total amounts of pEGFR-positive vesicles per cell in D. In PC9 cells (A), ligand-induced EGFR phosphorylation occurs efficiently in early endosomes or in plasma membrane (a, b, c). By contrast, only small fraction of pEGFR staining associated with early endosomal vesicles is seen in the cytoplasm of QG56 cells even after 15 min incubation (f).
Figure 5
Figure 5
Identification for an efficient pEGFR trafficking via early/late endocytic pathway in PC9 cells. The PC9 or QG56 cells were preincubated with EGF for 15 min on ice and then cell were chased at 37°C for 3, 6, or 15 min, and cells were fixed and double-stained for pEGFR (green) or LIMPII (red) as described in the Materials section. Superimposed images of pEGFR and LIMPII are shown. Each cell line was stained with DAPI (blue) to reveal nuclei. The white arrows indicate the colocalized LIMPII-positive late endosomes/lysosomes and pEGFR-positive cytoplasmic vesicular structures. The merged confocal images as yellow color as indicated by white arrows (c, d) at 15 min incubation were quantified and presented as the percentage of total amounts of LIMPII-positive vesicles per cell in B. It is notable that an efficient trafficking of pEGFR to late endosomes is seen in PC9 cells (a, b, c), but is not observed in QG56 cells (d).
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