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,Biborka Bereczky,Kazuyuki Itoh,Kiyoko Yoshioka

doi:10.1186/1476-4598-7-42

Yukio Nishimura, Biborka Bereczky + Show 2 more

Open Access

https://doi.org/10.1186/1476-4598-7-42

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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.

Highlights

The epidermal growth factor receptor (EGFR) is a prototypical member of the ErbB family of tyrosine kinases and plays an important role in the pathogenesis of different tumors; therapies directed at inhibiting EGFR function have potential as anticancer treatments [1,2]
We found novel evidence that an aberration in some steps of EGF-EGFR trafficking from the early endosomes to the late endosomes/lysosomes does occur in the gefitinib-resistant human lung cancer cell line derived from non-small cell lung cancer (NSCLC), whereas endocytosis of EGFR is normal in gefitinib-sensitive PC9 cells [13], suggesting that impairment in some steps of EGF-EGFR trafficking from early endosomes to late endosomes/lysosomes might confer gefitinib-resistance in NSCLC cell lines
In both PC9 and QG56 cells, it is notable that phosphorylated EGFR (pEGFR)-positive small punctate vesicles are spreading in the cytoplasm and these vesicles are not colocalized with the Lysosomal integral membrane protein II (LIMPII), or cathepsin D-positive vesicular structures, and pEGFR-positive punctate stainings are clearly seen in the nucleus

Summary

Introduction

The epidermal growth factor receptor (EGFR) is a prototypical member of the ErbB family of tyrosine kinases and plays an important role in the pathogenesis of different tumors; therapies directed at inhibiting EGFR function have potential as anticancer treatments [1,2]. A recent in vitro study demonstrated that of the 9 non-small cell lung cancer (NSCLC) cell lines examined, the PC9 cell line was most sensitive to the effect of gefitinib when assayed under basal growth conditions for EGFR phosphorylation and activation of EGFR downstream effectors such as AKT and those in the ERK1/2 pathway, which are required for its survival and proliferation [11]. This suggests that the mechanism underlying the sensitivity of the EGFR pathway could be useful in predicting the potential effectiveness of gefitinib in NSCLC patients. Inefficient EGFR down regulation was observed in the gefitinib-resistant cell line QG56, whereas rapid down regulation occurred in the gefitinib-sensitive cell line PC9, wherein the cells were in the exponential phase of growth, suggesting that a different unknown down-regulation mechanism operates in each cell type

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