Int J Oncol. 2015 Apr;46(4):1721-9. doi: 10.3892/ijo.2015.2871.

EGF‑stimulated AKT activation is mediated by EGFR recycling via an early endocytic pathway in a gefitinib‑resistant human lung cancer cell line.

 

Nishimura Y1, Takiguchi S2, Ito S3, Itoh K4.
  • 1Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812‑8582, Japan.
  • 2Institute for Clinical Research, National Kyushu Cancer Center, Fukuoka 811‑1395, Japan.
  • 3Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo 101‑0062, Japan.
  • 4Department of Biology, Osaka Medical Center for Cancer and Cardiovascular Diseases, Osaka 537‑8511, Japan.

 

Abstract

The receptor tyrosine kinase epidermal growth factor receptor (EGFR) and its ligand epidermal growth factor (EGF) are known to play important roles in malignant tumor cells, and the EGFR signaling pathway is one of the most important targets in various tumors, including non-small cell lung cancer (NSCLC). We reported recently that an aberration in certain steps of EGF-stimulated phosphorylated epidermal growth factor receptor (pEGFR) endocytic trafficking from the early endosomes to the late endosomes occurs in the gefitinib-resistant NSCLC cells, in which large amounts of sorting nexin 1 (SNX1) are colocalized with EGFR in the aggregated early endosomes where the internalized pEGFR is also accumulated of these cells. To further investigate the role of SNX1 in EGF‑stimulated pEGFR endocytosis, followed by downstream signaling leading to the activation of phosphatidylinositol 3-kinase (PI3K)–the serine/threonine kinase AKT pathway, we examined the effect of depletion of SNX1 knock-down expression by siRNA and an inhibition of targeting membrane recycling using monensin. Using immunofluorescence, we observed an efficient endocytic transport of pEGFR from early endosomes to late endosomes/lysosomes after EGF-stimulation in the cells transfected with siRNA‑SNX1, whereas the delayed endocytic delivery of pEGFR was evident in the siRNA-control-transfected cells. Furthermore, a large amount of endocytosed pEGFR was accumulated in the presence of monensin in the early endosomes of the SNX1 knock-down cells. In western blot analysis, EGF stimulation of both control and cells transfected with siRNA-SNX1 resulted in rapid phosphorylation of EGFR and enhanced AKT phosphorylation. Monensin-dependent inhibition of AKT phosphorylation was stronger in SNX1 knock-down cells than in controls. In contrast, however, monensin had no effect on AKT phosphorylation triggered by activation of the MET receptor tyrosine kinase. Collectively, we suggest that EGF-stimulated recycling of EGFR to the plasma membrane induces downstream signaling leading to AKT phosphorylation. Suppression of EGFR membrane recycling by SNX1 appears to be critical for the activation of EGFR/PI3K/AKT signaling pathway in human lung cancer cells.

PMID: 25653196

11-23-15€”õƒf[ƒ^ EGFR-recycling-WBF.pptx

Supplement Figure: Schematic diagram illustrates the regulation of EGFR/phosphorylated EGFR (pEGFR) endocytosis in the human NSCLC cells.

(a) In the gefitinib-sensitive cells, EGFR is phosphorylated and activated at the plasma membrane following EGF binding1 and then EGF-pEGFR complex is internalized, transported to the early endosomes2 where EGF-pEGFR is temporally associated with sorting nexin 1 (SNX1), a protein that interacts with EGFR and is localized to early endosomes through its phospholipid-binding motif [ref. 1]. Alternatively, EGFR is recycled back to the plasma membrane by the endosomal recycling pathways3.

(b) In the early endosomes, EGF-pEGFR complex is subsequently trafficked to the late endosome/lysosomes where degradation of EGF/pEGFR takes place, leading to the termination of the EGFR signaling4. By contrast, in the gefitinib-resistant cells, an accumulation of SNX1 occurs in the early endosomes, which leads to the suppression of the translocation of EGF-pEGFR toward the late endosomes/lysosomes. We recently demonstrated that silencing of endogenous SNX1 by siRNA stimulated endocytosis and the ligand-induced down-regulation of EGFR/pEGFR or MET/pMET in gefitinib-resistant cells [ref. 2, 3]. Based on these findings, we postulated that SNX1 negatively regulated EGF-dependent downregulation of EGFR and its phosphorylation via the early/late endocytic pathways in human lung cancer cells [ref. 2, 3].

(c) On the basis of these observations, we further designed the current study to investigate the role of SNX1 on EGF-stimulated pEGFR endocytosis. We examined the effect of SNX1 knockdown by siRNA and treatment with monensin, an inhibitor of membrane recycling, on EGF-stimulated EGFR endocytosis, and on phosphorylation of AKT. In the gefitinib-resistant NSCLC cells, we found a rapid phosphorylation of EGFR along with a substantial increase in pAKT after EGF stimulation. On the other hand, monensin considerably suppressed AKT phosphorylation in the EGF-treated cells [ref. 4]. We therefore infer that EGF-stimulated recycling of EGFR to the plasma membrane is an important step that activates the PI3K-AKT pathway, and that SNX1 is a negative regulator of EGFR recycling. Thus, it should be considered that the targeting the EGFR recycling pathway would be an important strategy in combination with EGFR-TKI therapy in lung cancer.

 

References:

  1. Worby CA, Dixon JE 2002 Sorting out the cellular function of sorting nexins. Nat Rev Mol Cell Biol 3: 919-931
  2. Nishimura Y, Takiguchi S, Yoshioka K, Nakabeppu Y, Itoh K 2012 Silencing of SNX1 by siRNA stimulates the ligand-induced endocytosis of EGFR and increases EGFR phosphorylation in gefitinib-resistant human lung cancer cell lines. Int J Oncol 41: 1520-1530
  3. Nishimura Y, Takiguchi S, Ito S, Itoh K 2014 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. Int J Oncol 44: 412-426
  4. Nishimura Y, Takiguchi S, Ito S, Itoh K 2015 EGF-stimulated AKT activation is mediated by EGFR recycling via an early endocytic pathway in a gefitinib-resistant
 human lung cancer cell line. Int J Oncol 46: 1721-1729

 

Corresponding to:

Yukio Nishimura, Ph.D.

Associate Professor

Division of Pharmaceutical Cell Biology

Graduate School of Pharmaceutical Sciences

Kyushu University

Fukuoka, 812-8582, Japan

ynkio443@gmail.com

 

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