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
TL;DR: Novel evidence is provided that extensive impairment in pEGFR endocytosis via the early endocytic pathway might confer gefitinib-resistance in NSCLC cell lines, and an aberration in some steps of EGF-EGFR trafficking from the early endsomes to late endosomes/lysosomes occurs in QG56 cells.
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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TL;DR: This study shows that mutant EGFRs in NSCLC cell lines are constitutively endocytosed and undergo aberrant traffic into the endocytic recycling compartment which allows mutant EG FRs to engage in a preferential interaction with Src, a critical partner for EGFR-mediated oncogenesis.
Abstract: Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and altered EGFR signaling contributes to human cancer. EGFR kinase domain mutants found in non-small cell lung cancer (NSCLC) are constitutively active, a trait critical for cell transformation through activation of downstream pathways. Endocytic trafficking of EGFR is a major regulatory mechanism as ligand-induced lysosomal degradation results in termination of signaling. While numerous studies have examined mutant EGFR signaling, the endocytic traffic of mutant EGFR within the NSCLC milieu remains less clear. This study shows that mutant EGFRs in NSCLC cell lines are constitutively endocytosed as shown by their colocalization with the early/recycling endosomal marker transferrin and the late endosomal/lysosomal marker LAMP1. Notably, mutant EGFRs, but not the wild-type EGFR, show a perinuclear accumulation and colocalization with recycling endosomal markers such as Rab11 and EHD1 upon treatment of cells with endocytic recycling inhibitor monensin, suggesting that mutant EGFRs preferentially traffic through the endocytic recycling compartments. Importantly, monensin treatment enhanced the mutant EGFR association and colocalization with Src, indicating that aberrant transit through the endocytic recycling compartment promotes mutant EGFR-Src association. The findings presented in this study show that mutant EGFRs undergo aberrant traffic into the endocytic recycling compartment which allows mutant EGFRs to engage in a preferential interaction with Src, a critical partner for EGFR-mediated oncogenesis.
77 citations
Cites background from "Evidence for efficient phosphorylat..."
...However, another study in an NSCLC cell line reported that mutant EGFR traffics into lysosomes upon EGF stimulation [54]....
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...Notably, the wtEGFR in the gefitinib-resistant cell line did not undergo ligand-induced lysosomal sorting, even though the receptor was found in endocytic vesicles [54]....
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TL;DR: Clinical experience with the EGFR-TKI gefitinib in Asian patients with NSCLC will be reviewed, both in patients who have previously failed chemotherapy and in the first-line setting (gefitinib is currently not licensed for first- line treatment).
Abstract: Clinical experience with the EGFR-TKI gefitinib in Asian patients with NSCLC will be reviewed, both in patients who have previously failed chemotherapy and in the first-line setting (gefitinib is currently not licensed for first-line treatment). Tolerability and specific adverse events in patients of Asian origin will be discussed. Differing objective response rates between patients of Asian and non-Asian origin when treated with gefitinib (and standard cytotoxics) will also be discussed along with EGFR mutations and drug resistance. Reports of Phase II/III clinical experience with gefitinib 250 mg/day in Asia were identified by searching in Medline and ASCO databases for publications between 1993 and 2008. Defined search criteria included (gefitinib OR Iressa OR ZD1839) AND NSCLC AND (Asia OR Japan OR China OR Taiwan OR Korea) or 'Clinical trial' type, with additional searches, including AND 'interstitial lung disease (ILD)' or 'EGFR mutation'. Numerous Phase II/III trials including patients of Asian origin with previously treated advanced NSCLC report a consistent clinical benefit of gefitinib. Gefitinib is generally well tolerated by patients with NSCLC although the incidence of ILD in Japanese patients must be noted. Studies analyzing EGFR mutations indicate that these mutations occur at a much higher rate in patients of Asian origin than in non-Asian patients. Data from several studies indicate that EGFR mutation-positive patients of Asian origin have better efficacy outcomes with first-line gefitinib when compared with those who are EGFR mutation-negative. Research is ongoing to evaluate the role of tailoring patients' treatment according to their genetic phenotype.
72 citations
TL;DR: A noninvasive, label-free, in vitro vibrational spectroscopic test to determine the integral physiologically relevant drug response in cell lines is developed and opens a new field of patient-centered drug testing that could deliver superior patient therapies.
Abstract: Predictions about the cellular efficacy of drugs tested in vitro are usually based on the measured responses of a few proteins or signal transduction pathways. However, cellular proteins are highly coupled in networks, and observations of single proteins may not adequately reflect the in vivo cellular response to drugs. This might explain some large discrepancies between in vitro drug studies and drug responses observed in patients. We present a novel in vitro marker-free approach that enables detection of cellular responses to a drug. We use Raman spectral imaging to measure the effect of the epidermal growth factor receptor (EGFR) inhibitor panitumumab on cell lines expressing wild-type Kirsten-Ras (K-Ras) and oncogenic K-Ras mutations. Oncogenic K-Ras mutation blocks the response to anti-EGFR therapy in patients, but this effect is not readily observed in vitro. The Raman studies detect large panitumumab-induced differences in vitro in cells harboring wild-type K-Ras as seen in A in red but not in cell...
59 citations
TL;DR: The interaction between EGFR and radiation including radiation induced EGFR signaling, the effect of EGFR on DNA damage repair, and potential mechanisms of radiosensitization are discussed and the potential pitfalls with scheduling EGFR targeted therapies with chemoradiation are examined.
43 citations
TL;DR: The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells.
40 citations
Cites background from "Evidence for efficient phosphorylat..."
...Interestingly, elevated accumulation of EGFR was observed in the nucleus of gefitinib-resistant cell line and gefitinib treated cells [29, 30]....
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TL;DR: A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib, and these mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor.
Abstract: BACKGROUND Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown. METHODS We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells. RESULTS Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib. CONCLUSIONS A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.
10,879 citations
"Evidence for efficient phosphorylat..." refers background in this paper
...It has been previously reported that gefitinib-sensitive lung tumors were found to express EGFR variants with a higher sensitivity for the drug than the wild-type receptor [29,30]....
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TL;DR: Results suggest that EGFR mutations may predict sensitivity to gefitinib, and treatment with the EGFR kinase inhibitor gefitsinib causes tumor regression in some patients with NSCLC, more frequently in Japan.
Abstract: Receptor tyrosine kinase genes were sequenced in nonsmall cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations of the epidermal growth factor receptor gene EGFR were found in 15 of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinibinsensitive tumors or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib. Protein kinase activation by somatic mutation or
9,265 citations
TL;DR: Cet article synthese montre comment des recepteurs membranaires a activite tyrosine kinase peuvent etre impliques dans la transduction and notamment jouent le role de signal de the transduction.
5,536 citations
"Evidence for efficient phosphorylat..." refers background in this paper
...These events lead to the recruitment and phosphorylation of several intracellular substrates and the subsequent transmission of extracellular signals to the nucleus via an intracellular signaling network [4,5]....
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TL;DR: Analysis of the multiple processes that modulate EGFR signal transduction has revealed new therapeutic opportunities and elucidated mechanisms contributing to the efficacy of existing anticancer treatments.
1,627 citations
"Evidence for efficient phosphorylat..." refers background in this paper
...Following ligand binding, the EGFR is dimerized and the intracellular tyrosine kinase region is activated, causing receptor tyrosine autophosphorylation and transphosphorylation of another receptor monomer [4]....
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...These events lead to the recruitment and phosphorylation of several intracellular substrates and the subsequent transmission of extracellular signals to the nucleus via an intracellular signaling network [4,5]....
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TL;DR: Article de synthese sur les lysosomes: biogenese, definition, role biologique, Les enzymes lysOSomales sont decrites ainsi that leur role dans l'exo et l'endocytose.
Abstract: Article de synthese sur les lysosomes: biogenese, definition, role biologique. Les enzymes lysosomales sont decrites ainsi que leur role dans l'exo et l'endocytose. La structure et le biogenese de la membrane lysosomale sont etudiees
1,559 citations
"Evidence for efficient phosphorylat..." refers background in this paper
...These proteins are distributed within endocytic organelles and are at the highest concentration in the late endosomes/lysosomes, as observed for other lysosomal glycoproteins, namely, lysosomal associated membrane protein-1 (LAMP-1) and LAMP-2 [14-17]....
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