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Journal ArticleDOI

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

21 May 2008-Molecular Cancer (BioMed Central)-Vol. 7, Iss: 1, pp 42-42

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.

AbstractGefitinib (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.

Topics: Endosome (66%), Endocytic cycle (61%), Endocytosis (55%), Epidermal growth factor (54%), Gefitinib (53%)

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Citations
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Journal ArticleDOI
TL;DR: An overview of the PX domain proteins is presented, incorporating recent functional and structural insights, and an updated classification of the proteins into distinct subfamilies is proposed on the basis of these insights.
Abstract: The mammalian genome encodes 49 proteins that possess a PX (phox-homology) domain, responsible for membrane attachment to organelles of the secretory and endocytic system via binding of phosphoinositide lipids. The PX domain proteins, most of which are classified as SNXs (sorting nexins), constitute an extremely diverse family of molecules that play varied roles in membrane trafficking, cell signalling, membrane remodelling and organelle motility. In the present review, we present an overview of the family, incorporating recent functional and structural insights, and propose an updated classification of the proteins into distinct subfamilies on the basis of these insights. Almost all PX domain proteins bind PtdIns3P and are recruited to early endosomal membranes. Although other specificities and localizations have been reported for a select few family members, the molecular basis for binding to other lipids is still not clear. The PX domain is also emerging as an important protein-protein interaction domain, binding endocytic and exocytic machinery, transmembrane proteins and many other molecules. A comprehensive survey of the molecular interactions governed by PX proteins highlights the functional diversity of the family as trafficking cargo adaptors and membrane-associated scaffolds regulating cell signalling. Finally, we examine the mounting evidence linking PX proteins to different disorders, in particular focusing on their emerging importance in both pathogen invasion and amyloid production in Alzheimer's disease.

220 citations


Journal Article
TL;DR: The current knowledge of the nuclear EGFR signaling network is summarized, including how it is trafficked to the nucleus, the functions it serves inThe nucleus, and how these functions impact cancer progression, survival, and response to chemotherapeutics.
Abstract: The epidermal growth factor receptor (EGFR) is a member of the EGFR family of receptor tyrosine kinases (RTKs). EGFR activation via ligand binding results in signaling through various pathways ultimately resulting in cellular proliferation, survival, angiogenesis, invasion, and metastasis. Aberrant expression or activity of EGFR has been strongly linked to the etiology of several human epithelial cancers including but not limited to head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer, pancreatic cancer, and brain cancer. Thus intense efforts have been made to inhibit the activity of EGFR by designing antibodies against the ligand binding domains (cetuximab and panitumumab) or small molecules against the tyrosine kinase domain (erlotinib, gefitinib, and lapatinib). Although targeting membrane-bound EGFR has shown benefit, a new and emerging role for EGFR is now being elucidated. In this review we will summarize the current knowledge of the nuclear EGFR signaling network, including how it is trafficked to the nucleus, the functions it serves in the nucleus, and how these functions impact cancer progression, survival, and response to chemotherapeutics.

195 citations


Journal ArticleDOI
TL;DR: Light is shed on the role of nuclear EGFR in the sensitivity of wtEGFR-expressing cancer cells to EGFR tyrosine kinase inhibitors and a putative molecular mechanism contributing to gefitinib resistance through BCRP/ABCG2 expression is deciphered.
Abstract: Epidermal growth factor receptor (EGFR), an aberrantly overexpressed or activated receptor-tyrosine kinase in many cancers, plays a pivotal role in cancer progression and has been an attractive target for cancer therapy. Gefitinib and erlotinib, two EGFR-tyrosine kinase inhibitors, have been approved for non-small cell lung cancer. However, durable clinical efficacy of these EGFR inhibitors is severely limited by the emergence of acquired resistance. For example, the expression of breast cancer-resistant protein (BCRP/ABCG2) has been shown to confer acquired resistance of wild-type EGFR (wtEGFR)-expressing cancer cells to gefitinib. However, the underlying molecular mechanisms still remain unclear. Here, we show that wtEGFR expression is elevated in the nucleus of acquired gefitinib-resistant cancer cells. Moreover, nuclear translocation of EGFR requires phosphorylation at Ser-229 by Akt. In the nucleus, EGFR then targets the proximal promoter of BCRP/ABCG2 and thereby enhances its gene transcription. The nuclear EGFR-mediated BCRP/ABCG2 expression may contribute at least in part to the acquired resistance of wtEGFR-expressing cancer cells to gefitinib. Our findings shed light on the role of nuclear EGFR in the sensitivity of wtEGFR-expressing cancer cells to EGFR tyrosine kinase inhibitors and also deciphered a putative molecular mechanism contributing to gefitinib resistance through BCRP/ABCG2 expression.

161 citations


Journal ArticleDOI
23 Jun 2011-PLOS ONE
TL;DR: It is shown that acquired resistance of wtEGFR-expressing cancer cells to an EGFR TKI, gefitinib, is associated with elevated expression of breast cancer resistance protein (BCRP/ABCG2), which in turn leads to gefITinib efflux from cells.
Abstract: Background The sensitivity of non-small cell lung cancer (NSCLC) patients to EGFR tyrosine kinase inhibitors (TKIs) is strongly associated with activating EGFR mutations. Although not as sensitive as patients harboring these mutations, some patients with wild-type EGFR (wtEGFR) remain responsive to EGFR TKIs, suggesting that the existence of unexplored mechanisms renders most of wtEGFR-expressing cancer cells insensitive.

76 citations


Additional excerpts

  • ...mutations [6,7,8,10,11,12,13], suggesting that other unknown...

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Journal ArticleDOI
TL;DR: An improved understanding of the molecular mechanisms of resistance to EGFR inhibitors may provide valuable indications to identify biomarkers that can be used clinically to predict response to EG FR blockade and to establish new treatment options to overcome resistance.
Abstract: Targeted therapy against the epidermal growth factor receptor (EGFR) is one of the most promising molecular therapeutics for head and neck squamous cell carcinoma (HNSCC). EGFR is overexpressed in a wide range of malignancies, including HNSCC, and initiates important signal transduction pathways in HNSCC carcinogenesis. However, primary and acquired resistance are serious problems and are responsible for low single-agent response rate and tumor recurrence. Therefore, an improved understanding of the molecular mechanisms of resistance to EGFR inhibitors may provide valuable indications to identify biomarkers that can be used clinically to predict response to EGFR blockade and to establish new treatment options to overcome resistance. To date, no predictive biomarker for HNSCC is available in the clinic. Therapeutic resistance to anti-EGFR therapy may arise from mechanisms that can compensate for reduced EGFR signaling and/or mechanisms that can modulate EGFR-dependent signaling. In this review, we will summarize some of these molecular mechanisms and describe strategies to overcome that resistance.

73 citations


References
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Journal ArticleDOI
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.

10,359 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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Journal ArticleDOI
04 Jun 2004-Science
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

8,825 citations


Journal ArticleDOI
20 Apr 1990-Cell
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.
Abstract: Cet article synthese montre comment des recepteurs membranaires a activite tyrosine kinase peuvent etre impliques dans la transduction et notamment jouent le role de signal de la transduction

5,463 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]....

    [...]


Journal ArticleDOI
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.
Abstract: Growth factors and their transmembrane receptor tyrosine kinases play important roles in cell proliferation, survival, migration and differentiation. One group of growth factors, comprising epidermal growth factor (EGF)-like proteins and neuregulins, stimulates cells to divide by activating members of the EGF receptor (EGFR) family, which consists of the EGFR itself and the receptors known as HER2-4. This highly conserved signalling module plays a fundamental role in the morphogenesis of a diverse spectrum of organisms, ranging from humans to nematodes, and has also been implicated in the development and growth of many types of human tumour cells. In humans, more than 30 ligands and the EGFR family of four receptors lie at the head of a complex, multi-layered signal-transduction network. Different activated receptor-ligand complexes vary in both the strength and type of cellular responses that they induce. Analysis of the multiple processes that modulate EGFR signal transduction, such as receptor heterodimerisation and endocytosis, has revealed new therapeutic opportunities and elucidated mechanisms contributing to the efficacy of existing anticancer treatments.

1,552 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]....

    [...]


Journal ArticleDOI
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

1,534 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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