A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells

We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGFR in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining were accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15–30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.     

LIM kinase 1: evidence for a role in the regulation of intracellular vesicle trafficking of lysosomes and endosomes in human breast cancer cells

LIM kinase (LIMK) plays a critical role in stimulus-induced remodeling of the actin cytoskeleton by linking signals from the Rho family GTPases to changes in cofilin activity. Recent studies have shown an important role for LIMK1 signaling in tumor cell invasion through regulating actin dynamics. In this study, we investigate the role of LIMK1 in intracellular vesicle trafficking of lysosomes/endosomes. We analyzed by confocal immunofluorescence microscopy the cellular distribution of lysosomal proteins and the endocytosis of an endocytic tracer, epidermal growth factor (EGF), in LIMK1-transfected cells. We found in these cells an abnormal dispersed translocation of lysosomes stained for LIMPII and cathepsin D throughout the cytoplasm. The small punctate structures that stained for these lysosomal proteins were redistributed to the periphery of the cell. Computational 3D-image analysis of confocal immunofluorescence micrographs further demonstrated that these vesicles did not colocalize with the transferrin receptor, an early endosomal marker. Furthermore, LIMPII-positive lysosomes did not colocalize with early endosomes labeled with endocytosed Texas red-transferrin. These results indicate that there is no mixing between dispersed lysosomes and early endosomes in the LIMK1-transfected cells. Moreover, LIMK1 overexpression resulted in a marked retardation in the receptor-mediated internalization of Texas red-labeled EGF in comparison with mock-transfected cells. At 30 min after internalization, most of the Texas red-EGF staining overlapped with LIMPII-positive late endosomes/lysosomes in mock-transfected cells, whereas in LIMK1 transfectants only a small fraction of internalized EGF colocalized with LIMPII-positive structures in the perinuclear region. Taken together, the findings presented in this paper suggest that LIMK1 has a role in regulating vesicle trafficking of lysosomes and endosomes in invasive tumor cells.     

"Synchronized" endocytosis and intracellular sorting in alveolar macrophages: the early sorting endosome is a transient organelle

Incubation of alveolar macrophages in hypoosmotic K(+)-containing buffers results in persistent cell swelling and an inability to undergo regulatory volume decrease. We demonstrate that cells incubated in hypo- K+ show an inhibition of endocytosis without any observed alteration in recycling. The inhibition of endocytosis affected all forms of membrane internalization, receptor and fluid phase. Both increased cell volume and the inhibition of endocytosis could be released upon return of cells to iso-Na+ buffers. The ability to synchronize the endocytic apparatus allowed us to examine hypotheses regarding the origin and maturation of endocytic vesicles. Incubation in hypo-K+ buffers had no effect on the delivery of ligands to degradative compartments or on the return of previously internalized receptors to the cell surface. Thus, membrane recycling and movement of internalized components to lysosomes occurred in the absence of continued membrane influx. We also demonstrate that fluorescent lipids, that had been incorporated into early endosomes, returned to the cell surface upon exposure of cells to hypo-K+ buffers. These results indicate that the early sorting endosome is a transient structure, whose existence depends upon continued membrane internalization. Our data supports the hypothesis that the transfer of material to lysosomes can best be explained by the continuous maturation of endosomes.     

The role of tyrosine kinase activity in endocytosis, compartmentation, and down-regulation of the epidermal growth factor receptor.

Occupancy-induced down-regulation of cell surface epidermal growth factor (EGF) receptors attenuates signal transduction. To define mechanisms through which down-regulation of this class of growth factor receptors occurs, we have investigated the relative roles of ligand-induced internalization and recycling in this process. Occupied, kinase-active EGF receptors were internalized through a high affinity, saturable endocytic system at rates up to 10-fold faster than empty receptors. In contrast, full length EGF receptors lacking tyrosine kinase activity underwent internalization at a rate independent of occupancy. This "kinase-independent" internalization rate appeared to reflect constitutive receptor internalization since it was similar to the internalization rate of both receptors lacking a cytoplasmic domain and of antibodies bound to empty receptors. EGF internalized by either kinase-active or kinase-inactive receptors was efficiently recycled and was found within endosomes containing recycling transferrin receptors. However, targeting of internalized receptors to lysosomes did not require receptor kinase activity. All receptors that displayed ligand-induced internalization also underwent down-regulation, indicating that the proximal cause of down-regulation is occupancy-induced endocytosis. Tyrosine kinase activity greatly enhances this process by stabilizing receptor association with the endocytic apparatus.     

Endocytosis and intracellular trafficking of ErbBs

This review article describes the pathways and mechanisms of endocytosis and post-endocytic sorting of the EGF receptor (EGFR/ErbB1) and other members of the ErbB family. Growth factor binding to EGFR accelerates its internalization through clathrin-coated pits which is followed by the efficient lysosomal targeting of internalized receptors and results in receptor down-regulation. The role of EGFR interaction with the Grb2 adaptor protein and Cbl ubiquitin ligase, and receptor ubiquitination in the clathrin-dependent internalization and sorting of EGFR in multivesicular endosomes is discussed. Activation and phosphorylation of ErbB2, ErbB3 and ErbB4 also results in their ubiquitination. However, these ErbBs are internalized and targeted to lysosomes less efficiently than EGFR. When overexpressed endocytosis-impaired ErbBs may inhibit the internalization and degradation of EGFR.     

Endocytosis and intracellular trafficking of ErbBs

This review article describes the pathways and mechanisms of endocytosis and post-endocytic sorting of the EGF receptor (EGFR/ErbB1) and other members of the ErbB family. Growth factor binding to EGFR accelerates its internalization through clathrin-coated pits which is followed by the efficient lysosomal targeting of internalized receptors and results in receptor down-regulation. The role of EGFR interaction with the Grb2 adaptor protein and Cbl ubiquitin ligase, and receptor ubiquitination in the clathrin-dependent internalization and sorting of EGFR in multivesicular endosomes is discussed. Activation and phosphorylation of ErbB2, ErbB3 and ErbB4 also results in their ubiquitination. However, these ErbBs are internalized and targeted to lysosomes less efficiently than EGFR. When overexpressed endocytosis-impaired ErbBs may inhibit the internalization and degradation of EGFR.     

Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor.

Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.     

Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of (125)I-EGF internalization, whereas internalization of (125)I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized (125)I-EGF was increased, while degradation of (125)I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.     

Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.     

Synaptic vesicles form by budding from tubular extensions of sorting endosomes in PC12 cells

The putative role of sorting early endosomes (EEs) in synaptic-like microvesicle (SLMV) formation in the neuroendocrine PC12 cell line was investigated by quantitative immunoelectron microscopy. By BSA-gold internalization kinetics, four distinct endosomal subcompartments were distinguished: primary endocytic vesicles, EEs, late endosomes, and lysosomes. As in other cells, EEs consisted of vacuolar and tubulovesicular subdomains. The SLMV marker proteins synaptophysin and vesicle-associated membrane protein 2 (VAMP-2) localized to both the EE vacuoles and associated tubulovesicles. Quantitative analysis showed that the transferrin receptor and SLMV proteins colocalized to a significantly higher degree in primary endocytic vesicles then in EE-associated tubulovesicles. By incubating PC12 cells expressing T antigen-tagged VAMP (VAMP-TAg) with antibodies against the luminal TAg, the recycling pathway of SLMV proteins was directly visualized. At 15 degrees C, internalized VAMP-TAg accumulated in the vacuolar domain of EEs. Upon rewarming to 37 degrees C, the labeling shifted to the tubular part of EEs and to newly formed SLMVs. Our data delineate a pathway in which SLMV proteins together with transferrin receptor are delivered to EEs, where they are sorted into SLMVs and recycling vesicles, respectively.     

Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

hSef potentiates EGF-mediated MAPK signaling through affecting EGFR trafficking and degradation

Sef (similar expression to fgf genes) was identified as an effective antagonist of fibroblast growth factor (FGF) in vertebrates. Previous reports have demonstrated that Sef interacts with FGF receptors (FGFRs) and inhibits FGF signaling, however, its role in regulating epidermal growth factor receptor (EGFR) signaling remains unclear. In this report, we found that hSef localizes to the plasma membrane (PM) and is subjected to rapid internalization and well localizes in early/recycling endosomes while poorly in late endosomes/lysosomes. We observed that hSef interacts and functionally colocalizes with EGFR in early endosomes in response to EGF stimulation. Importantly, we demonstrated that overexpression of hSef attenuates EGFR degradation and potentiates EGF-mediated mitogen-activated protein kinase (MAPK) signaling by interfering EGFR trafficking. Finally, our data showed that, with overexpression of hSef, elevated levels of Erk phosphorylation and differentiation of rat pheochromocytoma (PC12) cells occur in response to EGF stimulation. Taken together, these data suggest that hSef plays a positive role in the EGFR-mediated MAPK signaling pathway. This report, for the first time, reveals opposite roles for Sef in EGF and FGF signalings.     

Gefitinib resistance of cancer cells correlated with TM4SF5-mediated epithelial-mesenchymal transition

Although cancers can be initially treated with the epidermal growth factor receptor (EGFR) inhibitor, gefitinib, continued gefitinib therapy does not benefit the survival of patients due to acquired resistance through EGFR mutations, c-MET amplification, or epithelial-mesenchymal transition (EMT). It is of further interest to determine whether mesenchymal-like, but not epithelial-like, cancer cells can become resistant to gefitinib by bypassing EGFR signaling and acquiring alternative routes of proliferative and survival signaling. Here we examined whether gefitinib resistance of cancer cells can be caused by transmembrane 4 L six family member 5 (TM4SF5), which has been shown to induce EMT via cytosolic p27Kip1 stabilization. Gefitinib-resistant cells exhibited higher and/or sustained TM4SF5 expression, cytosolic p27Kip1 stabilization, and mesenchymal phenotypes, compared with gefitinib-sensitive cells. Conversion of gefitinib-sensitive to -resistant cells by introduction of the T790M EGFR mutation caused enhanced and sustained expression of TM4SF5, phosphorylation of p27Kip1 Ser10 (responsible for cytosolic location), loss of E-cadherin from cell-cell contacts, and gefitinib-resistant EGFR and survival signaling activities. Additionally, TM4SF5 overexpression lessened the sensitivity of NSCLC cells to gefitinib. Suppression of TM4SF5 or p27Kip1 in gefitinib-resistant cells via the T790M EGFR mutation or TM4SF5 expression rendered them gefitinib-sensitive, displaying more epithelial-like and less mesenchymal-like characteristics. Together, these results indicate that TM4SF5-mediated EMT may have an important function in the gefitinib resistance of cancer cells.     

Novel mechanism for regulation of epidermal growth factor receptor endocytosis revealed by protein kinase A inhibition

Current models put forward that the epidermal growth factor receptor (EGFR) is efficiently internalized via clathrin-coated pits only in response to ligand-induced activation of its intrinsic tyrosine kinase and is subsequently directed into a lysosomal-proteasomal degradation pathway by mechanisms that include receptor tyrosine phosphorylation and ubiquitylation. Herein, we report a novel mechanism of EGFR internalization that does not require ligand binding, receptor kinase activity, or ubiquitylation and does not direct the receptor into a degradative pathway. Inhibition of basal protein kinase A (PKA) activity by H89 and the cell-permeable substrate peptide Myr-PKI induced internalization of 40-60% unoccupied, inactive EGFR, and its accumulation into early endosomes without affecting endocytosis of transferrin and mu-opioid receptors. This effect was abrogated by interfering with clathrin function. Thus, the predominant distribution of inactive EGFR at the plasma membrane is not simply by default but involves a PKA-dependent restrictive condition resulting in receptor avoidance of endocytosis until it is stimulated by ligand. Furthermore, PKA inhibition may contribute to ligand-induced EGFR endocytosis because epidermal growth factor inhibited 26% of PKA basal activity. On the other hand, H89 did not alter ligand-induced internalization of EGFR but doubled its half-time of down-regulation by retarding its segregation into degradative compartments, seemingly due to a delay in the receptor tyrosine phosphorylation and ubiquitylation. Our results reveal that PKA basal activity controls EGFR function at two levels: 1) residence time of inactive EGFR at the cell surface by a process of "endocytic evasion," modulating the accessibility of receptors to stimuli; and 2) sorting events leading to the down-regulation pathway of ligand-activated EGFR, determining the length of its intracellular signaling. They add a new dimension to the fine-tuning of EGFR function in response to cellular demands and cross talk with other signaling receptors.     

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.     

A Dyn2–CIN85 complex mediates degradative traffic of the EGFR by regulation of late endosomal budding

The epidermal growth factor receptor (EGFR) is over‐expressed in a variety of human cancers. Downstream signalling of this receptor is tightly regulated both spatially and temporally by controlling its internalization and subsequent degradation. Internalization of the EGFR requires dynamin 2 (Dyn2), a large GTPase that deforms lipid bilayers, leading to vesicle scission. The adaptor protein CIN85 (cbl‐interacting protein of 85 kDa), which has been proposed to indirectly link the EGFR to the endocytic machinery at the plasma membrane, is also thought to be involved in receptor internalization. Here, we report a novel and direct interaction between Dyn2 and CIN85 that is induced by EGFR stimulation and, most surprisingly, occurs late in the endocytic process. Importantly, disruption of the CIN85–Dyn2 interaction results in accumulation of internalized EGFR in late endosomes that become aberrantly elongated into distended tubules. Consistent with the accumulation of this receptor is a sustention of downstream signalling cascades. These findings provide novel insights into a previously unknown protein complex that can regulate EGFR traffic at very late stages of the endocytic pathway.     

Decorin evokes protracted internalization and degradation of the epidermal growth factor receptor via caveolar endocytosis

Decorin inhibits the epidermal growth factor receptor (EGFR) by down-regulating its tyrosine kinase activity, thereby blocking the growth of a variety of transformed cells and tumor xenografts. In this study we provide evidence that decorin directly binds to the EGFR causing its dimerization, internalization, and ultimately its degradation. Using various pharmacological agents to disrupt clathrin-dependent and -independent endocytosis, we demonstrate that decorin evokes a protracted internalization of the EGFR primarily via caveolar-mediated endocytosis. In contrast to EGF, decorin targets the EGFR to caveolae, but not to early or recycling endosomes. Ultimately, however, both EGF- and decorin-induced pathways converge into late endosomes/lysosomes for final degradation. Thus, we have discovered a novel biological mechanism for decorin that could explain its anti-proliferative and anti-oncogenic mode of action.     

miR-138-5p reverses gefitinib resistance in non-small cell lung cancer cells via negatively regulating G protein-coupled receptor 124

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) such as gefitinib are clinically effective treatments for non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, therapeutic effect is ultimately limited by the development of acquired TKI resistance. MicroRNAs (miRNAs) represent a category of small non-coding RNAs commonly deregulated in human malignancies. The aim of this study was to investigate the role of miRNAs in gefitinib resistance. We established a gefitinib-resistant cell model (PC9GR) by continually exposing PC9 NSCLC cells to gefitinib for 6 months. MiRNA microarray screening revealed miR-138-5p showed the greatest downregulation in PC9GR cells. Re-expression of miR-138-5p was sufficient to sensitize PC9GR cells and another gefitinib-resistant NSCLC cell line, H1975, to gefitinib. Bioinformatics analysis and luciferase reporter assay showed that G protein-coupled receptor124 (GPR124) was a direct target of miR-138-5p. Experimental validation demonstrated that expression of GPR124 was suppressed by miR-138-5p on protein and mRNA levels in NSCLC cells. Furthermore, we observed an inverse correlation between the expression of miR-138-5p and GPR124 in lung adenocarcinoma specimens. Knockdown of GPR124 mimicked the effects of miR-138-5p on the sensitivity to gefitinib. Collectively, our results suggest that downregulation of miR-138-5p contributes to gefitinib resistance and that restoration of miR-138-5p or inhibition GPR124 might serve as potential therapeutic approach for overcoming NSCLC gefitinib resistance.     

MiR-181a contributes gefitinib resistance in non-small cell lung cancer cells by targeting GAS7

Tyrosine kinase inhibitors (TKIs) exert potent therapeutic efficacy in non-small cell lung cancers (NSCLC) harboring epidermal growth factor receptor (EGFR) activating mutations. However, a major impediment for the effective treatment is the development of drug resistance. Some evidence supports a role for miRNAs in modulating NSCLC TKIs resistance. Here we show that miR-181a is significantly up-regulated in gefitinib-resistant cells compared with gefitinib-sensitive cells. Upregulation of miR-181a caused resistance of gefitinib, whereas downregulation of miR-181a sensitized NSCLC cells to gefitinib. Furthermore, the miR-181a plasma levels were significantly increased in acquired gefitinib resistant NSCLC patients compared with the plasma levels prior to gefitinib treatment in each patient. Bioinformatics analysis and luciferase reporter assay showed that growth arrest-specific 7 (GAS7) was a direct target gene of miR-181a. A significant inverse correlation between the expression of miR-181a and GAS7 was identified in NSCLC tissues. Downregulation of GAS7 expression could antagonize gefitinib re-sensitivity in PC9GR mediated by knockdown of miR-181a via AKT/ERK pathways and epithelial-to-mesenchymal transition markers. Additionally, GAS7 expression was downregulated in a large cohort of NSCLC patients, and a high mRNA level of GAS7 was associated with improved overall survival. Collectively, our findings provide a novel basis for using miR-181a/GAS7-based therapeutic strategies to reverse gefitinib resistance in NSCLC.     

Immunocytochemical localization of Shc and activated EGF receptor in early endosomes after EGF stimulation of HeLa cells.

After binding of epidermal growth factor (EGF), the EGF receptor (EGFR) becomes autophosphorylated via tyrosine. The ligand-activated receptor is internalized by endocytosis and subsequently degraded in the lysosomal pathway. To follow EGFR activation after EGF stimulation, we generated antisera to the EGFR phosphotyrosine sites pY992 and pY1173. The SH2 region of Shc binds to both these sites. Both antisera identified EGFR after EGF binding and did not crossreact with the unactivated receptor. The intracellular distribution of phosphorylated EGFR after ligand binding was traced by two-color immunofluorescence confocal microscopy and immunoelectron microscopy. Before EGF stimulation EGFR was primarily located along the cell surface. When internalization of activated EGFR was inhibited by incubation with EGF on ice, Y992- and Y1173-phosphorylated EGFR were located along the plasma membrane. Ten minutes after internalization at 37C, Y992- and Y1173-phosphorylated EGFR were almost exclusively located in early endosomes, as shown by co-localization with EEA1. Immunoelectron microscopy confirmed that phosphorylated EGFR was located in intracellular vesicles resembling early endosomes. After EGF stimulation, the adaptor protein Shc redistributed to EGFR-containing early endosomes. Our results indicate that EGFR activation of Shc via tyrosine-phosphorylated Y992 and Y1173 occurred in early endocytic compartments, and support a role for membrane trafficking in intracellular signaling.