Ligand-stimulated Epidermal Growth Factor Receptor Research Articles

Aqueous extract of Polygonum bistorta modulates proteostasis by ROS-induced ER stress in human hepatoma cells

Hepatocellular carcinoma (HCC) remains the leading cause of cancer mortality with limited therapeutic targets. The endoplasmic reticulum (ER) plays a pivotal role in maintaining proteostasis in normal cells. However, alterations in proteostasis are often found in cancer cells, making it a potential target for therapy. Polygonum bistorta is used in traditional Chinese medicine owing to its anticancer activities, but the molecular and pharmacological mechanisms remain unclear. Using hepatoma cells as a model system, this study demonstrated that P. bistorta aqueous extract (PB) stimulated ER stress by increasing autophagosomes but by blocking degradation, followed by the accumulation of ubiquitinated proteins and cell apoptosis. In addition, an autophagy inhibitor did not enhance ubiquitinated protein accumulation whereas a reactive oxygen species (ROS) scavenger diminished both ubiquitinated protein accumulation and ligand-stimulated epidermal growth factor receptor (EGFR) expression, suggesting that ROS generation by PB may be upstream of PB-triggered cell death. Nevertheless, PB-exerted proteostasis impairment resulted in cytoskeletal changes, impairment of cell adhesion and motility, and inhibition of cell cycle progression. Oral administration of PB delayed tumour growth in a xenograft model without significant body weight loss. These findings indicate that PB may be a potential new alternative or complementary medicine for HCC.

Stress-Induced EGFR Trafficking: Mechanisms, Functions, and Therapeutic Implications.

Epidermal growth factor receptor (EGFR) has fundamental roles in normal physiology and cancer, making it a rational target for cancer therapy. Surprisingly, however, inhibitors that target canonical, ligand-stimulated EGFR signaling have proven to be largely ineffective in treating many EGFR-dependent cancers. Recent evidence indicates that both intrinsic and therapy-induced cellular stress triggers robust, noncanonical pathways of ligand-independent EGFR trafficking and signaling, which provides cancer cells with a survival advantage and resistance to therapeutics. Here, we review the mechanistic regulation of noncanonical EGFR trafficking and signaling, and the pathological and therapeutic stresses that activate it. We also discuss the implications of this pathway in clinical treatment of EGFR-overexpressing cancers.

EGF receptor trafficking: consequences for signaling and cancer

The ligand-stimulated epidermal growth factor receptor (EGFR) has been extensively studied in the analysis of molecular mechanisms regulating endocytic traffic and the role of that traffic in signal transduction. Although such studies have largely focused on mitogenic signaling and dysregulated traffic in tumorigenesis, there is growing interest in the potential role of EGFR traffic in cell survival and the consequent response to cancer therapy. Here we review recent advances in our understanding of molecular mechanisms regulating ligand-stimulated EGFR activation, internalization, and post-endocytic sorting. The role of EGFR overexpression/mutation and new modulators of EGFR traffic in cancer and the response to cancer therapeutics are also discussed. Finally, we speculate on the relationship between EGFR traffic and cell survival.

Ligand-dependent EGFR activation induces the co-expression of IL-6 and PAI-1 via the NFkB pathway in advanced-stage epithelial ovarian cancer

The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptor tyrosine kinases, is expressed in up to 70% of epithelial ovarian cancers (EOCs), where it correlates with poor prognosis. The majority of EOCs are diagnosed at an advanced stage, and at least 50% present malignant ascites. High levels of IL-6 have been found in the ascites of EOC patients and correlate with shorter survival. Herein, we investigated the signaling cascade led by EGFR activation in EOC and assessed whether EGFR activation could induce an EOC microenvironment characterized by pro-inflammatory molecules. In vitro analysis of EOC cell lines revealed that ligand-stimulated EGFR activated NFkB-dependent transcription and induced secretion of IL-6 and plasminogen activator inhibitor (PAI-1). IL-6/PAI-1 expression and secretion were strongly inhibited by the tyrosine kinase inhibitor AG1478 and EGFR silencing. A significant reduction of EGF-stimulated IL-6/PAI-1 secretion was also obtained with the NFkB inhibitor dehydroxymethylepoxyquinomicin. Of 23 primary EOC tumors from advanced-stage patients with malignant ascites at surgery, 12 co-expressed membrane EGFR, IL-6 and PAI-1 by immunohistochemistry; both IL-6 and PAI-1 were present in 83% of the corresponding ascites. Analysis of a publicly available gene-expression data set from 204 EOCs confirmed a significant correlation between IL-6 and PAI-1 expression, and patients with the highest IL-6 and PAI-1 co-expression showed a significantly shorter progression-free survival time (P=0.028). This suggests that EGFR/NFkB/IL-6-PAI-1 may have a significant impact on the therapy of a particular subset of EOC, and that IL-6/PAI-1 co-expression may be a novel prognostic marker.

Re-epithelialization from human skin explant cultures is promoted by ligand-activated HER3 receptor

Ligand-stimulated epidermal growth factor receptor (EGFR/HER1) plays a fundamental role in skin biology as potent transducer of mitotic and anti-apoptotic stimuli in keratinocytes. In human epidermis, at least two additional EGFR family members--HER2 and HER3--are expressed but their biological functions in normal and diseased human skin remain obscure. Here, we studied the expression and biological impact of HER3 in regenerating human epidermis formed from skin explants adhered to acellular dermis. Neoepidermal HER3 expression was examined by quantitative real-time reverse transcriptase polymerase chain reaction, immunohistochemistry and Western blot analysis. The dynamic effect of HER3 receptor stimulation by recombinant heregulin (HRG)-beta1 was assessed by fluorescence imaging of re-epithelialization. In the neoepidermis, HER3 mRNA and protein were detected with activated receptors being immunolocalized at basal and low suprabasal levels. Exogenous HRG-beta1 at 10-20 ng/ml increased the outgrowth rate corresponding to approximately 30% the response of exogenous EGF. The growth-promoting effect of HRG-beta1 was associated with enhanced HER3 phosphorylation, keratinocyte proliferation and thickening of viable neoepidermis whereas blockade of ligand-binding to HER3 delayed the outgrowth process and inhibited both constitutive and ligand-induced HER3 phosphorylation. HER2 antagonism using an anti-dimerization antibody, pertuzumab, impeded the re-epithelialization rate. In addition, a selective HER2 kinase inhibitor, CP654577, downregulated phospho-HER3 expression suggesting that transactivation of kinase-deficient HER3 was accomplished through dimerization with HER2. The study emphasizes the central role of EGFR in epidermal renewal and demonstrates that HRG-activated HER3 contributes to the outgrowth process of epidermis in vitro.

Mig-6 controls EGFR trafficking and suppresses gliomagenesis

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer that is driven by aberrant signaling of growth factor receptors, particularly the epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosome-mediated degradation, although the molecular mechanisms governing such regulation, particularly in the context of cancer, remain poorly delineated. Here, high-resolution genomic profiles of GBM identified a highly recurrent focal 1p36 deletion encompassing the putative tumor suppressor gene, Mig-6. We show that Mig-6 quells the malignant potential of GBM cells and dampens EGFR signaling by driving EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation. Mechanistically, this effect is mediated by the binding of Mig-6 to a SNARE protein STX8, a protein known to be required for late endosome trafficking. Thus, Mig-6 functions to ensure recruitment of internalized receptor to late endosomes and subsequently the lysosomal degradation compartment through its ability to specifically link EGFR and STX8 during ligand-stimulated EGFR trafficking. In GBM, the highly frequent loss of Mig-6 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, these data uncover a unique tumor suppression mechanism involving the regulation of receptor trafficking.

Epidermal growth factor receptor activation in an in vitro diabetes model

Diabetes is the fifth leading cause of death in the United States and a major cause of morbidity. One of the more troubling complications in diabetes are non‐healing skin wounds, where high glucose levels and poor blood flow contribute to the formation of infected sores and ulcers. Hyperglycemia leads to the accumulation of advanced glycation end products, which can alter receptor functions. Because the epidermal growth factor receptor (EGFR) plays a crucial role in keratinocyte migration and wound reepithelialization, we tested the impact of high glucose on EGFR signaling in a human keratinocyte cell line. Basal EGFR tyrosine phosphorylation was decreased in cells following chronic exposure to high glucose (4.5 g/l) compared to cells maintained in normal glucose (1.0 g/l). Acute exposure of cells with high glucose diminished ligand‐stimulated EGFR tyrosine phosphorylation. This response was evident within 2‐4 hrs of high glucose treatment and became more pronounced with extended exposure. Similarly, downstream EGFR signaling, as detected by ERK phosphorylation, was decreased with time following high glucose treatment. These experiments demonstrate that high glucose interferes with EGF receptor function in keratinocytes with predicted impacts on cell migration and other EGFR‐dependent functions in wound reepithelialization. Funded by NIH award R01 GM079381.

Abl Tyrosine Kinase Regulates Endocytosis of the Epidermal Growth Factor Receptor

Signal attenuation from ligand-activated epidermal growth factor receptor (EGFR) is mediated in part by receptor endocytosis and trafficking to the lysosomal degradative compartment. Uncoupling the activated EGFR from endocytosis and degradation has emerged as a mechanism for oncogenic activation of the EGFR. The Abl nonreceptor tyrosine kinase is activated by ligand-stimulated EGFR, but the role of Abl in EGFR signaling has not been defined. Here we uncovered a novel role for the activated Abl kinase in the regulation of EGFR endocytosis. We show that activated Abl impairs EGFR internalization. Moreover, we show that activated Abl phosphorylates the EGFR primarily on tyrosine 1173, and that mutation of this site to phenylalanine restores ligand-dependent endocytosis of the EGFR in the presence of activated Abl. Furthermore, we show that activated Abl allows the ligand-activated EGFR to escape Cbl-dependent down-regulation by inhibiting the accumulation of Cbl at the plasma membrane in response to epidermal growth factor stimulation and disrupting the formation of the EGFR.Cbl complex without affecting Cbl protein stability. These findings reveal a novel role for Abl in promoting increased cell-surface expression of the EGFR and suggest that Abl/EGFR signaling may cooperate in human tumors.

Distinct Turnover of Alternatively Spliced Isoforms of the RET Kinase Receptor Mediated by Differential Recruitment of the Cbl Ubiquitin Ligase

Alternative splicing of transcripts encoding the RET kinase receptor leads to isoforms differing in their cytoplasmic tail. Although in vitro studies have demonstrated a higher transforming activity of the long RET isoform (RET51), only the short isoform (RET9) can rescue the effects of a RET null mutation in the enteric nervous system and kidney development. The molecular basis underlying the distinct functions of the two RET isoforms is not understood. Here we demonstrated that activated RET51 associated more strongly with the ubiquitin ligase Cbl than did RET9, leading to increased ubiquitylation and faster turnover of RET51. The association of Cbl with RET was indirect and was mediated through Grb2. A constitutive complex of Grb2 and Cbl could be recruited to both receptor isoforms via docking of Shc to phosphorylated Tyr-1062 in RET. A mutant Shc protein unable to recruit the Grb2.Cbl complex decreased the turnover and prolonged the half-life of RET9, thus ascribing a previously unknown negative role to the Shc adaptor molecule. In addition, phosphorylation of Tyr-1096, which is present in RET51 but absent in RET9, endowed the longer isoform with a second route to recruit the Grb2.Cbl complex. These findings establish a mechanism for the differential down-regulation of RET9 and RET51 signaling that could explain the apparently paradoxical activities of these two RET isoforms. More generally, these results illustrate how alternative splicing can regulate the half-life and function of a growth factor receptor.

Stabilization of the E3 ubiquitin ligase Nrdp1 by the deubiquitinating enzyme USP8.

Nrdp1 is a RING finger-containing E3 ubiquitin ligase that physically interacts with and regulates steady-state cellular levels of the ErbB3 and ErbB4 receptor tyrosine kinases and has been implicated in the degradation of the inhibitor-of-apoptosis protein BRUCE. Here we demonstrate that the Nrdp1 protein undergoes efficient proteasome-dependent degradation and that mutations in its RING finger domain that disrupt ubiquitin ligase activity enhance stability. These observations suggest that Nrdp1 self-ubiquitination and stability could play an important role in regulating the activity of this protein. Using affinity chromatography, we identified the deubiquitinating enzyme USP8 (also called Ubpy) as a protein that physically interacts with Nrdp1. Nrdp1 and USP8 could be coimmunoprecipitated, and in transfected cells USP8 specifically bound to Nrdp1 but not cbl, a RING finger E3 ligase involved in ligand-stimulated epidermal growth factor receptor down-regulation. The USP8 rhodanese and catalytic domains mediated Nrdp1 binding. USP8 markedly enhanced the stability of Nrdp1, and a point mutant that disrupts USP8 catalytic activity destabilized endogenous Nrdp1. Our results indicate that Nrdp1 is a specific target for the USP8 deubiquitinating enzyme and are consistent with a model where USP8 augments Nrdp1 activity by mediating its stabilization.

Reversible G1 Arrest Induced by Inhibition of the Epidermal Growth Factor Receptor Tyrosine Kinase Requires Up-regulation of p27KIP1 Independent of MAPK Activity

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G(1) to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1-10 microM AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1-1 microM AG-1517 abrogated (125)I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 microM. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G(1) phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G(1) arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G(1) arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G(1). These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G(1) arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.

The RING Finger of c-Cbl Mediates Desensitization of the Epidermal Growth Factor Receptor

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both down-regulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of c-Cbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.