TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation.Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown.Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGFβ enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells.Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGFβ might be an important pathway of gastric cancer cell proliferation by TGFβ.     

Contact-dependent growth inhibition and apoptosis of epidermal growth factor (EGF) receptor-expressing cells by the membrane-anchored form of heparin-binding EGF-like growth factor.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) transduces mitogenic signals through the EGF receptor (EGFR). There are two forms of HB-EGF, the membrane-anchored form (pro-HB-EGF) and the soluble form (sHB-EGF). We studied the biological activity of pro-HB-EGF by using a model in which pro-HB-EGF-expressing effector cells was co-cultured with EGFR-expressing target cells. The DER cell, an EGFR-expressing derivative of the interleukin-3-dependent hematopoietic 32D cell line, grows well in the presence of EGF or sHB-EGF without IL-3. When DER cells were co-cultured on a monolayer of Vero-H cells overexpressing pro-HB-EGF, growth inhibition and subsequent apoptosis were induced in the DER cells even in the presence of excess amounts of EGF or sHB-EGF. Such growth inhibition of DER cells was abrogated when specific antagonists for pro-HB-EGF were added in the culture medium or when direct contact of DER cells with Vero-H cells was prevented, indicating that pro-HB-EGF is involved in this inhibitory effect. Pro-HB-EGF-induced apoptosis of DER cells was also observed even in the presence of IL-3. This rules out the possibility of simple competition between soluble EGFR ligands and pro-HB-EGF. Moreover, 32D cells expressing EGFR mutant composed of the extracellular and the transmembrane domain of EGFR and the cytoplasmic domain of erythropoietin receptor did not undergo apoptosis by co-culture with Vero-H cells, indicating that the inhibitory signal induced by pro-HB-EGF-expressing Vero-H cells is mediated to DER cells via EGFR and that the cytoplasmic domain of EGFR is essential for pro-HB-EGF-induced apoptosis. From these results, we concluded that pro-HB-EGF has unique biological activity through cell-cell contact that is distinct from the activity of sHB-EGF.     

Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway

The epidermal growth factor (EGF) transduces its actions via the EGF receptor (EGFR), which can traffic from the plasma membrane to either the cytoplasm or the nucleus. However, the mechanism by which EGFR reaches the nucleus is unclear. To investigate these questions, liver cells were analyzed by immunoblot of cell fractions, confocal immunofluorescence and real time confocal imaging. Cell fractionation studies showed that EGFR was detectable in the nucleus after EGF stimulation with a peak in nuclear receptor after 10 min. Movement of EGFR to the nucleus was confirmed by confocal immunofluorescence and labeled EGF moved with the receptor to the nucleus. Small interference RNA (siRNA) was used to knockdown clathrin in order to assess the first endocytic steps of EGFR nuclear translocation in liver cells. A mutant dynamin (dynamin K44A) was also used to determine the pathways for this traffic. Movement of labeled EGF or EGFR to the nucleus depended upon dynamin and clathrin. This identifies the pathway that mediates the first steps for EGFR nuclear translocation in liver cells.     

HDAC6 is required for epidermal growth factor-induced beta-catenin nuclear localization

Nuclear translocation of beta-catenin is a hallmark of Wnt signaling and is associated with various cancers. In addition to the canonical Wnt pathway activated by Wnt ligands, growth factors such as epidermal growth factor (EGF) also induce beta-catenin dissociation from the adherens junction complex, translocation into the nucleus, and activation of target genes such as c-myc. Here we report that EGF-induced beta-catenin nuclear localization and activation of c-myc are dependent on the deacetylase HDAC6. We show that EGF induces HDAC6 translocation to the caveolae membrane and association with beta-catenin. HDAC6 deacetylates beta-catenin at lysine 49, a site frequently mutated in anaplastic thyroid cancer, and inhibits beta-catenin phosphorylation at serine 45. HDAC6 inactivation blocks EGF-induced beta-catenin nuclear localization and decreases c-Myc expression, leading to inhibition of tumor cell proliferation. These results suggest that EGF-induced nuclear localization of beta-catenin is regulated by HDAC6-dependent deacetylation and provide a new mechanism by which HDAC inhibitors prevent tumor growth.     

Multiple EGFR ligands participate in guiding migrating border cells

Cell migration is an important feature of embryonic development as well as tumor metastasis. Border cells in the Drosophila ovary have emerged as a useful in vivo model for uncovering the molecular mechanisms that control many aspects of cell migration including guidance. It was previously shown that two receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and PDGF- and VEGF-related receptor (PVR), together contribute to border cell migration. Whereas the ligand for PVR, PVF1, is known to guide border cells, it is unclear which of the four activating EGFR ligands function in this process. We developed an assay to detect the ability of secreted factors to reroute migrating border cells in vivo and tested the activity of EGFR ligands compared to PVF1. Two ligands, Keren and Spitz, guided border cells whereas the other ligands, Gurken and Vein, did not. In addition, only Keren and Spitz were expressed at the appropriate stage in the oocyte, the target of border cell migration. Therefore, a complex combination of EGFR and PVR ligands together guide border cells to the oocyte.     

Reactive oxygen species mediate Met receptor transactivation by G protein-coupled receptors and the epidermal growth factor receptor in human carcinoma cells

Cross-communication between the Met receptor tyrosine kinase and the epidermal growth factor receptor (EGFR) has been proposed to involve direct association of both receptors and EGFR kinase-dependent phosphorylation. Here, we demonstrate that in human hepatocellular and pancreatic carcinoma cells the Met receptor becomes tyrosine phosphorylated not only upon EGF stimulation but also in response to G protein-coupled receptor (GPCR) agonists. Whereas specific inhibition of the EGFR kinase activity blocked EGF- but not GPCR agonist-induced Met receptor transactivation, it was abrogated in the presence of a reducing agent or treatment of cells with a NADPH oxidase inhibitor. Both GPCR ligands and EGF are further shown to increase the level of reactive oxygen species within the cell. Interestingly, stimulation of the Met receptor by either GPCR agonists, EGF or its cognate ligand HGF, resulted in release of Met-associated beta-catenin and in its Met-dependent translocation into the nucleus, as analyzed by small interfering RNA-mediated knockdown of the Met receptor. Our results provide a new molecular explanation for cell surface receptor cross-talk involving the Met receptor and thereby link the wide diversity of GPCRs and the EGFR to the oncogenic potential of Met signaling in human carcinoma cells.     

Nuclear translocation of EGF receptor regulated by Epstein-Barr virus encoded latent membrane protein 1

Epstein-Barrvirus(EBV),oneoftheDNAon-cogenicviruses,iscloselyassociatedwiththegenesisofBurkitt抯lymphoma,undifferentiatednasopharyn-gealcarcinoma(NPC),Hodgkin抯disease,gastriccancerand,lungcancer,etc.[1].EBVencodedlatentmembraneprotein1(LMP1)isconsideredtobethemajoroncogenicproteinofEBVencodedproteins.Biologicallyspeaking,LMP1isanintegralmembraneproteincontaining386aminoacids.Thethreedo-mains(CTAR1,CTAR2,CTAR3)intheC-terminusofLMP1havebeenshowntoinitiatethesignalingproc-ess.The…     

Helicobacter pylori protein HP0175 transactivates epidermal growth factor receptor through TLR4 in gastric epithelial cells

The pathophysiology of Helicobacter pylori-associated gastroduodenal diseases, ulcerogenesis, and carcinogenesis is intimately linked to activation of epidermal growth factor receptor (EGFR) and production of vascular endothelial growth factor (VEGF). Extracellular virulence factors, such as CagA and VacA, have been proposed to regulate EGFR activation and VEGF production in gastric epithelial cells. We demonstrate that the H. pylori secretory protein, HP0175, by virtue of its ability to bind TLR4, transactivates EGFR and stimulates EGFR-dependent VEGF production in the gastric cancer cell line AGS. Knock-out of the hp0175 gene attenuates the ability of the resultant H. pylori strain to activate EGFR or to induce VEGF production. HP0175-induced activation of EGFR is preceded by translocation of TLR4 into lipid rafts. In lipid rafts, the Src kinase family member Lyn interacts with TLR4, leading to tyrosine phosphorylation of TLR4. Knockdown of Lyn prevents HP0175-induced activation of EGFR and VEGF production. Tyrosine-phosphorylated TLR4 interacts with EGFR. This interaction is necessary for the activation of EGFR. Disruption of lipid rafts with methyl beta-cyclodextrin prevents HP0175-induced tyrosine phosphorylation of TLR4 and activation of EGFR. This mechanism of transactivation of EGFR is novel and distinct from that of metalloprotease-dependent shedding of EGF-like ligands, leading to autocrine activation of EGFR. It provides new insight into our understanding of the receptor cross-talk network.     

Epidermal growth factor receptor (EGFR) signaling in cancer

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptor tyrosine kinases (RTK). These trans-membrane proteins are activated following binding with peptide growth factors of the EGF-family of proteins. Evidence suggests that the EGFR is involved in the pathogenesis and progression of different carcinoma types. The EGFR and EGF-like peptides are often over-expressed in human carcinomas, and in vivo and in vitro studies have shown that these proteins are able to induce cell transformation. Amplification of the EGFR gene and mutations of the EGFR tyrosine kinase domain have been recently demonstrated to occur in carcinoma patients. Interestingly, both these genetic alterations of the EGFR are correlated with high probability to respond to anti-EGFR agents. However, ErbB proteins and their ligands form a complex system in which the interactions occurring between receptors and ligands affect the type and the duration of the intracellular signals that derive from receptor activation. In fact, proteins of the ErbB family form either homo- or hetero-dimers following ligand binding, each dimer showing different affinity for ligands and different signaling properties. In this regard, evidence suggests that cooperation of multiple ErbB receptors and cognate ligands is necessary to induce cell transformation. In particular, the growth and the survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. This phenomenon is also important for therapeutic approaches, since the response to anti-EGFR agents might depend on the total level of expression of ErbB receptors and ligands in tumor cells.     

Role of the EGFR ligand/receptor system in the secretion of angiogenic factors in mesenchymal stem cells

Increasing evidence suggests that bone marrow-derived mesenchymal stem cells (MSCs) are recruited into the stroma of developing tumors where they contribute to cancer progression. MSCs produce different growth factors that sustain tumor-associated neo-angiogenesis. Since the majority of carcinomas secrete ligands of the epidermal growth factor receptor (EGFR), we assessed the role of EGFR signaling in regulating the release of angiogenic factors in MSCs. Treatment of human primary MSCs and of the human osteoblastic cell line hFOB with transforming growth factor α (TGF-α), one of the main ligands of the EGFR, significantly induced activation of this receptor and of different intracellular signaling proteins, including the PI3K/AKT and the MEK/MAPK pathways. TGF-α induced a significant increase in the levels of secretion of vascular endothelial growth factor in both MSCs and hFOB. Conditioned medium from TGF-α treated MSCs showed an higher in vivo angiogenic effect as compared with medium from untreated cells. Treatment of MSCs with TGF-α also produced a significant increase in the secretion of other angiogenic growth factors such as angiopoietin-2, granulocyte-colony stimulating factor, hepatocyte growth factor, interleukin (IL)-6, IL-8, and platelet-derived growth factor-BB. Using selective MEK and PI3K inhibitors, we found that both MEK/MAPK and the PI3K/AKT signaling pathways mediate the ability of TGF-α to induce secretion of angiogenic factors in MSCs. Finally, stimulation with TGF-α increased the ability of MSCs to induce migration of MCF-7 breast cancer cells. These data suggest that EGFR signaling regulates the ability of MSCs to sustain cancer progression through the release of growth factors that promote neo-angiogenesis and tumor cell migration.     

Membrane-bound trafficking regulates nuclear transport of integral epidermal growth factor receptor (EGFR) and ErbB-2

Nuclear localization of multiple receptor-tyrosine kinases (RTKs), such as EGF receptor (EGFR), ErbB-2, FGF receptor (FGFR), and many others, has been reported by several groups. We previously showed that cell surface EGFR is trafficked to the nucleus through a retrograde pathway from the Golgi to the endoplasmic reticulum (ER) and that EGFR is then translocated to the inner nuclear membrane (INM) through the INTERNET (integral trafficking from the ER to the nuclear envelope transport) pathway. However, the nuclear trafficking mechanisms of other membrane RTKs, apart from EGFR, remain unclear. The purpose of this study was to compare the nuclear transport of EGFR family proteins with that of FGFR-1. Interestingly, we found that digitonin permeabilization, which selectively releases soluble nuclear transporters from the cytoplasm and has been shown to inhibit nuclear transport of FGFR-1, had no effects on EGFR nuclear transport, raising the possibility that EGFR and FGFR-1 use different pathways to be translocated into the nucleus. Using the subnuclear fractionation assay, we further demonstrated that biotinylated cell surface ErbB-2, but not FGFR-1, is targeted to the INM, associating with Sec61β in the INM, similar to the nuclear trafficking of EGFR. Thus, ErbB-2, but not FGFR-1, shows a similar trafficking pathway to EGFR for translocation to the nucleus, indicating that at least two different pathways of nuclear transport exist for cell surface receptors. This finding provides a new direction for investigating the trafficking mechanisms of various nuclear RTKs.     

Nuclear accumulation of epidermal growth factor receptor and acceleration of G1/S stage by Epstein-Barr-encoded oncoprotein latent membrane protein 1

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV-encoded proteins and has always been the core of the oncogenic mechanism of EBV. Advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly improved our knowledge of the biological function of cell surface receptors. In this study, we used the Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1-integrated nasopharyngeal carcinoma (NPC) cell line and the expression of LMP1 which could be regulated by the Tet system. We found that LMP1 could regulate the nuclear accumulation of EGFR in a dose-dependent manner quantitatively and qualitatively. We also demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation and EGFR in the nucleus could bind to the promoters of cyclinD1 and cyclinE, respectively. We further demonstrated that EGFR is involved in the acceleration of the G1/S phase transition by LMP1 through binding to cyclinD1 and cyclinE directly. These findings provided a novel view that the acceleration of LMP1 on the G1/S transition via the nuclear accumulation of EGFR was critical in the process of nasopharyngeal carcinoma.     

Ligand-independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signaling

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between (835)Ala and (918)Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events.