Interferon-γ–inducible Rab20 regulates endosomal morphology and EGFR degradation in macrophages

Little is known about the molecular players that regulate changes in the endocytic pathway during immune activation. Here we investigate the role of Rab20 in the endocytic pathway during activation of macrophages. Rab20 is associated with endocytic structures, but the function of this Rab GTPase in the endocytic pathway remains poorly characterized. We find that in macrophages, Rab20 expression and endosomal association significantly increase after interferon-γ (IFN-γ) treatment. Moreover, IFN-γ and Rab20 expression induce a dramatic enlargement of endosomes. These enlarged endosomes are the result of homotypic fusion promoted by Rab20 expression. The expression of Rab20 or the dominant-negative mutant Rab20T19N does not affect transferrin or dextran 70 kDa uptake. However, knockdown of Rab20 accelerates epidermal growth factor (EGF) trafficking to LAMP-2–positive compartments and EGF receptor degradation. Thus this work defines a function for Rab20 in the endocytic pathway during immune activation of macrophages.     

TNF-α induces upregulation of EGFR expression and signaling in human colonic myofibroblasts

The myofibroblast has recently been identified as an important mediator of tumor necrosis factor-α (TNF-α)-associated colitis and cancer, but the mechanism(s) involved remains incompletely understood. Recent evidence suggests that TNF-α is a central regulator of multiple inflammatory signaling cascades. One important target of TNF-α may be the signaling pathway downstream of the epidermal growth factor receptor (EGFR), which has been associated with many human cancers. Here, we show that long-term exposure of 18Co cells, a model of human colonic myofibroblasts, with TNF-α led to a striking increase in cell surface EGFR expression, an effect that was completely inhibited by cycloheximide. Subsequent EGFR binding by EGF and heparin binding (HB)-EGF was associated with enhanced EGFR tyrosine kinase activity, prolonged ERK activation, and a significant increase in cyclooxygenase-2 (COX-2) expression compared with 18Co cells treated with EGF and HB-EGF alone. TNF-α also increased EGFR expression and signaling in primary myofibroblasts isolated from human colon tissue. TNF-α-induced upregulation of EGFR may be a plausible mechanism to explain the exaggerated cellular responsiveness that characterizes inflammatory bowel disease and that may contribute to a microenvironment that predisposes to colitis-associated cancer through enhanced COX-2 expression.     

Desmoglein 1–dependent suppression of EGFR signaling promotes epidermal differentiation and morphogenesis

Dsg1 (desmoglein 1) is a member of the cadherin family of Ca²⁺-dependent cell adhesion molecules that is first expressed in the epidermis as keratinocytes transit out of the basal layer and becomes concentrated in the uppermost cell layers of this stratified epithelium. In this study, we show that Dsg1 is not only required for maintaining epidermal tissue integrity in the superficial layers but also supports keratinocyte differentiation and suprabasal morphogenesis. Dsg1 lacking N-terminal ectodomain residues required for adhesion remained capable of promoting keratinocyte differentiation. Moreover, this capability did not depend on cytodomain interactions with the armadillo protein plakoglobin or coexpression of its companion suprabasal cadherin, Dsc1 (desmocollin 1). Instead, Dsg1 was required for suppression of epidermal growth factor receptor–Erk1/2 (extracellular signal-regulated kinase 1/2) signaling, thereby facilitating keratinocyte progression through a terminal differentiation program. In addition to serving as a rigid anchor between adjacent cells, this study implicates desmosomal cadherins as key components of a signaling axis governing epithelial morphogenesis.     

Forced dimerization increases the activity of ΔEGFR/EGFRvIII and enhances its oncogenicity

Delta epidermal growth factor receptor (ΔEGFR), an in-frame deletion mutant of the extracellular ligand-binding domain, which occurs in about 30% of glioblastoma, is a potent oncogene that promotes tumor growth and progression. The signaling of ΔEGFR is ligand-independent and low intensity, allowing it to evade the normal mechanisms of internalization and degradation by the endocytic machinery and hence is persistent. The basis of the oncogenic potential of ΔEGFR remains incompletely understood, including whether dimerization plays an important role in its signal and whether its oncogenic potential is dependent on its relatively low intensity, when compared with the acutely activated wild-type receptor. To examine these two important questions, we have generated a chimeric ΔEGFR that allows forced dimerization via domains derived from variants of the FKBP12 protein that are brought together by FK506 derivatives. Forced dimerization of chimeric ΔEGFR significantly increased the intensity of its signal, as measured by receptor phosphorylation levels, suggesting that the naturally occurring ΔEGFR does not form strong or stable dimers as part of its low level signal. Interestingly, the increased activity of dimerized, chimeric ΔEGFR did not promote receptor internalization, implying that reduced rate of endocytic downregulation of ΔEGFR is an inherent characteristic. Significantly, forced dimerization enhanced the oncogenic signal of the receptor, implying that the ΔEGFR is a potent oncogene despite, not because of its low intensity.     

Efficient EGFR signaling and dorsal–ventral axis patterning requires syntaxin dependent Gurken trafficking

Vesicle trafficking plays a crucial role in the establishment of cell polarity in various cellular contexts, including axis-pattern formation in the developing egg chamber of Drosophila. The EGFR ligand, Gurken (Grk), is first localized at the posterior of young oocytes for anterior–posterior axis formation and later in the dorsal anterior region for induction of the dorsal–ventral (DV) axis, but regulation of Grk localization by membrane trafficking in the oocyte remains poorly understood. Here, we report that Syntaxin 1A (Syx1A) is required for efficient trafficking of Grk protein for DV patterning. We show that Syx1A is associated with the Golgi membrane and is required for the transportation of Grk-containing vesicles along the microtubules to their dorsal anterior destination in the oocyte. Our studies reveal that the Syx1A dependent trafficking of Grk protein is required for efficient EGFR signaling during DV patterning.     

Quinazoline-1-deoxynojirimycin hybrids as high active dual inhibitors of EGFR and α-glucosidase

A series of novel quinazoline-1-deoxynojirimycin hybrids were designed, synthesized and evaluated for their inhibitory activities against two drug target enzymes, epidermal growth factor receptor (EGFR) tyrosine kinase and α-glucosidase. Some synthesized compounds exhibited significantly inhibitory activities against the tested enzymes. Comparing with reference compounds gefitinib and lapatinib, compounds 7d, 8d, 9b and 9d showed higher inhibitory activities against EGFR (IC₅₀: 1.79–10.71 nM). Meanwhile the inhibitory activities of 7d, 8d and 9c against α-glucosidase (IC₅₀ = 0.14, 0.09 and 0.25 µM, respectively) were obvious higher than that of miglitol (IC₅₀ = 2.43 µM), a clinical using α-glucosidase inhibitor. Interestingly, compound 9d as a dual inhibitor showed high inhibitory activity to EGFR^wt tyrosine kinase (IC₅₀ = 1.79 nM), also to α-glucosidase (IC₅₀ = 0.39 µM). The work could be very useful starting point for developing a new series of enzyme inhibitors targeting EGFR and/or α-glucosidase.     

Inhibitory effects of metachromins L–Q and its related analogs against receptor tyrosine kinases EGFR and HER2

Metachromins are a series of sesquiterpenoid quinones isolated from Okinawan marine sponges. Inhibitory effects of metachromins L–Q (1–6), sesquiterpenoid quinones with an amino acid residue, and their related analogs (7–18) prepared from metachromins A (19) and C (20) against receptor tyrosine kinases EGFR and HER2 were investigated. Two analogs 11 and 12 showed relatively stronger inhibitory activity against EGFR, while metachromins L–Q (1-6) and seven analogs (8, 10, 11, and 15–18) showed inhibitory activities against HER2.     

人EGFR显性负性突变体负调控内源性EGFR功能的机制分析

通过定向克隆法构建真核表达载体pEGFPN1-DNEGFR,脂质体介导下转染体外培养的SGC-7901细胞,应用Western blotting检测DNEGFR-EGFP蛋白的表达,激光共聚焦显微镜对DNEGFR-EGFP亚细胞结构定位检测;并经RT-PCR、Western blotting检测DNEGFR-EGFP对内源性EGFRmRNA水平、蛋白及磷酸化水平的影响.成功检测到DNEGFR-EGFP蛋白的表达,DNEGFR-EGFP蛋白主要定位于细胞膜,DNEGFR-EGFP能降低内源性EGFR蛋白磷酸化水平,而对内源性EGFRmRNA水平及蛋白水平无影响.研究证明DNEGFR通过降低内源性EGFR蛋白磷酸化水平负调控EGFR功能,为靶向EGFR显性负性策略在肿瘤生物治疗中的进一步研究打下基础.     

EGFR在胃癌中的研究进展

表皮生长因子受体（EGFR）通过下游的信号传导途径参与肿瘤细胞增殖、血管生成、侵袭、转移及凋亡抑制等的基因调控,在多种肿瘤组织中有高表达。近几年在胃癌的研究中显示：EGFR在胃癌中有高表达,与胃癌的发生、发展及生物学行为密切相关,被认为是胃癌等肿瘤治疗的理想靶点。当前以EGFR为靶点的生物治疗成为胃癌新的研究热点,本文就其近几年EGFR在胃癌的表达、预后及靶向治疗等研究现状作一综述。     

Synthesis,antitumour and antioxidant activities of novel α,β-unsaturated ketones and related heterocyclic analogues: EGFR inhibition and molecular modelling study

New α,β-unsaturated ketones 4a,b; 5a–c; and 6a,b; as well as 4-H pyran 7; pyrazoline 8a,b; isoxazoline 9; pyridine 10–11; and quinoline-4-carboxylic acid 12a,b derivatives were synthesized and evaluated for in vitro antitumour activity against HepG2, MCF-7, HeLa, and PC-3 cancer cell lines. Antioxidant activity was investigated by the ability of these compounds to scavenge the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^?+). Compounds 6a, 6b, 7, and 8b exhibited potent antitumour activities against all tested cell lines with [IC₅₀] ?5.5–18.1 µΜ), in addition to significantly high ABTS^?+ scavenging activities. In vitro EGFR kinase assay for 6a, 6b, 7, and 8b as the most potent antitumour compounds showed that; compounds 6b, and 7 exhibited worthy EGFR inhibition activity with IC₅₀ values of 0.56 and 1.6?µM, respectively, while compounds 6a and 8b showed good inhibition activity with IC₅₀ values of 4.66 and 2.16?µM, respectively, compared with sorafenib reference drug (IC₅₀?=?1.28?µM). Molecular modelling studies for compounds 6b, 7, and 8b were conducted to exhibit the binding mode towards EGFR kinase, which showed similar interaction with erlotinib.     

Both EGFR kinase and Src-related tyrosine kinases regulate human ether-à-go-go-related gene potassium channels

Human ether-à-go-go-related gene (hERG or Kv11.1) encodes the rapidly activated delayed rectifier K(+) current (I(Kr)) in the human heart. Potential regulation of hERG channel by protein tyrosine kinases (PTKs) is not understood. The present study was designed to investigate whether this channel is modulated by PTKs using whole-cell patch clamp technique, and immunoprecipitation and Western blot analysis in HEK 293 cells stably expressing hERG gene. We found that the broad-spectrum PTK inhibitor genistein (30 muM), the selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 (10 muM) and the Src-family kinase inhibitor PP2 (10 muM) remarkably inhibited hERG channel current (I(hERG)), and the effects were significantly countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (1 mM). Immunoprecipitation and Western blot analysis demonstrated that membrane protein tyrosine phosphorylation of hERG channels was reduced by genistein, AG556, and PP2. The reduction of hERG channel phosphorylation level by genistein, AG556 or PP2 was antagonized by orthovanadate. Single point mutation(s) of Y475A and/or Y611A dramatically attenuated the inhibitory effect of I(hERG) by PP2 and/or AG556. Our results demonstrate the novel information that I(hERG) is modulated not only by Src-family kinases, but also by EGFR kinases. Y475 and/or Y611 are likely the preferred phosphorylation sites. Regulation of hERG channels by PTKs modifies the channel activity and thus likely alters electrophysiological properties including action potential duration and cell excitability in human heart and neurons.     

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.     

EGFR配体生物学效应的多样性

表皮生长因子受体（epidermal growth factor receptor, EGFR）的配体作为一类重要的信号分子参与了细胞功能的调节,并且和机体发育、器官形成、组织修复与稳态维持,以及疾病的发生密切相关。虽然这些信号分子具有序列和结构上的相似性,但由于这些信号分子结构上的细微差异以及它们受体信号传导上的复杂性,造成了这些信号分子（配体）生物学效应的多样性。目前,从结构和机制上,对于单个信号分子的生物学效应已有较为深入的研究,但这些信号分子之间以及信号分子与受体之间的调控网络较为复杂,并且这种调控网络对信号的精细、有序和多样化转导至关重要。本文对EGFR配体的结构及配体生物学效应多样性的分子机制进行回顾,并对未来的研究方向提出展望。     

Construction and humanization of a functional bispecific EGFR × CD16 diabody using a refolding system

We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti-EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR-expressing tumor cells. After we confirmed that an anti-EGFR?×?anti-CD16 bispecific diabody (Ex16) consisting of a previously humanized anti-EGFR variable fragment (Fv) and a mouse anti-CD16 Fv had growth inhibitory activity, we designed a humanized anti-CD16 Fv to construct the fully humanized Ex16 (hEx16). However, the humanized form had lower activity for inhibition of cancer growth. To restore its growth inhibitory activity, we introduced mutations into the Vernier zone, which is located near the complementarity-determining regions and is involved in their binding activity. We efficiently prepared 15 different hEx16 mutants by expressing each chimeric single-chain component for hEx16 separately. We then used our in vitro refolding system to select the most functional mutant, which had a growth inhibitory effect comparable with that of the commercially available chimeric anti-EGFR antibody, cetuximab. Our refolding system could aid in the efficient optimization of other proteins with heterodimeric structure.     

EGFR配体生物学效应的多样性

表皮生长因子受体（epidermal growth factor receptor, EGFR）的配体作为一类重要的信号分子参与了细胞功能的调节,并且和机体发育、器官形成、组织修复与稳态维持,以及疾病的发生密切相关。虽然这些信号分子具有序列和结构上的相似性,但由于这些信号分子结构上的细微差异以及它们受体信号传导上的复杂性,造成了这些信号分子（配体）生物学效应的多样性。目前,从结构和机制上,对于单个信号分子的生物学效应已有较为深入的研究,但这些信号分子之间以及信号分子与受体之间的调控网络较为复杂,并且这种调控网络对信号的精细、有序和多样化转导至关重要。本文对EGFR配体的结构及配体生物学效应多样性的分子机制进行回顾,并对未来的研究方向提出展望。     

抗EGFR抗体研究有望成功

2010年5月6日．在美国芝加哥召开的BIO International Convention（BIO2010）会场上．丹麦抗体医药风险企业Symphogen公司的Kristen Drejier首席执行官（CEO）应《日经生物技术产业》刊物的采访,关于2010年4月开始了I期临床试验的以EGFR（上皮细胞生长因子受体）为靶点的抗体医药Sym004发表了“从前临床试验结果来看．非常有希望”的谈话。     

Schiff’s base derivatives bearing nitroimidazole and quinoline nuclei: New class of anticancer agents and potential EGFR tyrosine kinase inhibitors

New Schiff’s base derivatives 5a–j have been synthesized by reaction between 2-phenoxyquinoline-3-carbaldehydes 3a–j and 2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide 4 in presence of nickel(II) nitrate as a catalyst in ethanol under reflux in good yield (78–92%). All compounds were tested for anticancer and inhibition of EGFR. Of the compounds studied, majority of the compounds showed effective antiproliferation and inhibition of EGFR and HER-2 activities. Compound 5h showed most effective inhibition (IC₅₀ = 0.12 ± 0.05 μM) by binding in to the active pocket of EGFR receptor with minimum binding energy (ΔG_b = −58.3691 kcal/mol). The binding was stabilized by two hydrogen bonds, two π–cation and one π–sigma interactions. Compound 5d showed most effective inhibition (IC₅₀ = 0.37 ± 0.04 μM).