The Constitutive Activity of Epidermal Growth Factor Receptor vIII Leads to Activation and Differential Trafficking of Wild-type Epidermal Growth Factor Receptor and erbB2

A constitutively active epidermal growth factor receptor (EGFR) mutant, EGFR variant III (EGFRvIII), has been detected at high frequencies in certain human cancers. This study evaluated transactivation and trafficking of erbB family members as a result of constitutive EGFR activity in a cancer cell line. Expression of EGFRvIII modulated erbB family members through different mechanisms; the erbB3 mRNA level was reduced, whereas wild-type EGFR (wtEGFR) and erbB2 protein levels were diminished, with no change in their mRNA levels, and there was no change in the erbB4 expression level. Both EGFR and erbB2 were internalized as a result of EGFRvIII''s activity and redistributed to the cell surface upon addition of AG1478, an inhibitor of wtEGFR/EGFRvIII catalytic activity. Acute activation of EGFRvIII by removing AG1478 from cells increased phosphorylation of both wtEGFR and erbB2 and caused differential trafficking of EGFRvIII''s activation partners; wtEGFR was directed primarily to lysosomal compartments and partially to recycling compartments, whereas erbB2 was directed primarily to recycling compartments and partially to lysosomal compartments. Our data demonstrate that the constitutive activity of EGFRvIII is sufficient to trigger endocytosis and trafficking of wtEGFR and erbB2, which may play a role in activating signaling pathways that are triggered during receptor endocytosis. (J Histochem Cytochem 58:529–541, 2010)     

EGFR突变种类与临床疗效关联

癌组织中表皮生长因子受体（epidermal growth factor receptor,EGFR）基因突变是应用靶向药物EGFR酪氨酸激酶抑制剂（tyrosine kinase inhibitor,TKI）治疗的一个重要相关因素及预测指标。对其突变的检测可以指导TKI类药物（TKIs）的最佳应用。该种突变常出现在非小细胞肺癌（NSCLC）中,尤其是在亚洲女性、肺腺癌、非吸烟者中,与非小细胞肺癌患者对TKIs治疗的敏感性密切相关。本文旨在探讨利用EGFR基因的已知突变热点的相关知识选择适合不同分子遗传学背景的群体或/和个体的＂个体化＂治疗方案,最终达到延长肺癌患者生存时间和提高生活质量的双重目的。     

Synthesis,Characterization, and Anti‐Amoebic Activity of N‐(Pyrimidin‐2‐yl)benzenesulfonamide Derivatives

A new series of N‐(pyrimidin‐2‐yl)benzenesulfonamide derivatives, 3a – 3i and 4a – 4i , was synthesized from pyrimidin‐2‐amines, 2a – 2i , with the aim to explore their effects on in vitro growth of Entamoeba histolytica. The chemical structures of the compounds were elucidated by elemental analysis, FT‐IR, ¹H‐ and ¹³C‐NMR, and ESI mass‐spectral data. In vitro anti‐amoebic activity was evaluated against HM1 : IMSS strain of Entamoeba histolytica. The IC₅₀ values were calculated by using the double dilution method. The results were compared with the IC₅₀ value of the standard drug ‘metronidazole’. The selected compounds were tested for their cytotoxic activities by cell‐viability assay using H9C2 cardiac myoblasts cell line, and the results indicated that all the compounds displayed remarkable >80% viabilities to a concentration of 100 μg/ml.     

Destabilization of the Epidermal Growth Factor Receptor (EGFR) by a Peptide That Inhibits EGFR Binding to Heat Shock Protein 90 and Receptor Dimerization

An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.     

Overexpression of RhoA-GTP induces activation of the Epidermal Growth Factor Receptor, dephosphorylation of focal adhesion kinase and increased motility in breast cancer cells

Rho GTPases are overexpressed in human tumors and are involved in a variety of cellular processes such as organization of the actin cytoskeleton, cell-cell contact and malignant transformation. EGFR activation plays a key role in the acquisition of motile properties in carcinoma cells, and it has been proposed that downregulation of FAK activity is one of its most relevant consequences. In the present study, using mammary MCF-7 cells, we demonstrated that overexpression of the active form of the small GTPase RhoA induced the activation of EGFR by a phenomenon that depends on the activity of a metalloproteinase (MMP), which presumably cleaves a membrane-bound EGFR ligand. The EGFR tyrosine phosphorylation correlates with ERK1,2 activation and the stimulation of urokinase production. An aggressive mammary cell line (MDA-MB-231) that overexpresses both RhoA and EGFR in their active forms also displayed an MMP-dependent activation mechanism of EGFR. RhoA-GTP-transfected cells showed a cortical array of F-actin, rounded morphology, reduced spreading potential and a dephosphorylation of FAK that was released by integrin-dependent fibronectin adhesion and a specific EGFR tyrosine kinase inhibitor. Our results suggest that the MMP-dependent EGFR activation observed in V14 RhoA cells represents the starting point of a signaling route that promotes cell motility by activation of ERK1,2 and further enhancement of proteases production.     

P2Y2 Nucleotide Receptors Mediate Metalloprotease-dependent Phosphorylation of Epidermal Growth Factor Receptor and ErbB3 in Human Salivary Gland Cells

The G protein-coupled receptor P2Y₂ nucleotide receptor (P2Y₂R) has been shown to be up-regulated in a variety of tissues in response to stress or injury. Recent studies have suggested that P2Y₂Rs may play a role in immune responses, wound healing, and tissue regeneration via their ability to activate multiple signaling pathways, including activation of growth factor receptors. Here, we demonstrate that in human salivary gland (HSG) cells, activation of the P2Y₂R by its agonist induces phosphorylation of ERK1/2 via two distinct mechanisms, a rapid, protein kinase C-dependent pathway and a slower and prolonged, epidermal growth factor receptor (EGFR)-dependent pathway. The EGFR-dependent stimulation of UTP-induced ERK1/2 phosphorylation in HSG cells is inhibited by the adamalysin inhibitor tumor necrosis factor-α protease inhibitor or by small interfering RNA that selectively silences ADAM10 and ADAM17 expression, suggesting that ADAM metalloproteases are required for P2Y₂R-mediated activation of the EGFR. G protein-coupled receptors have been shown to promote proteolytic release of EGFR ligands; however, neutralizing antibodies to known ligands of the EGFR did not inhibit UTP-induced EGFR phosphorylation. Immunoprecipitation experiments indicated that UTP causes association of the EGFR with another member of the EGF receptor family, ErbB3. Furthermore, stimulation of HSG cells with UTP induced phosphorylation of ErbB3, and silencing of ErbB3 expression inhibited UTP-induced phosphorylation of both ErbB3 and EGFR. UTP-induced phosphorylation of ErbB3 and EGFR was also inhibited by silencing the expression of the ErbB3 ligand neuregulin 1 (NRG1). These results suggest that P2Y₂R activation in salivary gland cells promotes the formation of EGFR/ErbB3 heterodimers and metalloprotease-dependent neuregulin 1 release, resulting in the activation of both EGFR and ErbB3.     

Different Epidermal Growth Factor Receptor (EGFR) Agonists Produce Unique Signatures for the Recruitment of Downstream Signaling Proteins

The EGF receptor can bind seven different agonist ligands. Although each agonist appears to stimulate the same suite of downstream signaling proteins, different agonists are capable of inducing distinct responses in the same cell. To determine the basis for these differences, we used luciferase fragment complementation imaging to monitor the recruitment of Cbl, CrkL, Gab1, Grb2, PI3K, p52 Shc, p66 Shc, and Shp2 to the EGF receptor when stimulated by the seven EGF receptor ligands. Recruitment of all eight proteins was rapid, dose-dependent, and inhibited by erlotinib and lapatinib, although to differing extents. Comparison of the time course of recruitment of the eight proteins in response to a fixed concentration of each growth factor revealed differences among the growth factors that could contribute to their differing biological effects. Principal component analysis of the resulting data set confirmed that the recruitment of these proteins differed between agonists and also between different doses of the same agonist. Ensemble clustering of the overall response to the different growth factors suggests that these EGF receptor ligands fall into two major groups as follows: (i) EGF, amphiregulin, and EPR; and (ii) betacellulin, TGFα, and epigen. Heparin-binding EGF is distantly related to both clusters. Our data identify differences in network utilization by different EGF receptor agonists and highlight the need to characterize network interactions under conditions other than high dose EGF.     

Larvicidal and Structure–Activity Studies of Natural Phenylpropanoids and Their Semisynthetic Derivatives against the Tobacco Armyworm Spodoptera litura (Fab.) (Lepidoptera: Noctuidae)

The larvicidal activity of 18 phenylpropanoids, 1 – 18 , including phenylpropenoate, phenylpropenal, phenylpropene, and their semisynthetic analogues, were evaluated against the tobacco armyworm, Spodoptera litura (Fab .), to identify promising structures with insecticidal activity. Amongst various phenylpropanoids, isosafrole, a phenylpropene, showed the best activity, with an LC₅₀ value of 0.6 μg/leaf cm², followed by its hydrogenated derivative dihydrosafrole (LC₅₀=2.7 μg/leaf cm²). The overall larvicidal activity of various phenylpropene derivatives was observed in the following order: isosafrole ( 6 )>dihydrosafrole ( 16 )>safrole ( 12 )>anethole ( 4 )>methyl eugenol ( 11 )>eugenol ( 13 )>β‐asarone ( 8 )>dihydroasarone ( 18 )>dihydroanethole ( 15 ). Dihydrosafrole might be a promising compound, although presenting a lower larvicidal activity than isosafrole, because of its better stability and resistance to oxidative degradation (due to the removal of the extremely reactive olefinic bond) in comparison to isosafrole. Such structure–activity relationship studies promote the identification of lead structures from natural sources for the development of larvicidal products against S. litura and related insect pests.     

Constitutive Activation of Epidermal Growth Factor Receptor Promotes Tumorigenesis of Cr(VI)-transformed Cells through Decreased Reactive Oxygen Species and Apoptosis Resistance Development

Hexavalent chromium (Cr(VI)) compounds are well-established lung carcinogens. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor that regulates cell survival, tumor invasion, and angiogenesis. Our results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) is able to cause malignant cell transformation. These transformed cells exhibit apoptosis resistance with reduced poly ADP-ribose polymerase cleavage (C-PARP) and Bax expression and enhanced expressions of Bcl-2 and Bcl-xL. These transformed cells also exhibit reduced capacity of reactive oxygen species (ROS) generation along with elevated expression of antioxidant manganese superoxide dismutase 2 (SOD2). The expression of this antioxidant was also elevated in lung tumor tissue from a worker exposed to Cr(VI) for 19 years. EGFR was activated in Cr(VI)-transformed BEAS-2B cells, lung tissue from animals exposed to Cr(VI) particles, and human lung tumor tissue. Further study indicates that constitutive activation of EGFR in Cr(VI)-transformed cells was due to increased binding to its ligand amphiregulin (AREG). Inhibition of EGFR or AREG increased Bax expression and reduced Bcl-2 expression, resulting in reduced apoptosis resistance. Furthermore, inhibition of AREG or EGFR restored capacity of ROS generation and decreased SOD2 expression. PI3K/AKT was activated, which depended on EGFR in Cr(VI)-transformed BEAS-2B cells. Inhibition of PI3K/AKT increased ROS generation and reduced SOD2 expression, resulting in reduced apoptosis resistance with commitment increase in Bax expression and reduction of Bcl-2 expression. Xenograft mouse tumor study further demonstrates the essential role of EGFR in tumorigenesis of Cr(VI)-transformed cells. In summary, the present study suggests that ligand-dependent constitutive activation of EGFR causes reduced ROS generation and increased antioxidant expression, leading to development of apoptosis resistance, contributing to Cr(VI)-induced tumorigenesis.     

Antitumor‐Promoting Effects and Cytotoxic Activities of Dammar Resin Triterpenoids and Their Derivatives

Nineteen known triterpenoids, 1 – 19 , and one known sesquiterpenoid, 20 , were isolated from dammar resin obtained from Shorea javanica K. & V. (Dipterocarpaceae). One of the acidic triterpenoids, dammarenolic acid ( 1 ), was converted to fourteen derivatives, namely, an alcohol, 21 , an aldehyde, 22 , and twelve L ‐amino acid conjugates, 23 – 34 . Compounds 1 – 34 were examined for their inhibitory effects on the induction of Epstein–Barr virus early antigen (EBV‐EA) by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Raji cells, a known primary screening test for antitumor promoters. All of the compounds tested, except for compounds 4, 5, 12 – 14, 16 , and 17 , showed inhibitory effects against EBV‐EA activation with potencies either comparable with or stronger than that of β‐carotene, a known natural antitumor promoter. In addition, (20S)‐20‐hydroxy‐3,4‐secodammara‐4(28),24‐dien‐3‐al ( 22 ) exhibited inhibitory effects on skin tumor promotion in an in vivo two‐stage mouse skin carcinogenesis test based on 7,12‐dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter. Furthermore, evaluation of the cytotoxic activities of compounds 1 – 34 against human cancer cell lines showed that reduction (i.e., 21 and 22 ) or conjugation with L ‐amino acids (i.e., 23 – 34 ) of compound 1 enhanced the cytotoxicity against human melanoma cell line CRL1579.     

Probing the Ubiquinone Reduction Site in Bovine Mitochondrial Complex I Using a Series of Synthetic Ubiquinones and Inhibitors

Studies of the structure–activity relationships of ubiquinones and specific inhibitors are helpful to probe the structural and functional features of the ubiquinone reduction site of bovine heart mitochondrial complex I. Bulky exogenous short-chain ubiquinones serve as sufficient electron acceptors from the physiological ubiquinone reduction site of bovine complex I. This feature is in marked contrast to other respiratory enzymes such as mitochondrial complexes II and III. For various complex I inhibitors, including the most potent inhibitors, acetogenins, the essential structural factors that markedly affect the inhibitory potency are not necessarily obvious. Thus, the loose recognition by the enzyme of substrate and inhibitor structures may reflect the large cavitylike structure of the ubiquinone (or inhibitor) binding domain in the enzyme. On the other hand, several phenomena are difficult to explain by a simple one-catalytic site model for ubiquinone.     

乳腺癌组织表皮生长因子受体、细胞角蛋白5/6、上皮钙黏蛋白表达与预后的关系研究

摘要 目的：探讨乳腺癌组织表皮生长因子受体(EGFR)、细胞角蛋白5/6(CK5/6)、上皮钙黏蛋白(E-Cad)表达与预后的关系。方法：选取我院(2015年1月～2017年12月)收治的100例接受乳腺外科手术治疗的乳腺癌患者,免疫组织化学检测乳腺癌组织和癌旁组织中EGFR、CK5/6、E-Cad表达。比较乳腺癌组织与癌旁组织中EGFR、CK5/6、E-Cad的阳性表达率,分析乳腺癌组织中EGFR、CK5/6、E-Cad表达与临床病理特征的关系,Kaplan-Meier曲线分析EGFR、CK5/6、E-Cad不同表达患者的无病生存期(DFS)和总生存期(OS)曲线,多因素Cox回归分析乳腺癌预后的影响因素。结果：乳腺癌组织中EGFR、CK5/6的阳性表达率明显高于癌旁组织,E-Cad的阳性表达率明显低于癌旁组织(P＜0.05)。乳腺癌组织中EGFR、E-Cad阳性表达率与分化程度、TNM分期相关(P＜0.05),与年龄、肿瘤直径、病理类型无关(P＞0.05);CK5/6阳性表达率与年龄、肿瘤直径、分化程度、TNM分期无关(P＞0.05),与病理类型相关(P＜0.05)。Kaplan-Meier生存曲线显示,EGFR、CK5/6阳性表达患者3年DFS和OS明显低于阴性表达患者,E-Cad阳性表达患者3年DFS和OS明显高于阴性表达患者(P＜0.05)。多因素Cox回归分析显示,TNM分期Ⅲ期(HR=5.756,95%CI：1.535～21.591)、EGFR阳性(HR=8.090,95%CI：0.954～68.616)、CK5/6阳性(HR=4.507,95%CI：0.466～43.593)为乳腺癌预后独立危险因素,E-Cad阳性(HR=0.221,95%CI：0.048～1.020)为乳腺癌预后独立保护因素(P＜0.05)。结论：乳腺癌组织中EGFR、CK5/6表达明显升高,E-Cad表达明显降低,三者为乳腺癌患者预后独立影响因素。     

UTP Controls Cell Surface Distribution and Vasomotor Activity of the Human P2Y2 Receptor through an Epidermal Growth Factor Receptor-transregulated Mechanism

Extracellular nucleotides transmit signals into the cells through the P2 family of cell surface receptors. These receptors are amply expressed in human blood vessels and participate in vascular tone control; however, their signaling mechanisms remain unknown. Here we show that in smooth muscle cells of isolated human chorionic arteries, the activation of the P2Y₂ receptor (P2Y₂R) induces not only its partition into membrane rafts but also its rapid internalization. Cholesterol depletion with methyl-β-cyclodextrin reduced the association of the agonist-activated receptor into membrane rafts but did not affect either the UTP-mediated vasoconstrictions or the vasomotor responses elicited by both serotonin and KCl. Ex vivo perfusion of human chorionic artery segments with 1–10 μm UTP, a selective P2Y₂R agonist, displaced the P2Y₂R localization into membrane rafts within 1 min, a process preceded by the activation of both RhoA and Rac1 GTPases. AG1478, a selective and potent inhibitor of the epidermal growth factor receptor tyrosine kinase activity, not only blocked the UTP-induced vasomotor activity but also abrogated both RhoA and Rac1 activation, the P2Y₂R association with membrane rafts, and its internalization. Altogether, these results show for the first time that the plasma membrane distribution of the P2Y₂R is transregulated by the epidermal growth factor receptor, revealing an unsuspected functional interplay that controls both the membrane distribution and the vasomotor activity of the P2Y₂R in intact human blood vessels.