EGF receptor activation by heterologous mechanisms

A myriad of growth factor-dependent and -independent mechanisms for activating the EGF receptor and the related ErbB receptors underscore the importance of receptor inhibitors in the treatment of some solid tumors.     

Monoclonal antibodies directed against the EGF receptor show differential bindings of amphiregulin and EGF to the EGF receptor

Amphiregulin (AR), a new member of the EGF family of ligand, is a glycoprotein containing a 78 or 84 amino acid core polypeptide that was originally purified from the conditioned medium of the breast carcinoma cell line MCF-7 after treatment with phorbol 12-myristate 13-acetate. The aim of the present study was to determine whether, like EGF, TGF alpha, heparin binding EGF-related growth factor (HB-EGF) and betacellulin (ETC), the recombinant 78 amino acid form of mature human AR transmits its biological effects following binding to the EGF receptor (EGFR). We show that unlike EGF, TGF alpha, HB-EGF and BTC, the mature AR is not effective in blocking the binding of I-125-EGF or the iodinated anti-EGFR antibodies (mAbs) I-125-ICR62 and I-125-ICR80 to the external domain of the EGF receptor on EJ cells. Again, in contrast to other EGF ligands, AR is not effective in enhancing the binding of another anti-EGFR mAb ICR9 to the EGFR on EJ cells. Like EGF, TGF alpha and HB-EGF, AR could inhibit the growth in culture of EGFR overexpressing tumour cell lines, namely HN5, HSC-1 and MDA-MB468 cells, and again compared to other ligands AR was moderately effective at low concentration. Despite these differences, we show that like EGF, AR could induce the tyrosine phosphorylation of the 170 kDa EGF receptor on HN5 cells and that this effect could be blocked in the presence of anti-EGFR mAbs ICR62 and ICR80. Moreover, like EGF, the AR-induced growth inhibition of MDA-MB468 cells could also be reversed in the presence of anti-EGFR mAbs ICR62 and ICR80. On the basis of our results we conclude that, unlike the EGF, TGF alpha, HB-EGF and BTC, the AR-induced activation of the EGFR may involve another receptor.     

Absence of constitutive EGF receptor activation in ovarian cancer cell lines.

Previous investigators have noted that certain ovarian cancer cell lines secrete and respond to transforming growth factor-alpha (TGF-alpha), suggesting that endogenous activation of the epidermal growth factor (EGF) receptor through autocrine or paracrine mechanisms might contribute to the proliferative response. In order to determine whether autocrine stimulation was partly responsible for the proliferative response in ovarian cancer, we investigated whether the EGF receptor expressed by ovarian cancer cell lines was constitutively activated as assessed by the presence of tyrosine phosphorylation. A specific anti-phosphotyrosine antibody was used in conjunction with an immunoblotting technique in order to detect EGF receptor phosphorylation in ovarian cancer cell lines in the absence and presence of exogenous EGF. The effects of neutralising anti-EGF receptor antibody on the proliferation of ovarian cancer cell lines was also examined. We found no evidence for constitutive tyrosine phosphorylation of the p170 EGF receptor in eight epithelial ovarian cancer cell lines tested, although each line demonstrated inducible phosphorylation in response to exogenous EGF. The absence of constitutive EGF receptor activation was also noted when cells were grown under high density conditions, thus excluding a role for membrane-bound EGF or TGF-alpha in this process. Media conditioned by five ovarian cancer cell lines, as well as malignant ascites obtained from 12 different ovarian cancer patients, were not capable of stimulating EGF receptor phosphorylation. Finally, the proliferation of ovarian cancer cell lines was not significantly inhibited in the presence of neutralising anti-EGF receptor antibody. These data suggest that EGF receptor activation through autocrine pathways is not a major mechanism for the growth of many ovarian cancer cell lines. Other pathways of signal transduction which bypass the requirement for EGF receptor activation may be important in the proliferation for ovarian cancer cells. Such EGF receptor-independent pathways may limit the effectiveness of strategies designed to inhibit ovarian cancer cell growth through disruption of EGF receptor function.     

Azoxymethane enhances ligand-induced activation of EGF receptor tyrosine kinase in the colonic mucosa of rats

Recent observations suggest that transforming growth facto     

The EGF receptor Hokey-Cokey

In cancer, the epidermal growth factor (EGF) receptor (EGFR) can be activated by mutations that disrupt the inactive conformation and allow the active conformation to predominate. Structural studies have elucidated the molecular events that lead to EGFR activation and shown that small-molecule anti-EGFR drugs can bind to either the inactive or the active conformation of the kinase domain. In this issue of Cancer Cell, Yun et al. present 12 crystal structures of the wild-type or mutant forms of the EGFR kinase domain bound to four different ligands. This study will prove invaluable to those developing novel anti-EGFR drugs.     

Defective cleavage of membrane bound TGFalpha leads to enhanced activation of the EGF receptor in malignant cells

Transforming growth factor alpha (TGFalpha) is widely expressed in malignant as well as normal cells and is involved in regulating cell growth and differentiation. Although processing of TGFalpha has been extensively studied in normal cells, there is little information regarding TGFalpha cleavage in malignant cells. Therefore, we compared the processing of TGFalpha in two human colon carcinoma cell lines. We found that there was a defective cleavage pattern for the TGFalpha precursor resulting in retention of partially processed TGFalpha on the cell surface of both the HCT116a2alphaS3 and CBS4alphaS2 cell lines. This raised the possibility that signaling from the resulting defective cleavage species could differ from that of soluble TGFalpha. The membrane-associated TGFalpha induced higher phosphorylation of EGFR on the cell surface of adjacent cells than equivalent levels of mature TGFalpha. The interaction of membrane bound TGFalpha precursor with the EGFR caused a slower internalization of activated EGFR relative to the internalization of the soluble TGFalpha/EGFR complexes. In addition, the tethered TGFalpha was resistant to the ability of protein-tyrosine phosphatases (PTPs) to reduce EGFR tyrosine phosphorylation, also contributing to higher activation of EGFR. The enhanced activation of EGFR by the tethered form of TGFalpha was reflected by higher activation of Grb2, SHC and Erk downstream mediators of EGF receptor signaling. The higher activation of EGFR by membrane tethered TGFalpha indicates that defective TGFalpha processing provides a mechanism whereby malignant cells can obtain a growth advantage over normal cells.     

Cisplatin induces PKB/Akt activation and p38(MAPK) phosphorylation of the EGF receptor

Cisplatin is an effective DNA-damaging antitumor agent employed for the treatment of various human cancers. In this study, we report that Cisplatin activates PKB/Akt in several cancer cell lines and that this activation is mediated by EGFR, Src and PI3-kinase. Inhibition of PI3-kinase activity decreases the survival of the cells exposed to Cisplatin, suggesting that Cisplatin-induced PKB/Akt activation may lead to Cisplatin resistance. While investigating the EGFR-dependent PKB/Akt activation in MDA-MB-468 cells, we found that the EGFR receptor undergoes a gel mobility shift upon Cisplatin treatment, which is mediated by p38(MAPK). An EGFR, in which threonine 669 was mutated to alanine (A669), is phosphorylated by p38(MAPK) to a much lesser extent, suggesting that threonine 669 is a p38 phosphorylation site. We found that Cisplatin induces EGFR internalization, which is mediated by p38(MAPK-)dependent phosphorylation of the receptor on threonine 669. Our results identify the EGFR as a new substrate of p38 and identify threonine 669 as a new phosphorylation site that regulates EGFR internalization. Together, these results suggest that Cisplatin has side effects, which may alter the signaling pattern of cancer cells and modulate the desired effects of Cisplatin treatment.     

The erbB gene and the EGF receptor

The epidermal growth factor (EGF) receptor is a plasma membrane glycoprotein. It contains four distinct segments: an N-terminal EGF binding domain which is exposed at the cell surface; a short transmembrane segment; a cytoplasmic domain with protein-tyrosine kinase activity; and a C-terminal regulatory segment. Binding of EGF to the external domain of the receptor activates the protein-tyrosine kinase activity of the receptor, and this elevated kinase activity is presumed to be involved in the activation of cell growth. The v-erbB transforming gene of avian erythroblastosis virus is derived, by retroviral transduction, from the gene (c-erbB) which encodes the avian EGF receptor. The transforming capacity of v-erbB appears to result from truncation of the receptor. In erythroid cells, truncation of the N-terminal ligand binding domain is sufficient for transformation, whereas in fibroblasts removal of an additional C-terminal segment is required for transformation. The EGF receptor is subject to complex regulatory controls, including ligand activation, downregulation by internalization, autophosphorylation and autoregulation and transmodulation involving phosphorylation by kinase C. This review is centered around the hypothesis that the transforming capacity of the truncated v-erbB gene product results from a loss in sensitivity to regulators and the consequent activation of protein kinase activity.     

ABINs inhibit EGF receptor-mediated NF-kappaB activation and growth of EGF receptor-overexpressing tumour cells

The epidermal growth factor receptor (EGFR) is frequently overexpressed in various tumours of epidermal origin and is held responsible for tumourigenicity and tumour persistence. Increased nuclear factor (NF)-kappaB activity has been suggested to be involved in the malignant behaviour of EGFR-overexpressing cells. However, the mechanisms that regulate EGF-induced NF-kappaB activation are still largely unknown. Here we show that EGF can induce NF-kappaB-dependent gene expression independently from IkappaBalpha degradation or p100 processing in EGFR-overexpressing HEK293T cells. Moreover, EGF-induced NF-kappaB activation could be inhibited by overexpression of ABINs, which were previously identified as intracellular inhibitors of tumour necrosis factor, interleukin-1 and lipopolysaccharide-induced NF-kappaB activation. Knockdown of ABIN-1 by RNA interference boosted the NF-kappaB response upon EGF stimulation. The C-terminal ubiquitin-binding domain containing region of ABINs was crucial and sufficient for NF-kappaB inhibition. Adenoviral gene transfer of ABINs reduced constitutive NF-kappaB activity as well as the proliferation of EGFR-overexpressing A431 and DU145 human carcinoma cells. Altogether, these results demonstrate an important role for an ABIN-sensitive non-classical NF-kappaB signalling pathway in the proliferation of EGFR-overexpressing tumour cells, and indicate a potential use for ABIN gene therapy in the treatment of cancer.     

Targeting the EGF receptor in breast cancer treatment

Summary Immunotoxins are a relatively new class of cytotoxic agents consisting of a catalytic toxin linked to an appropriate targeting ligand. The ligand directs the toxin to the surface of a tumor cell, whereupon the toxin enters the cell and catalytically inactivates the ribosome, thus disrupting protein synthesis and effecting cell death. Monoclonal antibodies (or their fragments) have been most commonly used to carry chemically conjugated toxins to proteins or antigens overexposed on the tumor cell surface, but specific ligands for tumor cell surface receptors could also provide effective targeting.The receptor for epidermal growth factor (EGFR) is overexpressed primarily in poor prognosis breast cancers that do not respond well to traditional therapies. Because EGFR is frequently overexpressed in breast cancer tissue and is associated with a poor prognosis, it is an attractive target for antitumor therapy.DAB ₃₈₉ EGF is an EGFR specific fusion toxin produced with recombinant DNA techniques consisting of sequences for the enzymatically active and membrane translocation domains of diphtheria toxin plus sequences for human epidermal growth factor. DAB ₃₈₉ EGF is a potent, EGFR specific, cytotoxic agent which rapidly inhibits protein synthesis by a mechanism of action similar to that of diphtheria itself. Preclinical studies in the laboratory and in animals now suggest the feasibility of investigating such an agent in the targeted therapy of patients with human breast cancer.     

EGF receptor in neoplasia and metastasis

Summary EGFR is a member of the tyrosine kinase family of cell surface receptors with a wide range of expression throughout development and in a variety of different cell types. The receptor can transmit signals to cells: i) upon interaction with ligands such as EGF, TGF, amphiregulin or heparin binding EGF, ii) upon truncation or mutation of extracellular and/or intracellular domains, iii) upon amplification of a basal receptor activity (in the absence of ligand) through cooperation with other cellular signaling pathways or nuclear events (e.g. expression of v-erbA). The activated EGFR can exert pleiotropic functions on cells, depending on their tissue origin and state of differentiation. Under certain conditions it can also contribute to neoplasia and development of metastases. Such conditions can exist upon aberrant receptor/ligand expression and activation (e.g. in the wrong cell; at the wrong time; in the wrong amounts). Aberrant signalling can also occur through constitutive EGFR activation. Oncogenic potential of EGFR has been demonstrated in a wide range of experimental animals. EGFR is also implicated in human cancer, where it may contribute both to the initiation (glioblastoma) and progression (epithelial tumors) of the disease. EGFR may influence key steps in the processes of tumor invasion and dissemination. Involvement of EGFR in tumor spread may indicate a potential use of this receptor as a target for antimetastatic therapy.     

EGF receptor as a therapeutic target

Surgery alone is currently still accepted "standard of care" for patients with operable NSCLC, this includes stages IA and IIB, as well as selected early subsets of IIIA disease. In more advanced and inoperable stage III disease, combinations of chemotherapy and radiotherapy remain the standard treatment approach for patients with good performance status. The role of surgery following induction therapy in these advanced stage III patients is at the moment not conclusively defined. More evidence from randomized trials is clearly needed to tailor treatment for the large number of patients that present in these locally advanced stages. Enrollment of patients into ongoing prospective clinical trials should be encouraged, whenever possible, to further define prognostic factors and improve multimodality strategies in this clinical setting.     

Expression of herstatin, an autoinhibitor of HER-2/neu, inhibits transactivation of HER-3 by HER-2 and blocks EGF activation of the EGF receptor

The four members of the EGF receptor family are capable of homomeric as well as heteromeric interactions. HER-2/neu (erbB-2) dominates as the preferred coreceptor that amplifies mitogenic signaling. An alternative HER-2/neu product, herstatin, consists of a segment of the ectodomain of p185HER-2 and an intron-encoded C-terminus. Recombinant herstatin was found to bind with nM affinity and inhibit p185HER-2. To further examine the impact on receptor activity, herstatin was expressed with various receptor tyrosine kinases. In CHO cells that overexpressed HER-2, herstatin caused a sevenfold inhibition of colony formation that corresponded to a reduction in the tyrosine phosphorylation of p185HER-2. Herstatin also prevented HER-2 mediated transactivation of the kinase impaired HER-3 as reflected in transphosphorylation of HER-3 and heteromers between HER-2 and HER-3. In EGF receptor-overexpressing cells, EGF induction of receptor dimerization and tyrosine phosphorylation were reduced more than 90%, and receptor down-regulation as well as colony formation were also suppressed by coexpression with herstatin. Inhibition was selective for the EGF receptor family since herstatin expression did not reduce tyrosine phosphorylation mediated by the FGF receptor-2 or by insulin-like growth factor -1. Herstatin bound to the EGF receptor as well as to p185HER-2 in pull-down assays suggesting that complex formation may be involved in receptor inhibition. Our findings indicate that herstatin has the capability to negatively regulate combinations of interactions between group I receptor tyrosine kinases that confer synergistic growth signals.     

Osteopontin-induced migration of human mammary epithelial cells involves activation of EGF receptor and multiple signal transduction pathways

Osteopontin (OPN) is a secreted, integrin-binding glycophosphoprotein that has been implicated in breast cancer. We previously showed that OPN-induced cell migration of mammary epithelial cells (MEC) depends on binding to cell surface integrins and involves activation of the hepatocyte growth factor (HGF) receptor, Met. Here, we show that OPN-induced migration of MEC also requires activation of the epidermal growth factor (EGF) pathway. Synergism was seen between EGF and OPN in inducing cell migration. Furthermore, incubation of cells with exogenous OPN increased ligand (TGFalpha> EGF) and EGF receptor (EGFR) mRNA expression, as well as EGFR kinase activity. Treatment of cells with anti-TGFalpha or anti-EGFR antibody, or with tyrphostin-25 (EGFR inhibitor), significantly impaired the cell migration response to OPN. Other more broad-spectrum tyrosine kinase inhibitors and the growth factor/ receptor interaction inhibitor, suramin, also inhibited OPN-induced migration. Using specific signal transduction pathway inhibitors, we have screened for involvement of MEK (MAP kinase kinase), phosphatidylinositol 3-kinase, phospholipase C (PLC), and protein kinase C (PKC). Results implicated all of these pathways in OPN-induced cell migration, the most pronounced effect being seen with PLC and PKC inhibitors. These results suggest that induction of MEC migration by OPN involves a cascade of events including at least two growth factor/receptor pathways and multiple downstream signal transduction pathways. A number of potential targets are thus provided for strategies aimed at blocking the malignancy-promoting effects of OPN.     

Dexamethasone protects against Cisplatin-induced activation of the mitochondrial apoptotic pathway in human osteosarcoma cells

BACKGROUND: Dexamethasone (Dx) is often used to alleviate the acute emetic toxicity associated with high dose cisplatin (cDDP) in osteosarcoma patients. However, in other tumour cell types, Dx has been reported to induce partial resistance to anticancer drugs. MATERIALS AND METHODS: We examined the effect of Dx on cDDP-induced apoptosis in the HOS human osteosarcoma cell line. RESULTS: Exposure to cDDP, induced apoptosis via the mitochondrial apoptotic pathway as evidenced by cytochrome c release and caspase activation. Pre and cotreatment of HOS cells with Dx reduced cDDP induced apoptosis by 10-25%. Investigation of the mechanisms of this protective effect indicated both the upregulation of the survival factor Akt and a possible direct receptor-mediated action of Dx to attenuate the activation of the mitochondrial apoptotic pathway components. CONCLUSIONS: These data indicate the need to carefully address the timing of glucocorticoid use in the clinical management of cancer patients.     

EGF receptor mediates adhesion-dependent activation of the Rac GTPase: a role for phosphatidylinositol 3-kinase and Vav2

Organization of the actin cytoskeleton in eucaryotic cells is controlled by small GTPases of the Rho family. Rac becomes activated by growth factor stimulation and integrin-mediated cell adhesion to extracellular matrix and is known to have a crucial role in lamellipodia formation, cell spreading and migration. At present, the intracellular pathways that connect cell surface receptors to Rac activation are poorly characterized. It has been reported previously that integrin-mediated cell attachment induces activation of the EGF receptor (EGFR) in the absence of EGF. We demonstrate here that this activation is instrumental for integrin-dependent Rac activation. Thus, we found that cells in which EGFR activity had been inhibited failed to spread and form lamellipodia on fibronectin. Failure to spread coincided with inhibition of adhesion-induced GTP loading of Rac and also with inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Subsequent studies demonstrated that an activated form of PI 3-kinase restored Rac GTP loading in the presence of EGFR inhibition, while a dominant-negative form of PI 3-kinase blocked Rac GTP loading in fibronectin-adherent cells. Our further functional studies identified Vav2, a known exchange factor for Rac, as a crucial downstream component in EGFR- and PI 3-kinase-dependent Rac activation upon integrin-mediated cell adhesion. Our results provide a mechanistic insight into integrin-dependent Rac activation, and identify a novel role for EGFR, PI 3-kinase and Vav2 in this pathway.     

Role of basal calcium in the EGF activation of MAP kinases

The role of intracellular Ca2+ pools in the regulation of growth factor signal transduction pathways and mitogenesis is not well understood. We have examined the roles of basal and transiently mobilized Ca2+ in the regulation of MAP kinases by EGF. To assess the influence of Ca2+ transients we utilized Plcg1-/- and Plcg1+/+ mouse embryonic fibroblasts, while BAPTA/AM was employed to chelate total intracellular Ca2+ in the same cell lines. The MAP kinases erk-1, erk-2 and erk-5 exhibited similar patterns of activation in wild-type and Plcg1-/- cells treated with EGF. However, pretreatment with BAPTA/AM significantly increased and prolonged erk-1 and erk-2 activation in both cell types. In contrast, BAPTA/AM prevented the EGF activation of erk-5 in wild-type and Plcg1-/- cells. These data indicate that basal Ca2+, but not growth factor provoked Ca2+ transients, has a significant influence on the activation of these MAP kinases. AG1478, a specific EGF receptor kinase inhibitor, abolished the prolonged erk-1 and erk-2 activation produced by EGF in cells pretreated with BAPTA/AM. This indicates that the prolonged activation of erk-1 and erk-2 produced in the presence of BAPTA/AM requires continuous signaling from the EGF receptor kinase.     

Abnormalities of the EGF receptor system in human thyroid neoplasia

The epidermal-growth-factor receptor (EGF-r) and its ligands are involved in the control of proliferation of both normal and neoplastic thyroid epithelium. Autocrine stimulation of growth involving this receptor system has been identified in several types of human neoplasia and we were interested to determine whether it might occur in human thyroid tumours. We have therefore examined an archival series of thyroid tumours and non-neoplastic pathologies for expression of the EGF-r and 2 of its ligands, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF), using immunohistochemistry. We found evidence of expression of both the EGF-r and the TGF-alpha in the majority of thyroid tumours, with a trend to higher expression in more malignant neoplasms. We also found variable levels of expression of EGF-r and TGF-alpha in all cases of thyroiditis examined. We conclude that there is a potential autocrine loop involving the EGF-r system in both neoplastic and non-neoplastic conditions of the human thyroid.     

Direct inhibition of EGF receptor activation in vascular endothelial cells by gefitinib ('Iressa', ZD1839)

The development of gefitinib ('Iressa', ZD1839) by targeting the EGFR tyrosine kinase is a recent therapeutic highlight. We have reported that gefitinib is antiangiogenic in vitro, as well as in vivo. In this study, we asked if the anti-angiogenic action of gefitinib is due to a direct effect on activation of vascular endothelial cells by EGF. EGF, as well as VEGF, caused pronounced angiogenesis in an avascular area of the mouse cornea, and i.p. administration of gefitinib almost completely blocked the response to EGF, but not to VEGF. Immunohistochemical analysis demonstrated phosphorylation of EGFR by EGF in the neovasculature, and gefitinib markedly reduced this effect. Gefitinib also inhibited downstream activation of ERK 1/2 via EGFR in cultured microvascular endothelial (HMVE) cells. These findings suggest that the anti-angiogenic effect of gefitinib in the vascular endothelial cells of neo-vasculature is partly attributable to direct inhibition of EGFR activation, and that endothelial cells in malignant tumors play a critical role in the cancer therapeutic efficacy of gefitinib.