EGFR INHIBITORS
Signal transduction and oncogenesis by ErbB/HER receptors

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Abstract

Growth factors enable cells to escape irradiation-induced death (apoptosis). One important family of growth factors share an epidermal growth factor motif, and all bind to ErbB transmembrane receptors. In response to growth factor ligands, ErbB receptor tyrosine kinases induce a variety of cellular responses, including proliferation, differentiation and motility. Signal transduction pathways are initiated upon ligand-induced receptor homo- or heterodimerization and activation of tyrosine kinase activity. The complement of induced signaling pathways, as well as their magnitude and duration, determines the biological outcome of signaling, and in turn, is regulated by the identity of the ligand and the receptor composition. Recent insights into the structural basis for receptor dimerization, as provided by crystallographic analysis, are described, as is the differential activation of signaling pathways and downregulatory mechanisms. Further, dysregulation of the ErbB network is implicated in a vareity of human cancers, and the nature of aberrant signaling through ErbB proteins, as well as current therapeutic approaches, are discussed, highlighting the role of the highly oncogenic ErbB-2 molecule.

Introduction

The ErbB family of receptor tyrosine kinases (RTKs) couples binding of extracellular growth factor ligands to intracellular signaling pathways regulating diverse biologic responses, including proliferation, differentiation, cell motility, and survival. The four closely related members of this RTK family—epidermal growth factor receptor (EGFR, also known as ErbB-1 or HER1), ErbB-2 (HER2), ErbB-3 (HER3), and ErbB-4 (HER4)—are activated upon ligand-induced receptor homo- and heterodimerization. The cellular outcome of activation depends upon the complement of signaling pathways induced, as well as their magnitude and duration, which in turn are determined by the composition of the receptor pair and the identity of the ligand. This complex network of 4 receptors and more than 10 ligands has evolved through diversification from invertebrates, where 1 ErbB homolog and 1 ligand are found in the worm C. elegans(1), and 1 receptor with 4 ligands is found in the fruit fly D. melanogaster(2). The multilayered network found in higher organisms allows for the combinatorial interactions of ligands, receptors, effectors, and transcription factors, allowing for a high degree of adaptability and signal diversification, with multiple regulatory levels controlling biologic responses (reviewed in Ref. 3).

ErbB RTKs have a broad expression pattern on epithelial, mesenchymal, and neuronal cells, and signaling through these receptors plays a critical developmental role in inductive cell fate determination in many organ systems, as exemplified by the perinatal (ErbB-1) or early embryonic lethality (ErbB-2, -3, and -4) of knockout mice because of insufficient heart and nervous system development (reviewed in Ref. 4). Furthermore, ErbB RTKs are involved in mammary gland development during puberty and pregnancy, as well as in the maintenance of tissue homeostasis. Conversely, dysregulation of the ErbB signaling network is implicated in multiple human pathologies, of which the role of ErbBs in cancer is the best characterized, particularly for ErbB-1 and ErbB-2. This review will outline the structure and signaling functions of ErbB RTKs and highlight their involvement in human neoplasia.

The ligands of ErbB receptors are generated upon cleavage of transmembrane precursors (5) and are characterized by an epidermal growth factor (EGF)–like domain composed of 3 disulfide-bonded intramolecular loops (6). The specificity of these growth factors for ErbB RTKs is depicted in Fig. 1. ErbB receptors contain an extracellular ligand-binding domain and a single hydrophobic transmembrane domain. The intracellular portion of ErbBs consists of a highly conserved tyrosine kinase domain, although ErbB-3 contains substitutions of critical amino acids within this domain and lacks kinase activity (7). Ligand binding induces the homo- or heterodimerization of ErbBs, resulting in receptor transphosphorylation on tyrosine residues within the activation loop, which significantly enhances kinase activity 8, 9. Subsequent tyrosine phosphorylation on residues within the carboxyl terminal tail of the receptors enables the recruitment and activation of signaling effectors containing Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains.

The crystal structures of the extracellular domains of ErbB-1 bound to EGF and transforming growth factor alpha (TGFα) and unoccupied ErbB-3 were solved recently 10, 11, 12. These structures, in conjunction with all the biochemical data gathered over the years, have helped build a convincing picture of ligand-induced receptor activation. Of the 4 subdomains in the extracellular domain, subdomains I and III (also called L1 and L2) have a beta helical fold, and they bind ligands in a bivalent manner. The cysteine-rich subdomains II and IV (S1 and S2) have an extended structure held together by disulfide bonds. The structures of ErbB-1 bound to EGF and TGFα revealed a unique back-to-back mode of dimerization mediated by a beta hairpin, termed the “dimerization loop,” which protrudes from the S1 domains of each ligand bound monomer 11, 12. The dimerization loop penetrates deep into the dimer partner and makes many complementary stabilizing contacts. In contrast, in the structure of the extracellular domain of ErbB-3 with no ligand bound (10), an autoinhibited conformation mediated by the dimerization loop and a beta hairpin in the S2 domain was observed. The interaction between the dimerization loop and the S2 domain holds L1 and L2 far apart. Comparing the relative orientations of the different domains in the structures of ErbB-1 and ErbB-3, ligand binding would cause a drastic conformational change, resulting in an extended conformation of the dimerization loop, and render the receptors capable of dimerizing with another ligand-bound molecule. Though the crystal structures suggest that the dimerization loop is the major contributor, other regions of ErbBs, including S2, the transmembrane domain, and the kinase domain, have been implicated in dimerization and may stabilize receptor dimers 13, 14, 15.

ErbB-2 is a unique member of the ErbB family in that it does not bind any of the known ligands with high affinity, but it is the preferred heterodimeric partner for other ErbB receptors 16, 17, 18, 19. The structure of ErbB-2 provides mechanistic insight as to its preferred inclusion in ErbB heterodimers. Though the basic structure is similar, the structure of ErbB-2 nonetheless differs significantly from that of the unengaged ErbB-3 (A. W. Burgess, personal communication). A strong interaction was observed between the L1 and the L2 domain of ErbB-2 that mimics the ligand bound conformation seen in ErbB-1 and results in a pre-extended conformation of the dimerization loop of ErbB-2. Thus, ErbB-2 exists in a dimerization-favorable conformation on the cell surface, which likely underlies its enhanced capacity for heterodimerization with other ErbB family members. However, mechanisms must be in place to prevent spontaneous dimerization of ErbB-2 20, 21.

Although both homo- and heterodimerization result in activation of the ErbB network, heterodimers are more potent and mitogenic. Heterodimerization can provide additional phosphotyrosine residues for the recruitment of binding partners, as well as induce distinct patterns of receptor phosphorylation and downstream signaling. In addition, the attenuation of signaling through receptor endocytosis and subsequent lysosomal degradation differs between receptor dimers 22, 23. The physiologic role of ErbB-2 as an activation partner for other ErbB RTKs results in increased signaling potency through several means, including increased ligand affinity through a decelerated rate of ligand dissociation, efficient coupling to signaling pathways, and a decreased rate of receptor downregulation 16, 24, 25, 26. Furthermore, signaling through the kinase-deficient ErbB-3 requires heterodimerization with a kinase-active partner. Through heterodimerization, the ligand-less ErbB-2 and the kinase-inactive ErbB-3 provide the most potent mitogenic 16, 27 and angiogenic signal (28). Knockout mice provide in vivo evidence for the requirement for heterodimerization of ErbB receptors. NRG-1–, ErbB-2–, and ErbB-4–deficient mice die in utero of insufficient heart development 29, 30, 31, 32. Hence, ErbB-4 homodimers cannot functionally replace the contribution of ErbB-2/ErbB-4 heterodimerization.

Receptor dimerization is also regulated by the identity of the ligand, in addition to regulation by the complement of ErbB RTKs expressed on the cell surface. Furthermore, signal strength and duration are influenced by ligand affinity. However, low-affinity ligands may function more potently as a result of decreased receptor downregulation, as was observed with low-affinity virally encoded ligands (33). Furthermore, receptor trafficking is influenced by the properties of the ligand–receptor interaction. The interaction of ErbB-1 with EGF is stable at endosomal pH and results in lysosomal targeting and degradation, whereas pH-sensitive interactions with TGFα and NRG-1 result in dissociation in the endosome, favoring receptor recycling and enhanced signaling 34, 35. These differences can translate into distinct biologic responses.

The ErbB network is implicated in multiple human cancers, and dysregulation of the many signaling pathways induced through ErbB RTKs can promote multiple properties of neoplastic cells, including proliferation, migration, angiogenesis, stromal invasion, and resistance to apoptosis. The role of ErbBs in cancer has been extensively reviewed 36, 37, 38 and is summarized below. Hyperactivation of the ErbB network can occur via an autocrine secretory loop involving overproduction of ligands and receptors by the tumor cells, or paracrine growth dependent on ErbB ligands produced by adjacent stromal cells (39). Alternatively, aberrant growth can ensue from constitutive receptor activation (40).

Overexpression, gene amplification, rearrangements, or mutations of ErbB-1 are found in multiple human malignancies, including cancers of the breast, head and neck, and lung, with a particularly high incidence of ErbB-1 overexpression in gliomas (41). Accumulating evidence suggests that overexpression of ErbB-1 contributes to cell transformation in a ligand-dependent manner, and several tumors show coexpression of ErbB-1 and its ligands TGFα or EGF. However, a mutant of ErbB-1 (type III) lacking a portion of its extracellular domain is commonly found in breast, ovary, and lung carcinomas 42, 43 and is constitutively active in a ligand-independent manner 44, 45.

The tumor-promoting effects of ErbB-2 have been best characterized (reviewed in Ref. 46); although mutations have been found only rarely, if at all, in human cancers, ErbB-2 is overexpressed in many types of cancer. Most notably, ErbB-2 is overexpressed due to gene amplification in 20%–30% of breast and ovarian tumors, and its overexpression correlates with tumor chemoresistance and poor patient prognosis. The oncogenic effect of ErbB-2 may relate to its high basal autophosphorylation (40). High levels of ErbB-2 expression may result in its constitutive homodimerization and promote transformation in tissue culture models 47, 48. In addition, ligand-independent homodimerization was observed in rats treated with carcinogen, in which the NeuT mutation within the transmembrane domain (V664E) results in constitutive receptor activation and the formation of neuroglioblastomas 49, 50, 51. Although not naturally occurring, a comparable mutation in human ErbB-2 leads to increased dimerization and transforming ability (52). Alternatively, overexpressed ErbB-2 may promote tumor formation as a result of spontaneous or ligand-induced heterodimerization with other ErbBs (53), and the resultant signal potentiation. Indeed, coexpression of an additional ErbB receptor is required for NeuT-mediated transformation 54, 55. Furthermore, many ErbB-2–expressing tumors also exhibit an autocrine loop involving expression of both ErbB-1 and one of its ligands, and loss of ErbB-2 function inhibits the proliferation of these tumor cells. Most breast, skin, lung, ovary, and gastrointestinal tract tumors express ErbB-3 and/or ErbB-4, and heterodimerization of these receptors with ErbB-2 may be involved in some cancers, such as oral squamous cell cancer and childhood medulloblastoma 56, 57.

The transforming potential of ErbB-2 within ErbB heterodimers most likely relates to signal potentiation through decreases in attenuation pathways, as mentioned above. ErbB-2 has a relatively slow rate of endocytosis and can reduce the rate of ErbB-1 internalization and subsequent degradation 58, 59. Moreover, ErbB-2 can enhance the rate of recycling of ErbB-1 and reduce its lysosomal targeting 22, 23. Furthermore, ErbB-2 results in increased ligand affinity by decreasing the rate of ligand dissociation from heterodimers (25). Together, these attributes of ErbB-2/HER2 underlie the sustained signals induced through ErbB-2 containing heterodimers.

Signal transduction pathways are initiated upon the recruitment and assembly of complexes of signaling effectors to activated ErbB RTKs. The pattern of tyrosine autophosphorylation dictates the signaling pathways induced through ErbBs, because the 5–8 amino acids surrounding the phosphotyrosine residue confer the specificity of SH2 and PTB domain binding (60). Thus, individual ErbBs bind a distinct subset of signaling molecules, which is also influenced by the composition of the dimer, and a given receptor can be differentially transphosphorylated in distinct ErbB dimers (61). Furthermore, the identity of the stimulating ligand influences the tyrosine phosphorylation pattern. For example, binding of betacellulin or several neuregulins to ErbB-4 induced similar levels of receptor dimerization and phosphorylation, however with different patterns of tyrosine phosphorylation. This, in turn, leads to the recruitment of a distinct set of signaling molecules and different potencies in the induction of cell growth (62).

The three best characterized signaling pathways induced through ErbBs are Ras–mitogen-activated protein kinase (Ras-MAPK), phosphatidylinositol 3′ kinase-protein kinase B (PI3K-PKB/Akt), and phospholipase C–protein kinase C (PLC-PKC) pathways and are schematically depicted in Fig. 2 (reviewed in Refs. 63, 64, 65). All ErbB ligands and receptors couple to activation of the Ras-MAPK pathway, either directly through SH2 domain–mediated recruitment of Grb-2 or indirectly through PTB domain–mediated binding of the Shc adaptor. The Grb-2 associated guanine nucleotide exchange factor Sos activates Ras through the exchange of GDP for GTP. Among other effectors, active Ras binds and activates the Raf kinase, initiating a kinase cascade involving serine phosphorylation of MEK1/2 (MAPKK) and tyrosine and threonine phosphorylation of Erk1/2 (MAPK). Erk phosphorylates multiple cytoplasmic and cytoskeletal proteins, including MAPK-activated protein kinases and the ribosomal p70-S6 kinase. Erk also undergoes rapid translocation into the nucleus, where it phosphorylates and activates a variety of transcription factor targets, including Sp1, E2F, Elk-1, and AP1.

ErbB RTKs couple to activation of PI3K through binding of GTP-Ras to the p110 catalytic subunit of PI3K. In addition, SH2-mediated recruitment of the p85 regulatory subunit to activated receptors results in allosteric activation of the lipid kinase. PI3K signaling is induced with differing potencies and kinetics through ErbB RTKs, because p85 binding to ErbB-1 and ErbB-2 is indirect and mediated by adaptor proteins, whereas ErbB-3 and ErbB-4 contain 6 and 1 putative p85 binding sites, respectively (66). Active PI3K phosphorylates phosphatidylinositol 4,5-bisphosphate at the 3′ position, resulting in the recruitment of multiple signaling effectors containing the lipid-binding FYVE or pleckstrin homology (PH) domains. The Akt kinase (also called PKB) is a key effector of PI3K, which is recruited to the membrane through its PH domain and activated upon phosphorylation by PDK1, another PH domain–containing kinase and a second as yet unknown kinase (67). Akt also undergoes nuclear translocation and has many cytoplasmic and nuclear targets. ErbB-mediated phosphorylation of Bad by Akt promotes survival by blocking the interaction of this pro-apoptotic molecule with Bcl-2 and Bcl-X proteins (68). Akt negatively regulates the Raf and GSK-3 kinases and the cell cycle regulatory transcription factor FKHR, while promoting initiation of protein translation through mTOR, eukaryotic initiation factor 4E, and the ribosomal p70-S6 kinase. Furthermore, Akt promotes cell cycle progression through downregulation of the cyclin dependent kinase inhibitor p27KIP1. Thus, PI3K mediates many proliferation and cell survival signals, which is reflected by the tumor suppressive effects of PTEN, a lipid phosphatase that dephosphorylates the 3′ position of PI3K products and is frequently mutationally inactivated in human cancers (69).

Phospholipase Cγ (PLCγ) is recruited to the membrane through SH2 domain-mediated binding to activated ErbB-1 and ErbB-2, as well as through binding of its PH domain to PI3K products. Subsequent PLCγ phosphorylation by RTKs results in its activation. PLCγ hydrolyzes phosphatidylinositol 4′, 5′ bisphosphate to generate the second messengers diacylglycerol and inositol trisphosphate. Binding of inositol trisphosphate to receptors on the endoplasmic reticulum results in calcium release and increased intracellular calcium concentrations, which lead to the activation of calcium/calmodulin-dependent protein kinases and phosphatases, including Pyk2 and calcineurin. Furthermore, calcium and diacylglycerol activate protein kinase C, resulting in the phosphorylation of a large variety of substrates.

Multiple additional signaling pathways are induced through ErbB RTKs. c-Src is activated upon stimulation with EGF and phosphorylates additional tyrosine docking sites within the ErbB-1, as well as cytoskeletal and endocytic proteins 70, 71. The transcription factors STAT1, STAT 3, and STAT5 are directly phosphorylated by ErbB-1, subsequent to which they dimerize through phosphotyrosine–SH2 domain interactions and translocate to the nucleus to activate gene transcription critical for proliferation. GTP-bound Ras couples to activation of the Rho, Rac, and Cdc42 small GTPases, which control cytoskeleton rearrangements. Furthermore, nuclear translocation of proteolytic fragments of ErbB-1, ErbB-3, and ErbB-4 has been detected, where these receptors may function as transcription factors (72).

The ErbB network is implicated also in signaling downstream of other cellular stimuli, including cell adhesion, lymphokines, or stress signals (73). Notably, agonists of G-protein coupled receptors may require ErbB-1–induced signaling to couple to activation of the Ras-MAPK pathway (74). Transactivation of ErbBs may involve phosphorylation by nonreceptor tyrosine kinases such as FAK, Src, Pyk2, or JAKs, enabling the recruitment of effector molecules such as Grb2/Sos or resulting in catalytic activation of the RTK. Alternatively, direct activation of ErbB RTKs may result from binding of cognate ligands, generated upon cleavage of transmembrane precursors by membrane metalloproteinases stimulated by G-protein coupled receptors (74).

ErbB RTKs may also be regulated by subcellular localization. ErbB-1, ErbB-2, and ErbB-4 are enriched in membrane microdomains called caveolae (reviewed in Refs. 75, 76). Caveolae are a subset of lipid rafts enriched in caveolin proteins, glycosphingolipids, and cholesterol, as well as in multiple signaling molecules, including Src family kinases and H-Ras. Caveolin-1 has been shown to interact with ErbBs, which results in an inhibition of their kinase activity 77, 78. Although some ErbB-induced signaling pathways, including Ras-MAPK, were observed in caveolae (79), disruption of these microdomains results in enhanced MAPK activation (80). Furthermore, ErbB-1 and ErbB-4 were shown to undergo ligand-induced migration out of caveolae 81, 82, which may remove the inhibitory effect of the interaction with caveolin. Indeed, caveolin has been shown to have tumor suppressive effects, and its expression is negatively correlated with tumorigenicity and with ErbB-2 signaling 83, 84. Thus, caveolae may serve to maintain low basal activity of ErbB RTKs, in addition to regulating signaling through the compartmentalized preassembly of signaling molecules. Moreover, caveolae have been implicated in nonclathrin-mediated internalization (85); however, the precise function of caveolae in regulating the signaling and potentially the endocytosis of ErbB RTKs remains to be fully elucidated.

Signaling pathways induced through ErbB RTKs converge on the cell cycle machinery to induce proliferation (reviewed in Refs. 86, 87). A key cell cycle regulator downstream of ErbBs is cyclin D1, which activates the cyclin-dependent kinases CDK4 and CDK-6 to promote G1/S phase cell cycle progression. ErbB-induced MAPK activation results in the transcriptional upregulation of cyclin D1 through the SP1 and E2F transcription factors (88) and stabilization of the protein upon its phosphorylation by Akt (89). Furthermore, Akt and Ras-MAPK signaling mediate additional cell cycle regulatory effects downstream of ErbBs, through the CDK inhibitors p27KIP1 and p21Waf190, 91, 92 and through promoting p53 ubiquitylation by MDM2 (93). The precise mode of ErbB-induced cell cycle progression remains to be fully elucidated; however, dysregulation of cell cycle checkpoints as a result of aberrant ErbB signaling can contribute to oncogenic transformation.

A critical aspect of signaling through ErbBs is promoting cell survival (reviewed in Ref. 68). Apoptosis or programmed cell death is an active process induced in response to extracellular or intracellular-derived signals, which is regulated by members of the Bcl-2 family and executed by caspase proteases. Apoptosis constitutes an important regulatory mechanism preventing cancer, and dysregulated proliferation due to overexpression of cell cycle regulatory proteins, such as E2F, Myc, or cyclin D1, is linked to the induction of programmed cell death. Thus, the contribution of ErbB receptors to oncogenesis is also mediated in part through effects on cell survival, which antagonize apoptotic checkpoints. PI-3K, MAPK, and STAT3 are implicated in survival signaling through ErbBs and induce the transcription of several antiapoptotic proteins, including Bcl-2 and Bcl-x, as well as inhibitors of apoptosis, which bind to and inactivate caspases. In addition, ErbB RTKs lead to decreased levels of pro-apoptotic Bax and the inactivation of Bad through serine phosphorylation by Akt or MAPK activated kinases. ErbB receptors and their ligands have been shown to prevent tumor cell apoptosis in animal models and in human cancer cells through PI3K/Akt survival pathways (94). Furthermore, hyperactivation of ErbB-1 and ErbB-2 results in resistance to apoptosis induced by several chemotherapeutic agents and seems to be mediated through upregulation of p21Waf195, 96, 97.

Multiple negative regulatory pathways function to attenuate signaling through ErbB receptors and ensure the appropriate tuning of signals in development and adult tissues. The principal process of signal attenuation is through downregulation of surface receptor levels by ligand-induced receptor endocytosis (reviewed in Ref. 98). Receptor dimers are internalized through clathrin-coated regions of plasma membrane that invaginate to form endocytic vesicles. Subsequent sorting steps result in either receptor recycling back to the cell surface or targeting to lysosomes and degradation.

ErbB-1 undergoes efficient ligand-induced downregulation after phosphorylation-dependent binding to proteins, such as Eps15, involved in the endocytic machinery. Eps15 and other endocytic adaptors mediate accelerated receptor recruitment to AP-2 complexes, which drives the assembly of clathrin-coated vesicles (99). Fission of the vesicle from the plasma membrane involves the GTPase dynamin. Furthermore, phosphorylated EGFR recruits and activates c-Cbl, a RING finger domain–containing E3 ubiquitin ligase that enhances receptor ubiquitylation, thus directing the receptor to lysosomal degradation 100, 101. In contrast to the rapid internalization and degradation of ErbB-1, ligand-induced endocytosis and ubiquitylation of other ErbB receptors are impaired, likely the result of weak coupling to c-Cbl 18, 102, 103. Furthermore, sorting in the early endosome or multivesicular body leads to the recycling of other ErbBs back to the plasma membrane and signal potentiation (35). Thus, as for the induction of signaling pathways, signal attenuation differs among ErbB receptors and can also be regulated by ligand. For example, Cbl couples to EGF- but not NRG-activated ErbB-1 (18), resulting in lysosomal degradation, with a corresponding decrease in mitogenicity.

Signaling through ErbB RTKs is modulated by additional negative regulatory pathways (reviewed in Ref. 104). Ras-GAP binds ErbB-1 and inhibits MAPK activation by increasing the GTPase activity of Ras. RALT was shown to act as a MAPK-induced feedback inhibitor that binds to ErbB-2 and inhibits ErbB-2–induced signaling and proliferation 105, 106. In addition, alternative splice variants of ErbBs that can inhibit signaling have been characterized (107). Herstatin is a splice variant of ErbB-2 composed of the extracellular domain and a novel C-terminus that binds ErbB-2 and blocks its activity and heterodimerization (108). A secreted protein, p85-s, composed of the extracellular domain of ErbB-3, binds NRG and blocks its binding to full-length ErbB-3 and ErbB-4 (109). Studies in invertebrates have uncovered additional negative regulators. In D. melanogaster, Argos was shown to be an EGF-like molecule that inhibits ligand binding to Drosophila EGF receptor (DER) (110), whereas Kekkon1 is a transmembrane leucine-rich repeat protein that inhibits DER by interfering with ligand activation (111). Furthermore, the cytosolic protein Sprouty binds Cbl and inhibits DER-induced activation of the Ras-MAPK pathway (112). Genetic evidence in C. elegans demonstrated that the Ark-1 kinase is a negative regulator of LET-23/ErbB-1 signaling (113). The function of mammalian homologs of Kekkon (Lig-1) and Ark-1 (ACK-1) remains to be fully characterized, whereas mammalian Sprouty proteins may enhance ErbB-1 signaling through binding of Cbl, resulting in decreased receptor ubiquitination and downregulation (114).

The many signaling pathways induced through ErbBs can lead to tumor cell growth and antagonize cell cycle and apoptotic checkpoints, as described above. Furthermore, ErbBs can promote tumor cell invasiveness through MAPK-induced activation of the MMP2 and MMP9 proteases (115) and can result in increased expression of the angiogenesis promoting factor VEGF 116, 117. Furthermore, enhanced ErbB function is associated with poor prognosis, shorter disease-free intervals, increased risk of metastasis, and resistance to chemotherapy, rendering the ErbB network highly attractive for therapeutic interventions in cancer treatment 3, 118.

Several approaches targeting ErbBs are in clinical use or development 119, 120. Herceptin/Trastuzumab and Cetuximab are chimeric or humanized ErbB-2–specific and ErbB-1–specific monoclonal antibodies currently in use for the treatment of breast cancer patients. The antitumor effects of these mAbs have been reviewed extensively 121, 122 and include Herceptin-induced endocytic downregulation of ErbB-2 through a mechanism that may involve c-Cbl 123, 124, as well as targeting of immune effector cells to the tumor (125). Furthermore, Herceptin induces partial tyrosine phosphorylation of ErbB-2, and although it does not activate the MAPK pathway, it does induce the CDK inhibitor p27KIP1, which may mediate growth inhibitory effects (126). Other immunologic approaches targeting the ErbB network include coupling antibodies to toxins, mAb-mediated inhibition of ErbB-2 heterodimerization with other ErbB family members (127), and specific mAb-mediated targeting of the constitutively active ErbB1 mutant EGFRvIII (128). Furthermore, small molecule inhibitors are in development, such as ErbB-specific tyrosine kinase inhibitors. For example, Iressa is a small molecule ATP-binding site inhibitor of ErbB-1 that mediates tumor inhibitory effects (129). Finally, derivatives of the antibiotic geldanamycin, which antagonize the function of the ErbB-2 chaperone Hsp90 and lead to ErbB2 degradation, block the growth of ErbB-2–overexpressing cells 130, 131.

Section snippets

Perspectives

The ErbB receptor signaling network induces a wide variety of biologic responses and is involved in many physiologic and pathologic conditions. The complex network involving receptor homo- and heterodimers allows for multiple levels of regulation by the spatial and temporal expression of receptor and ligand combinations. Thus, the biologic outcome of signaling through ErbBs depends on the cellular and environmental context. Moreover, cells exist in a complex milieu, and the involvement of ErbB

Acknowledgements

We thank the members of our group for insightful discussions.

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    Dr. Marmor is the recipient of a European Molecular Biology Organization Long-term Fellowship. Our laboratory is supported by grants from the National Cancer Institute, the U.S. Army, the European Commission, and the Israel Academy of Sciences and Humanities.

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