ErbB receptors: new insights on mechanisms and biology

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TRENDS in Cell Biology

Vol.16 No.12

ErbB receptors: new insights on mechanisms and biology Bryan Linggi and Graham Carpenter Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA

The ErbB family of four receptor tyrosine kinases occupies a central role in a wide variety of biological processes from neuronal development to breast cancer. New information continues to expand their biologic significance and to unravel the molecular mechanisms that underlie the signaling capacity of these receptors. Here, we review several aspects of ErbB receptor physiology for which new and significant information is available. These include ligand-dependent receptor dimerization and kinase activation, which is a prerequisite for all subsequent growth factor-dependent cell responses. We also address novel roles of receptor fragments in signaling, trafficking to intracellular sites, such as the nucleus, and ErbB roles in non-cancer disease processes, including schizophrenia, chronic renal disease, hypertension, and the cellular entry of infectious pathogens. Introduction Receptors for growth factors mediate a variety of cellular responses to the environment. The epidermal growth factor (EGF) receptor ErbB-1 and three family members (ErbB-2, ErbB-3 and ErbB-4) continue to yield important biological and mechanistic insights that are informative for the entire receptor tyrosine kinase (RTK) field. The four ErbB receptors recognize 11 different but structurally related growth factors (Box 1) and mediate processes in development, homeostasis and pathologies. Each of the receptors is a type I transmembrane protein consisting of a heavily glycosylated and disulfide-bonded ectodomain that provides a ligand-binding site, a single transmembrane domain and a large cytoplasmic region that encodes a tyrosine kinase and multiple phosphorylation sites (Box 2). ErbB-2 does not bind to a known ligand but instead functions as a co-receptor for each of the other three. Growth factor binding to the ectodomain activates the cytoplasmic tyrosine kinase, stimulating signaling pathways that direct cellular responses. Receptor activation is initiated by dimerization events in multiple regions of the proteins (Figure 1a). It is well established that, except for certain constitutively active mutants, dimerization is provoked by ligand binding and is essential for kinase activation [1]. Receptor dimerization includes both homo- and heterodimerization, particularly of ErbB-1, -3 and -4 with ErbB-2. ErbB-3 does not contain an active tyrosine kinase and thus relies on interaction with ErbB-2 for signaling. Corresponding author: Carpenter, G. ([email protected]). Available online 7 November 2006. www.sciencedirect.com

Within the context of a cellular environment, the level of activated ErbB receptors is modulated by an increasing number of negative regulators and is positively influenced by other cellular components, such as adhesion molecules. These modulators and their mechanisms are only beginning to be understood. Post-receptor signaling by activated ErbBs includes pathways [Ras/MAP kinase, phospholipase Cg, signal transducer and activation of transcription (STATs) and phosphatidylinositol (PtdIns) 3-kinase] that are common to nearly all RTKs [2]. In addition, ErbB receptors are processed from the cell surface by a well-described endocytic pathway leading to the lysosome and rapid receptor degradation [3]. Before the lysosomal delivery, however, receptor signal transduction events take place from intracellular endocytic compartments [4]. Related to the intracellular trafficking of ErbB receptors is the finding that each of these receptors is reported to be present in the nucleus and, in the instance of ErbB-1 and ErbB-4, this relocalization is ligand dependent and influences gene expression [5], thus revealing a novel signaling pathway. Here, we discuss recent data that relate to the function of ErbB receptors. Space limitations and the availability of other reviews constrain the scope and background information presented. The newer data discussed here represent advances in understanding dimerization and kinase activation mechanisms, including kinase mutations relevant to clinical treatment, the function of receptor fragments, receptor trafficking to novel intracellular sites, and new disease implications. We do not cover advances in signal transduction pathways, mechanisms of endocytosis, and the physiology of ErbB ligands and their precursors. Dimerization and activation Although ligand-induced ErbB dimerization has been recognized for about 20 years as a crucial event for receptor activation, the relevant protein–protein interactions were unknown until 2002. Significant insight into the molecular mechanism of ErbB dimerization comes from reports detailing the high-resolution structures of the soluble ectodomains of ErbB-1 [6–8], ErbB-2 [9], ErbB-3 [10] and ErbB-4 [11]. These revealed that in the unliganded state, ErbB-1, ErbB-3 and ErbB-4 exist in a ‘tethered’ intramolecular conformation, in which the dimerization motif is unable to mediate monomer–monomer interactions. By contrast, ligand-bound ErbB-1 and unliganded ErbB-2 exhibit a dimerization competent conformation in which the dimerization arm (a 10-residue sequence) is exposed on the receptor surface (Figure 1b). Ectodomain

0962-8924/$ – see front matter ß 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tcb.2006.10.008

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Box 1. Elements of the ErbB system There are 11 growth factors, each the product of a single gene, that serve as specific agonists for ErbB receptors. Each of these ligands is a soluble, small (6–10 kDa) protein that shares an EGF-like motif of three disulfide bonds generated by the conserved positioning of six cysteine residues. The neuregulins are also frequently referred to as heregulins, whereas the ErbB receptors are also designated HER-1, HER-2, HER-3, HER-4 and the EGF receptor (ErbB-1). Splicing generates alternative forms of NRG-1, NRG-2 and ErbB-4. The specificity of each growth factor for ErbB receptors is shown in Table I. The diffusible ligands that activate ErbB receptors are produced by the cell surface proteolysis of transmembrane precursors, as depicted in Figure I. Available data indicate that precursor cleavage is executed by members of the ADAM family of metalloproteases [74]. This proteolysis step is a point of acute regulation but the underlying molecular mechanisms are not clear.

Table I. Specificity of ErbB receptors and ligands Ligand EGF TGF-a HB-EGF Amphiregulin Betacellulin Epigen Epiregulin Neuregulin-1 Neuregulin-2 Neuregulin-3 Neuregulin-4

ErbB-1 + + + + + + +    

Receptor ErbB-2 ErbB-3                +  +    

ErbB-4   +  +  + + + + + Figure 1. ErbB receptor dimerization and activation. (a) A general scheme for ligand-dependent dimerization and activation of an ErbB receptor. (b) The contribution of the dimerization arm to receptor association within the ectodomain. Before ligand binding, the arm is sequestered within a monomer by interactions with subdomain IV. Ligand binding alters this interaction such that the arm is now exposed to facilitate dimerization by intermonomer associations between dimerization arms. From the data available, the likely consequence of ectodomain dimerization is the asymmetric interaction of kinase domains such that activation occurs (c). P, phosphorylation; Y, tyrosine.

Figure I. Tyrosine precursor forms of ErbB growth factors are present on the cell surface. Activation of ErbB receptors requires cleavage of the precursor to liberate the diffusible ligand. The enzyme that executes cleavage is a membrane of the ADAM family of transmembrane Zn2+ metalloproteases.

contacts between two monomers occur mainly through this dimerization arm, although other contact regions exist. Mutagenesis of the arm [6,7] or an adjacent loop [12] abrogates receptor activation but not ligand binding. In the absence of bound ligand, this arm is tethered by intramolecular contacts, preventing intermolecular association of monomers. Introduction of mutations thought to release the tethered arm do increase ligand binding affinity but do not provoke receptor dimerization or activation [8,13,14]. This indicates that ligand-induced exposure of the dimerization arm is necessary, but not sufficient, for receptor dimerization. Ligand-induced dimerization is postulated to mediate kinase activation by positioning two cytoplasmic domains such that transphosphorylation can occur. A recent paper [15] has indicated that kinase activation is, not surprisingly, a more intricate process. All protein kinase structures show that the kinase is divided into two lobes, www.sciencedirect.com

termed N and C, that cooperate to form the active site. Based on the analysis of intermolecular contacts observed in crystals of the isolated kinase domain and directed mutagenesis of the contact residues in the full-length receptor expressed in cells, the authors conclude that initiation of the active conformation of the one kinase domain is explained within the context of an asymmetric kinase dimer. In this mechanism the C-lobe of one kinase allosterically activates a second kinase molecule by contacting the N-lobe of that kinase and repositioning the activation loop such that catalysis is facilitated (Figure 1c). Left unresolved from the current data is the ultimate structure of the dimer in which both kinase domains are active. This structure addresses two other questions about ErbB kinase activation. One is the ErbB-3 conundrum. The ErbB-3 kinase domain is enzymatically inactive because of point mutations (relative to other ErbB kinases) and its heterodimerization with ErbB-2 is considered essential for ligand-bound ErbB-3 to activate signaling. However, if reciprocal transphosphorylation is required, this cannot occur when one kinase monomer is inactive. The model predicts that the C-lobe of the ErbB-3 kinase is capable of interacting with the N-lobe of an ErbB-2 kinase and allosterically initiating its activation.

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Box 2. ErbB receptor modifications The general structure of the mature ErbB receptors is represented in Figure I (numbering is for ErbB-1). The receptors (170–185 kDa) have an ectodomain that is heavily N-glycosylated at about 12 sites and characterized by two cysteine-rich subregions (domains II and IV) that produce 25 disulfide bonds. The ligand-binding site is formed by domains I and III. There is a single transmembrane domain. The cytoplasmic region encodes a tyrosine kinase domain, a C-terminal (CT) region containing all known autophosphorylation sites, and a juxtamembrane (JM) region. The ErbB-3 kinase domain has mutations that abrogate its kinase activity. Splice variants for ErbB-4 occur in the ectodomain (region IV) that allow (Jma) or disallow (Jmb) ectodomain cleavage and in the CT region that include (CYT-1) or exclude (CYT-2) a 16-residue motif that contains a PtdIns 3-kinase binding site. The ErbB receptors are subject to extensive co-translational (Nglycosylation) or post-translational modifications (phosphorylation and ubiquitination). These modifications have been mapped most extensively for ErbB-1 and are shown in Table I. Although conventional sequencing identified many phosphotyrosine and a few phosphoserine/threonine sites, the recent application of mass spectrometry has increased the list of modified sites and has also been useful to quantitatively map ligand and time-specific modifications [75–77]. In the case of phosphotyrosine residues, function is assigned in terms of the association of signaling proteins, as shown Table II. A comparison of the relative capacity of each ErbB receptor to associate with proteins through phosphotyrosine residues has recently been described for the ErbB family [78]. Many of the associated proteins are tyrosine phosphorylated by the receptor. Others, such as GRB-2, are not phosphorylated but function as adaptors to allow the receptor to influence pathways, such as Ras, without tyrosine phosphorylation of an exogenous substrate. In contrast to some other RTKs, such as the platelet-derived growth factor (PDGF) receptor, there is not a stringent requirement between one ErbB receptor phosphotyrosine site and a particular protein that associates with the receptor. Instead, there seem to be primary and secondary association sites.

Table I. Sites modified in ErbB-1a P-Tyr [76,79,80] 845 891 920 992 1045 1068 1086 1114 1148 1173

P-Ser [76] P-Thr [76] K-Ubiquitin [81] N-Glycosylated [76] 671 654 692 32 967 669 713 104 1002 730 151 1046 843 172 1047 905 328 1057 946 337 1142 389 420 504 544 579 599

a

The numbers shown are residue numbers

Table II. Proteins recruited to ErbB-1 P-Tyr sitesa Recruited Y891/920 Y992 Y1045 Y1068 protein + GRB-2 + SHC + PTP-1B + PTP-2C SHP-1 + SHP-2 + SRC + CBL DOK-R + PLC-g ABL

Y1086 Y1148 Y1173 + +

+ + +

+ +

+

+

+ +

+

a

Data taken from Refs [78,82].

Figure I. Using the numbering for ErbB-1, this diagram illustrates the structural features of each ErbB extracellular and intracellular region.

A second explanation derived from this structure deals the use of ErbB-1-specific kinase inhibitors, such as Iressa, for treatment of patients with lung cancer. Surprisingly, in a large trial, only a small fraction of these patients responded to treatment with Iressa. Only later was it recognized that patients who have mutations in the ErbB-1 kinase domain were responsive to the drug, whereas those without mutations were not [16]. These mutations promote survival signaling when placed in a mouse background and are oncogenic [17,18]. The study by Zhang et al. [15] shows that the ErbB-1 L834R mutation, which occurs in the kinase domain activation loop, increases the catalytic activity of the isolated kinase domain and suggests that this mutant intramolecularly destabilizes the inactive conformation of the kinase domain, probably allowing the inhibitor to bind more readily. However, the mutant still requires ligand stimulation www.sciencedirect.com

for maximal activity. The dimerization model reveals that because the mutation partially facilitates the active conformation, the mutant receptor should be more sensitive to clinically used kinase inhibitors, such as Iressa, that preferentially bind to the active state of the kinase. The biological activity of these clinically important ErbB-1 kinase domain mutations must also be interpreted in light of the influence of other ErbB receptors present in tumor cells. Evidence has been presented that ErbB-3 is required for survival signaling by these mutants [19], as ErbB-1 is by itself a poor activator of PtdIns-3 kinase. In addition, oncogenic kinase domain mutations in ErbB-2 provoke activation of ErbB-1 and resistance to certain ErbB-1 kinase inhibitors [20]. As these mutations are in the N-lobe of the ErbB-2 kinase, the model for allosteric activation of the ErbB-1 kinase by the C-lobe of ErbB-2 does not, however, provide a ready explanation.

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Although these structural studies are certainly enlightening, there is as yet no clear picture of how these dimerization and activation mechanisms operate together in the context of the membrane-bound intact receptor. Also unaccounted for in any of the crystal structures are the transmembrane domain (TM), the cytojuxtamembrane region (JM) and the C-terminal domain (CT), which are present in each of the ErbB receptors (Box 2). There is evidence that the unphosphorylated 225-residue CT domain of ErbB-1 negatively influences receptor activation [15,21] and, when tyrosine is phosphorylated, the CT is displaced from the kinase domain [22,23]. The JM region in other kinases, such as c-Kit, is known to contact the kinase domain and contains residues that, when mutated, activate kinase activity [24]. In ErbB receptors there are non-tyrosine phosphorylation sites in the 35-residue JM region. Threonine 654, a protein kinase C (PKC) site in the JM region of ErbB-1, is known to modulate kinase activity by a mechanism that has not been investigated. In addition, the JM region of each ErbB contains an unusually high number of basic residues that are reported to associate with acidic phospholipids, bind calmodulin, and influence dimerization [25–28]. The potential involvement of the TM domains is illustrated by the Neu mutation in the ErbB-2 TM that provokes constitutive dimerization and kinase activation. Finally, although the mechanistic focus is, at this point, on receptor dimerization, ligand-dependent receptor tetramerization occurs and might be part of the receptor activation process [29]. Given the presence of multiple negative regulators (phosphatases, ubiquitin ligases, adaptors) in the ErbB system [30,31], it is plausible that kinase activation also involves release from negative intermolecular constraints. The negative regulator RALT (Mig6, gene 33), a protein with no other known function, binds to all ErbB kinase domains and attenuates kinase activity [32]. RALT expression is induced by ErbB growth factors and might therefore act as a feedback inhibitor to reduce kinase activation. Depletion or knockout of RALT activates ErbB receptors and facilitates tumor formation [33,34]. Similarly, targeted overexpression of RALT in the skin blocks ErbB-1 signaling and generates a phenotype similar to a hypomorphic Egfr allele [35]. Although RALT seems to interact directly with ErbB kinase domains, the contact regions and mechanism of kinase inhibition have not been reported. Receptor trafficking to novel sites and receptor fragments The canonical endocytic pathway for trafficking dimerized and activated ErbB-1 from the cell surface to the lysosome was viewed for many years as a desensitization mechanism. However, more recent data implicate this pathway in assisting the delivery of signaling molecules to the nucleus and as an organizing center for the activation of signaling pathways (such as MAP kinase) [3,4]. A variation of the endocytic pathway is proposed as part of the mechanism for the translocation of activated intact ErbB-1 and ErbB-2 to the nucleus [36,37], where these receptors are implicated in the induction of specific genes, such as cyclin D, www.sciencedirect.com

iNOS, c-myb and COX-2 [38] (Figure 2). The presence of ErbB-3 in the nucleus has also been described [5]. There are two outstanding issues in these studies. The first is the absence of a demonstrable mechanism to extract these transmembrane molecules from a lipid bilayer into the nucleoplasm, as non-membranous molecules. The second is whether nuclear translocation is required for EGF action. The only evidence for this latter point is a mutant ErbB-1 receptor that exhibits neither nuclear translocation nor induction of certain target genes, such as iNOS, following the addition of EGF [39]. As the mutations used are within the basic region of the JM, pleiotropic effects are possible. Importantly, control experiments show that this mutant retains the ability to mediate EGF-dependent activation of Erk and induction of an Elk reporter. This result is consistent with a nuclear ErbB-1 requirement for iNOS induction by EGF, but does not prove the issue. In the absence of more mechanistic and direct information, this issue will continue to evoke skepticism. A different scenario exists for the ligand-dependent relocalization of ErbB-4 to the nucleus. In this case, growth factor-dependent secretase cleavage liberates the ErbB-4 intracellular domain (ICD, also known as membranebound m80 or soluble s80) from the plasma membrane, allowing it to localize to the cytoplasm and nucleus [40] (Figure 2). The ICD associates with several transcription factors [41–47] and it seems that the ICD is required to shuttle transcription factors into the nucleus or to regulate transcription factor function. There is less compelling evidence that the ICD functions as an essential part of a transcription complex at target gene promoters. Consistent with the ability of ErbB-4 to regulate STAT5, a key protein in mammary development, mammary cell studies suggest that ErbB-4 cleavage might be part of a mammary differentiation program [48,49]. Paradoxically, the ICD can also provoke cell death [48,50] or cell proliferation in mammary cells [46,51]. The cell death response to the ICD is suggested to be a consequence of ICD translocation to the mitochondria [48,50]. These studies, however, involve exogenous expression of the ICD, with the attendant concern that the level of expression could drive an artifactual response. Although it is clear that ErbB-4 has an important function in mammary differentiation [52], evidence for the requirement of the ICD fragment is only supportive at this time. ErbB-4 has been shown to regulate neural development [51] and more recently to regulate astrogenesis in the developing mouse cerebral cortex [47]. Mechanistically, the experiments demonstrate that the ICD fragment associates with a TAB-2–N-COR transcriptional repression complex. The function of the ICD in this system is to act as a carrier of TAB-2 and NCOR into the nucleus, reminiscent of data showing that the ICD is required for STAT5 nuclear localization in mammary cells [44]. Similarly, another group reports the association of the ICD with ETO-2, which is usually present with N-COR in a repression complex [45]. The ICD fragment is an active kinase that can form dimers [53]. The ICD phosphorylates tyrosine residues in Mdm2 [54] and increases p53 levels, which might be responsible, in part, for ErbB-4-dependent apoptotic and

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Figure 2. Processing of ErbB-1 and ErbB-4 from the cell surface to the nucleus. The steps in this process for each receptor are labelled (i–v). Following ligand binding, ErbB-1 (1, with the ICD in blue) is internalized by coated pits and released into the cytoplasm, by a mechanism not yet identified. ErbB-1 is known to associate with importin-b1, an interaction required for ErbB-1 nuclear localization. In the case of ErbB-4 (4, with the ICD in yellow), the translocation mechanism is clearer. The ligand occupied receptor is subject to two sequential cleavage events. The first cleavage is executed by ADAM17 to release the ectodomain and produce a cell-associated transmembrane (TM)-ICD fragment. This fragment is then cleaved within the TM domain by g-secretase to liberate the ICD from the plasma membrane and allow its translocation to the nucleus.

differentiation responses. Surprisingly, no other ICD phosphorylation substrates have been reported. It has been known for many years that in some cells secreted ErbB-1 or ErbB-3 ectodomain fragments are generated by a discrete mRNA; however, no function has been attributed to these fragments. In addition, ErbB-2 fragments are generated by several mechanism(s) that are not yet clearly understood. Many studies have reported that an ectodomain fragment of ErbB2 is found in the fluids of cancer patients [55]. This ectodomain fragment can be produced by proteolysis [56] or an aberrant transcriptional mechanism [57,58]. As the fragment can attenuate the activity of ErbB receptors, its clinical use is being explored [59]. Additionally, a 95-kDa ErbB-2 cytoplasmic domain fragment is produced by two distinct mechanisms: ectodomain cleavage [56] or an alternate reading frame mechanism [60]. Although there is a clinical association of disease progression with this fragment [61], its cellular function is not known, nor is it known if this fragment is present in the nucleus. www.sciencedirect.com

ErbB pathology The role of ErbB receptors in development and cancer has been described elsewhere and is not recounted here [2]. However, the relevance of ErbBs to other pathologies is evidenced by recent papers, as described here. ErbB-1 is known to be transactivated by a substantial variety of heterologous agonists, such as ligands that activate Gprotein-coupled receptors (GPCRs) [62–64]. In many cases, this requires the increased cleavage of an ErbB-1 ligand from its plasma membrane precursor, such that the diffusible ligand then dimerizes and activates ErbB-1 (Box 1). This ‘transactivation’ pathway allows GPCRs to influence cell growth and proliferation. One such transactivator is angiotensin II (AngII), which promotes a variety of lesions in the kidney that manifest as chronic renal disease. Lautrette et al. [65] have now shown that the capacity of AngII to promote kidney lesions in mice requires ErbB-1 transactivation. In this instance, AngII promotes the cleavage of protransforming growth factor (proTGF) by the transmembrane protease ADAM17

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(TACE), resulting in increased levels of free TGF-a and activation of ErbB-1. Similarly, adrenergic receptor transactivation of ErbB-1, mediated by the release of heparin-binding (HB)-EGF, is reported to be a significant factor in vasoconstriction and hypertension [66]. Although a role for ErbB-4 in neural development is clear [52], it was a surprising finding that ErbB-4 and its ligand neuregulin-1 (NRG-1) are involved in the pathogenesis of schizophrenia. The initial evidence came from human population studies that identified the NRG-1 gene as one of a small number of candidate genes for this disorder [67]. The schizophrenia haplotypes are mostly in the 50 region of the NRG-1 gene and might influence transcription factor association [68]; however, this has not been well characterized. Subsequent evidence has appeared that connects NRG-1 with ErbB-4 in schizophrenia [69]. Tissue studies show a large amount of activated ErbB-4 in schizophrenic brain tissue and an increased level of ErbB-4 association with PSD-95, an adaptor protein that interacts with the C-terminus of ErbB-4 and is known to mediate protein– protein scaffolding in neural synapses. One target of the ErbB-4–PSD-95 complex is the N-methyl-D-aspartate (NMDA) receptor and hypofunction of this receptor is implicated in schizophrenia. Although NRG-1 decreases NMDA receptor activation in normal brain tissue, the effect is greater in schizophrenic tissue, indicating that NRG-activated ErbB-4 might mediate the hypofunction of NMDA receptors in schizophrenia. It is interesting to note behavioral studies concluding that mice heterozygous for the NRG-1 or ErbB-4 gene exhibit behaviors akin to mouse models of schizophrenia [70,71]. ErbB-1 and ErbB-2 have recently been identified as receptors for viruses and bacteria, respectively. ErbB-1 is used by cytomegalovirus to enter target cells and ErbB-2 functions as a receptor for Mycobacterium leprae, the causative agent of leprosy. In each of these cases, strong evidence is presented that the ErbB in question is activated by pathogen binding to the cell surface and that this activation is required for biological activity of the pathogen. In the case of cytomegalovirus, evidence is presented to show that envelope glycoprotein gB interacts directly with ErbB-1 [72]. The data for M. leprae directly associating with ErbB-2, however, are less convincing, although consistent with that conclusion [73]. This could be an important issue because, despite intensive efforts, no direct ligand for ErbB-2 has been identified. Concluding remarks Investigations of ErbB receptors have led to numerous insights into general cell biology mechanisms, such as endocytosis, and pathologies derived from aberrations in these mechanisms. In addition, because of the early availability of reagents, lessons learned have been applicable to most other RTKs, of which there are now close to 100. From the recent data reviewed here, it seems that the influence of this receptor family continues to expand and provide new avenues for future investigation. It also seems likely that the mechanisms discussed are ‘works in progress’ and will be subject to additional layers of complexity. www.sciencedirect.com

Acknowledgements We thank Sue Carpenter for preparation of the manuscript. Support from Department of Defense grants BC043057 and BC04152 and NIH grant CA 75195 is acknowledged.

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