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Receptor tyrosine kinases as targets for anticancer drugs
Review
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Receptor tyrosine kinases as targets for anticancer drugs Esther Zwick, Johannes Bange and Axel Ullrich Receptor tyrosine kinases (RTKs) are the primary mediators of the signaling network that transmit extracellular signals into the cell. Gene amplification and/or overexpression of RTK proteins or functional alterations caused by mutations in the corresponding genes or abnormal autocrine–paracrine growth factor loops contribute to constitutive RTK signaling, ultimately resulting in the manifestation of dysregulated cell growth and cancer. The mechanism of uncontrolled RTK signaling that leads to cancer has provided the rationale for anti-RTK drug development. Strategies towards the prevention and interception of RTK signaling include monoclonal antibodies, small-molecule inhibitors, immunotoxins and antisense oligonucleotides.
Esther Zwick Johannes Bange Axel Ullrich* Max-Planck-Institut of Biochemistry, Dept of Molecular Biology, Am Klopferspitz 18a, 82152 Martinsried, Germany. *e-mail: [email protected]
Receptor tyrosine kinases (RTKs) are important regulators of intercellular communication controlling cell growth, proliferation, differentiation, survival and metabolism. Approximately 20 different RTK families have been identified that share a similar structure: an extracellular binding site for polypeptide growth factors, a transmembrane-spanning region and an intracellular tyrosine kinase domain [1]. Extracellular ligand binding induces or stabilizes receptor dimerization leading to increased RTK kinase activity. The intracellular catalytic domain displays the highest level of conservation between RTKs and includes the ATP-binding site that catalyzes receptor autophosphorylation of cytoplasmic tyrosine residues, which serve as docking sites for Src homology 2 (SH2)- and phosphotyrosine-binding (PTB) domain-containing proteins such as Shc, Grb2, Src, Cbl or phospholipase C γ. These proteins then recruit additional effector molecules containing SH2, SH3, PTB and pleckstrin-homology (PH) domains to the activated receptor, which results in the assembly of signaling complexes at the membrane and the activation of a cascade of intracellular biochemical signals. The most important downstream signaling cascades activated by RTKs include the Ras–extracellular regulated kinase (ERK)–mitogen activated (MAP) kinase pathway, the phosphoinositide 3-kinase (PI 3-kinase)–Akt and the JAK/STAT pathway (Fig. 1). Ultimately, the complex signaling network triggered by RTKs leads either to activation or repression of various subsets of genes and thus defines the biological response to a given signal. Role of RTKs in human cancer and other diseases
In normal cells, the activity of RTKs and their mediated cellular signaling is precisely co-ordinated and tightly controlled. Deregulation of this RTK signaling system, either by stimulation through autocrine–paracrine growth factor loops and/or http://tmm.trends.com
genetic alteration, result in deregulated tyrosine kinase activity. What most of these aberrations have in common is that they result in RTKs with constitutive or strongly enhanced signaling capacity, which leads to malignant transformation. Therefore, they are frequently linked to human cancer and also to other hyperproliferative diseases such as psoriasis. Moreover, certain genetic alterations are associated with distinct inherited and spontaneous human developmental syndromes, for example dwarfism [2]. The various mechanisms by which RTKs become potent oncogenes with transforming potential will be discussed below and are schematically shown in Fig. 2. Gene amplification and overexpression of RTKs
In many human cancers, gene amplification and/or overexpression of RTKs occurs, which might increase the response of cancer cells to normal growth factor levels. Additionally, overexpression of a specific RTK on the cell surface increases the incidence of receptor dimerization even in the absence of an activating ligand. In many cases this results in constitutive activation of the RTK leading to aberrant and uncontrolled cell proliferation and tumor formation. An important example for such a scenario is HER2, also known as ErbB2, that belongs to the epidermal growth factor (EGF) receptor family of RTKs. Overexpression and/or gene amplification of HER2 was found in various types of human cancers, especially in human breast and ovarian carcinomas [3]. Most importantly, Slamon et al. demonstrated that aberrantly elevated levels of HER2 correlate with more aggressive progression of disease and reduced patient survival time [4]. EGFR, which was the first receptor tyrosine kinase to be molecularly cloned [5], also plays a fundamental role in tumorigenesis. EGFR is frequently overexpressed in non-small-cell lung, bladder, cervical, ovarian, kidney and pancreatic cancer and in squamous-cell carcinomas of the head and neck [6]. The predominant mechanism leading to EGFR overexpression is gene amplification with up to 60 copies per cell reported in certain tumors [7]. In general, elevated levels of EGFR expression are associated with high metastatic rate and increased tumor proliferation [8]. Gene mutations
An important mechanism leading to deregulation of tyrosine kinases are genetic alterations including
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Ligand
Ligand
P
Extracellular domain
P
PLC
Grb 2
Intracellular catalytic/tyrosine kinase domain with ATP-binding site
Grb 2 Src
Shc Cbl
intermolecular disulfide bonding [11], or in the activation loop of the kinase domain were identified in human multiple myeloma [12]. Moreover, somatic mutations in FGFR 2 and 3 have been associated with human bladder and cervical carcinomas [13,14]. Multiple endocrine neoplasia type 2 (MEN2) is a dominant autosomal inherited cancer syndrome, which is characterized by the development of medullary thyroid carcinoma. The gene responsible for MEN2 was identified as rearranged during transformation (RET ) RTK, which is expressed during embryogenesis in the peripheral nervous system and is involved in neural crest and kidney development [15]. MEN2 cancer syndromes are caused by mutations at five different cysteine in the extracellular region of RET which result in intermolecular disulfide bonding, leading to constitutively activated receptors [16]. Mutations in the hepatocyte growth factor receptor have been identified in patients with inherited predisposition to develop multiple papillary renal cell carcinomas [17]. Most of these mutations are located adjacent to the kinase domain, leading to enhanced enzymatic activity, transformation of fibroblasts and invasive growth as shown by in vitro experiments [18]. Autocrine–paracrine stimulation
PI3 kinase/Akt
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JAK/STAT
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Cell cycle progression TRENDS in Molecular Medicine
Fig. 1. Receptor tyrosine kinase (RTK) signaling. Upon ligand binding and activation, the intracellular catalytic domain catalyzes receptor autophosphorylation of cytoplasmic tyrosine residues serving as docking sites for Src homology (SH)2- and phosphotyrosine-binding (PTB)-containing molecules. Assembly of activated protein complexes at the membrane trigger several signaling cascades which ultimately define the biological response. Abbreviations: ERK, extracellular-regulated kinase; JAK, Janus kinase; P, phosphorylated tyrosine residue; PI3 kinase, phosphatidylinositol-3 kinase; PLCγ, phospholipase C γ; STAT, signal transducer and activator of transcription.
deletion or mutations within the extracellular domain and alterations of the catalytic domain especially of the ATP-binding motif. The EGFRvIII mutant, for example, lacks amino acids 6–273 of the extracellular domain and gives rise to a constitutively active receptor tyrosine kinase that induces cell proliferation in the absence of ligand [9]. The EGFRvIII mutant has a strong transforming capacity and has been detected in glioblastomas, ovarian tumors, non-small-cell lung and breast carcinomas [10]. Mutations in another RTK family, the fibroblast growth factor receptor (FGFR) family, are connected to a number of human cancers. For example, point mutations either in the extracellular domain of the FGFR 3 resulting in an unpaired cysteine residue allowing abnormal receptor dimerization through http://tmm.trends.com
An important mechanism of constitutive RTK signaling involves autocrine–paracrine stimulation through growth factor loops and has been described for the EGFR and insulin-like growth factor-I receptor (IGF-IR) family [19,20]. This potent mechanism of activation occurs when a RTK is aberrantly expressed or overexpressed in the presence of its cognate ligand, or when overexpression of the ligand occurs in the presence of its associated receptor. For example, it has been shown in many solid tumors that elevated levels of both growth factor receptor and its ligand are expressed concomitantly [21]. In recent years, it was shown that the IGF-R1 and its cognate ligands insulin-like growth factor-I (IGF-I) and IGF-II are involved in the pathogenesis of a variety of human tumors, particularly breast, prostate and colorectal cancer. In breast and colorectal cancer cells the IGF-IR was found to be overexpressed and showed enhanced tyrosine kinase activity [22]. In addition, IGF-1 and IGF-II are predominantly expressed in stromal fibroblasts surrounding the normal and malignant breast epithelium [23], and it was demonstrated that high plasma IGF-1 levels correlate with an elevated prostate cancer risk [24]. These findings indicate that IGFs are able to stimulate breast and prostate carcinoma growth in a paracrine manner and therefore might stimulate and promote tumor formation. One of the ligands most investigated for its involvement in autocrine EGFR activation is transforming growth factor α (TGFα) [25]. Co-expression of TGFα and EGFR is frequently observed in glioblastomas and squamous-cell carcinomas of the head and neck and is correlated with poor prognosis [26].
Review
Fig. 2. Constitutive activation of RTKs. The most important mechanisms leading to constitutive RTK signaling include: overexpression and/or gene amplification of RTKs, genetic alterations such as deletions and mutations within the extracellular domain as well as alterations of the catalytic site, or autocrine–paracrine stimulation through aberrant growth factor loops. Abbreviations: P, phosphorylated tyrosine residue; RTK, receptor tyrosine kinase.
TRENDS in Molecular Medicine Vol.8 No.1 January 2002
Overexpression of RTKs
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Truncation or other gene mutation
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Furthermore, recent findings suggest that overexpression of G-protein-coupled receptor (GPCR) ligands, which often occurs in human tumor cells, might contribute to autocrine EGFR stimulation. Prenzel et al. demonstrated that GPCR-induced EGF-like growth factor precursor processing is an important step in EGFR transactivation [27]. This process of GPCR-induced release of growth factors, which can bind either in a paracrine or autocrine manner to the extracellular EGFR domain, involves an unknown metalloprotease. This process is known as the triple-membrane-passing signal (TMPS) mechanism [28,29]. Importantly, inhibition of the metalloprotease function by batimastat blocks bombesin-induced EGFR transactivation in PC3 human prostate cancer cells and significantly reduces the high constitutive level of EGFR tyrosine phosphorylation [27]. In addition, it was reported that batimastat reduces cell proliferation of a human mammary epithelial cell line by interfering with the release of EGFR ligand [30]. This suggests an important role for GPCR–EGFR cross-talk in the development and progression of human cancers known to depend on autocrine–paracrine signaling. Concepts in the design of RTK inhibitors
Since tyrosine kinases have been implicated in a variety of cancer indications, RTKs and the activated signaling cascades represent promising areas for the development of target-selective anticancer drugs. Because the mechanism of signal generation by RTKs http://tmm.trends.com
is rather well understood and the crystallographic structure of many RTKs is known, several approaches towards the prevention or interception of cancer-relevant signaling have been persued. Strategies include the development of selective components that target either the extracellular ligand-binding domain, the intracellular tyrosine kinase or the substrate-binding region. The variety of pharmacological agents, such as monoclonal antibodies, antibody conjugates, antisense oligonucleotides and small chemical compounds that can be used in such therapeutic strategies are discussed (Fig. 3). Monoclonal antibodies as anti-RTK drugs
An effective strategy to selectively kill tumor cells is the usage of monoclonal antibodies (mAbs) that are directed against the extracellular domain of RTKs [31]. Recombinant antibody technology has enabled the design, selection and production of humanized or human antibodies, human-mouse chimeric or bispecific antibodies for targeted cancer therapy [32,33]. For example, based on the discovery of the importance of HER2 gene amplification in breast cancer Herceptin, a mAb against HER2, was developed. Herceptin was accepted by the Food and Drug Administration (FDA) in 1998 for the treatment of HER2-overexpressing breast cancer. Moreover, herceptin represents the first genomics-based therapeutic agent that is applied selectively on the basis of genetic characteristics of the tumor [34,35]. In patients treated with herceptin as single therapy,
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altering the intracellular signaling pattern inside the targeted tumor cell. It appears that by cross-linking or binding to membrane receptors mAbs mimic or modulate receptor signaling activity. These mAbs-generated transmembrane signals might cause apoptosis and/or growth inhibition [42].
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Fig. 3. Target-selective intervention of RTK signaling. Strategies towards the prevention and interception of RTK signaling. Abbreviations: MAb, monoclonal antibody; ODN, oligodeoxynucleotides; P, phosphorylated tyrosine residue; RTK, receptor tyrosine kinase; TKI, tyrosine kinase inhibitor.
disease stabilization was observed in about 14% of all of the evaluable patients [36]. When combined with antracycline or paclitaxel, herceptin increased the overall response rate to about 45% and improved the survival time by approximately 25% [37]. The monoclonal antibody cetuximab (C225) represents a promising drug for anti-EGFR therapies. C225, which is directed against the extracellular domain of the EGFR, has shown growth-inhibiting and anti-tumor effect in a variety of human cancer cells including pancreatic, renal and breast carcinomas [38,39]. It is now being tested in a number of clinical trials, either alone or in combination with chemotherapeutic treatments such as cisplatin, paclitaxel or gemcitabine [40,41]. In addition to herceptin and cetuximab, other promising mAbs directed against HER2 and EGFR are currently in clinical development and are listed in Table 1. Mechanistically, anti-RTK mAbs have been shown to work by blocking the ligand–receptor interaction and therefore inhibiting ligand-induced RTK signaling and increase RTK downregulation and internalization. Moreover, by binding to certain epitopes on the cancer cells mAbs induce immune-mediated responses such as complement-mediated lysis and trigger antibodydependent cellular cytotoxicity by macrophages or natural killer cells. In recent years, it has become evident that certain mAbs influence tumor growth by http://tmm.trends.com
Another promising approach to inhibit aberrant RTK signaling is the development of small-molecule drugs that selectively interfere with their intrinsic tyrosine kinase activity and thereby block receptor autophosphorylation and activation of downstream signal transducers [43]. Protein tyrosine kinase inhibitors such as genistein and herbimycin A, isolated from fungal extracts, have served as a starting point for the generation of many types of synthetic small-molecule tyrosine kinase inhibitors (TKIs), particularly for the EGFR family. The most advanced of these compounds are ATP analogues of the quinazoline and pyridopyrimidine family that compete with ATP for the ATP-binding site at the receptor tyrosine kinase domain and thus block RTK activation [44]. The quinazoline ZD-1839 (Iressa) shows a significant anti-tumor effect on human breast, ovarian and colon cancer cells that co-express EGFR and its ligand TGFα. In combination with various cytotoxic agents it produced a synergistic enhancement of growth inhibition in tumor cells [45]. For its potential clinical utility ZD-1839, is currently evaluated in Phase II clinical trials for the treatment of solid tumors. The small-molecule Tarceva (OSI-774) is another potent EGFR inhibitor and is currently tested in phase II clinical trials against solid tumors [46]. The tyrosine kinase inhibitor gleevec (STI 571) belongs to the class of 2-phenylaminopyrimidine pharmacophores and exhibits a high selectivity for the non-receptor tyrosine kinase abl, but also affects the activity of two related RTKs, PDGFR and c-Kit [47,48]. The compound is now in stage III clinical trials for the treatment of chronical myeloid leukemia, a disease caused by the expression of a bcr–abl fusion protein, which is the result of a translocation between chromosomes 9 and 22. As STI 571 has shown encouraging results in stage I, II clinical trials, the FDA approved gleevec in May 2001 before phase III clinical trials are completed [49]. Another class of synthetic RTK inhibitors targets molecules that are not found on tumor but on endothelial cells. For example, SU5416, which specifically blocks Flk-1/VEGFR-2 RTK, is a promising anti-angiogenic drug preventing the formation of new blood vessels and thereby inhibiting the further expansion of tumors [50]. SU5416 is currently evaluated in Phase III clinical trials for the treatment of a variety of solid tumors. Additionally, it has been shown that SU6668, a synthetic RTK inhibitor for FGFR, vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor
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Table 1. RTK-based drugs in clinical trialsa RTK
Drug
HER2
Trastuzumab Genentech Herceptin BsAb 2B-1, Chiron NSC-673928
Approved by the FDA 1998 Phase Ib/II (03/98)
HER2
APC8024
Phase I
EGFR
C225 Cetuximab MDX-447
HER2
EGFR EGFR EGFR EGFR
ABX-EGF ZD18539 Iressa DAB389EGF
EGFR
OSI-774 Tarceva Abl/ STI 571 PDGFR/ Gleevec c-Kit VEGFR2 SU5416 VEGFR2 IMC-1C11 VEGFR1 RPI.4610 Angiozyme VEGFR/ FGFR/ IGF1R
SU6668
TRK
CEP-701
INX-4437
Company
Description
MAb directed against HER2 Bispecific MAb inducing lysis of HER2-expressing tumor cells Dendreon Vaccine against HER2overexpressing tumors ImClone MAb directed against Systems EGFR Medarex Bispecific Mab against EGFR Abgenix MAb against EGFR AstraZeneca TKI that inhibits EGFR signalling Seragen Recombinant diphtheria toxin-hEGF fusion protein OSI Small-molecule that Pharmaceuticals directly inhibits EGFR Novartis TKI that interferes with Abl, PDGFR and c-Kit
SUGEN TKI that inhibits VEGFR2 ImClone MAb against VEGFR2 Systems Ribozyme Nuclease-stabilized Pharmaceuticals hairpin ribozyme targeting VEGFR1 mRNA SUGEN RTK inhibition of VEGFR, FGFR and PDGFR INEX USA Antisense ODN targeting IGF1R mRNA Cephalon TKI of TRK receptor kinase
Status
•
•
Phase II
•
Phase II Phase III
•
Phase II
What will be the influence of new methods of genomic diagnosis such as microarray gene expression analysis of RTK genes on patient risk management? Will inhibition of RTKs have a role in chemoprevention of cancer? What is the function of RTKs in other hyperproliferative and inflammatory disorders? Can single nucleotide polymorphisms (SNPs) in RTKs be associated with increased risk of cancer formation and progression? Will pharmacogenomics accelerate the development of new and more RTK-specific drugs?
Phase III Phase III Approved by the FDA 2001 Phase II Phase I Phase I/II
Phase I Phase I Phase II
aAbbreviations: FDA, Food and Drug Administration; FGFR, fibroblast growth factor
receptor; MAb, monoclonal antibody; ODN, oligodeoxynucleotide; PDGFR, platelet derived growth factor receptor; RTK, receptor tyrosine kinase; TKI, tyrosine kinase inhibitor; VEGFR, vascular endothelial growth factor receptor.
receptor (PDGFR) proteins involved in endothelial cell growth and neovascularization, shows striking regression of large established human tumor xenografts, and phase I clinical trials are ongoing [51]. Using the advantage of structure-based drug design, combinatorial chemistry and high-throughput screening, new structural classes of TKIs with increased potency and selectivity, higher in vitro and in vivo efficacy and decreased toxicity have emerged [52]. One successful example is the discovery of site-directed irreversible inhibitors of the EGFR family with unique pharmacological properties and high efficacy that appear to represent a promising new generation of TKIs for the anti-cancer therapy. Alternative strategies: immunotoxins and antisense oligos
Additional strategies for the inhibition of receptor tyrosine kinase signaling include immunotoxins and antisense oligonucleotides. Immunotoxin conjugates and ligand-binding cytotoxic agents
Existing immunotoxins either contain the bacterial toxins Pseudomonas exotoxin A and diphtheria toxin http://tmm.trends.com
Outstanding questions
• Phase III
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or the plant toxins ricin A and clavin. They are fused or chemically conjugated to a specific ligand, such as the variable domains of the heavy and light chains of monoclonal antibodies, or to a growth factor. Upon binding to specific cell surface receptors, the toxin is rapidly internalized into an endosome and translocated to the cytosol where it directly inhibits protein synthesis leading to concomitant induction of apoptosis [53]. Cells that do not contain a cancer cell-specific antigen on the cell surface are not recognized and thus survive. One promising immunotoxin is the EGF fusion protein DAB389EGF, which contains the enzymatically active and membrane translocation domains of diphtheria toxin and sequences for human EGF. Preclinical studies have shown that a variety of EGFR-expressing tumors, such as breast cancer and non-small cell lung cancer cells, are sensitive to DAB389EGF, and this recombinant immunotoxin is now being evaluated in Phase II clinical trials [54]. Antisense strategies
Antisense oligodeoxynucleotides are short pieces of synthetic DNA or RNA that are designed to interact with the mRNA to block the transcription and thus the expression of specific target proteins. These compounds interact with the mRNA by Watson–Crick base-pairing and are therefore highly specific for the target protein. Preclinical studies have shown that antisense oligodeoxynucleotides (ODN) targeting the insulin-like growth factor-1 receptor (IGF-1R) induces not only apoptosis of malignant melanoma cells, but also causes reversal of epidermal hyperproliferation which occurs in psoriasis [55,56]. Because of the potential antitumor effect of IGF-1R ODN in malignant astrocytomas, this compound is now being evaluated in Phase I clinical trials for the treatment of brain cancer. Antisense ODN directed against the tyrosine kinase HER2 encapsulated in a transmembrane carrier system are also analyzed in preclinical studies for the treatment of breast cancer.
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Prospects for the future
Empirical observations and experimental studies in the past 20 years have led to an emerging consensus that several classes of receptor tyrosine kinases and polypeptide growth factors play an important role in the initiation and progression of human cancer. Moreover, following rapid advances in the characterization of cellular signaling mechanisms and pathways activated by receptor tyrosine kinases in normal and malignant cells the potential of RTKs as selective anti-cancer targets for therapeutic References 1 Ullrich, A. and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203–212 2 Robertson, S.C. et al. (2000) RTK mutations and human syndromes when good receptors turn bad. Trends Genet. 16, 265–271 3 Slamon, D.J. et al. (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER2/neu oncogene. Science 235, 77–82 4 Paik, S. et al. (1990) Pathologic findings from the national surgical adjuvant breast and bowel project: Prognostic significance of erbB-2 protein overexpression in primary breast cancer. J. Clin. Oncol. 8, 103–112 5 Ullrich, A. et al. (1984) Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309, 418–425 6 Hong, W.K. and Ullrich, A. (2000) The role of EGFR in solid tumors and implications for therapy. Oncol. Biother. 1, 1–29 7 Libermann, T.A. et al. (1985) Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature 313, 144–147 8 Pavelic, K. et al. (1993) Evidence for a role of EGF receptor in the progression of human lung carcinoma. Anticancer Res. 13, 1133–1138 9 Ekstrand, A.J. et al. (1992) Functional characterization of an EGF receptor with a truncated extracellular domain expressed in glioblastomas with EGFR gene amplification. Oncogene 9, 2313–2320 10 Nishikawa, R. et al. (1994) A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. U. S. A. 91, 7727–7731 11 Kannan, K. and Givol, D. (2000) FGF receptor mutations: dimerization syndromes, cell growth suppression, and animal models. IUBMB Life 49, 197–205 12 Chesi, M. et al. (1997) Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat. Genet. 16, 260–264 13 Cappellen, D. et al. (1999) Frequent activating mutations of FGFR3 in human bladder and cervix carcinomas. Nat. Genet. 23, 18–20 14 Jang, J.H. et al. (2001) Mutations in fibroblast growth factor receptor 2 and fibroblast growth factor receptor 3 genes associated with human gastric and colorectal cancers. Cancer Res. 61, 3541–3543 15 Edery, P. et al. (1997) Ret in human development and oncogenesis. BioEssays 19, 389–395 http://tmm.trends.com
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53 Frankel, A.E. et al. (2000) Targeted Toxins. Clin. Cancer Res. 6, 326–334 54 Murphy, J.R. and vanderSpek, J.C. (1995) Targeting diphteria toxin to growth factor receptors. Semin. Cancer Biol. 6, 259–267 55 Andrews, D.W. et al. (2001) Results of a pilot study involving the use of an antisense oligodeoxynucleotide directed against the insulinlike growth factor type I receptor in malignant astrocytomas. J. Clin. Oncol. 19, 2189–2200 56 Wraight, C.J. et al. (2000) Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I antisense oligonucleotides. Nat. Biotechnol. 18, 521–526
Rab GTPases, intracellular traffic and disease Miguel C. Seabra, Emilie H. Mules and Alistair N. Hume Membrane and protein traffic in the secretory and endocytic pathways is mediated by vesicular transport. Recent studies of certain key regulators of vesicular transport, the Rab GTPases, have linked Rab dysfunction to human disease. Mutations in Rab27a result in Griscelli syndrome, caused by defects in melanosome transport in melanocytes and loss of cytotoxic killing activity in T cells. Other genetic diseases are caused by partial dysfunction of multiple Rab proteins resulting from mutations in general regulators of Rab activity; Rab escort protein-1 (choroideremia), Rab geranylgeranyl transferase α (X-linked (Hermansky–Pudlak syndrome) and Rab GDP dissociation inhibitor-α mental retardation). In infectious diseases caused by intracellular microorganisms, the function of endocytic Rabs is altered either as part of host defences or as part of survival strategy of the pathogen. The human genome is predicted to contain 60 RAB genes, suggesting that future work could reveal further links between Rab dysfunction and disease.
Miguel C. Seabra* Emilie H. Mules Alistair N. Hume Cell and Molecular Biology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Exhibition Road, London, UK SW7 2AZ. *e-mail: [email protected]
Intracellular protein and lipid traffic is a fundamental process required for the generation of specialized membranous organelles and the communication between them. One form of communication between organelles is through vesicular transport. This is a multi-step process, including formation of a vesicular or tubular carrier from the donor organelle membrane, movement of the carrier towards the acceptor, tethering/docking of the carrier with the acceptor and finally fusion of the carrier and acceptor compartment (Fig. 1). Transport pathways in cells have been traditionally divided into a secretory pathway starting at the endoplasmic reticulum and finishing at the plasma membrane, and an endocytic pathway starting at the plasma membrane and ending in the lysosome. Within each main pathway anterograde and retrograde routes exist and both pathways communicate at multiple junctures. Defects in intracellular trafficking underlie a large variety of human diseases [1,2]. In heritable http://tmm.trends.com
diseases, genes that control and/or encode protein machinery involved in vesicular transport can be affected by mutations. In addition, certain acquired diseases including infections, cancer and autoimmunity alter membrane trafficking pathways [2–6]. This review focuses on recent advances in understanding human disease through the study of a family of crucial regulators of vesicular transport, Rab proteins. Rab GTPases
Rab proteins (also known as Ypt in yeasts and plants) are monomeric GTPases of the Ras superfamily (reviewed in Refs [7–9]). Recent analyses indicate that Rabs are present in all eukaryotes and the Rab families in the genomes of several species have been reported [10–12]. Currently 60 human RAB genes are known (see Table 1). However, the complexity of the Rab protein family might be even greater as there is evidence that alternative splicing of Rab genes results in the production of functionally distinct isoforms [13]. Although intrinsically soluble, post-translational addition of isoprenoid moieties allows Rabs to associate with the cytoplasmic face of membranebound intracellular organelles and vesicles (Fig. 2) (reviewed in Ref. [14]). Lipid-modified Rabs appear to be targeted to specific membranes and thus display a characteristic pattern of subcellular localization. Evolutionarily conserved Rabs tend to be expressed in all cell and tissue types and regulate fundamental transport pathways whereas less conserved family members function in the many specialized pathways found in different mammalian cell types (Table 1).
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Receptor tyrosine kinases as targets for anticancer drugs Esther Zwick, Johannes Bange and Axel Ullrich Receptor tyrosine kinases (RTKs) are the primary mediators of the signaling network that transmit extracellular signals into the cell. Gene amplification and/or overexpression of RTK proteins or functional alterations caused by mutations in the corresponding genes or abnormal autocrine–paracrine growth factor loops contribute to constitutive RTK signaling, ultimately resulting in the manifestation of dysregulated cell growth and cancer. The mechanism of uncontrolled RTK signaling that leads to cancer has provided the rationale for anti-RTK drug development. Strategies towards the prevention and interception of RTK signaling include monoclonal antibodies, small-molecule inhibitors, immunotoxins and antisense oligonucleotides.
Esther Zwick Johannes Bange Axel Ullrich* Max-Planck-Institut of Biochemistry, Dept of Molecular Biology, Am Klopferspitz 18a, 82152 Martinsried, Germany. *e-mail: [email protected]
Receptor tyrosine kinases (RTKs) are important regulators of intercellular communication controlling cell growth, proliferation, differentiation, survival and metabolism. Approximately 20 different RTK families have been identified that share a similar structure: an extracellular binding site for polypeptide growth factors, a transmembrane-spanning region and an intracellular tyrosine kinase domain [1]. Extracellular ligand binding induces or stabilizes receptor dimerization leading to increased RTK kinase activity. The intracellular catalytic domain displays the highest level of conservation between RTKs and includes the ATP-binding site that catalyzes receptor autophosphorylation of cytoplasmic tyrosine residues, which serve as docking sites for Src homology 2 (SH2)- and phosphotyrosine-binding (PTB) domain-containing proteins such as Shc, Grb2, Src, Cbl or phospholipase C γ. These proteins then recruit additional effector molecules containing SH2, SH3, PTB and pleckstrin-homology (PH) domains to the activated receptor, which results in the assembly of signaling complexes at the membrane and the activation of a cascade of intracellular biochemical signals. The most important downstream signaling cascades activated by RTKs include the Ras–extracellular regulated kinase (ERK)–mitogen activated (MAP) kinase pathway, the phosphoinositide 3-kinase (PI 3-kinase)–Akt and the JAK/STAT pathway (Fig. 1). Ultimately, the complex signaling network triggered by RTKs leads either to activation or repression of various subsets of genes and thus defines the biological response to a given signal. Role of RTKs in human cancer and other diseases
In normal cells, the activity of RTKs and their mediated cellular signaling is precisely co-ordinated and tightly controlled. Deregulation of this RTK signaling system, either by stimulation through autocrine–paracrine growth factor loops and/or http://tmm.trends.com
genetic alteration, result in deregulated tyrosine kinase activity. What most of these aberrations have in common is that they result in RTKs with constitutive or strongly enhanced signaling capacity, which leads to malignant transformation. Therefore, they are frequently linked to human cancer and also to other hyperproliferative diseases such as psoriasis. Moreover, certain genetic alterations are associated with distinct inherited and spontaneous human developmental syndromes, for example dwarfism [2]. The various mechanisms by which RTKs become potent oncogenes with transforming potential will be discussed below and are schematically shown in Fig. 2. Gene amplification and overexpression of RTKs
In many human cancers, gene amplification and/or overexpression of RTKs occurs, which might increase the response of cancer cells to normal growth factor levels. Additionally, overexpression of a specific RTK on the cell surface increases the incidence of receptor dimerization even in the absence of an activating ligand. In many cases this results in constitutive activation of the RTK leading to aberrant and uncontrolled cell proliferation and tumor formation. An important example for such a scenario is HER2, also known as ErbB2, that belongs to the epidermal growth factor (EGF) receptor family of RTKs. Overexpression and/or gene amplification of HER2 was found in various types of human cancers, especially in human breast and ovarian carcinomas [3]. Most importantly, Slamon et al. demonstrated that aberrantly elevated levels of HER2 correlate with more aggressive progression of disease and reduced patient survival time [4]. EGFR, which was the first receptor tyrosine kinase to be molecularly cloned [5], also plays a fundamental role in tumorigenesis. EGFR is frequently overexpressed in non-small-cell lung, bladder, cervical, ovarian, kidney and pancreatic cancer and in squamous-cell carcinomas of the head and neck [6]. The predominant mechanism leading to EGFR overexpression is gene amplification with up to 60 copies per cell reported in certain tumors [7]. In general, elevated levels of EGFR expression are associated with high metastatic rate and increased tumor proliferation [8]. Gene mutations
An important mechanism leading to deregulation of tyrosine kinases are genetic alterations including
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Ligand
Ligand
P
Extracellular domain
P
PLC
Grb 2
Intracellular catalytic/tyrosine kinase domain with ATP-binding site
Grb 2 Src
Shc Cbl
intermolecular disulfide bonding [11], or in the activation loop of the kinase domain were identified in human multiple myeloma [12]. Moreover, somatic mutations in FGFR 2 and 3 have been associated with human bladder and cervical carcinomas [13,14]. Multiple endocrine neoplasia type 2 (MEN2) is a dominant autosomal inherited cancer syndrome, which is characterized by the development of medullary thyroid carcinoma. The gene responsible for MEN2 was identified as rearranged during transformation (RET ) RTK, which is expressed during embryogenesis in the peripheral nervous system and is involved in neural crest and kidney development [15]. MEN2 cancer syndromes are caused by mutations at five different cysteine in the extracellular region of RET which result in intermolecular disulfide bonding, leading to constitutively activated receptors [16]. Mutations in the hepatocyte growth factor receptor have been identified in patients with inherited predisposition to develop multiple papillary renal cell carcinomas [17]. Most of these mutations are located adjacent to the kinase domain, leading to enhanced enzymatic activity, transformation of fibroblasts and invasive growth as shown by in vitro experiments [18]. Autocrine–paracrine stimulation
PI3 kinase/Akt
Migration
Ras/ERK
JAK/STAT
Survival
Proliferation Gene transcription
Cell cycle progression TRENDS in Molecular Medicine
Fig. 1. Receptor tyrosine kinase (RTK) signaling. Upon ligand binding and activation, the intracellular catalytic domain catalyzes receptor autophosphorylation of cytoplasmic tyrosine residues serving as docking sites for Src homology (SH)2- and phosphotyrosine-binding (PTB)-containing molecules. Assembly of activated protein complexes at the membrane trigger several signaling cascades which ultimately define the biological response. Abbreviations: ERK, extracellular-regulated kinase; JAK, Janus kinase; P, phosphorylated tyrosine residue; PI3 kinase, phosphatidylinositol-3 kinase; PLCγ, phospholipase C γ; STAT, signal transducer and activator of transcription.
deletion or mutations within the extracellular domain and alterations of the catalytic domain especially of the ATP-binding motif. The EGFRvIII mutant, for example, lacks amino acids 6–273 of the extracellular domain and gives rise to a constitutively active receptor tyrosine kinase that induces cell proliferation in the absence of ligand [9]. The EGFRvIII mutant has a strong transforming capacity and has been detected in glioblastomas, ovarian tumors, non-small-cell lung and breast carcinomas [10]. Mutations in another RTK family, the fibroblast growth factor receptor (FGFR) family, are connected to a number of human cancers. For example, point mutations either in the extracellular domain of the FGFR 3 resulting in an unpaired cysteine residue allowing abnormal receptor dimerization through http://tmm.trends.com
An important mechanism of constitutive RTK signaling involves autocrine–paracrine stimulation through growth factor loops and has been described for the EGFR and insulin-like growth factor-I receptor (IGF-IR) family [19,20]. This potent mechanism of activation occurs when a RTK is aberrantly expressed or overexpressed in the presence of its cognate ligand, or when overexpression of the ligand occurs in the presence of its associated receptor. For example, it has been shown in many solid tumors that elevated levels of both growth factor receptor and its ligand are expressed concomitantly [21]. In recent years, it was shown that the IGF-R1 and its cognate ligands insulin-like growth factor-I (IGF-I) and IGF-II are involved in the pathogenesis of a variety of human tumors, particularly breast, prostate and colorectal cancer. In breast and colorectal cancer cells the IGF-IR was found to be overexpressed and showed enhanced tyrosine kinase activity [22]. In addition, IGF-1 and IGF-II are predominantly expressed in stromal fibroblasts surrounding the normal and malignant breast epithelium [23], and it was demonstrated that high plasma IGF-1 levels correlate with an elevated prostate cancer risk [24]. These findings indicate that IGFs are able to stimulate breast and prostate carcinoma growth in a paracrine manner and therefore might stimulate and promote tumor formation. One of the ligands most investigated for its involvement in autocrine EGFR activation is transforming growth factor α (TGFα) [25]. Co-expression of TGFα and EGFR is frequently observed in glioblastomas and squamous-cell carcinomas of the head and neck and is correlated with poor prognosis [26].
Review
Fig. 2. Constitutive activation of RTKs. The most important mechanisms leading to constitutive RTK signaling include: overexpression and/or gene amplification of RTKs, genetic alterations such as deletions and mutations within the extracellular domain as well as alterations of the catalytic site, or autocrine–paracrine stimulation through aberrant growth factor loops. Abbreviations: P, phosphorylated tyrosine residue; RTK, receptor tyrosine kinase.
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Overexpression of RTKs
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Truncation or other gene mutation
Autocrine growth factor loops
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Ligand
Ligand
Ligand
Ligand Ligand
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P
P
P
P
P
P
P
P
P
P
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Survival TRENDS in Molecular Medicine
Furthermore, recent findings suggest that overexpression of G-protein-coupled receptor (GPCR) ligands, which often occurs in human tumor cells, might contribute to autocrine EGFR stimulation. Prenzel et al. demonstrated that GPCR-induced EGF-like growth factor precursor processing is an important step in EGFR transactivation [27]. This process of GPCR-induced release of growth factors, which can bind either in a paracrine or autocrine manner to the extracellular EGFR domain, involves an unknown metalloprotease. This process is known as the triple-membrane-passing signal (TMPS) mechanism [28,29]. Importantly, inhibition of the metalloprotease function by batimastat blocks bombesin-induced EGFR transactivation in PC3 human prostate cancer cells and significantly reduces the high constitutive level of EGFR tyrosine phosphorylation [27]. In addition, it was reported that batimastat reduces cell proliferation of a human mammary epithelial cell line by interfering with the release of EGFR ligand [30]. This suggests an important role for GPCR–EGFR cross-talk in the development and progression of human cancers known to depend on autocrine–paracrine signaling. Concepts in the design of RTK inhibitors
Since tyrosine kinases have been implicated in a variety of cancer indications, RTKs and the activated signaling cascades represent promising areas for the development of target-selective anticancer drugs. Because the mechanism of signal generation by RTKs http://tmm.trends.com
is rather well understood and the crystallographic structure of many RTKs is known, several approaches towards the prevention or interception of cancer-relevant signaling have been persued. Strategies include the development of selective components that target either the extracellular ligand-binding domain, the intracellular tyrosine kinase or the substrate-binding region. The variety of pharmacological agents, such as monoclonal antibodies, antibody conjugates, antisense oligonucleotides and small chemical compounds that can be used in such therapeutic strategies are discussed (Fig. 3). Monoclonal antibodies as anti-RTK drugs
An effective strategy to selectively kill tumor cells is the usage of monoclonal antibodies (mAbs) that are directed against the extracellular domain of RTKs [31]. Recombinant antibody technology has enabled the design, selection and production of humanized or human antibodies, human-mouse chimeric or bispecific antibodies for targeted cancer therapy [32,33]. For example, based on the discovery of the importance of HER2 gene amplification in breast cancer Herceptin, a mAb against HER2, was developed. Herceptin was accepted by the Food and Drug Administration (FDA) in 1998 for the treatment of HER2-overexpressing breast cancer. Moreover, herceptin represents the first genomics-based therapeutic agent that is applied selectively on the basis of genetic characteristics of the tumor [34,35]. In patients treated with herceptin as single therapy,
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altering the intracellular signaling pattern inside the targeted tumor cell. It appears that by cross-linking or binding to membrane receptors mAbs mimic or modulate receptor signaling activity. These mAbs-generated transmembrane signals might cause apoptosis and/or growth inhibition [42].
Immunotoxin Ligand
Ligand toxin
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Small-molecule tyrosine kinase inhibitors in cancer therapy TKI
P
P
P
P
Disease-relevant signaling of RTKs
Gene transcription
Antisense ODN
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Fig. 3. Target-selective intervention of RTK signaling. Strategies towards the prevention and interception of RTK signaling. Abbreviations: MAb, monoclonal antibody; ODN, oligodeoxynucleotides; P, phosphorylated tyrosine residue; RTK, receptor tyrosine kinase; TKI, tyrosine kinase inhibitor.
disease stabilization was observed in about 14% of all of the evaluable patients [36]. When combined with antracycline or paclitaxel, herceptin increased the overall response rate to about 45% and improved the survival time by approximately 25% [37]. The monoclonal antibody cetuximab (C225) represents a promising drug for anti-EGFR therapies. C225, which is directed against the extracellular domain of the EGFR, has shown growth-inhibiting and anti-tumor effect in a variety of human cancer cells including pancreatic, renal and breast carcinomas [38,39]. It is now being tested in a number of clinical trials, either alone or in combination with chemotherapeutic treatments such as cisplatin, paclitaxel or gemcitabine [40,41]. In addition to herceptin and cetuximab, other promising mAbs directed against HER2 and EGFR are currently in clinical development and are listed in Table 1. Mechanistically, anti-RTK mAbs have been shown to work by blocking the ligand–receptor interaction and therefore inhibiting ligand-induced RTK signaling and increase RTK downregulation and internalization. Moreover, by binding to certain epitopes on the cancer cells mAbs induce immune-mediated responses such as complement-mediated lysis and trigger antibodydependent cellular cytotoxicity by macrophages or natural killer cells. In recent years, it has become evident that certain mAbs influence tumor growth by http://tmm.trends.com
Another promising approach to inhibit aberrant RTK signaling is the development of small-molecule drugs that selectively interfere with their intrinsic tyrosine kinase activity and thereby block receptor autophosphorylation and activation of downstream signal transducers [43]. Protein tyrosine kinase inhibitors such as genistein and herbimycin A, isolated from fungal extracts, have served as a starting point for the generation of many types of synthetic small-molecule tyrosine kinase inhibitors (TKIs), particularly for the EGFR family. The most advanced of these compounds are ATP analogues of the quinazoline and pyridopyrimidine family that compete with ATP for the ATP-binding site at the receptor tyrosine kinase domain and thus block RTK activation [44]. The quinazoline ZD-1839 (Iressa) shows a significant anti-tumor effect on human breast, ovarian and colon cancer cells that co-express EGFR and its ligand TGFα. In combination with various cytotoxic agents it produced a synergistic enhancement of growth inhibition in tumor cells [45]. For its potential clinical utility ZD-1839, is currently evaluated in Phase II clinical trials for the treatment of solid tumors. The small-molecule Tarceva (OSI-774) is another potent EGFR inhibitor and is currently tested in phase II clinical trials against solid tumors [46]. The tyrosine kinase inhibitor gleevec (STI 571) belongs to the class of 2-phenylaminopyrimidine pharmacophores and exhibits a high selectivity for the non-receptor tyrosine kinase abl, but also affects the activity of two related RTKs, PDGFR and c-Kit [47,48]. The compound is now in stage III clinical trials for the treatment of chronical myeloid leukemia, a disease caused by the expression of a bcr–abl fusion protein, which is the result of a translocation between chromosomes 9 and 22. As STI 571 has shown encouraging results in stage I, II clinical trials, the FDA approved gleevec in May 2001 before phase III clinical trials are completed [49]. Another class of synthetic RTK inhibitors targets molecules that are not found on tumor but on endothelial cells. For example, SU5416, which specifically blocks Flk-1/VEGFR-2 RTK, is a promising anti-angiogenic drug preventing the formation of new blood vessels and thereby inhibiting the further expansion of tumors [50]. SU5416 is currently evaluated in Phase III clinical trials for the treatment of a variety of solid tumors. Additionally, it has been shown that SU6668, a synthetic RTK inhibitor for FGFR, vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor
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Table 1. RTK-based drugs in clinical trialsa RTK
Drug
HER2
Trastuzumab Genentech Herceptin BsAb 2B-1, Chiron NSC-673928
Approved by the FDA 1998 Phase Ib/II (03/98)
HER2
APC8024
Phase I
EGFR
C225 Cetuximab MDX-447
HER2
EGFR EGFR EGFR EGFR
ABX-EGF ZD18539 Iressa DAB389EGF
EGFR
OSI-774 Tarceva Abl/ STI 571 PDGFR/ Gleevec c-Kit VEGFR2 SU5416 VEGFR2 IMC-1C11 VEGFR1 RPI.4610 Angiozyme VEGFR/ FGFR/ IGF1R
SU6668
TRK
CEP-701
INX-4437
Company
Description
MAb directed against HER2 Bispecific MAb inducing lysis of HER2-expressing tumor cells Dendreon Vaccine against HER2overexpressing tumors ImClone MAb directed against Systems EGFR Medarex Bispecific Mab against EGFR Abgenix MAb against EGFR AstraZeneca TKI that inhibits EGFR signalling Seragen Recombinant diphtheria toxin-hEGF fusion protein OSI Small-molecule that Pharmaceuticals directly inhibits EGFR Novartis TKI that interferes with Abl, PDGFR and c-Kit
SUGEN TKI that inhibits VEGFR2 ImClone MAb against VEGFR2 Systems Ribozyme Nuclease-stabilized Pharmaceuticals hairpin ribozyme targeting VEGFR1 mRNA SUGEN RTK inhibition of VEGFR, FGFR and PDGFR INEX USA Antisense ODN targeting IGF1R mRNA Cephalon TKI of TRK receptor kinase
Status
•
•
Phase II
•
Phase II Phase III
•
Phase II
What will be the influence of new methods of genomic diagnosis such as microarray gene expression analysis of RTK genes on patient risk management? Will inhibition of RTKs have a role in chemoprevention of cancer? What is the function of RTKs in other hyperproliferative and inflammatory disorders? Can single nucleotide polymorphisms (SNPs) in RTKs be associated with increased risk of cancer formation and progression? Will pharmacogenomics accelerate the development of new and more RTK-specific drugs?
Phase III Phase III Approved by the FDA 2001 Phase II Phase I Phase I/II
Phase I Phase I Phase II
aAbbreviations: FDA, Food and Drug Administration; FGFR, fibroblast growth factor
receptor; MAb, monoclonal antibody; ODN, oligodeoxynucleotide; PDGFR, platelet derived growth factor receptor; RTK, receptor tyrosine kinase; TKI, tyrosine kinase inhibitor; VEGFR, vascular endothelial growth factor receptor.
receptor (PDGFR) proteins involved in endothelial cell growth and neovascularization, shows striking regression of large established human tumor xenografts, and phase I clinical trials are ongoing [51]. Using the advantage of structure-based drug design, combinatorial chemistry and high-throughput screening, new structural classes of TKIs with increased potency and selectivity, higher in vitro and in vivo efficacy and decreased toxicity have emerged [52]. One successful example is the discovery of site-directed irreversible inhibitors of the EGFR family with unique pharmacological properties and high efficacy that appear to represent a promising new generation of TKIs for the anti-cancer therapy. Alternative strategies: immunotoxins and antisense oligos
Additional strategies for the inhibition of receptor tyrosine kinase signaling include immunotoxins and antisense oligonucleotides. Immunotoxin conjugates and ligand-binding cytotoxic agents
Existing immunotoxins either contain the bacterial toxins Pseudomonas exotoxin A and diphtheria toxin http://tmm.trends.com
Outstanding questions
• Phase III
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or the plant toxins ricin A and clavin. They are fused or chemically conjugated to a specific ligand, such as the variable domains of the heavy and light chains of monoclonal antibodies, or to a growth factor. Upon binding to specific cell surface receptors, the toxin is rapidly internalized into an endosome and translocated to the cytosol where it directly inhibits protein synthesis leading to concomitant induction of apoptosis [53]. Cells that do not contain a cancer cell-specific antigen on the cell surface are not recognized and thus survive. One promising immunotoxin is the EGF fusion protein DAB389EGF, which contains the enzymatically active and membrane translocation domains of diphtheria toxin and sequences for human EGF. Preclinical studies have shown that a variety of EGFR-expressing tumors, such as breast cancer and non-small cell lung cancer cells, are sensitive to DAB389EGF, and this recombinant immunotoxin is now being evaluated in Phase II clinical trials [54]. Antisense strategies
Antisense oligodeoxynucleotides are short pieces of synthetic DNA or RNA that are designed to interact with the mRNA to block the transcription and thus the expression of specific target proteins. These compounds interact with the mRNA by Watson–Crick base-pairing and are therefore highly specific for the target protein. Preclinical studies have shown that antisense oligodeoxynucleotides (ODN) targeting the insulin-like growth factor-1 receptor (IGF-1R) induces not only apoptosis of malignant melanoma cells, but also causes reversal of epidermal hyperproliferation which occurs in psoriasis [55,56]. Because of the potential antitumor effect of IGF-1R ODN in malignant astrocytomas, this compound is now being evaluated in Phase I clinical trials for the treatment of brain cancer. Antisense ODN directed against the tyrosine kinase HER2 encapsulated in a transmembrane carrier system are also analyzed in preclinical studies for the treatment of breast cancer.
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Prospects for the future
Empirical observations and experimental studies in the past 20 years have led to an emerging consensus that several classes of receptor tyrosine kinases and polypeptide growth factors play an important role in the initiation and progression of human cancer. Moreover, following rapid advances in the characterization of cellular signaling mechanisms and pathways activated by receptor tyrosine kinases in normal and malignant cells the potential of RTKs as selective anti-cancer targets for therapeutic References 1 Ullrich, A. and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203–212 2 Robertson, S.C. et al. (2000) RTK mutations and human syndromes when good receptors turn bad. Trends Genet. 16, 265–271 3 Slamon, D.J. et al. (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER2/neu oncogene. Science 235, 77–82 4 Paik, S. et al. (1990) Pathologic findings from the national surgical adjuvant breast and bowel project: Prognostic significance of erbB-2 protein overexpression in primary breast cancer. J. Clin. Oncol. 8, 103–112 5 Ullrich, A. et al. (1984) Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature 309, 418–425 6 Hong, W.K. and Ullrich, A. (2000) The role of EGFR in solid tumors and implications for therapy. Oncol. Biother. 1, 1–29 7 Libermann, T.A. et al. (1985) Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature 313, 144–147 8 Pavelic, K. et al. (1993) Evidence for a role of EGF receptor in the progression of human lung carcinoma. Anticancer Res. 13, 1133–1138 9 Ekstrand, A.J. et al. (1992) Functional characterization of an EGF receptor with a truncated extracellular domain expressed in glioblastomas with EGFR gene amplification. Oncogene 9, 2313–2320 10 Nishikawa, R. et al. (1994) A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc. Natl. Acad. Sci. U. S. A. 91, 7727–7731 11 Kannan, K. and Givol, D. (2000) FGF receptor mutations: dimerization syndromes, cell growth suppression, and animal models. IUBMB Life 49, 197–205 12 Chesi, M. et al. (1997) Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat. Genet. 16, 260–264 13 Cappellen, D. et al. (1999) Frequent activating mutations of FGFR3 in human bladder and cervix carcinomas. Nat. Genet. 23, 18–20 14 Jang, J.H. et al. (2001) Mutations in fibroblast growth factor receptor 2 and fibroblast growth factor receptor 3 genes associated with human gastric and colorectal cancers. Cancer Res. 61, 3541–3543 15 Edery, P. et al. (1997) Ret in human development and oncogenesis. BioEssays 19, 389–395 http://tmm.trends.com
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Rab GTPases, intracellular traffic and disease Miguel C. Seabra, Emilie H. Mules and Alistair N. Hume Membrane and protein traffic in the secretory and endocytic pathways is mediated by vesicular transport. Recent studies of certain key regulators of vesicular transport, the Rab GTPases, have linked Rab dysfunction to human disease. Mutations in Rab27a result in Griscelli syndrome, caused by defects in melanosome transport in melanocytes and loss of cytotoxic killing activity in T cells. Other genetic diseases are caused by partial dysfunction of multiple Rab proteins resulting from mutations in general regulators of Rab activity; Rab escort protein-1 (choroideremia), Rab geranylgeranyl transferase α (X-linked (Hermansky–Pudlak syndrome) and Rab GDP dissociation inhibitor-α mental retardation). In infectious diseases caused by intracellular microorganisms, the function of endocytic Rabs is altered either as part of host defences or as part of survival strategy of the pathogen. The human genome is predicted to contain 60 RAB genes, suggesting that future work could reveal further links between Rab dysfunction and disease.
Miguel C. Seabra* Emilie H. Mules Alistair N. Hume Cell and Molecular Biology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College, Exhibition Road, London, UK SW7 2AZ. *e-mail: [email protected]
Intracellular protein and lipid traffic is a fundamental process required for the generation of specialized membranous organelles and the communication between them. One form of communication between organelles is through vesicular transport. This is a multi-step process, including formation of a vesicular or tubular carrier from the donor organelle membrane, movement of the carrier towards the acceptor, tethering/docking of the carrier with the acceptor and finally fusion of the carrier and acceptor compartment (Fig. 1). Transport pathways in cells have been traditionally divided into a secretory pathway starting at the endoplasmic reticulum and finishing at the plasma membrane, and an endocytic pathway starting at the plasma membrane and ending in the lysosome. Within each main pathway anterograde and retrograde routes exist and both pathways communicate at multiple junctures. Defects in intracellular trafficking underlie a large variety of human diseases [1,2]. In heritable http://tmm.trends.com
diseases, genes that control and/or encode protein machinery involved in vesicular transport can be affected by mutations. In addition, certain acquired diseases including infections, cancer and autoimmunity alter membrane trafficking pathways [2–6]. This review focuses on recent advances in understanding human disease through the study of a family of crucial regulators of vesicular transport, Rab proteins. Rab GTPases
Rab proteins (also known as Ypt in yeasts and plants) are monomeric GTPases of the Ras superfamily (reviewed in Refs [7–9]). Recent analyses indicate that Rabs are present in all eukaryotes and the Rab families in the genomes of several species have been reported [10–12]. Currently 60 human RAB genes are known (see Table 1). However, the complexity of the Rab protein family might be even greater as there is evidence that alternative splicing of Rab genes results in the production of functionally distinct isoforms [13]. Although intrinsically soluble, post-translational addition of isoprenoid moieties allows Rabs to associate with the cytoplasmic face of membranebound intracellular organelles and vesicles (Fig. 2) (reviewed in Ref. [14]). Lipid-modified Rabs appear to be targeted to specific membranes and thus display a characteristic pattern of subcellular localization. Evolutionarily conserved Rabs tend to be expressed in all cell and tissue types and regulate fundamental transport pathways whereas less conserved family members function in the many specialized pathways found in different mammalian cell types (Table 1).
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