Critical Reviews in Oncology/Hematology 50 (2004) 23–38

Tyrosine kinase receptors as attractive targets of cancer therapy

Amar Bennasroune, Anne Gardin1 , Dominique Aunis, Gérard Crémel, Pierre Hubert∗
INSERM Unit 575, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France

Accepted 11 August 2003

Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2. RTK signaling: ligand-binding, dimerization, kinase activity, substrate phosphorylation, protein docking 25
2.1. RTK structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.2. Aberrations affecting growth factor receptors in tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.3. Signaling pathways of tyrosine kinase receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.4. Regulation of RTK activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
3. RTKs and cancer: experimental tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4. RTK as targets in clinical research: what is going on? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.1. Antagonists of ligand-binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.2. Antibodies to RTKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
4.3. Tyrosine kinase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4.4. Protein–protein interaction inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4.4.1. Inhibitors of signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4.4.2. Inhibitors of dimerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5. Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Reviewers (MA 496) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Abstract

Receptor tyrosine kinases (RTKs) are the main mediators of the signaling network that transmit extracellular signals into the cell, and
control cellular differentiation and proliferation. Recent and rapid advances in our understanding of cellular signaling by receptor tyrosine
kinases, in normal and malignant cells, have brought to light the potential of RTKs as selective anti-cancer targets. Their activity is normally
tightly controlled and regulated. Overexpression of RTK proteins or functional alterations caused by mutations in the corresponding genes
or abnormal stimulation by autocrine growth factor loops contribute to constitutive RTK signaling, resulting in dysregulated cell growth
and cancer. The mechanisms of uncontrolled RTK signaling that leads to cancer has provided the rationale for anti-RTK drug development.
Herceptin, Gleevec, and Iressa are the first examples of drugs which have successfully translated basic research on oncogenes into cancer
therapeutics. RTKs can be viewed as multifunctional targets, and strategies towards the prevention and inhibition of RTK signaling include
antibodies, antagonist ligands, small molecule inhibitors of protein kinase activity, and inhibitors of protein–protein interactions. Progresses
in the field of rational drug design and computational chemistry will vastly benefit from the availability of increasing structural knowledge of
both the kinase domains and the ligand-binding sites of these receptors.
© 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Tyrosine protein kinase; Receptors; Oncology; Signaling; Therapy

∗ Corresponding author. Tel.: +33-3-88-45-67-03; fax: +33-3-88-60-08-06.

E-mail address: hubert@neurochem.u-strasbg.fr (P. Hubert).
1 Present address: Clinical Pharmacology Department, Novartis Pharma AG, Lichtstrasse 35, CH-4056 Basel, Switzerland.

1040-8428/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.critrevonc.2003.08.004
24 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38
1. Introduction
To this day (early 2003), Herceptin (trastuzumab) for the
treatment of advanced breast cancer, Gleevec (Imatinib Me-
sylate, STI-571) for the therapy of patients with Philadel-
phia chromosome-positive chronic myelogenous leukemia
and GIST (gastrointestinal stromal tumors), and Iressa (Gefi-
tinib, ZD1839, in Japan) for the treatment of lung cancer are
the first examples of drugs which have successfully trans-
lated basic research on oncogenes into cancer therapeutics.
They have been heralded as the most significant develop-
ment toward a new era of target-directed therapies [1]. These
agents provide at last clinical validation of the emerging
field of molecular oncology, specifically therapies targeting
deregulated cellular pathways that play a critical role in tu-
morigenesis, which were anticipated at the very beginning
of our understanding of molecular oncogenesis in the 1980s
[2–4]. Very interestingly, these three drugs are inhibitors
of protein tyrosine kinases, a class of enzymes which have
been at the early onset of the discovery of oncogenes [5–7].
Furthermore, they inhibit a subclass of these oncogenes, the
receptor tyrosine kinases (RTKs), which present distinctive
advantages as potential drug targets. This article reviews the
links between the still growing field of our basic understand-
ing of the functions of these RTKs and their deregulation
in cancer, and the ongoing developments of new drugs tar-
geting RTKs which will hopefully be as successful as Her-
ceptin and Gleevec [8,9]. The development of the pertain-
ing literature is so fast that we have had to operate a very
subjective selection. We have chosen to present the RTKs
as a whole family, stressing the common features that make
them attractive targets, rather than discussing details about
each receptor or subgroup of receptors.
The mutation and deregulation of cancer genes lead to
a wide range of changes in cellular structure and func-
tion, all of which contribute to the malignant phenotype
and pathological behavior of human cancer. Hanahan and
Weinberg [10] have usefully characterized cancer in terms
of six hallmark traits: (1) self-sufficiency in growth signals;
(2) insensitivity to growth inhibitory signals; (3) evasion of
apoptosis; (4) limitless replicative potential; (5) induction
of sustained angiogenesis; and (6) invasion and metastasis.
At least half of these traits involve RTKs and their signaling
pathways: clearly, activating mutations and/or overexpres-
sion of RTKs and their ligands can drive deregulated cell
growth; RTKs such as the insulin-like growth factor 1
(IGF1) receptor are key components of the cell death control
machinery; and, last but not least, angiogenesis-initiating
signaling involves RTKs such as the vascular endothe-
lium growth factor (VEGF), platelet-derived growth factor
(PDGF) and fibroblast growth factor (FGF) receptors. As
all of these traits can be targeted by the new generation of
molecular therapeutic agents, this should place RTKs at a
very prominent place among targets to attack malignant dis-
ease. Indeed, Sir Philip Cohen has very recently proposed
that protein kinases in general may prove to be the major
drug targets for the 21st century [11], in cancer and other
diseases.
The breakthrough discovery of Varmus and Bishop that
cancer-inducing genes of animal retroviruses such as v-src
represent mutated host genes that were recombined into the
viral genome raised the question of whether the oncogene
concept was also relevant to human cancer [12]. This first
connection between a human gene product that regulates
normal cell proliferation and a viral oncogene strongly sug-
gested that human cancer development may also involve ab-
normalities in the expression and structure of endogenous
genes that have regulatory roles in cell proliferation. The
first cloning and sequence analysis of a cDNA encoding
a cell surface protein, the human epidermal growth factor
(EGF) receptor, provided a partial answer to this question
by revealing a close relationship with the v-erbB oncogene
[13,14]. Interestingly, this cloning was performed from a
cell line which had been established from a human cancer,
an epidermoı̈d sarcoma termed A431. A search for simi-
lar genetic aberrations in tumor tissues using cDNA probes
of EGFR and an accidentally cloned EGFR-related gene,
termed human EGFR-related gene (HER2), resulted in the
discovery that the gene encoding the HER2/neu receptor ty-
rosine kinase is amplified up to 100-fold in the tumor cells
of about 30% of patients with invasive breast cancer. A sig-
nificant clinical correlation was shown between HER2/neu
gene amplification and overexpression and parameters of
malignancy, including reduced survival and reduced time to
relapse, relative to patients with normal receptor levels [15].
This explains the current emphasis on the ErbB/HER family
as promising targets for new drugs.
The importance of discovering new targets and path-
ways for therapeutic intervention in cancer is clear, and
criteria for validation and selection of new drug targets
can be summarized as follows: (1) frequency of genetic or
epigenetic deregulation of the target or pathway in human
cancer; (2) demonstration in a model system that the target
contributes to the malignant phenotype; (3) evidence of
the reversal of the malignant phenotype by expression of
dominant negative protein, antisense strategies, antibodies,
inhibitory peptides or drug leads; (4) practical feasibility,
tractability or drugability of the target; (5) availability of a
robust, efficient biological test cascade to support the drug
discovery program; (6) ability to run a robust cost-effective
high-throughput screen; (7) availability of a structure-based
drug design approach [9]. As a family, oncogenic RTKs
do fulfill these criteria. Proof-of-concept for the drugability
of these molecular targets is, of course, already established
with the regulatory approval of the anti-ErbB2 antibody
Herceptin and the tyrosine kinase inhibitors Gleevec and
Iressa. Although many efforts in designing new anti-cancer
drugs have so far been mostly directed at the ErbB
sub-family, one may reasonably believe that the established
proof-of-concept will hold true for most if not all oncogenic
RTKs. Most noteworthy is the fact that structural knowledge
of the crystal structure of many tyrosine kinase domains
 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38 25

Fig. 1. Schematic structure of receptor tyrosine kinases, and their mechanism of activation: (a) extracellular ligand-binding domain; (b) unique
transmembrane domain; (c) juxtamembrane region; (d) kinase domain; (e) C-terminus. (1) The first step in receptor activation consists in ligand-induced
dimerization/conformational change; (2) this brings the catalytic domains in close proximity for transphosphorylation, yielding full receptor activation.

has already been instrumental in the very rapid molecular
understanding of clinical resistance to STI-571 [16].
Furthermore, RTKs are multifunctional transmembrane
proteins, expressed at the cell surface. These characteristics
make them potential multisite targets, which can be inhibited
by water soluble molecules such as antibodies or binding
antagonists, as well as by cell permeant substances such as
kinase inhibitors.
2. RTK signaling: ligand-binding, dimerization, kinase
activity, substrate phosphorylation, protein docking
2.1. RTK structure
Growth factors mediate their diverse biologic responses
(control of cellular proliferation, differentiation, migration
and metabolism) by binding to and activating cell-surface
receptors with intrinsic protein kinase activity [17,18]. To
date, about 60 RTKs, which belong to ∼16 different re-
ceptor families, have been identified [19]. This catalog has
recently been confirmed by analysis of the human genome
[20]. All RTKs contain a large, glycosylated, extracellu-
lar ligand-binding domain, a single transmembrane region,
and a cytoplasmic portion with a conserved protein tyro-
sine kinase domain (Fig. 1). In addition to the catalytic do-
main, a juxtamembrane region and a carboxyl-terminal tail
can be identified in the cytoplasmic portion. They further
contain regulatory sub-domains which negatively or posi-
tively influence substrate-binding and phosphorylation, as
well as sub-domains involved in the obligatory dimeriza-
tion and/or structural changes during kinase activation af-
ter ligand-binding. Each of these structural domains, and
their functions, are potential targets for drug developments
(Fig. 2), and have indeed been addressed in a number of ex-
perimental studies (see below). Because of their structure,
RTKs can be visualized as membrane-associated allosteric
enzymes with the ligand-binding and protein tyrosine ki-
nase domains separated by the plasma membrane [5,17,21].
Their role is to catalyze the transfer of the gamma-phosphate

Fig. 2. Receptor tyrosine kinases as multisite targets. The different steps involved in RTK activation, regulation and synthesis are schematically depicted.
Each step is associated with actual (bold face) or potential (italicized) means of inhibition for cancer therapy.
26 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38
of adenosine triphosphate (ATP) to tyrosine residues within
their own polypeptide chain and to that of exogenous sub-
strates.
On the basis of sequence similarity, it is possible to
classify these receptors into related groups [17,18,21].
Characteristic structural features of the extracellular do-
mains of these groups include, among others, cysteine-rich
motifs, immunoglobulin-like repeats, fibronectin type III
repeats, and EGF motifs that can be present singly or in
different combinations. These different domains determine
specificity for ligand-binding. It is beyond the scope of this
review to provide detailed information on specific RTKs or
RTK families. A number of excellent reviews is published
each year on these topics (e.g. EGFR-ErbB family [22–24],
platelet-derived growth factor receptors (PDGFRs) [25], in-
sulin receptor (IR) family [26,27], FGFRs [28], c-Kit [29],
Met [30], VEGFRs [28,31], Trks [32], Ret [33,34], ephrin
receptors [35], etc.).
There is substantial evidence that ligand-induced activa-
tion of the kinase domain and its signaling potential are me-
diated by receptor dimerization [36–38]. This event stabi-
lizes interactions between adjacent cytoplasmic domains and
controls the activation of kinase activity. Receptor oligomer-
ization appears to be a common phenomenon among growth
factor receptors. Dimerization can take place between two
identical receptors (homodimerization), or between differ-
ent members of the same receptor family (heterodimeriza-
tion) [17]. Heterodimerization of RTKs has been shown to
increase the repertoire of ligands that can be recognized by
each receptor alone and, on the other hand, to expand the
diversity of signaling pathways that can be recruited by a
given receptor [17,22,23].
How ligands bind to the receptors and induce oligomer-
ization is specific for each class of RTKs [5,36–38]. PDGF,
for example, induces receptor dimerization by virtue of its
dimeric nature [25]. Interestingly, the insulin receptor family
exists as preformed disulfide-bonded homo- or heterodimers
of receptor subunits. Thus, in this case, ligand-binding does
not induce receptor dimerization but presumably causes a
conformational change in the preformed dimeric receptor,
which leads to receptor activation. The existence of pre-
formed, but inactive, receptor dimers has also been evoked
for other RTKs, indicating that ligand-induced conforma-
tional alterations may be the universal mechanism of activa-
tion. In any case, it is now well established that dimerization
is necessary for RTK activation. Nevertheless, recent data
have shown that receptor dimerization may not be sufficient
for activation. This notion stems mostly from experimental
modifications of the transmembrane domain of some RTKs
and other related receptors [39]. The main function of the
transmembrane domain is to anchor the receptor in the plane
of the plasma membrane, thereby connecting the extracel-
lular environment with internal compartments of the cell. It
was initially thought that this domain represented a passive
anchor of the receptor to the membrane. However, a point
mutation in the transmembrane domain of one receptor, the
neu/ErbB2, enhances its transforming properties [40]. The
transmembrane mutation in neu may have a stabilizing ef-
fect on its conformation, which results in dimerization and
constitutive activation of receptor signaling [41]. Genetic al-
terations in this domain have, in fact, demonstrated an active
role of this region in RTK dimerization and demonstrated
that dimerization is not sufficient, but proper alignment must
also occur for activation and signaling [42,43].
The activation of intrinsic protein kinase activity results in
autophosphorylation of specific tyrosine residues in the cyto-
plasmic portion of the RTK. Moreover, tyrosine phosphory-
lation in the kinase domain stimulates the intrinsic catalytic
activity of the receptor. Recently, biochemical and structural
studies have revealed some of the molecular mechanisms
that mediate such activation [44,45]. There is substantial ev-
idence that autophosphorylation occurs in trans by a second
receptor tyrosine kinase after dimerization and/or conforma-
tional changes induced by ligand-binding. In the unphospho-
rylated state, the receptor possesses a low catalytic activity
due to the particular conformation of a specific domain in
the kinase region (the activation loop), which interferes with
the phosphotransfer event. Phosphorylation of the kinase
domain removes this inhibition, and the catalytic activity is
enhanced and persists for some time independently of the
presence of the ligand. Although kinase activity is at a low
basal level in the monomeric state, this activity is sufficient
to induce trans-autophosphorylation once the dimer forms.
Autophosphorylation also occurs outside the kinase domain
and serves the important function of creating docking sites
for downstream signal transduction molecules.
The juxtamembrane sequence that separates the trans-
membrane and cytoplasmic domains is not well conserved
between different families of receptors. However, jux-
tamembrane sequences are highly similar among members
of the same family, and some studies indicate that this
domain plays a role in modulation of receptor functions
by heterologous stimuli [46]. This region may also play a
role in signaling, as has been suggested by the capacity to
bind specific substrates in a ligand-dependent manner. For
example, it has been shown that eps8 directly binds to the
juxtamembrane domain of EGFR in a phosphotyrosine- and
SH2-independent manner.
The tyrosine kinase domain is the best conserved among
tyrosine kinase receptors, and an intact protein tyrosine ki-
nase domain is absolutely required for receptor signaling.
For example, mutation of a single lysine in the ATP-binding
site, which blocks the ability of the receptor to phosphory-
late tyrosine residues, completely inactivates receptor bio-
logic function. The kinase domain of some receptor tyrosine
kinases (e.g. PDGF and FGF receptors) is split by inser-
tions of up to 100 amino acid residues. Kinase insertion se-
quences are highly conserved between species, and contain
autophosphorylation sites that have the function of coupling
with signal-transducing molecules.
The carboxyl-terminal tail sequences are the most diver-
gent between known RTKs. The carboxy-terminal domain is
 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38 27

thought to play an important role in regulating kinase activ-
ity. This region typically contains several tyrosine residues,
which are phosphorylated by the activated kinase. In fact,
the receptor itself is often the major tyrosine-phosphorylated
species observed following ligand stimulation. The pres-
ence of specific amino acid sequences in this domain plays
an important role in determining activation of specific
signal-transducing molecules by RTKs.
Although membrane proteins in general are major drug
targets and represent an important class of proteins en-
coded by about 30% of all genes, little progress has been
achieved in elucidating their detailed structures at the
atomic level [47]. No complete structure of a RTK has
yet been established. Nevertheless, the modular nature of
RTKs has allowed the determination of a few extracellular
domain and a larger number of kinase domain structure
through X-ray crystallography [44] (Fig. 3). Among others,
the detailed structures of the EGF receptor extracellular
domain complexed with its ligands (TGFalpha or EGF)
has very recently been published [48,49]. These structures
show that ligand-induced dimerization is achieved by direct
receptor–receptor interactions between a loop protruding
from two neighboring monomers. A rather similar configu-
ration has also been found for the ligand-binding region of
ErbB3/HER3 [50]. Other examples of resolved extracellular
domain structures include the neurotrophin-binding domain
of TrkA and TrkB [51,52], the complex between VEGF and
the second domain of Flt-1 [53], or the complex formed
between the receptor EphB2 and ephrin-B2 [54]. As for the
intracellular kinase domain, several structures have been
reported, demonstrating a common overall architecture sim-
ilar to that of other protein kinases, as had been inferred
from the strong sequence homology. Data obtained with
the inactive and active form of the insulin receptor kinase
have provided a molecular explanation for the repression of
the catalytic activity in the absence of ligand [55,56]. They
have confirmed the importance of dimerization, and shown
that an activation loop in the kinase domain is blocking
the ATP-binding site in the inactive form. Phosphorylation
of this loop allows for full kinase activity. Role of such
an activation loop has been confirmed for other RTKs (see
Hubbard and Till [45]). These are extremely important
progresses, since they will allow for rational design of bind-
ing, dimerization, and kinase inhibitors. Strikingly, such

Fig. 3. An overview of selected structures of RTK domains and related inhibitors and signaling proteins. Structures were extracted from the Protein Data
Bank (PDB, http://www.pdb.org/, [153]), and drawn with Rasmol [154]. EGF receptor: (A) extracellular domains I–IV (green ribbon diagram) in complex
with egf (orange spacefill display) (PDB access code: 1IVO [48]); (B) tyrosine kinase domain (blue ribbon) with inhibitor Erlotinib (Tarceva, red spacefill
representation) (PDB 1M17 [155]). ErbB2: (C) extracellular domain of human ErbB2/HER2 (green ribbon) in complex with Herceptin Fab fragment (red
spacefill display); (D) helical membrane spanning fragment (PDB 1IIJ [156]). FGFR: (E) complex of the extracellular domain of FGFR2 (green) with
FGF1 (orange) and heparin (blue spacefill display) (PDB 1E0O [157]); (F) kinase domain of FGFR1 (blue) in complex with SU4984 inhibitor (red) (PDB
1AGW [158]). Adaptor protein and signaling domains (purple ribbons): (G) Grb2 (PDB 1GRI [159]; (H) Pleckstrin homology–phosphotyrosine-binding
(PH–PTB) domain of IRS-1 (PDB 1QQG [160]); (I) SH2 domain of the regulatory subunit of PI3 kinase (PDB 1BFI [161]). Dotted lines denote the
fragments of unknown structure (usually, transmembrane and juxtamembrane domains, as well as distal N- an C-terminus).
28 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38
structural data have already been instrumental in the molec-
ular understanding of clinical resistance to Gleevec [16,57].
Also, the binding domain of Herceptin on the extracellular
region of HER2 has been solved very recently [58].
2.2. Aberrations affecting growth factor receptors in tumor
cells
Recent reviews discuss the mechanisms by which RTKs
participate in malignant transformation and tumor prolif-
eration [18,59,60]. Growth factor receptors can be consti-
tutively activated by autocrine loops, a number of other
mechanisms have been identified by which growth factor
receptors can become transforming, among which muta-
tions and overexpression represent prominent anomalies.
Retroviral transduction of proto-oncogenes can result in
mutation of the normal version of the gene.
The paradigm for such alterations is v-erbB, the onco-
genic counterpart of the EGFR receptor, transduced as the
viral oncogene of avian erythroblastosis virus [61]. The
mechanism of v-ErbB activation involves deletion of its
ligand-binding domain, resulting in a truncated EGFR. An-
other example of extracellular deletion is the avian v-ros
oncogene which differs from its cellular counterpart, c-ros,
in that this domain is deleted with the exception of six
amino acids and contains a three amino acid insertion in the
middle of the transmembrane domain. This results in con-
stitutive activation of the molecule [62]. Neu (the murine
homologue of ErbB2/HER2) was initially identified as an
oncogene by NIH-3T3 transfection analysis of cDNA from
ethylnitrosourea-induced rat neuroblastomas. The trans-
forming gene was identified as having a specific mutation
in its transmembrane domain responsible for oncogenic ac-
tivation, again responsible for constitutive activation of the
kinase [40].
In human malignancies, overexpression of a normal re-
ceptor, with or without the concomitant presence of the
ligand, contributes to neoplastic transformation. Examples
include the EGFR, ErbB2, the insulin-like growth factor 1
receptor, etc. The best characterized mechanisms involve
EGFR family members [38,63,64]. ErbB2/HER2 was ini-
tially identified as an amplified gene in a primary human
breast carcinoma and a salivary gland tumor. ErbB2 overex-
pression beyond some critical threshold level in NIH/3T3 fi-
broblasts was shown to be sufficient to induce the malignant
phenotype. Clinical studies have indicated that the normal
erbB2 gene is frequently amplified and/or overexpressed in
human breast carcinomas and in ovarian carcinomas, and
detection in breast carcinomas of high ErbB2 levels is a
prognostic indicator of poor survival. Thus, ErbB2 appears
to be most commonly altered in human malignancies by
mechanisms leading to its overexpression. Whereas ErbB2
overexpression has been observed primarily in adenocarci-
nomas, overexpression of an apparently normal EGFR has
been reported frequently in squamous cell carcinomas and
glioblastomas. In many cases, the EGFR appears to be ac-
tivated by autocrine stimulation by one of its ligands, most
commonly TGFalpha.
2.3. Signaling pathways of tyrosine kinase receptors
Knowledge of the cascade of biochemical events trig-
gered by ligand stimulation of tyrosine kinase receptors has
increased rapidly in recent years and provides further evi-
dence of the importance of these signaling pathways in can-
cer. In order to effectively coordinate signaling cascades,
nature has created a variety of molecules known as adap-
tor and scaffolding proteins [61]. These proteins play a role
in intracellular signaling by both recruiting various pro-
teins to specific locations and by assembling networks of
proteins particular to a cascade. Adaptor proteins, through
protein–protein interactions via specific motifs, provide a
link between molecules of a signaling cascade and proteins
such as RTKs. One such adaptor, Grb2, is important in the
activation of downstream signaling pathways such as the
ras/raf/MAPK pathway. These adaptor proteins often con-
tain a variety of motifs that mediate protein–protein interac-
tions. Src homology 2 (SH2) and phosphotyrosine-binding
(PTB) domains are protein motifs that bind to specific phos-
phorylated tyrosine-containing sequences, dictating partic-
ular binding partners. SH3 domains recognize and bind to
proline-rich sequences in target proteins. Thus, as in the case
of Grb2 which contains both SH2 and SH3 sequences, an
adaptor protein can bring a cytoplasmic protein via its SH3
domain to an activated RTK via an SH2 domain-binding to
phosphorylated tyrosine residues of the receptor.
Recently, new signaling pathways have been described
for the RTKs, in addition to the classical plasma membrane
effectors which activate the ras/MAPK and PI3kinase/akt
pathways [65] (schematized in Fig. 4). Notably, EGFR has
been shown to stimulate directly phosphorylation and nu-
clear translocation of signal transducer and activator of tran-
scription (STATs) proteins. RTKs or their proteolytic frag-
ments may also be active inside cells, and surprisingly in
the nucleus where they could act directly as transcription
factors.
Again, it is beyond the scope of this review to discuss in
detail the diverse signaling cascades of RTKs, the reader is
referred to recent reviews [17,60,66–70].
2.4. Regulation of RTK activity
Because of the critical roles played by RTKs in cellu-
lar signaling processes, their catalytic activity has to be
normally under tight control. This is achieved by a vari-
ety of means, which can be briefly summarized as follows:
a first level of regulation is constituted by the kinase do-
main itself where the state of phosphorylation controls di-
rectly kinase activity [71]. Phosphorylation of tyrosines on
the receptor will also dictate the binding of SH2 and PTB
domains-containing substrates and adapters. Obviously, the
phosphorylation state of a RTK will also be controlled in
 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38 29

Fig. 4. A very schematic view of RTK signaling. The main pathways are indicated, with kinase names italicized. Elements of these pathways for
which inhibitors are under development are also underlined. Abbreviations (pathways from left to right): PI3K, phosphatidylinositol 3-kinase; Pdk1,
phosphoinositide-dependent protein kinase-1; Akt, oncogenic kinase initially isolated from a transforming mouse retrovirus (also named PKB, or related to
A and C protein kinase: RAC-PK); p70S6K , ribosomal S6 kinase; Shc, (src homology collagen); Sos, (son of sevenless); Grb2, (growth factor receptor-bound
protein 2) are adaptor proteins; Ras, oncogene first isolated in rat sarcomas; Raf, oncogenic kinase initially isolated from a transforming mouse virus;
Mek, Map/Erk kinase (or Mkk: map kinase kinase); Erk/MapK, extracellular signal-regulated kinase/mitogen-activated protein kinase; Jak, janus kinase;
Stat, signal transducer and activator of transcription; PLC, phospholipase C; PKC, protein kinase C; Src, oncogene of the chicken Rous sarcoma
virus.

a specific manner by the activity of protein tyrosine phos-
phatases, a number of which have recently been identified
[72,73].
Another important means of regulation in cells is
“down-regulation” whereby ligand-binding leads to disap-
pearance of receptors from cell surface. This phenomenon
is due to accelerated endocytosis of ligand–receptor com-
plexes, and has been extensively studied for the ErbB/HER
receptors [74]. Endocytosis occurs through clathrin-
dependent and independent mechanisms, although the pre-
cise nature of clathrin-independent pathways remains un-
clear. Quite recently a role for ubiquitinylation of receptors
has been evidenced [75–77].
All these findings may yield new developments in drugs
aiming at attenuating RTK signaling, for instance by acti-
vating specific phosphatases, or accelerating receptor down-
regulation, or preventing receptor nuclear translocation.
3. RTKs and cancer: experimental tools
Historically, discovery and preclinical development of
anti-cancer drugs have mostly relied on empiric cell-based
screening for inhibition of proliferation. Together with the
use of xenografts of human cancers in immunologically
deficient mice, this approach has yielded much information
and allowed the discovery of many useful therapeutics. Re-
cently, targeting specific molecules such as oncogenes has
provided a more mechanistically-based approach. Many
cell lines are available that express mutated RTKs, or over-
express normal receptors, or such cell lines can be easily
constructed by transfection. Although these cell lines can-
not replicate the full spectrum of characteristics of any
cancer, especially because they do not allow for the study
of interactions with the tumor host, their use is certainly
still justified [78]. More sophisticated models can also be
developed from genetically engineered cells. For instance,
Brandt et al. [79] have described an elegant model of breast
cancer in the mouse, by injecting mammary fat pads with
cells expressing an oncogenic ErbB2 receptor.
Laboratory mice have also been a central player in cancer
research. The techniques of gene targeting and transgenesis
have allowed the creation and study of many transgenic
and knock-out strains. More recently, genetic systems al-
lowing for precise conditional control of the expression
of specific genes have been introduced [80]. The benefits
and disadvantages of these animal models have recently
been discussed [81–84]. As far as RTKs are concerned,
numerous transgenic mice have been created (see Table 1
for some selected examples). The first such transgenic mice
carried an activated neu/erbB2 oncogene driven by a mouse
mammary tumor virus (MMTV) promoter, and developed
mammary adenocarcinomas [85]. Besides providing direct
evidence supporting the role of RTKs and their ligands in
tumorigenesis, these models have allowed and will allow for
increasing our understanding of the development of tumors
in a whole organism, and for in vivo preclinical drug testing
in a more sophisticated setting than cultured cells [86].
High-throughput discovery tools are always desirable in
the quest for effective new drugs. Peptide chips represent
a very recent development in the search for performant as-
says to discover kinase inhibitors. Houseman et al. [103]
have chemically immobilized a short peptide substrate of
the tyrosine kinase src on gold surfaces. Phosphorylation of
the peptide in the presence of the enzyme can be quanti-
tatively analyzed by plasmon resonance, fluorescence and
30 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38

Table 1
Selected examples of transgenic mouse overexpressing one RTK as cancer models
Type of cancer Oncogenic RTK or ligand Method Phenotype Reference

Breast Transforming c-neu MMTV promoter Synchronous early mammary [85]

adeno-carcinoma + glandular
dysplasias
Breast Transforming c-neu MMTV promoter Stochastic late mammary [87]
adenocarcinoma + glandular
dysplasias
Breast ErbB2/neu + TGFalpha MMTV promoter Progressive mammary tumors [88,89]
Breast ErbB2 Neu promoter [90]
Breast ErbB2/neu MMTV promoter Late focal mammary tumors [91]
+ lung metastasis
Breast Transforming c-neu MMTV promoter Synchronous mammary [92]
adenocarcinoma in each gland
+ glandular dysplasias
Breast ErbB2/neu MMTV promoter Late unifocal mammary tumors [93]
Breast Mutated met Metallothionein promoter Metastatic mammary carcinoma [94]
Thyroid RET/PTC chimera Thryroglobulin promoter Papillary carcinomas [95]
Thyroid RET with MEN2 mutation Calcitonin promoter Medullary carinoma [96]
Angiogenesis VEGF Keratin promoter Skin papilloma [97]
Diverse RET-Men2B Dopamine hydroxylase Ganglioneuromas + renal [98]
promoter malformations
Skin and other epithelia Activated erbB2 Keratin promoter Epithelial hyperplasia, [99]
+ tetracyclin-induction up-regulation of TGFalpha
Skin ErbB2 Keratin promoter [100]
Brain tumor Transforming c-neu MBP promoter Low incidence of brain tumors [101]
Pituitary adenoma Truncated FGFR4 Prolactin promoter Lactotroph pituitary tumors [102]

phosphorimaging. Such a system can easily be used for
high-throughput assays monitoring the activity of protein ki-
nases, and should be instrumental in the discovery of new
kinase inhibitors.
4. RTK as targets in clinical research: what is
going on?
In this section, we aim at presenting an necessarily
non-exhaustive overview of current development of drugs
targeting RTKs. As schematized in Fig. 2, RTKs can be
viewed as multisite targets, and we chose to follow the same
logical order of presentation, from the extracellular-binding
site to the intracellular events. Gene therapies will not be
discussed here, as they are still in their prime infancy for
RTKs. Table 2 presents a short overview of some drugs cur-
rently in clinical trials, belonging to the two largest groups,
namely antibodies and small kinase inhibitors.
4.1. Antagonists of ligand-binding
As ligand-binding is the first step in RTK activation, tar-
geting the ligands themselves, or the extracellular-binding
site on the receptor, represent a first straightforward ap-
proach. The relatively large size of peptidic growth factors
represents a major obstacle in the development of small an-
tagonists. Nevertheless, quite a few RTK antagonists have
been developed.
Suramin was introduced in 1920 for the treatment of try-
panosomiasis, and was later found to be a potent inhibitor
of the reverse transcriptase of retroviruses, including the
HTLV-III. It was also found to possess anti-neoplasic prop-
erties, notably by inhibiting angiogenesis [104]. Suramin
does so by inhibiting the action of aFGF and bFGF at the re-
ceptor level. Recent clinical studies have shown little bene-
fits in prostate cancer, with neurotoxicity being a significant
complication [105].
Another way to disrupt the interaction of key growth
factors with their RTK targets in the cell membrane has
been developed by Sebti and Hamilton [106]. They used
an entirely novel strategy based on the design of synthetic
agents which contain a large and functionalized surface
area that selectively and strongly bind to the complemen-
tary surface of the growth factor and so block its oncogenic
signaling function. Sebti and Hamilton [106] have placed
particular focus on platelet-derived growth factor and its
complementary RTK, platelet-derived growth factor recep-
tor because its overexpression is seen in many carcinomas
and some cancer patients have high serum levels of PDGF.
This approach to growth factor binding agents involves the
attachment of several peptide loops onto a core scaffold in
direct analogy to the six hypervariable loops that are found
in the antigen recognition region of an antibody Fab frag-
ment [106], The synthesis of a library of binding agents has
permitted to identify a molecule, GFB-111 that targets the
regions of PDGF (loops I and III) that are involved in bind-
ing to its receptor. By preventing PDGF from binding to its
 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38 31

Table 2
Some examples of drugs targeting RTKs (monoclonal antibody (MAb))
RTK Drug Company Description Status Indication

HER2 Trastuzumab/Herceptin Genentech MAb directed against Approved Approved for breast
HER2 Phases I cancer; other solid
and II tumours
EGFR/HER2 PKI 166 Novartis Inhibitor of EGFR and Phase I Prostate cancer,
HER2 renal cell carcinoma
EGFR OSI-774 (Tarceva) Roche/Genentech/OSI Inhibitor of EGFR Phase III NSCLC, head and
neck, ovarian cancer
EGFR C225/cetuximab ImClone Systems MAb directed against Phase III Colorectal, head
EGFR and neck, pancreas
cancers
EGFR ABX-EGF Abgenix MAb against EGFR Phase II NSCLC
EGFR ZD1839 (Iressa) AstraZeneca Inhibitor of EGFR Phase II Approved in Japan
signalling for NSCLC many
cancers
Abl/PDGFR/c-Kit STI-571 (Gleevec) Novartis Inhibitor of PDGFR, Approved Approved by the
c-Kit (and abl) Phase IV FDA for GIST (and
CML) glioma,
sarcoma
VEGFR SU5416 SUGEN Inhibitor of VEGFR-2 Phase III Angiogenesis
VEGFR/FGFR/PDGFR SU6668 SUGEN Inhibitor Phase I Angiogenesis
Trk CEP2563 Cephalon Inhibitor of Trk Phase II Ovarian, prostate,
pancreas cancers

RTK, GFB-111 blocks PDGF-induced receptor autophos-
phorylation, activation of Erk1 and Erk2 kinases, and DNA
synthesis. This molecule is highly potent (IC50 = 250 nM)
and selective for PDGF over EGF, IGF-1, aFGF, and bFGF
(IC50 values > 100 ␮M) but inhibits also VEGF-induced
Flk-1 tyrosine phosphorylation and Erk1/Erk2 activation
with an IC50 of 10 ␮M. Furthermore, GFB-111 treatment
of nude mice bearing human tumors resulted in significant
inhibition of tumor growth and angiogenesis. Thus, these
results demonstrate that selective PDGF-binding molecules
such as GFB-111 that are capable of blocking signaling,
tumor growth, and angiogenesis may lead to the develop-
ment of a new class of anti-cancer drugs capable of treating
a wide spectrum of human cancers [106,107].
4.2. Antibodies to RTKs
Today, more than 70 monoclonal antibodies (MAb) are
currently in commercial trials beyond Phases I and II. These
include reagents against cancer, transplant rejection, a vari-
ety of immunological conditions such as Crohn’s disease,
antiviral prophylaxis, and thrombosis [108].
Herceptin (trastuzumab, Genentech) is the first commer-
cially available antibody targeting a RTK. Herceptin is a hu-
manized monoclonal antibody, so called because all the orig-
inal mouse-derived components except the antigen-binding
region have been replaced by human counterparts. Several
mechanisms have been defined to explain its anti-tumor ef-
fects: trastuzumab down-regulates HER2 expression by ac-
celerating receptor endocytosis and degradation [109], act-
ing as a partial agonist of the ligand of this receptor. It can
also inhibit cell-cycle progression preceded by a rapid de-
phosphorylation of ErbB2 [110]. Recently, it has been pro-
posed that the anti-tumor effects of trastuzumab can be ex-
plained by inhibition of HER2 cleavage and prevention of
the production of an active truncated HER2 fragment [111].
As for ErbB2/HER2, there is a relationship between over-
expression of epidermal growth factor receptor and aggres-
sive behavior of tumor cells. The anti-EGFR monoclonal
antibody C225 (cetuximab) was generated and has been
shown to have anti-tumor activity in vitro and in xenograft
models [112]. This antibody has been chimerized with
human IgG1 in its constant region to increase its clinical
utility by decreasing the potential for generation of human
anti-mouse in recipients [113]. In fact, the anti-tumor ac-
tivity of cetuximab has been attributed to several distinct
mechanisms: a therapeutic strategy combining C225 and
chemotherapeutic agents such as doxorubicin or cisplatin
showed that anti-EGFR MAbs substantially enhance the ef-
fects of these agents against well-established xenografts of
tumor cells expressing high levels of EGF receptors. Com-
bination of chemotherapeutic agents and C225 may block
activation of receptor tyrosine kinase and induce EGFR
down-regulation [114]. Another mechanism of action is that
both cetuximab and M225, its murine progenitor, inhibit
cell-cycle progression in many cell lines, causing cells to
arrest in the G1 gap phase that occurs prior to DNA syn-
thesis. A series of experiments has shown that anti-EGFR
antibody treatment causes an increase in the expression of
the cell-cycle inhibitor p27kip1 . This in turn results in an
increase in the formation of inhibitory p27kip1 –Cdk2 com-
plexes which prevent cells from exiting the G1 phase of the
cell cycle [115]. Many data show that cetuximab has also
anti-angiogenic properties, which were first reported by Petit
32 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38
et al. [116]. This group found that established A431 tumor
xenografts treated with cetuximab displayed a significant
decrease in the production of angiogenic factors, and these
data have since been confirmed with additional tumor cell
lines. Cetuximab has also been shown to influence apop-
tosis. Indeed, in DiFi human colon carcinoma cells which
express high level of EGFR, cetuximab treatment-induced
programmed cell death by activation of multiple apoptotic
caspases [117]. A Phase Ib study of C225 in combination
with cisplatin in patients with recurrent squamous cell car-
cinoma of the head and neck has been conducted in order
to determine the optimal biological dose of C225 and to
establish a safety profile of C225 in a different range of
dose levels in combination with cisplatin [118].
ABX-EGF (Abgenix), a fully human IgG2 monoclonal
antibody specific for human EGFR has been generated.
ABX-EGF binds EGFR with high affinity (5 × 10−11 M),
blocks the binding of both EGF and TGFalpha to var-
ious EGFR-expressing human carcinoma cell lines, and
inhibits tumor cell activation and cell proliferation. In vivo,
ABX-EGF prevents completely the formation of human
epidermoı̈d carcinoma A431 xenografts in athymic mice,
and administration of ABX-EGF without concomitant
chemotherapy results in complete eradication of established
tumors. Human pancreatic, renal, breast and prostate tu-
mor xenografts which express more than 17,000 EGFR
molecules per cell showed significant growth inhibition
when treated with ABX-EGF. Thus, Yang et al. [119] have
demonstrated a potent anti-tumor activity of ABX-EGF and
its therapeutic potential for the treatment of multiple human
solid tumors that overexpress EGFR.
A very original approach, exocyclic mimetics of antibod-
ies, has been recently proposed [120]. A short peptide (called
AHNP, 12 aminoacids) was rationally designed to mimic the
structure of one loop of Herceptin, and was found to mimic
many effects of the native antibody. Analogues of this pep-
tide with better activity have been developed [121]. Such
short and stable antibody analogs may offer potentially sig-
nificant therapeutic advantages.
In general, antibodies have started to fulfill their promise
as anti-cancer therapeutics with four antibodies now mar-
keted in the United States: Rituxan (IDEC Pharmaceuticals,
Genentech) used against non-Hodgkin’s lymphoma, Her-
ceptin, Mylotarg (Wyeth Laboratories) used against acute
myelogenous leukemia, and Campath (Alemtuzumab, Mil-
lenium and ILEX Partners) used against B-cell chronic lym-
phocytic leukemia, and other MAbs are now in advanced
oncology trials [122].
It should also be stressed that RTKs being membrane
proteins, they represent also a target for combination therapy
coupling toxic drugs to ligands or antibodies [123].
4.3. Tyrosine kinase inhibitors
The design of small molecule inhibitors of kinases in gen-
eral, and RTKs in particular, seems now to be very promising
[124]. Although small molecules and enzymes are most pop-
ular among drug designers, the idea of developing specific
inhibitors of oncogenic RTKs was initially met with skep-
ticism, mostly for fear of poor specificity. This was due to
the nature of most inhibitors, which are competitive for the
well conserved ATP-binding site in the kinase domain. Nev-
ertheless, as exemplified by Gleevec and Iressa, this class
of molecules may prove to be much more useful than was
earlier expected (see [11]).
STI-571 (GleevecTM ) is a protein-tyrosine kinase in-
hibitor that inhibits the Bcr-Abl tyrosine kinase, the constitu-
tive abnormal tyrosine kinase created by Philadelphia chro-
mosome abnormality in chronic myeloid leukemia (CML)
[125]. STI-571 inhibits proliferation and induces apoptosis
in Bcr-Abl positive cell lines as well as in fresh leukemic
cells from Philadelphia chromosome-positive CML. In fact,
STI-571, first known as CGP 57148, was initially described
as an inhibitor of both Abl and platelet-derived growth fac-
tor receptor [126]. It inhibits at nanomolar concentrations
the kinase activity of v-abl, bcr-abl, c-Kit and PDGFR, and
this was found to translate into both in vivo anti-tumor ac-
tivity [127]. Small cell lung cancer (SCLC) is an aggressive
cancer characterized by deregulation of several autocrine
growth mechanisms including stem cell factor and its re-
ceptor c-Kit. It has been shown that STI-571 also inhibits
growth of SCLC cells through a mechanism that involves
inactivation of the tyrosine kinase c-Kit. The effectiveness
of STI-571 in this study suggests this drug may be use-
ful for patients with SCLC [128]. STI-571 has also been
approved for treatment of gastro-intestinal stromal cancer
[129,130].
Anilinoquinazolines are potent and selective inhibitors
of the ErbB family of receptor tyrosine kinases, and three
analogues (two reversible and one irreversible inhibitor)
have been evaluated clinically as anti-cancer drugs [131].
Among these quinazoline-derived agents that have been
tested as anti-cancer drugs in vitro and in preclinical mod-
els, ZD1839 (Gefitinib, Iressa, AstraZeneca) is a highly
specific EGF receptor tyrosine kinase inhibitor. Inhibition
of EGFR transphosphorylation by ZD 1839 blocks signal
transduction at its first step, thus providing anti-proliferative
effects on several human cancer cell lines overexpressing
EGFR, including ovarian, breast and colon cancer [132].
Preclinical studies demonstrated efficacy and good bioavail-
ability. In Phase I studies, toxicity was manageable. More
Phases II and III studies are ongoing, and Iressa has already
been approved for treatment of NSCLC in Japan.
Vascular endothelial growth factor, fibroblast growth fac-
tor and platelet-derived growth factor and their cognate re-
ceptor tyrosine kinases are strongly implicated in angiogen-
esis associated with solid tumors; the sustained growth of
solid tumors is dependent on angiogenesis, the growth of
new blood vessels from existing host vasculature [133]. Us-
ing rational drug design coupled with traditional screening
technologies, a novel inhibitor of these receptors has been
discovered: SU6668 (Sugen/Pharmacia). SU6668 is a small
 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38
molecule synthetic kinase inhibitor of the tyrosine activity of
Flk-1/KDR or VEGF receptor, PDGFR, an FGFR. Indeed,
in several cellular systems, it inhibits tyrosine phosphoryla-
tion and mitogenesis after stimulation of cells with appro-
priate ligands. Administration of SU6668 in athymic mice
resulted in significant growth inhibition of a diverse panel of
human tumor xenografts of glioma, melanoma, lung, colon,
ovarian and epidermoid origin. The attractive and validated
targets of SU6668, coupled with its broad activity in tumor
xenograft models have motivated its entry into clinical de-
velopment. Accordingly, SU6668 has recently entered Phase
I clinical trials, and its safety and efficacy profile in humans
will emerge in the near future [134].
Other small molecule inhibitors targeted against tyrosine
kinase are currently in clinical trials [1].
4.4. Protein–protein interaction inhibitors
In living cells, many regulated processes are initiated
or inhibited through specific protein–protein complex for-
mation. That is why peptides and small molecules that
modify the overall quaternary structure of a selection of
receptor–ligand interactions and oligomeric viral enzymes
have been developed recently [135]. This part presents sev-
eral approaches by which RTK can be inhibited by peptide
and peptidominetics which interfere with protein–protein
interactions.
4.4.1. Inhibitors of signaling
The small adaptor protein Grb2 is essential in the Ras
signaling pathway. It is made up of one SH2 domain of
approximately 100 aminoacids surrounded by two SH3 do-
mains, each containing about 60 aminoacids. The SH2 do-
main bind to target proteins at the level of phosphotyrosine
motifs, whereas the SH3 domains bind to proline-rich mo-
tifs of these target proteins [136]. The binding of growth
factor on its receptor triggers dimerization and phosphoryla-
tion on its carboxyl-terminal tyrosine residues. This allows
recruitment under the membrane near the Ras protein of
the Grb2–Sos complex. Sos, the exchange factor of Ras, is
thus able to activate Ras under its GTP form. Then, Ras can
recruit Raf and activate the MAPK cascade which induce
cell division and/or differentiation [137]. As overexpression
of ErbB receptors induce a downstream signaling deregula-
tion, and particularly of Ras signaling pathway [138], Grb2
constitutes a target for the design of therapeutic agents as
anti-tumor agents. Two possible ways of inhibiting the Ras
signaling pathway at the level of Grb2 have been described
by Garbay et al. [137]: they first describe a strategy to in-
hibit Grb2–Sos interaction at the level of its SH3 domains
and then report the results obtained in blocking Grb2 SH2
domain interaction with EGFR or Shc.
Concerning the interruption of the growth factor-
stimulated Ras signaling pathway at the level of the
Grb2–Sos interaction by inhibition of Grb2 SH3 domains,
a “peptidimer” made of two identical proline-rich sequence
33
from Sos linked by a lysine spacer was designed using struc-
tural data from Grb2 and a proline-rich peptide complexed
with its SH3 domains. This proline-rich “peptidimer” which
has two VPPPVPPRRR sequences, which specifically rec-
ognize Grb2, blocks selectively Grb2–Sos complexation in
ER22 (CCL39 fibroblasts overexpressing EGFR) cellular
extracts. Moreover, this “peptidimer” was modified to en-
ter by coupling it to penetratin, a peptide consisting of 16
aminoacids issued from the Antennapedia homeodomain.
The results showed that at 10 ␮M, the conjugate inhibits the
Grb2–Sos interaction (100%) and MAPK phosphorylation
(ERK1 and ERK2) was largely decreased (60%) in ER22
without modifying cellular growth. When tested for its
anti-proliferative effect, the conjugate was an efficient in-
hibitor of colony formation of transformed NIH 3T3/HER2
cells grown in soft agar, with an IC50 of 1 ␮M. Thus, as
these “peptidimers” do not seem to develop toxicity in sys-
tem which do not overexpress the Ras signaling pathway,
their design appear to be interesting leads to investigate
signaling and intracellular processes and for designing se-
lective inhibitors of tumorigenic Ras-dependent processes
[137,139].
Concerning the possibility of inhibiting Grb2 at the
level of its SH2 domain, this approach was expected to
be easier because small phosphopeptides were reported to
have affinities for SH2 domains in the 10−8 to 10−7 M
range. However, these peptides are negatively charged and
do not enter cells easily; moreover, cellular phosphatases
degrade the phosphotyrosine group which is necessary
for phosphopeptide activity. To design peptidomimetic
inhibitors, the three-dimensional structure of the Grb2
SH2 domain complexed with a phosphopeptide has been
used. Then, a series of small peptides with the sequence
mAZ-pTyr-Xaa-Asn-NH2, where Xaa denotes methylphos-
photyrosine or its carboxylic mimetics, were synthesized as
inhibitors of the Grb2 SH2 domains. Peptide with (Me)pTyr
as Xaa has the highest affinity for Grb2 (Kd = 3 ± 1 nM)
and exhibits to date the best inhibitory activity (IC50 =
11 ± 1 nM) to displace PSpYVNVQN–Grb2 interaction in
an ELISA test [137,140]. These compound which showed
the highest affinity for Grb2 was chosen in order to prepare
a prodrug in which the phosphate groups were protected
with S-acetyl thioethyl ester (SATE). The prodrugs obtained
are very hydrophobic and can easily enter cells, where they
are degraded by esterases, and the chemically unstable in-
termediates liberate the active groups which can inhibit the
growth of NIH3T3 cells transfected by the oncogene HER2
in colonies on soft agar [137].
4.4.2. Inhibitors of dimerization
Reports have shown that short peptides (10–20 amino
acids) can compete with target proteins [141]. In the context
of cancer, a few works have shown that small peptidomimet-
ics can be used to inhibit dimerization of RTKs which are
implicated by a mutation or overexpression in this disease.
In fact, these peptides act as antagonists of RTKs.
34 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38
Appropriate ligand-induced dimerization of RTKs implies
the participation of different interaction loops on the recep-
tor surface. One such dimerization interface has recently
been characterized for the EGF receptor (see above, and
[142]). Berezov et al. [143] have designed a series of 12–30
amino acids long peptides derived from potential such sur-
faces in ErbB/HER receptors [143]. These peptides are ef-
fective binders of ErbB receptor ectodomains, inhibit ErbB2
dimerization and growth of cells overexpressing these recep-
tors. Such small peptides may be developed as therapeutic
agents.
Another example is given by the activation of the rat
neu/erbB2 oncogene by a point mutation within its trans-
membrane domain that results in the substitution of glu-
tamic acid for valine at position 664, and is associated with
constitutive activation of the tyrosine kinase [40]. In order
to investigate the role of the alpha-helical transmembrane
sequence in the function of neu, Lofts et al. [144] have con-
structed an expression vector to produce a variety of short
transmembrane neu proteins, lacking ligand-binding or in-
tracellular kinase domain. Such sequences should interact
with full-length receptors and prevent receptor dimeriza-
tion. They showed that the short transmembrane molecules
are expressed at the cell surface and can retard the growth
of neu-transformed cells in monolayers, as colonies in soft
agar and as tumors in animals. We have recently extended
this work and shown that short hydrophobic peptides are
able to inhibit both EGFR and ErbB2/HER2 kinases in cul-
tured human cancer cells (Bennasroune et al., manuscript
submitted). In an another work, Qian et al. [145] have
shown that the transforming activity of oncogenic p185neu
(also termed Tneu) can be inhibited in vitro and in vivo
by co-expression of a truncated neu protein with a large
cytoplasmic deletion which includes the tyrosine kinase
domain. Indeed, in cell lines co-expressing full-length and
truncated neu proteins, they observed co-dimerization be-
tween full-length p185neu and truncated peptides resulting
in the formation of a kinase-inactive heteromeric complex
[145]. These peptides may be important for the design
of future therapies directed against ErbB family oncopro-
teins.
5. Conclusion and future directions
One should not overlook the possibility for RTK-targeting
drugs to potentially target more than one RTK (as exempli-
fied by Gleevec, and other molecules in development), and
thus more than one type of cancer, and even more than one
disease [146]. The position of RTKs at the very heart of
many of the cellular processes that are deregulated in can-
cer makes them even more attractive since inhibition of one
RTK pathway may yield effects on more than simply cell
proliferation. For instance, Herceptin has very recently been
shown in a mouse model of breast cancer to exert also its
beneficial effects by inhibiting angiogenesis [147]. In this
respect, relative lack of specificity of some effective drugs
may turn out to be one more advantage for this class of
new molecules. Indeed, although initially developed as an
inhibitor of the abl tyrosine kinase in CML, STI-571 has al-
ready proved as an effective inhibitor of the mutated c-kit
RTK in GIST [148], and has been approved for this indica-
tion. Of course, such a poor specificity is much less desir-
able in purely experimental settings, where kinase inhibitors
are very often used in cell-based assays for the dissection of
signaling pathways [149].
One possible drawback for strategies aiming at RTKs in-
hibition is the necessity for molecular characterization of
individual tumors in order to ascertain the target status. This
holds true for any drug aiming at specific targets deregu-
lated in cancer, whether be it a RTK, or any component
of the ras/MAP kinase pathway, or an anti-oncogene. Nev-
ertheless, this is not a trivial issue, as the experience with
Herceptin has already proved that only those patients who
show overexpression of HER2/ErbB2 would benefit from
treatment. However, it is not yet clear whether responsive
patients can be identified by target expression levels, as
is the case with Herceptin, which is approved for use in
ErbB2-positive cancer and requires the use of an assay to
measure expression of this receptor for patient selection.
Standardization of reproducible procedures to evaluate the
precise status of the tumor of individual patients will most
probably benefit from the ongoing progresses in gene anal-
ysis techniques such as microarrays, as well as more classi-
cal immuno-cytological methods. On the other hand, there
is a possibility that inhibition of an oncogenic pathway at
a level not obviously altered may be beneficial. For in-
stance, inhibition of the met RTK is effective in a cellular
system with a ras mutation [150]. In reverse, the use of a
farnesyl transferase inhibitor, targeting oncogenic ras, does
inhibit proliferation of an ErbB2 overexpressing cell line
[151].
Another serious problem has very recently emerged when
the use of Gleevec and Herceptin has been extended to more
patients. Resistance to these molecules has been observed,
as with any anti-cancer drug. The extent of the problem has
yet to be assessed, and it is most probably a witness of the
enormous genetic instability of cancerous cells. Indeed, a
point mutation in the kinase domain of bcr-abl has been
demonstrated in leukemic patients who became resistant to
Gleevec [16,152].
Nevertheless, the various implications of RTKs through
mutations and overexpression in cancer, together with the
ever increasing knowledge about their structure, mechanism
of activation and regulation, allows for reasonable hopes in
future developments of efficient treatments targeting these
oncogenes.
Reviewers (MA 496)
Prof. Jacques Robert, Institute Bergonié, 229, Cours de
l’Argonne, F-33076 Bordeaux Cedex, France.
 A. Bennasroune et al. / Critical Reviews in Oncology/Hematology 50 (2004) 23–38
Prof. Christiane Garbay, Laboratoire de Pharmacochimie
Moléculaire et Structurale, UFR des Sciences Pharmaceu-
tiques & Biologiques, 4 av. de l’Observatoire, F-75270 Paris
Cedex 06, France.
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Biography
Pierre Hubert obtained his M.D. in 1979 and a Ph.D.
in cell biology in 1992 at the University Louis Pasteur,
Strasbourg, France. He is currently Chargé de Recherche
at the National Institute for Health and Medical Research
(INSERM, Unit 575). He heads a small research group on
the role of membrane domains in receptor tyrosine kinase
activation.