ERBB RECEPTORS AND CANCER: THE COMPLEXITY OF TARGETED INHIBITORS
Nancy E. Hynes
* and Heidi A. Lane‡
Abstract ERBB receptor tyrosine kinases have important roles in human cancer. In particular,
the expression or activation of epidermal growth factor receptor and ERBB2 are altered in many
epithelial tumours, and clinical studies indicate that they have important roles in tumour aetiology
and progression. Accordingly, these receptors have been intensely studied to understand
their importance in cancer biology and as therapeutic targets, and many ERBB inhibitors are
now used in the clinic. We will discuss the significance of these receptors as clinical targets, in
particular the molecular mechanisms underlying response.
Subclass I of the receptor tyrosine kinase (RTK) super-
receptors tend to have a more aggressive disease, and
family consists of the ERBB or epidermal growth fac-
one that is associated with factors that predict a poor
tor (EGF) receptors and comprises four members:
clinical outcome, so ERBB receptors have been
EGFR/ERBB1, ERBB2, ERBB3 and ERBB4. All mem-
intensely pursued as therapeutic targets (reviewed in
bers have an extracellular ligand-binding region, a
REF. 5). There are two major classes of anti-ERBB
single membrane-spanning region and a cytoplasmic
therapeutics: ectodomain-binding antibodies and
tyrosine-kinase-containing domain. The ERBB recep-
small-molecule tyrosine-kinase inhibitors (TKIs) that
tors are expressed in various tissues of epithelial, mes-
compete with ATP in the tyrosine-kinase domain
enchymal and neuronal origin. Under normal
BOX 2. Many of these therapies are either in clinical
physiological conditions, activation of the ERBB
use or in advanced clinical development and these will
receptors is controlled by the spatial and temporal
be a main topic of this review.
expression of their ligands, which are members of the
We will outline our understanding of how ERBB
EGF family of growth factors (reviewed in REFS 1,2)
receptors contribute to cancer and, in particular,
(FIG. 1). Ligand binding to ERBB receptors induces the
how targeted therapeutics affect the transformed
formation of receptor homo- and heterodimers and
phenotype. Using data gleaned from preclinical
activation of the intrinsic kinase domain, resulting in
models, and where possible from the clinic, we will
phosphorylation on specific tyrosine residues within
discuss potential molecular mechanisms that under-
the cytoplasmic tail. These phosphorylated residues
lie a successful response to the blockade of ERBB
Institute for Biomedical
serve as docking sites for a range of proteins, the recruit-
signalling. We will also discuss mechanisms that
ment of which leads to the activation of intracellular
allow tumour cells to escape from anti-ERBB thera-
66, CH-4058 Basel,
signalling pathways (reviewed in REFS 24).
pies and suggest alternative strategies that might
‡Novartis Institutes for
The importance of ERBB receptors during develop-
lead to more effective treatment in the clinic.
BioMedical Research Basel,
ment and in normal adult physiology is evident
Oncology, K125.13.17, CH-
from analyses of genetically modified mice BOX 1.
The ERBB receptors and their ligands
4002 Basel, Switzerland.
Furthermore, EGFR and ERBB2 have been implicated
With respect to ERBB-receptor binding, the EGF family
Correspondence to: N.E.H.
e-mail: [email protected]
in the development of many human cancers. Patients
of ligands can be divided into three groups: the first
with cancer whose tumours have alterations in ERBB
includes EGF, transforming growth factor-α and
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2005 Nature Publishing Group
human tumours (for a timeline on EGFR and cancer
see REF. 12). ERBB receptors undergo various types of
• The family of ERBB or epidermal growth factor (EGF) receptors includes four
alteration in human tumours.
members: EGFR/ERBB1, ERBB2, ERBB3 and ERBB4. EGFR and ERBB2 are
Gene amplification leading to EGFR overexpression
involved in development of numerous types of human cancer and they have been
is often found in human cancers13,14. Furthermore, in
intensely pursued as therapeutic targets.
many tumours EGF-related growth factors are pro-
• Two important types of ERBB inhibitor are in clinical use: humanized antibodies
duced either by the tumour cells themselves or are
directed against the extracellular domain of EGFR or ERBB2, and small-molecule
available from surrounding stromal cells, leading to
tyrosine-kinase inhibitors (TKIs) that compete with ATP in the tyrosine-kinase
constitutive EGFR activation (FIG. 2) (reviewed in
domain of the receptor.
REF. 15). In gliomas, EGFR
amplification is often accom-
• In preclinical models, treatment of tumour cells with ERBB-directed TKIs and
panied by structural rearrangements that cause in-
antibodies rapidly downregulates phosphatidylinositol-3-kinase–AKT, mitogen-
frame deletions in the extracellular domain of the
activated protein kinase, SRC, and signal transducer and activator of transcription
receptor, the most frequent being the EGFRvIII vari-
(STAT) signalling, and blocks the proliferation of tumour cells. In the clinic, skin
ant16. Carcinomas of the breast, lung and ovaries have
biopsies (surrogate tissue), and to a limited extent tumours, have been analysed for
also been reported to express this variant17, although
the molecular consequences of treatment with ERBB inhibitors.
these data await further confirmation. Somatic muta-
• ERBB-directed therapeutics have demonstrated clinical efficacy; however, the
tions in the tyrosine-kinase domain of EGFR were
antitumour effects are often not as strong as predicted from preclinical studies.
recently identified in non-small-cell lung cancers
There are likely to be various reasons why this is so, an important one being that
(NSCLCs) in a subgroup of patients that showed clini-
other tumour-cell alterations influence the tumour response to ERBB-targeted
cal responses to treatment with the TKIs gefitinib18,19
inhibitors. Therefore, rational drug-combination strategies have great potential to
and erlotinib20. The functional properties of these
combat the complexity of tumour biology.
mutant receptors will be discussed below.
Amplification of ERBB2
leading to overexpression
of the receptor, originally detected in a subset of breast
amphiregulin, which bind specifically to EGFR; and
tumours21, occurs in other human cancers such as
the second includes betacellulin, heparin-binding EGF
ovarian, gastric and salivary cancers (reviewed in
(HB-EGF) and epiregulin, which show dual specificity,
REFS 5,22). Intriguingly, mutations in the kinase domain
binding both EGFR and ERBB4. The third group is
have been identified in a small number of
composed of the neuregulins (NRGs) and forms two
NSCLCs23. The impact of these mutations on ERBB2
subgroups based on their capacity to bind ERBB3 and
activity remains to be explored.
ERBB4 (NRG1 and NRG2) or only ERBB4 (NRG3 and NRG4) (FIG. 1a). None of the EGF family of peptides bind
Structural studies on ERBB receptors
ERBB2; however, MUC4, a member of the mucin family,
Publications describing the crystal structure of the
acts as an intramembrane modulator of ERBB2 activity6.
EGFR, ERBB2 and ERBB3 ectodomains (reviewed
Despite having no soluble ligand, ERBB2 is important
in REF. 24) have led to new insights into some intrigu-
because it is the preferred heterodimerization partner of
ing questions concerning the process of ligand-
the other ligand-bound family members7 (FIG. 1a).
induced receptor dimerization and biological
Activated ERBBs stimulate many intracellular sig-
activity of ERBB2-targeted antibodies. The extracel-
nalling pathways and, despite extensive overlap in the
lular region of each ERBB receptor consists of four
molecules that are recruited to the different active
domains (I−IV; FIG. 3). Determination of the struc-
receptors, different ERBBs preferentially modulate cer-
ture of ligand-bound EGFR has confirmed earlier
tain signalling pathways, owing to the ability of indi-
studies (reviewed in REF. 24) that show the impor-
vidual ERBBs to bind specific effector proteins (FIG. 1b).
tance of domains I and III in peptide binding.
Two of the main pathways activated by the receptors
Moreover, these studies also revealed that there is a
are the mitogen-activated protein kinase (MAPK) and
direct receptor−receptor interaction promoted by
the phosphatidylinositol 3-kinase (PI3K)–AKT path-
the domain II dimerization arm; the ligands are not
ways (reviewed in REFS 24). Other important ERBB
involved in the receptor−receptor interaction25,26. In
signalling effectors are the signal transducer and acti-
unliganded ERBB3 REF. 27 or ligand-bound inactive
vator of transcription proteins (STATs; reviewed in
EGFR28 the receptors assume the so-called tethered
REF. 8), which, in cancer, have often been associated
structure, in which the domain II dimerization
with EGFR activation9; SRC tyrosine kinase, the activ-
interface is blocked by intramolecular interactions
ity of which is increased in response to EGFR and
between domains II−IV. The EGFRvIII variant is
ERBB2 signalling (reviewed in REF. 10); and mamma-
missing exons 1−7 REF. 16 and, consequently, the
lian target of rapamycin (mTOR), a serine/threonine
domain II dimerization arm, and cannot assume the
A large family of receptors that
kinase activated downstream of PI3K−AKT and other
closed tethered structure, perhaps explaining its
span the membrane seven times and couple to G proteins, which
growth regulators (reviewed in REF. 11) (FIG. 2).
are composed of α-, β- and
The structure of ERBB2's extracellular region is
γ-subunits. The α-subunit
ERBB receptors and cancer
radically different from the others. ERBB2 has a fixed
contains the nucleotide (GTP
The ERBB receptors are implicated in the develop-
conformation that resembles the ligand-activated
or GDP) binding site, and the β- and γ-subunits behave as a
ment of many types of cancer, and EGFR was the first
state: the domain II−IV interaction is absent and the
tyrosine-kinase receptor to be linked directly to
dimerization loop in domain II is exposed30,31. This
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2005 Nature Publishing Group
structure is consistent with the data that indicate that
ERBB2 is the preferred partner for the other activated
ERBBs, as it is permanently poised for interaction with another ligand-bound receptor. Furthermore, this structure explains why no soluble EGF-related ligand has been found. It predicts that ERBB2 possesses a
unique subdomain I−III interaction that makes ligand binding impossible because the site is buried and not accessible for interaction.
ERBB-receptor transactivation in cancer
The EGF family of growth factors are produced as
transmembrane precursors that can be cleaved by cell-
surface proteases (reviewed in REFS 32,33), a step that
leads to the release of soluble ligands. This cleavage,
referred to as ectodomain shedding, is an important
step in the control of ligand availability and receptor
activation34 (reviewed in REF. 32). ERBB receptors are
often constitutively stimulated in cancer owing to the presence of EGF ligands in the tumours15 (FIG. 2).
Therefore, it is essential to understand the mechanisms
that control ligand processing, as novel therapeutic
targets might be discovered.
The proteases involved in ectodomain shedding
belong to the metalloproteinase family, in particular the
ADAM (a disintigrin and metalloprotease) family and
matrix metalloproteinases (MMPs). The production of
soluble EGF family ligands through ectodomain shed-
ding occurs in response to diverse stimuli and was first
described following activation of GPROTEINCOUPLED
RECEPTORS (GPCRs)35. In cells treated with a receptor
agonist, GPCR stimulates a batimastat-sensitive
metalloproteinase that induces cleavage and release of
Figure 1 ERBB receptors, ligands, dimers and downstream signalling pathways.
HB-EGF, leading to the rapid phosphorylation of
Members of the epidermal growth factor (EGF) family of growth factors are ligands
EGFR36. This process, termed EGFR transactivation,
for the ERBB receptors. Ligand binding to ERBB receptors induces the formation of
has important biological implications, as it leads to
receptor homo- and heterodimers and the activation of the intrinsic kinase domain,
stimulation of intracellular pathways such as MAPK
resulting in phosphorylation on specific tyrosine residues within the cytoplasmic tail. These
signalling37. The proteases involved in ectodomain shed-
phosphorylated residues serve as docking sites for a range of proteins, the recruitment of
ding have also been examined in tumour cells. ADAMs,
which leads to the activation of intracellular signalling pathways. None of the ligands bind ERBB2, but ERBB2 is the preferred dimerization partner for all the other ERBB receptors.
including ADAM9, ADAM10, ADAM15 and ADAM17
ERBB3 has impaired kinase activity and only acquires signalling potential when it is dimerized
REFS 38,39 have been associated with the shedding of
with another ERBB receptor, such as ERBB2. Overexpression of ERBB2 in tumours leads to
distinct EGF-related ligands in cancer cells. In primary
constitutive activation of ERBB2, presumably because of increased receptor concentrations
breast tumours, there is a correlation between high
at the plasma membrane. Many of these tumours contain phosphorylated ERBB3, which
EGFR activity and high ADAM17 levels34.
couples ERBB2 to the phosphatidylinositol 3-kinase (PI3K)−AKT pathway128. b
It is now widely accepted that diverse GPCR ago-
representation of the main autophosphorylation sites in EGF receptor (EGFR), ERBB2 and
nists transactivate ERBBs in both normal and cancer
ERBB3 and of the signalling molecules associated with these sites. Despite extensive overlap in the molecules recruited to the active receptors, there is some preferential modulation
cells. Although EGFR and ERBB2 have usually been
of signalling pathways. Tumour cells that express EGFR with kinase-domain mutations
monitored following GPCR stimulation, it is impor-
preferentially activate the pro-survival PI3K−AKT and signal transducer and activator of
tant to keep in mind that NRGs ⎯ the ligands for
transcription (STAT) pathways67. Although EGFR has no consensus sequence for the p85
ERBB3 and/or ERBB4 ⎯ are processed by the same
adaptor subunit of PI3K, it couples to this pathway through GAB1, which binds growth-factor-
metalloproteinases40 (reviewed in REF. 33). For certain
receptor-bound protein 2 (GRB2). Although no direct binding data have been published,
cancer types, such as prostate cancer, the deregulated
STATs have been proposed to couple to EGFR through tyrosine-1068 and tyrosine-1086 REF. 137. Additional EGFR binding partners are discussed in a recent review137. ERBB2
expression of GPCRs and their ligands has been linked
couples to the mitogen-activated protein kinase pathway through GRB2, SHC, downstream
to tumour development (reviewed in REF. 41), and
of kinase related (DOK-R)138 and CRK; phospholipase Cγ (PLCγ) binding has recently been
chronic EGFR activation is well described in prostate
described139. Although ERBB3 is able to bind neuregulins (NRGs), it has impaired kinase
tumours42, indicating a potential link between the two
activity owing to substitutions in crucial residues in the tyrosine-kinase domain. Therefore,
ERBB3 only becomes phosphorylated and functions as a signalling entity when it is dimerized
More recently, ERBB transactivation has been
with another ERBB receptor140, ERBB2 being its preferred partner7. ERBB3 contains six docking sites for the p85 adaptor subunit of PI3K and couples very efficiently to this pathway
shown to involve other physiological ligands (FIG. 2).
(reviewed in REF. 3). AR, amphiregulin; BTC, betacellulin; EPR, epiregulin; HB-EGF, heparin-
The binding of WNT to its seven-pass membrane
binding EGF; NRGs, neuregulins; TGFα, transforming growth factor-α.
receptor Frizzled (FZD) transactivates EGFR43. The
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Box 1 The toxicity of ERBB-directed therapeutics is related to their physiological roles
Mice lacking epidermal growth factor receptor (EGFR) usually die during the first postnatal week owing to respiratory
problems. They also show gastrointestinal phenotypes, thin skin, and hair-follicle defects that result in brittle hair
These observations help explain the most common side effects associated with EGFR inhibition in the clinic: rash
and acneic skin reactions (including folliculitis) and diarrhoea
58,148. Indeed, cutaneous skin rash has been proposed
as a surrogate marker of clinical benefit for many EGFR-targeted agents
149. Although infrequent (1% globally
interstitial lung disease (interstitial pneumonia) has also been associated with gefitinib treatment in patients with non-
small-cell lung cancer; patients with lung comorbidities, such as idiopathic pulmonary fibrosis, seem to be particularly
150,151. This is consistent with the demonstration that gefitinib augments bleomycin-induced pulmonary
fibrosis in a murine model, supporting a role for EGFR in the regenerative epithelial-cell proliferation associated with
ERBB2 has an essential role in the developing heart
153. Embryos that lack the receptor die owing to improper
formation of the ventricular trabeculea, the myocardium responsible for maintaining blood flow. Conditional
ablation of ERBB2 in postnatal cardiac-muscle cell lineages revealed a role for ERBB2 in the adult heart. In its absence,
ventricular enlargement of both chambers was observed, which is consistent with dilated cardiomyopathy
154. In the
clinic, some trastuzumab-treated breast cancer patients displayed cardiac phenotypes, including cardiomyopathy,
congestive heart failure and decreased left ventricular ejection fraction. This was particularly true for patients treated
concurrently with anthracyclines
155. Considering that ERBB2-null cardiomyocytes showed an increased sensitivity
to adriamycin-induced toxicity
154, it is possible that in the clinical setting trastuzumab-mediated effects on cardiac
ERBB2 signalling might aggravate anthracycline-induced toxicity. The heart phenotype observed in Erbb2-knockout
mice is identical to that observed in mice lacking ERBB4
REF. 156 or for neuregulin-1 (NRG1)
57, demonstrating the
importance of the ligand-induced ERBB2
−ERBB4 heterodimer in heart development. As both ERBB2 and ERBB4
are expressed in adult cardiomyocytes
158 and NRG1 promotes survival of isolated cardiomyocytes
159, it is possible
that ERBB4 has a role in trastuzumab-induced cardiotoxicity. It should be noted, however, that the antibody does not
interfere with the NRG1-induced ERBB2
−ERBB4 heterodimererization (see main text).
mechanism seems to be similar to that described for
to plasma-membrane-associated oestrogen receptor
GPCRs, as it is rapid and blocked by metalloprotein-
(ER) has also been shown to rapidly transactivate
ase inhibitors; however, the target ligand has not
ERBBs. According to one report, E2-stimulated acti-
been identified. WNT−FZD-mediated transactiva-
vation of MMP2 and MMP9 leads to the release of
tion has been observed in normal mammary cells43
HB-EGF44. Tamoxifen, a selective ER modifier
and in breast cancer cells (T. Schlange and N.E.H.,
(SERM) was shown to transactivate EGFR and
unpublished observations). Oestradiol (E2) binding
ERBB2, and in ERBB2-overexpressing breast cancer cells this reduced the antiproliferative activity of the SERM45. This has important clinical implications that
Box 2 Background on ERBB-targeted antibodies and kinase inhibitors
will be discussed below.
The first epidermal growth factor receptor (EGFR)-specific monoclonal antibodies
Considering that many GPCR agonists stimulate
(mAbs) were isolated using partially purified receptor
160 and A431-EGFR-
protein kinase C (PKC) and SRC (reviewed in REF. 46),
overexpressing cancer cells
161. Specific antibodies were detected by
these kinase families might have widespread func-
inhibition. Cetuximab is a chimeric human:murine derivative of mAb225, isolated
tions in ERBB transactivation, by providing the link
by Mendelsohn and colleagues
160, and is a potent inhibitor of cancer cells that have
between, for example, GPCR agonists, metalloprotei-
autocrine EGFR activation and human tumour xenografts that overexpress the
nases and ligand processing. It has been observed
162. Cetuximab was approved for treatment of patients with advanced colorectal
that PKCδ is recruited to and phosphorylates
cancer in 2003. Turning to ERBB2, mAb4D5, isolated by Ullrich and colleagues
ADAM9, resulting in proHB-EGF processing38. The
and trastuzumab — its humanized (human IgG1 backbone, murine complementary-
SH3 domain of SRC and other family members has
determining regions) variant
163 — block proliferation of ERBB2-overexpressing breast
been shown to interact with proline-rich motifs in
cancer cells. Trastuzumab was approved for the treatment of ERBB2-overexpressing
the cytoplasmic tail of ADAMs47. Once recruited,
metastatic breast cancer in 1998. The mechanism underlying trastuzumab's clinical
SRC might phosphorylate specific tyrosine residues
efficacy is still under debate and seems to be multifaceted
in the cytoplasmic domain of ADAMs, thereby influ-
Mutation of the kinase domain of EGFR blocks its biological activity
164, providing a
encing the ability of the ADAM to cleave proEGF-
rationale for developing tyrosine-kinase inhibitors for cancer treatment. Many years of
related peptides. How PKC or SRC direct specific
medicinal chemistry, together with progress in protein-kinase crystallization, has
metalloproteinases to cleave their substrates remains
subsequently proven that the ATP-binding domains of kinases are attractive targets for
to be explored. For example, it might involve the
rational drug design. Therefore, the development of ATP-site-directed, low-molecular-
relocalization of a protease to specialized membrane
weight tyrosine-kinase inhibitors (TKIs) has taken centre stage in modern cancer
regions, as recently described for ADAM19 and
165. Levitzki and colleagues did some of the pioneering work in designing EGFR
TKIs, which they named tyrphostins
166. Subsequently, optimization of various lead
β1 REF. 40, or the clustering of a protease with
structures (including quinazolines, pyrrolopyrimidines, phenylaminopyrimidines) led
its substrate48. A consistent increase in the level of
to the development of several ERBB-directed TKIs, some of which are already
SRC kinase activity in primary tumours of the colon49
registered for the treatment of cancer patients or are well advanced in clinical
and breast50 was described several years ago. It will
165 TABLE 1.
be interesting to explore the effects of SRC inhibition
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ERBB receptors as targets for cancer therapy
The ERBB receptors are aberrantly activated in a wide
range of human tumours, and as such they are excellent
candidates for selective anticancer therapies. Several antibodies directed against the extracellular domain of
ERBBs and TKIs that target the kinase domain are in
clinical use or at advanced developmental stages TABLE 1. The treatment of tumour cells with these agents affects many of the intracellular pathways that
are essential for cancer development and progression
(FIG. 2). In preclinical models, treatment of tumour cells
with ERBB-targeted TKIs and antibodies rapidly down-
regulates PI3K−AKT, MAPK, SRC and STAT signalling and, as a consequence, blocks the proliferation of
tumour cell lines and XENOGRAFTS in nude mice8,10,51–57.
Is there any evidence that in the clinical setting anti-
ERBB drugs function by decreasing the activity of sig-
nalling pathways? Current clinical practice concentrates
SURROGATE TISSUE to analyse the molecular
consequences of treatment with EGFR inhibitors. In sur-
rogate tissue, downregulation of EGFR phosphorylation
is associated with the downregulation of MAPK signal-ling; the increased expression of the cyclin-dependent
kinase (CDK) inhibitor p27 (also known as KIP1);
changes in STAT3 activity; and a decreased proliferation
index, associated in some cases with increased apopto-
sis58–61. Of course, the ideal tissue to use in these phar-macodynamic studies is the tumour62 and a few studies
Figure 2 Active ERBB receptors and downstream signalling pathways in a tumour
have shown that these pathways are downregulated in
In tumour cells, ERBB receptor tyrosine kinases are activated by various mechanisms,
tumours from treated patients63,64. In this context, it is
including mutation, overexpression, and autocrine or paracrine production of epidermal growth
important to note that the toxicity reported for ERBB-
factor (EGF) family ligands. a
Paracrine ERBB ligands (green circles) are released from stromal
targeted drugs is correlated with known functions of
Autocrine ligand (blue circles) production results from the activation of G-protein-coupled
EGFR and ERBB2 in normal physiology BOX 1.
receptors (GPCRs), Frizzled (FZD) or oestrogen receptor (ER), which causes the metalloproteinase-mediated cleavage and release of pro-EGF-related ligands (a process known as ectodomain shedding). The mechanisms controlling ectodomain shedding are still largely
Response to ERBB-targeted therapeutics
unknown, although SRC kinase has been implicated. c
Active ERBB receptors stimulate
When considering how ERBB-targeted therapeutics
numerous signalling pathways by recruiting proteins to specific phosphorylated tyrosine residues
function, it is important to mention that, in contrast to
in their carboxy-terminal domain. d
The phosphatidylinositol 3-kinase (PI3K)−AKT pathway is
the TKIs, antibodies targeting EGFR and ERBB2 have
stimulated through recruitment of the p85 adaptor subunit of PI3K to the receptor. Mammalian
the inherent ability to recruit immune effector cells
target of rapamycin (mTOR) acts as a central sensor for nutrient/energy availability, and can also be modulated by PI3K
such as macrophages and monocytes to the tumour
−AKT-dependent mechanisms11,92. e
The mitogen-activated protein
kinase (MAPK) pathway is activated by recruitment of growth-factor-receptor-bound protein 2
through the binding of the antibody constant Fc
(GRB2) or SHC to the receptor. f
SRC kinase is activated by ERBB receptors and by GPCRs (b
domain to specific receptors on these cells. In xenograft
and ER. There are many nuclear effectors of ERBBs in tumour cells. g
One of these is the cyclin-
models at least, this mechanism is relevant for the anti-
dependent kinase inhibitor p27 (also known as KIP1), which has an important role in the control of
tumour activity of ERBB2-targeted trastuzumab65.
proliferation. In tumour cells with overexpressed ERBB2, p27 is sequestered from cyclin E (Cyc
Whether this mechanism has a role in clinical efficacy
E)−CDK2 complexes and cells progress through the cell cycle54. h
Signal transducer and
activator of transcription (STAT) is another nuclear effector. i
Binding of STAT to ERBB leads to its
in cancer patients remains unclear.
tyrosine phosphorylation, dimerization and nuclear entry, resulting in STAT binding to specific DNA
Most NSCLC patients who showed clinical
sequences in promoter regions of target genes encoding, for example, pro-survival factors (h
responses to treatment with gefitinib and erlotinib
Nuclear ER and oestradiol (E2) controls transcription of cell-cycle regulators that are particularly
(both of which are TKIs) had tumours with somatic
important for breast cancer cell proliferation102. k
ERBB receptors also stimulate transcription of
mutations in the EGFR kinase domain18–20. However, it
vascular endothelial growth factor (VEGF) through the MAPK pathway141. VEGF has a role in
should be noted that some responding patients had
induction of tumour-associated angiogenesis. Active EGFR receptors have been detected on
tumours with no kinase-domain mutation. Therefore,
tumour-associated endothelial cells, which has been proposed to result from tumour release of ERBB ligands142. EGFR, EGF receptor; VEGFR, VEGR receptor.
although this observation is very exciting, the clinical significance of wild-type EGFR versus mutated EGFR for response to TKIs needs further examination and is
on ectodomain shedding. A detailed discussion of
discussed in detail in a recent review66. In vitro
metalloproteinases and ectodomain shedding is
of tumour cells that express EGFRs with kinase-domain
beyond the scope of this article. However, it is evi-
mutations have indicated that these mutations increase
dent that the process is complex; several proteases
the sensitivity of the receptor to activation by lig-
Commonly refers to the growth of tumour cells as tumours in
can process an individual pro-ligand and a specific
ands18,19,67. Moreover, tumour cells with mutant EGFR
protease has several substrates (reviewed in REF. 48).
preferentially activate the pro-survival PI3K−AKT and
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A Extracellular domain of ERBB2 and ERBB3
STAT pathways, and treatment of these cells with a TKI induces apoptosis67. AUTOCRINE EGFR activation is an
early event in the development of head and neck squa-
mous-cell carcinoma (HNSCC)9. Considering the
documented role of STAT3-mediated survival in pre-
clincal models of HNSCC, this tumour type might be
particularly susceptible to anti-EGFR therapies.
Preclinical studies showed that the murine precursor
of trastuzumab 4D5 blocks tumour cells that overexpress ERBB2, but not those expressing low levels of the recep-
tor51,68. Accordingly, trastuzumab is prescribed to breast cancer patients whose tumours overexpress that recep-tor. Clinical trials showed that the addition of trastuzu-mab to standard chemotherapy prolonged relapse-free
survival, leading to the approval of the drug for treat-
ment of ERBB2-overexpressing metastatic breast cancer
patients. The mechanism underlying trastuzumab's
clinical efficacy is likely to be multifacted51. In addition
to the Fc-mediated functions mentioned above, pre-clinical studies have shown that the antibody downregu-lates ERBB2 levels68 and ERBB2-mediated signalling pathways54,55. Furthermore, metalloproteinase-mediated ERBB2 ectodomain shedding has been proposed to cause constitutive ERBB2 signalling and trastuzumab also blocks this process70.
Resistance to ERBB-directed therapeutics
During the process of cancer development, cells acquire
multiple mutations, each of which contribute to and
are necessary for full malignancy (reviewed in REF. 71).
Therefore, it is unlikely that targeting only one altera-tion will be sufficient to kill metastatic tumour cells. For example, for ERBB2-overexpressing metastatic breast cancer, response rates of approximately 35% were observed in the trastuzumab-treated patients72. This is likely to be for various reasons, including resist-ance to the targeted therapy or, more broadly, because the malignant phenotype is unlikely to be due to
B Extracellular domain of ERBB2
ERBB2 activation alone. We will discuss the data indi-cating that other tumour-cell alterations do impact on
response to ERBB inhibitors and present rational strat-
egies for combining ERBB-targeted agents with other
signal-transduction inhibitors or with cytotoxics.
Acquired resistance to EGFR-targeted TKIs.
cussed above, tumours of lung cancer patients who
responded to gefitinib and erlotinib expressed EGFRs
that had gain-of-function mutations in the kinase
domain18–20. The recent identification of additional
mutations in NSCLC patients whose tumours displayed
To examine the in vivo
efficacy of tyrosine-kinase inhibitors
drug-sensitive mutations and who initially responded to
Figure 3 ERBB-receptor ectodomain structures. A
targeted at epidermal growth
TKI treatment might explain some of the acquired
extracellular region of each ERBB receptor consists of four
factor receptor (EGFR) in
domains (I−IV). It has been proposed that in the absence of
resistance to EGFR inhibitors73. Resistance to TKIs has
cancer patients, skin biopsies of
ligand, ERBB3 and epidermal growth factor receptor (EGFR;
emerged as a significant clinical problem, initially in the
treated patients have been
not shown) assume a tethered structure27,28 (a
). Domains I
context of chronic myelogenous leukaemia (CML), a
examined for downregulation of
and III are involved in neuregulin (NRG) binding and, following
disease that is associated with the BCR−ABL oncopro-
this, the dimerization arm in domain II is exposed (b
tein. CML patients treated with the BCL−ABL-
promotes receptor−receptor interaction (c
)25,26. ERBB2 has a
targeted TKI imatinib often experience complete
A form of bioregulation in
fixed conformation that resembles the ligand-activated state
of EGFR and ERBB3 REFS. 30,31. B
remission. However, imatinib-resistance can arise and
which a secreted peptide affects only the cell from which it is
antibodies trastuzumab and pertuzumab bind domains IV and
has been associated with acquired mutations in the
BCR−ABL kinase domain74.
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Table 1 ERBB-targeted therapeutics in clinical use
Status and comments
Approved for the treatment of ERBB2-
overexpressing breast cancer; ongoing trials for use in combination with various other drugs
Phase II trials to treat ovarian cancer, breast
cancer, prostate cancer and NSCLC; based on its ability to block ERBB2 dimerization, trials are ongoing in cancer that express low ERBB2 levels
Approved for the treatment of CRC; ongoing trials
in combination with various drugs for treatment of pancreatic cancer, HNSCC and NSCLC
Phase II trials for NSCLC, gynaecological cancer,
pancreatic cancer and oesophageal cancer
Trials are ongoing for CRC, RCC and NSCLC
Approved for the treatment of NSCLC after failure on other available treatments; ongoing trials in HNSCC, gastrointestinal cancer and breast cancer
Approved for the treatment of NSCLC after failure
on other available treatments; ongoing trials in many cancer types
Phase III trial underway on breast cancer patients who are refractory to trastuzumab and chemotherapy
Phase I trials underway — first multifunction
EGFR/ERBB2/VEGFR inhibitor, and there are many potential indications
Phase II trials underway in breast and NSCLC
Phase II trials underway in NSCLC
EXEL 7647/EXEL 0999
Phase I trials underway
CRC, colorectal cancer; EGFR, epidermal growth factor receptor; HNSCC, nead and neck squamous-cell cancer; mAb, monoclonal antibody; NSCLC, non-small-cell lung cancer; RCC, renal-cell cancer; TKI, tyrosine-kinase inhibitor; VEGFR, vascular endothelial growth factor receptor.
How do ERBB2-overexpressing cancer cells escape from
ERBB2-containing heterodimers78. This characteristic
Only approximately one-third of patients
might partly explain why pertuzumab inhibits the
with ERBB2-overexpressing metastatic breast cancer
growth of tumours that express low ERBB2 levels,
respond to trastuzumab69,72,75. These clinical results
whereas trastuzumab does not76. The impact that the
indicate that many ERBB2-overexpressing tumours are
different characteristics of pertuzumab have on its
resistant to this agent. Several theories, ranging from
clinical efficacy remains to be uncovered.
the existence of compensatory pathways to signalling
The potential for compensatory pathways to confer
aberrations downstream of ERBB2, have been pro-
resistance to anti-ERBB therapeutics is not restricted
posed to explain the clinical results. Considering the
to ERBB2 inhibitors, as bypassing the effects of an
first, ERBB ligands, either PARACRINE or autocrine, might
EGFR-directed TKI through ligand-mediated activa-
facilitate escape from trastuzumab through the activa-
tion of other ERBBs has also been observed and was
tion of alternative ERBB receptor homo- and het-
circumvented by the use of TKIs that target many dif-
erodimers. In fact, it has been shown experimentally
ferent ERBB receptors55. Clearly, the relevance of these
that trastuzumab cannot block the proliferation of
observations to the trastuzumab- or TKI-treated
tumour cells that have autocrine EGFR activation54,
patient will only become apparent when more detailed
and it cannot prevent the ligand-induced formation of
epidemiology has been carried out, correlating the
ERBB2-containing heterodimers or the activation of
molecular characteristics of a tumour with clinical
downstream signalling pathways55,76 (FIG. 4a). As trastu-
response. However, considering that many tumours
zumab binds to domain IV of ERBB2, a region not
express multiple ERBB receptors and co-express one
involved in receptor dimerization31 (FIG. 3), this explains
or more ERBB ligand15,79, the potential for their
why ERBB ligands can induce the formation of ERBB2-
involvement in resistance should be kept in mind.
containing heterodimers in the presence of the anti-
body. By contrast, pertuzumab binds ERBB2 near the
Resistance to ERBB-directed therapeutics through
A form of bioregulation in which a secreted peptide affects
centre of the domain II dimerization arm77 (FIG. 3),
activation of other RTKs.
Aberrant activation of
a neighbouring cell.
thereby preventing the formation of ligand-induced
other RTKs, for example, insulin like growth factor-1
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receptor (IGF1R)80 or fibroblast growth factor recep-
tor family members81, occurs in various types of cancer. These alterations might also impact on
response to ERBB-targeted agents (FIG. 4b). Indeed, the trastuzumab-sensitive ERBB2-overexpressing SKBR3 human breast cancer cell line was rendered resistant to the antibody following ectopic IGF1R expression82. In comparison to parental cells, these cells expressed low levels of the CDK inhibitor p27
REF. 83. The antiproliferative effect of trastuzumab has been linked to an increased association of p27 with the cyclin-E−CDK2 complex, resulting in
decreased kinase activity and a G1 block54. So the IGF1R-expressing cells might be trastuzumab insen-sitive because of downregulation of this important
negative regulator of cyclin-E−CDK2. A link to p27
has also been established in trastuzumab-resistant
SKBR3 cells, in which it was shown that continuous growth in trastuzumab resulted in cells with low p27
levels and high CDK2 kinase activity. Reintroduction of p27 into these cells restored trastuzumab sensitiv-
ity84. It is interesting that co-targeting ERBB2 and
IGF1R revealed a synergistic effect on cell growth in
ERBB2-overexpressing MCF7 breast cancer cells85,
an observation that prompts further investigation.
Loss of PTEN and resistance to ERBB-targeted thera-
The antiproliferative effect of ERBB-targeted
therapeutics often correlates with the downregulation of MAPK and PI3K−AKT pathways. It has been sug-gested that persistent activation of these pathways
caused by aberrations downstream of the receptors
might also have a role in resistance to trastuzumab, as
well as EGFR-directed inhibitors86–88. In fact, activation
of AKT, or loss or mutation of the dual-specificity pro-tein and lipid phosphatase PTEN, the negative regula-
Figure 4 Mechanisms of resistance to anti-ERBB therapeutics. a
tor of PI3K, have been found to be important causes of
tumour cells to trastuzumab through ligand-induced activation of ERBB2 dimers.
tumour-cell resistance87 (FIG. 4b). The main role of
of trastuzumab to overexpressed ERBB2 leads to downregulation of receptor signalling
PTEN is to dephosphorylate position D3 of phosphati-
potential, resulting in a block in tumour-cell proliferation. Preclinical results have shown that
dylinositol-3,4,5 triphosphate, and thereby antagonize
ERBB ligands can circumvent trastuzumab's ability to block downstream signalling and proliferation of ERBB2-overexpressing tumour cells. There are several likely explanations
PI3K function, leading to downregulation of AKT
including the inability of trastuzumab to prevent the formation of ligand-induced ERBB2-
activity. In a small panel of ERBB2-overexpressing pri-
containing heterodimers55,76. b
Multiple mechanisms have the potential to allow tumour
mary breast tumours it was shown that the expression
cells to escape from ERBB-targeted therapeutics. Treatment of tumour cells with
level of PTEN was positively correlated with trastuzu-
monoclonal antibodies (mAbs) or tyrosine-kinase inhibitors (TKIs), the two classes of
mab's clinical efficacy56. In this respect, constitutive
ERBB-targeted therapeutics discussed in the main text, interferes with ERBB-receptor
PI3K−AKT signalling through loss of PTEN expres-
signalling. PTEN dephosphorylates position D3 of phosphatidylinositol-3,4,5 triphosphate,
sion89, amplification of chromosomal loci encoding
and thereby antagonizes phosphatidylinositol 3-kinase (PI3K)–AKT pathway signalling. Loss or mutation of PTEN might cause tumour-cell resistance to ERBB therapeutics, because
AKT or PI3K (reviewed in REF. 90), or gain-of-function
in cells with low PTEN levels activation of the PI3K−AKT pathway becomes independent
mutations in PIK3CA
91 are common in solid tumours
of ERBB-receptor activation. Other classes of receptor tyrosine kinases, such as the
and might have an important role in modulating the
insulin-like growth factor 1 receptor (IGF1R) might be constitutively active in tumour cells.
efficacy of ERBB-directed therapies. Consequently,
As IGF1R signalling potential is not blocked by ERBB-targeted therapeutics, constitutive
logical combination strategies to alleviate this potential
activation of this receptor can promote strong activation of intracellular signalling pathways
resistance mechanism might be required.
(such as PI3K–AKT), even in the presence of ERBB-targeted therapeutics. The epidermal growth factor receptor variant III (EGFRvarIII) cannot bind the EGFR-targeted monoclonal antibodies cetuximab or matuzumab, and has been reported to be resistant to gefitinib143.
Drug combinations: the key to success?
Strong PI3K−AKT signalling interferes with ability of the cyclin-dependent kinase inhibitor
During the course of tumour development, genetic
(CKI) p27 (also known as KIP1) to block tumour proliferation in response to ERBB-targeted
alterations arise that contribute to the processes
therapeutics. AKT phosphorylates p27 on Thr157, leading to its cytoplasmic retention144.
linked to metastatic cancer71. Aberrantly activated
Furthermore, low levels of p27 and concomitant upregulation of cyclin E (Cyc E)−CDK2
ERBB receptors contribute to many of the processes5.
kinase activity in the nucleus have been reported in cells with high PI3K−AKT signalling84. In each case, p27 would not be able to function as a negative regulator of Cyc-E
However, it is very unlikely that inhibiting only
tumour cells would proliferate.
these receptors will block the malignant process.
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2005 Nature Publishing Group
A combination of signal-transduction inhibitors will
Recently, it has become evident that oestrogen−ER
probably have a stronger inhibitory effect.
signalling is far more complex than was initially antici-pated, and has pleiotropic effects through non-genomic
Rationale for combination of ERBB and mTOR inhib-
interactions with growth-factor signalling pathways106.
An important mediator of the PI3K−AKT path-
Several levels of interaction between the ER and ERBB
way with respect to tumour-cell growth and
RTKs have been documented (FIG. 5B). As mentioned
proliferation is the mTOR kinase. mTOR is a member
above, in vitro
E2 treatment transactivates EGFR and
of the phosphoinositide-kinase-related kinase family,
ERBB2. Moreover, ER physically interacts with ERBB2
which also includes PI3K11. The mTOR pathway acts
REF. 107. Strikingly, in many preclinical studies, upreg-
as a central sensor for nutrient/energy availability,
ulation of EGFR and ERBB2 expression has been asso-
and can also be modulated by PI3K−AKT-dependent
ciated with resistance to endocrine therapies (reviewed
mechanisms11,92 (FIG. 2). In the presence of mitogenic
in REFS 108,109). Indeed, tamoxifen can act as an oestro-
stimuli and sufficient nutrients and energy, mTOR
gen agonist in breast cancer cells that have increased
relays a positive signal to the translational machinery,
ERBB2 levels45 and reduction in tamoxifen resistance
facilitating events that drive cell growth11. The impor-
is associated with ERBB2 downregulation110. E2 treat-
tance of mTOR signalling in tumour biology is now
ment also activates the PI3K−AKT and MAPK path-
ways in oestrogen-sensitive breast cancer cells111 and
Consequently, several agents that selectively tar-
ER directly interacts with the p85 regulatory subunit
get mTOR (that is, the rapamycin derivatives
of PI3K112. Furthermore, the converse occurs: AKT,
RAD001 and CCI-779) are being developed as onco-
MAPK and p38 MAPK phosphorylate ER on key resi-
logical treatments11,93. Considering that ERBB recep-
dues that are involved in the induction of ligand-inde-
tors signal through the PI3K−AKT pathway, it is not
pendent activation by growth-factor receptors
surprising that mTOR activity can be influenced by
(reviewed in REF. 113). The observation that long-term
ERBB activation94,95. However, there is accumulating
oestrogen-deprived MCF7 cells (with increased sensi-
evidence that mTOR also signals independently
tivity to oestrogen) show upregulation of ERBB2
from these RTKs96,97. This indicates that targeting
REF. 114 indicates that ERBB-receptor signalling is also
mTOR in combination with anti-ERBB therapeutics
fundamental to the adaptation of cultured breast can-
might lead to more profound effects on tumour-cell
cer cells to low oestrogen levels, a situation that could
biology than could be achieved through individual
be said to mimic therapy with aromatase inhibitors.
targeting of the proteins (FIG. 5A). As mentioned
Based on the extensive crosstalk between the
above, loss of PTEN has been demonstrated to coun-
oestrogen−ER and ERBB signalling pathways, drug-
teract the antitumour action of gefitinib87,88. There is
combination approaches targeting both pathways would
also a clear association between PI3K−AKT activa-
seem to be a rational clinical strategy to improve the
tion and ERBB2 overexpression in breast cancer98
efficacy of endocrine therapies, as well as to potentially
and PTEN loss or activation of the PI3K−AKT path-
circumvent or delay the development of resistance.
way has been associated with a poorer response and
Indeed, NEOADJUVANT studies showed that primary breast
resistance to trastuzumab56,82,83,99. Bearing in mind
tumours derived from ER-positive patients exhibiting
that loss of PTEN or hyperactivation of AKT has
ERBB2-overexpression had an impeded antiprolifera-
been suggested to sensitize tumours to the effects of
tive response to endocrine therapy115, and tamoxifen
mTOR inhibition11,100,101, clinical investigation into
treatment resulted in increased ERBB2 activation in
combination treatment with mTOR and ERBB
tumours at relapse116. Moreover, although the use of a
inhibitors is warranted.
single prognostic factor should be viewed with caution in the heterogeneous setting of cancer117, in both the
Rationale for combining anti-oestrogens or aromatase
advanced and adjuvant setting, ER-positive patients
inhibitors with ERBB inhibitors.
with ERBB2-overexpressing breast tumours do seem to
interacts with oestrogen-responsive elements to stimu-
have a poorer clinical outcome to endocrine therapy118
late the transcription of target genes involved in cell-
(reviewed in REF. 108). Interestingly, in a neoadjuvant
cycle progression and survival, a process implicated in
study, advanced-disease patients with EGFR- or ERBB2-
the deregulated cell proliferation associated with breast
positive breast tumours responded well to the aromatase
cancer102. Although therapeutics that interfere with ER
inhibitor letrozole but poorly to the SERM tamoxifen119.
function, including the SERMs tamoxifen and fulves-
This observation indicates that oestrogen deprivation
trant (a partial ER agonist and a complete ER antagonist,
might be a more effective neoadjuvant therapy than
respectively) have significantly contributed to a reduc-
SERMs in this patient population, and is presumably
tion in breast cancer mortality, at best 50−60% of ER-
related to the different mode of action of aromatase
positive breast cancers respond to anti-oestrogen
inhibitors as opposed to SERMs. However, whether
therapy103 (reviewed in REF. 104). Consequently, several
oestrogen deprivation would be the preferred strategy
aromatase inhibitors that reduce oestrogen biosynthesis,
in this patient population awaits further clarification.
such as letrozole and anastrozole, have also been devel-
Preclinical efforts to examine combinations of
oped as part of a therapeutic strategy aimed at expand-
tamoxifen with the EGFR inhibitor gefitinib in vitro
A therapy that is given before the main treatment, which could
ing on the clinical success of anti-oestrogens (reviewed
in experimental tumour models have demonstrated the
be, for example, surgery.
in REF. 105).
potential for improved antitumour effects even in the
NATURE REVIEWS C ANCER
VOLUME 5 MAY 2005 349
2005 Nature Publishing Group
Figure 5 Combination strategies to potentiate cellular response and overcome resistance. A
Mammalian target of
rapamycin (mTOR) and ERBB inhibitors. An important mediator of the phosphatidylinositol 3-kinase (PI3K)−AKT pathway, with
respect to tumour-cell growth and proliferation, is mTOR. mTOR acts as a central sensor for nutrient/energy availability, and is
also modulated by the PI3K−AKT pathway. Although it has been shown that ERBB receptors signal through mTOR in cancer
cells94,95, there is accumulating evidence that mTOR can signal independently from these receptor tyrosine kinases (RTKs)96,97,
for example in tumour cells with low levels of PTEN. This indicates that targeting mTOR with a specific inhibitor such as RAD001
in combination with anti-ERBB therapeutics such as tyrosine-kinase inhibitors (TKIs) or antibodies (such as trastuzumab or
cetuximab), might have more profound antitumour activity than could be achieved through individual targeting of the proteins. B
Anti-oestrogens, aromatase inhibitors and ERBB inhibitors. Several levels of interaction between ERBB receptors and
oestrogen receptor (ER) have been documented. First (a
), oestradiol (E2) treatment transactivates ERBB receptors (mainly
EGFR and ERBB2 have been studied), very likely mediated through metalloproteinase activation leading to pro-ERBB ligand
cleavage and ectodomain shedding (a
). ERBB RTK-induced signalling to downstream effectors (b
), in particular AKT, mitogen-
activated protein kinase (MAPK) and p38 MAPK, leads to direct phosphorylation of ER on key residues involved in ligand-
independent activation of the steroid-hormone receptor. Based on this extensive crosstalk, drug combinations targeting both
pathways would seem to be a rational clinical strategy. These could include ERBB-targeted monoclonal antibodies (c
) or TKIs
), in combination with selective ER modifiers (SERMs; e
) such as tamoxifen, which acts as an antagonist in breast cancer
cells, or aromatase inhibitors, which lower the content of E2 available to the tumour cell.
background of tamoxifen resistance45,109. However, a
crosslinking drug cisplatin121. The activity of ERBB2-
study showing antagonism when using trastuzumab
targeted trastuzumab was also enhanced when
in combination with tamoxifen in the ERBB2-over-
combined with cisplatin122 or docetaxel123; the latter
expressing BT474 breast cell line120 indicates that
was registered with trastuzumab for cancer treat-
more experiments will be needed to determine the
ment. Mechanistic studies revealed that in the pres-
feasibility of this combination approach. But it should
ence of the ERBB2-targeted antibody, cancer cells
be noted that promising preliminary data, which indi-
treated with platinating agents showed a reduction
cate an increased objective response to the combina-
in unscheduled DNA synthesis, a sign of DNA
tion, are emerging from a Phase II clinical trial of
repair122,124. Therefore, downregulation of ERBB2
trastuzumab combined with letrozole in patients with
interferes with the ability of tumour cells to repair
ER- and ERBB2-positive advanced breast cancer.
DNA adducts, thereby causing tumour-cell death.
Moreover, Phase III clinical trials have been initiated
More recently, larger studies revealed synergistic
with trastuzumab or lapatinib TABLE 1 in combina-
interactions between trastuzumab and carboplatin,
tion with anastrozole, as well as a randomized Phase
4-hydroxycyclophosphamide, docetaxel or vinorel-
II study evaluating gefitinib with either fulvestrant or
bine in a panel of ERBB2-overexpressing breast
anastrozole (reviewed in REF. 108). Clearly, the results
cancer cells124. At present, the combination of trastu-
of these clinical studies, together with more in-depth
zumab, docetaxel and platinum salts is being ana-
preclinical analyses, will help to define the future of
lysed in clinical trials125. Preclinical studies with
this promising combination strategy.
EGFR-directed TKIs in combination with chemo-therapeutics looked promising126. Regrettably, Phase
Chemotherapy and ERBB inhibitors.
III trials evaluating the addition of the TKIs gefitinib
experiments it was shown that the antitumour effect
or erlotinib to chemotherapy as first-line therapy in
of EGFR-targeted monoclonal antibodies was
patients with metastatic NSCLC failed to show an
strengthened when combined with the DNA-
advantage in response rate, progression-free survival
MAY 2005 VOLUME 5
2005 Nature Publishing Group
or overall survival compared with standard treat-
important when attempting to evaluate the effects of
ment66,127. Elucidating why this combination approach
drug-combination strategies and for establishing
was unsuccessful might provide valuable information
effective criteria for patient selection.
pertinent to the design of drug-combination trials in
Turning to combination strategies, the importance
of IGF1R in maintaining strong activation of the PI3K−AKT pathway, and its potential to interfere with
Perspectives and future directions
ERBB-targeted inhibitors, indicates that it is logical to
Our increased understanding of the molecular,
consider combining anti-ERBB agents with an IGF1R
structural and biological characteristics of the ERBB
inhibitor129. More generally, the PI3K−AKT pathway
RTK family has been essential for the rational devel-
can be activated by many different mechanisms and
opment of ERBB-targeted inhibitors. As we discuss
these could also be targeted together with ERBB RTKs.
here, ectodomain-targeted antibodies and TKIs are
As discussed above, mTOR inhibitors are very appeal-
in clinical use and show efficacy. Nevertheless, one
ing. However, there are inhibitors targeting other
of the key goals for future work will be the develop-
kinases on the PI3K−AKT pathway (reviewed in REFS
ment of accurate predictors of response to ERBB-
130,131) that might also be effective in combination.
targeted therapies. Considering, for example, that
Furthermore, targeting the MAPK pathway (reviewed
only about one-third of the pre-selected group of
in REF. 132), specifically the RAF kinase (reviewed in
ERBB2-overexpressing breast cancer patients
REF. 133), would also seem to be appropriate.
respond to trastuzumab75, it becomes obvious that
Initially, the development of a TKI with a very spe-
other factors must be considered before choosing a
cific target was an important goal in the field.
patient for this treatment. These predictors should
Considering our increased understanding of how the
help in the design of better clinical trials for drug
tumour microenvironment impacts on the progres-
testing, thereby allowing the more rapid approval of
sion of an initially well-encapsulated tumour into
metastatic cancer, the concept of targeting several key
The recent discovery of kinase-domain mutations
kinases important in this progress has emerged. Based
in EGFR and ERBB2 and their impact on response
on the importance of tumour vasculature in the proc-
to ERBB-targeted therapeutics awaits further clini-
ess of cancer growth and spread, inhibitors that block
cal and basic research. It will be important to under-
endothelial-cell survival have gained in importance.
stand how the mutated receptors contribute to
Preclinical studies have demonstrated encouraging
tumour biology. Moreover, the intriguing results
combination effects with ERBB- and vascular
indicating that mutant EGFR couples to pro-survival
endothelial growth factor receptor (VEGFR)-directed
pathways more efficiently than the wild-type recep-
inhibitors in experimental animal tumour mod-
tor needs to be confirmed in patients. Finally, it will
els134,135. It will be very interesting to see how multi-
be essential to determine if kinase-domain muta-
targeted inhibitors such as AEE788 and EXEL7647
tions will be a useful tool for patient selection. In
TABLE 1 that block both ERBB and VEGFRs fare in
particular, are mutations in the ERBB2 kinase
the clinic in comparison to other ERBB inhibitors. In
domain predictive for clinical response? Considering
several preclinical models, AEE788 was as effective as
the proven molecular role of the ERBB2−ERBB3 het-
the combination of the ERBB inhibitor PKI166 and
erodimer in breast tumour cell lines that overexpress
the VEGFR inhibitor PTK787/ZK22584 REF. 136.
ERBB2 REF. 128, it will be interesting to see if acti-
Clearly, agents that have dual activity in one molecule
vating ERBB3 mutations are uncovered in tumours
present a ‘combination strategy in one', which could
that have low ERBB2 levels. The role of ERBB4 in
provide more flexibility in terms of the range of
cancer biology needs more study, one important rea-
potential tumour indications and facilitate advanced
son being to determine how blockade of ERBB4 by
multitargeted ERBB-kinase inhibitors impacts on
In the future, we are confident that by continuing
clinical response. In this context, it will be essential
the exchange of information between basic and clinical
to determine whether targeting multiple ERBB
studies we will uncover further factors that underlie
receptors will lead to unacceptable toxicity. Lastly,
clinical response to ERBB-targeted therapeutics. We
although there is evidence supporting the use of skin
also hope that the continued translation of knowledge
as a surrogate tissue to evaluate molecular responses
that is emerging from the field of signal transduction
to ERBB inhibitors62, the development of biomarker
will contribute not only to the development of novel
analyses to directly assess tumour response should
therapeutics, but also allow us to optimally use those
be given high priority. This will become especially
already in the clinic.
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Network Spinal Analysis Care - Literature Review The following is a list of peer-reviewed publications involving Network Spinal Analysis Care. Further information regarding Network Spinal Analysis Research currently in process or programs where information on Network Spinal Analysis Research has been presented is available at On a standing wave Central Pattern Generator and the coherence problem Jonckheere E, Lohsoonthorn P, Musuvathy S, Mahajan V, Stefanovic M. Biomedical Signal Processing and Control 5 (2010) 336–347. doi:10.1016/j.bspc.2010.04.002 An electrophysiological phenomenon running up and down the spine, elicited by light pressure contact at very precise points and thereafter taking the external appearance of an undulatory motion of the spine, is analyzed from its standing wave, coherence, and synchronization-at-a-distance properties. This standing spinal wave can be elicited in both normal and quadriplegic subjects, which demonstrates that the neuronal circuitry is embedded in the spine. The latter, along with the inherent rhythmicity of the motion, its wave properties, and the absence of external sensory input once the phenomenon is elicited reveal a Central Pattern Generator (CPG). The major investigative tool is surface electromyographic (sEMG) wavelet signal analysis at various points along the paraspinal muscles. Statistical correlation among the various points is used to establish the standing wave phenomenon on a specific subband of the Daubechies wavelet decomposition of the sEMG signals. More precisely, ∼10 Hz coherent bursts reveal synchronization between sensory-motor loops at a distance larger, and a frequency slower, than those already reported. As a potential therapeutic application, it is shown that partial recovery from spinal cord injury can be assessed by the correlation between the sEMG signals on both sides of the injury. Reorganizational Healing: A Paradigm for the Advancement of Wellness, Behavior Change, Holistic Practice, and Healing Epstein DM, Senzon SA, Lemberger D. Journal of Alternative and Complimentary Medicine. May 2009;15(5):461-64. PMID: 19450165 Reorganizational Healing, (ROH), is an emerging wellness, growth and behavioral change paradigm. Through its three central elements (the Four Seasons of Wellbeing, the Triad of Change, and the Five Energetic Intelligences) Reorganizational Healing takes an approach to help create a map for individuals to self-assess and draw on strengths to create sustainable change. Reorganizational Healing gives individuals concrete tools to explore and use the meanings of their symptoms, problems, and life-stressors as catalysts to taking new and sustained action to create a more fulfilling and resilient life. Editorial: Reorganizational Healing: A Health Change Model Whose Time Has Come Blanks RH. Journal of Alternative and Complimentary Medicine. May 2009;15(5):461-64. PMID: 19450161 No Abstract Available. Letter to the Editor: Network Spinal Analysis Jonckheere EA. Journal of Alternative and Complimentary Medicine. May 2009;15(5):469-70. PMID: 19450163 No Abstract Available.
Government of Nepal Ministry of Physical Planning and Works Department of Roads Environmental & Social Management Framework A guide to the environmental and social issues associated with new road construction and upgrading (Final Version) April, 2007 ANNEXES Annex 1 The Consultant's Terms of Reference for Preparing the ESMF