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.
Most often, when a man first has problems with lowered potency or with sexual dysfunction cialis australia shipping to pharmacies, paying purveyors, training personnel, or having a huge advertising budget.
Nrc0505-hynes.inddERBB 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- *Friedrich Miescher
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 Research, Maulbeerstrasse
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 NATURE REVIEWS C ANCER VOLUME 5 MAY 2005 341
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 of ERBB2 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 GPROTEINCOUPLED 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 single entity.
tyrosine-kinase receptor to be linked directly to dimerization loop in domain II is exposed30,31. This 342 MAY 2005 VOLUME 5
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
Overexpressed ERBB2 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 a 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 Schematic
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, receptor classes.
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 NATURE REVIEWS C ANCER VOLUME 5 MAY 2005 343
2005 Nature Publishing Group
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 hair145–147.
These observations help explain the most common side effects associated with EGFR inhibition in the clinic: rash
and acneic skin reactions (including folliculitis) and diarrhoea58,148. Indeed, cutaneous skin rash has been proposed
as a surrogate marker of clinical benefit for many EGFR-targeted agents149. Although infrequent (1% globally150),
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
at risk150,151. 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 heart153. 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 cardiomyopathy154. 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 anthracyclines155. Considering that ERBB2-null cardiomyocytes showed an increased sensitivity
to adriamycin-induced toxicity154, 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 cardiomyocytes158 and NRG1 promotes survival of isolated cardiomyocytes159, 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 receptor160 and A431-EGFR-
protein kinase C (PKC) and SRC (reviewed in REF. 46), overexpressing cancer cells161. Specific antibodies were detected by 125I-EGF-binding
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 colleagues160, 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 receptor162. 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 colleagues68,
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) variant163 — 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 multifaceted51.
in the cytoplasmic domain of ADAMs, thereby influ- Mutation of the kinase domain of EGFR blocks its biological activity164, 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 therapy165. Levitzki and colleagues did some of the pioneering work in designing EGFR
TKIs, which they named tyrphostins166. 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 development165 TABLE 1.
be interesting to explore the effects of SRC inhibition 344 MAY 2005 VOLUME 5
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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 Pro-survival genes kinase (CDK) inhibitor p27 (also known as KIP1); changes in STAT3 activity; and a decreased proliferation index, associated in some cases with increased apopto- stimulating genes 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 setting. 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 cells. b 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 j 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 analyses 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 NATURE REVIEWS C ANCER VOLUME 5 MAY 2005 345
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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. As dis-
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 The
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) and
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 The ERBB2-directed
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 II, respectively31,77. BCR−ABL kinase domain74.
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Table 1 ERBB-targeted therapeutics in clinical use
Status and comments
Trastuzumab (Herceptin) Approved for the treatment of ERBB2- overexpressing breast cancer; ongoing trials for use in combination with various other drugs Pertuzumab (Omnitarg) 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 Cetuximab (Erbitux) ImClone/Merck KgaA Approved for the treatment of CRC; ongoing trials Bristol-Myers Squibb 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 Gefitinib (Iressa) Approved for the treatment of NSCLC after failure on other available treatments; ongoing trials in HNSCC, gastrointestinal cancer and breast cancer Erlotinib (Tarceva) 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 Irreversible TKI Phase II trials underway in breast and NSCLC Irreversible TKI 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 trastuzumab? 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 NATURE REVIEWS C ANCER VOLUME 5 MAY 2005 347
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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-
peutics. 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 Resistance of
tor of PI3K, have been found to be important causes of tumour cells to trastuzumab through ligand-induced activation of ERBB2 dimers. Binding 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 PIK3CA91 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. 348 MAY 2005 VOLUME 5
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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. itors. 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- widely accepted.
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. Oestrogen-bound ER
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 and 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
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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
(d), 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. In preclinical
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 350 MAY 2005 VOLUME 5
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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 novel therapeutics.
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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