Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials.
ABSTRACT The epidermal growth factor receptor (EGFR) is a growth factor receptor that induces cell differentiation and proliferation upon activation through the binding of one of its ligands. The receptor is located at the cell surface, where the binding of a ligand activates a tyrosine kinase in the intracellular region of the receptor. This tyrosine kinase phosphorylates a number of intracellular substrates that activates pathways leading to cell growth, DNA synthesis and the expression of oncogenes such as fos and jun. EGFR is thought to be involved the development of cancer, as the EGFR gene is often amplified, and/or mutated in cancer cells. In this review we will focus on: (I) the structure and function of EGFR, (II) implications of receptor/ligand coexpression and EGFR mutations or overexpression, (III) its effect on cancer cells, (IV) the development of the malignant phenotype and (V) the clinical aspects of therapeutic targeting of EGFR.
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ABSTRACT: Absolute quantification of protein expression and posttranslational modifications by mass spectrometry has been challenging due to a variety of factors, including the potentially large dynamic range of phosphorylation response. To address these issues, we have developed MARQUIS-Multiplex Absolute Regressed Quantification with Internal Standards-a novel mass spectrometry-based approach using a combination of isobaric tags and heavy-labelled standard peptides, to construct internal standard curves for peptides derived from key nodes in signal transduction networks. We applied MARQUIS to quantify phosphorylation dynamics within the EGFR network at multiple time points following stimulation with several ligands, enabling a quantitative comparison of EGFR phosphorylation sites and demonstrating that receptor phosphorylation is qualitatively similar but quantitatively distinct for each EGFR ligand tested. MARQUIS was also applied to quantify the effect of EGFR kinase inhibition on glioblastoma patient-derived xenografts. MARQUIS is a versatile method, broadly applicable and extendable to multiple mass spectrometric platforms.Nature Communications 01/2015; 6:5924. · 10.74 Impact Factor
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ABSTRACT: Abstract EGFR tyrosine kinase has been reported mainly in 40-80% of non-small lung cancers, in addition to colon and breast cancers. In this study, we illustrate the synthesis of a highly potent antitumor agent. The synthesized compound 4 was screened at NCI, USA, for antitumor activity against non-small lung cancer, colon cancer and breast cancer cell lines. Results indicated that this compound is more potent antitumor agent compared to erlotinib against all tested cell lines except breast cancer (MDA-MB-468) cell line. In addition, it was tested initially at a single dose concentration of 100 µM over 11 different kinases. At this concentration, 94.45% inhibition of the enzymatic activity of EGFR kinase was observed, while the inhibition in activity was below 55% in all other kinases. Compound 4 was further tested in a 10-dose IC50 mode and showed IC50 value of 0.239 µM for EGFR kinase. In vivo acute toxicity of this compound was also tested.Journal of Enzyme Inhibition and Medicinal Chemistry 03/2014; · 1.50 Impact Factor
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ABSTRACT: Preclinical studies demonstrate that epidermal growth factor receptor (EGFR) signals through both kinase-dependent and independent pathways and that combining a small-molecule EGFR inhibitor, EGFR antibody, and/or anti-angiogenic agent is synergistic. We conducted a dose-escalation, phase I study combining erlotinib, cetuximab, and bevacizumab. The subset of patients with metastatic colorectal cancer was analyzed for safety and antitumor activity. Forty-one patients with heavily pretreated metastatic colorectal cancer received treatment on a range of dose levels. The most common treatment-related grade ≥2 adverse events were rash (68%), hypomagnesemia (37%), and fatigue (15%). Thirty of 34 patients (88%) treated at the full FDA-approved doses of all three drugs tolerated treatment without drug-related dose-limiting effects. Eleven patients (27%) achieved stable disease (SD) ≥6 months and three (7%) achieved a partial response (PR) (total SD>6 months/PR= 14 (34%)). Of the 14 patients with SD≥6 months/PR, eight (57%) had received prior sequential bevacizumab and cetuximab, two (5%) had received bevacizumab and cetuximab concurrently, and four (29%) had received prior bevacizumab but not cetuximab or erlotinib (though three had received prior panitumumab). The combination of bevacizumab, cetuximab, and erlotinib was well tolerated and demonstrated antitumor activity in heavily pretreated patients with metastatic colorectal cancer.Oncoscience. 01/2014; 1(8):540-9.
Annals of Oncology 8: 1197-1206,1997.
© 1997 Kluwer Academic Publishers. Printed in the Netherlands.
Epidermal growth factor receptor (EGFR) and EGFR mutations, function
and possible role in clinical trials
B. Rude Voldborg,1 L. Damstrup,1 M. Spang-Thomsen2 & H. Skovgaard Poulsen1
^Section for Radiation Biology, The Finsen Centre, Rigshospitalel, 2 Tumourpathological Laboratory, Institute of Molecular Pathology, University of
Copenhagen, Copenhagen, Denmark
The epidermal growth factor receptor (EGFR) is a growth
factor receptor that induces cell differentiation and prolifera-
tion upon activation through the binding of one of its ligands.
The receptor is located at the cell surface, where the binding of
a ligand activates a tyrosine kinase in the intracellular region
of the receptor. This tyrosine kinase phosphorylates a number
of intracellular substrates that activates pathways leading to
cell growth, DNA synthesis and the expression of oncogenes
such as^os andyun.
EGFR is thought to be involved the development of cancer,
as the EGFR gene is often amplified, and/or mutated in
In this review we will focus on: (I) the structure and
function of EGFR, (II) implications of receptor/ligand coex-
pression and EGFR mutations or overexpression, (III) its
effect on cancer cells, (IV) the development of the malignant
phenotype and (V) the clinical aspects of therapeutic targeting
Key words: cancer, epidermal growth factor receptor, signal-
ling, tyrosine kinase
Abbreviations: AR - amphiregulin; bp - basepairs; BTC -
betaceOulin; cAMP - cyclic adenosine monophosphate; CDK
- cyclin dependent kinase; DAPH - dianilinophtalimides;
E-cadherin - epithelial cadherin; EGF - epidermal growth
factor; EGFR - epidermal growth factor receptor; GAP -
GTPase-activating protein; HB-EGF - Heparin-bmding
EGF-like growth factor; MAPK - mitogen-activated protein
kinase; PKC - protein kinase C; PLC-y - phospholipase C-y;
RB - retinoblastoma protein; RT-PCR - reverse transcriptase
polymerase chain reaction; She - sre homology and collagen
protein; S-oligos - phosphothiorate oligos; TGF-a - trans-
forming growth factor-a; wtEGFR - wildtype EGFR
Growth factors belongs to a family of polypeptides
which have been shown to stimulate proliferation and/
or differentiation in both normal and malignant cells.
One of the first growth factors discovered was epidermal
growth factor (EGF) . Later studies have shown that
this protein binds to a cell surface growth factor recep-
tor, epidermal growth factor receptor (EGFR). Through
binding to the receptor, EGF either induces cell pro-
liferation or differentiation in mammalian cells .
The binding of a ligand to the EGFR, induces con-
formational changes within the receptor which increases
the catalytic activity of its intrinsic tyrosine kinase,
resulting in autophosphorylation which is necessary for
the biological activity [3,4].
The activated EGFR kinase phosphorylates tyrosine
residues on a number of cellular substrates including
phospholipase C-y, (PLC-y), mitogen-activated protein
kinase (MAPK) and the ras GTPase-activating protein
(GAP) [5,6], which leads to an increase in catalytical
The activated receptor/ligand complex is endocytosed
and either degraded within the lysosomes , or recycled
to the plasma-membrane . Endocytosis and degra-
dation induces down-regulation of the growth factor
Thus, the activity of EGFR is normally under pos-
itive, as well as negative regulation, through regulatory
mechanisms and feed-back information.
Structure of EGFR
The EGFR consists of a single polypeptide chain of
1186 aminoacids, Mr 170 kDaltons (kDa) , and is
expressed on the surface of the majority of normal cells.
The receptor consists of three regions, the extracellular
ligand binding region, the intracellular region with tyro-
sine kinase activity and a transmembrane region with a
single hydrophobic anchor sequence, by which the recep-
tor traverses the cell membrane a single time (Figure 1).
The extracellular aminoterminal end can be divided
into four domains with domain III responsible for ligand
binding. The ligands binding to EGFR are, besides
EGF, transforming growth factor-a (TGF-a) , am-
phiregulin (AR) , Heparin-binding EGF-like growth
factor (HB-EGF) , and betacellulin (BTC) .
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Domain ID, (ligand binding domain)
Domain V (transmembrane domain)
Tyrosine kinase domain
_] Ca +regulatory/internalisation domain
Figure 1. Schematic structure of EGFR, with the extra- and intra-
The cytoplasmic carboxy-terminal region of the
EGFR is the region responsible for the tyrosine kinase
activity and carboxyterminal regulatory functions. Just
inside the cell membrane the juxtamembrane region is
followed by the protein tyrosine kinase and autophos-
phorylation domains. The protein tyrosine kinase activ-
ity plays a key role in the regulation of cell proliferation
EGFR deletion mutations
A large number of deletions of the EGFR mRNA has
been observed in a number of neoplasia, first in glio-
blastoma , but recently also in non-small-cell lung
carcinomas , breast cancer , paediatric gliomas,
medulloblastomas, and ovarian carcinomas .
These deletions are found both in the part of the
mRNA that encodes the extracellular region of EGFR
and in the part that encodes the intracellular region of
the EGFR. A large number of these deletions are the
result of genomic rearrangements, resulting in alterna-
tive splicing of the mRNA .
No deletion mutants have been found in the trans-
membrane or the tyrosine kinase domains. The removal
of the transmembrane domain would make it impossible
for the receptor to be positioned across the membrane,
which would abolish the interaction with the cell mem-
brane associated substrates for the tyrosine kinase.
The loss of the tyrosine kinase domain would com-
pletely abolish the function of EGFR, and therefore not
lead to ligand-induced signal transduction, even if growth
factors were available. Thus, it is in the ligand binding
and the regulatory domains where deletions seems to
have an activating effect on signal transduction.
Deletion mutations in the extracellular domain
Three different deletions of the extracellular domain of
EGFR have been observed, type I, II and III (EGFRvI,
II and III).
EGFRvI is total deletion of the extracellular domain
and resembles the v-erb-B oncoprotein [19, 20]. It is
constitutivally active, and cannot be regulated by EGF
[21, 22]. This deletion has only been observed in a single
tumour cell line, a xenograft derived from a malignant
human glioma .
EGFRvII, found in gliomas with amplified rearranged
EGFR genes , contains a deletion of 83 aminoacids
in domain IV of the extracellular domain. The 83 amino-
acids deleted represents only 7% of the total polypeptide
backbone mass of the EGFR. The deleted region is part
of the cysteine-rich domain IV, lying between the ligand
binding domain III and the transmembrane domain V.
The EGFRvII is capable of transducing EGF stimula-
tion of cell proliferation and invasion in vitro , and it
responds very similar to the wildtype EGFR (wtEGFR)
to growth factors . EGFRvII does not seem to have
any influence on the malignant phenotype of the glio-
blastoma, that might rather be the result of the EGFR
gene amplification. Due to the structural rigidity of
domain IV by disulphide bonds the conformation of
this domain might not be affected by a small deletion,
thereby leaving the ligand binding domain intact.
The best described and the most common of the three
mutants found in human cancer is EGFRvIII. This
mutation is the result of intragene rearrangements that
result in overexpression of transcripts lacking exons 2-7,
which represents 801 basepairs (bp). In some cases this
mutant does not arise from gene rearrangement, but
rather from alternative splicing of the mRNA. The
receptor lack aminoacids 6-273, which constitutes a
large portion of the extracellular domain. The alterna-
tive splicing results in the insertion of a glycine residue
at the deletion point, thereby replacing aminoacids
6-273, without altering the reading frame. The truncated
EGFRvIII lacks domain I and II of the extracellular
domain [14, 18, 24, 25]. The rearranged EGFRvIII gene
is often amplified, thus resulting in tumour cells over-
expressing the EGFRvIII [16,19]. The overexpression of
EGFRvIII does not exclude a possible overexpression of
wtEGFR. This situation might occur when only one
allele of the gene is rearranged, but both alleles are
amplified, or when EGFRvIII arises from alternative
splicing. EGFRvIII has been found in more than 50% of
high and low grade gliomas , in 5 of 32 lung carcino-
mas , in 21 of 27 breast carcinomas [16, 17], in 4 of 6
paediatric gliomas, in 6 of 7 medulloblastomas, and in
24 of 32 ovarian carcinomas  (table 1).
Most of the studies concerning EGFRvIII expression
in carcinomas have been performed using antibodies,
only to determine whether or not the mutant was ex-
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pressed. These studies do not determine the genetic
origin of the mutant, i.e., gene-rearrangement or alter-
native splicing, nor do they determine the influence of
the EGFRvIII on the maligne phenotype of the cancer.
EGFRvIII is not capable of ligand binding as the
deletion destroys the ligand-binding site and has a con-
stitutively activated tyrosine kinase similar to EGFRvI
[27, 28]. The EGFRvIII stimulates cell proliferation
independently of ligand interaction  and enhances
the tumourigenicity of transfected human glioma cells in
nude mice .
The tyrosine kinase of EGFRvIII is much less auto-
phosphorylated when compared to wtEGFR . The
level of the constitutive activation of EGFRvIII is there-
fore lower than the activation level seen in ligand-acti-
vated wtEGFR. It has been shown that EGFRvIII is not
internalised , which couples changes in the extracel-
lular domain with changes in the internalisation domain
in the intracellular region. It appears that the conforma-
tion of the intracellular region is different in EGFRvIII
when compared to ligand activated wtEGFR.
Thus, the mitogenic activity of EGFRvIII might be
the result of the overrepresentation of the receptor at
the cell surface rather than its constitutive active tyro-
sine kinase. Persistence of EGFRvIII at the cell surface
prolongs and enhances its low level of activity.
Many studies have focused on the use of this mutant
as a target for tumour specific antibodies. The inserted
glycine residue at the splice site creates a new epitope,
which is specific for EGFRvIII [26, 30]. Targeting this
new epitope would circumvent problems occurring if
wtEGFR antibodies were to be used, as most cells
express wtEGFR. In addition, EGFRvIII is specifically
found in malignancies and has not been found in normal
The current development of specific antibodies against
EGFRvIII [16, 31] might result in a powerful therapy
tool for highly specific and efficient delivery of anti-
bodies coupled with radioactive isotopes, gene vectors
or cytotoxins directly to the tumour cells.
Transfection studies using a SV40 based expression
vector carrying the EGFRvIII cDNA in Chinese hamster
ovary cells, revealed an altered subcellular location of
EGFRvIII, when compared to wtEGFR . EGFRvIII
was found primarily in the endoplasmatic reticulum,
whereas wtEGFR was expressed on the cell surface.
This intracellular localisation has not been observed in
human tumours, frozen sections of xenografts or other
EGFRvIII transfected cell lines and might be restricted
to these particular transfected cells.
Mutations in the cytoplasmic domain
Mutations in the cytoplasmic domain, have been inves-
tigated to a lesser extend than the extracellular deletions.
A study of eight glioblastomas expressing transcripts
that did not encode large C-terminal, intracellular por-
tions of the receptor, revealed that all the deletions were
located in the intracellular inhibitory and Ca2+ regula-
tory/internalisation domains of the EGFR . No
deletions were found in the tyrosine kinase domain. All
deletions started at the same point, but the size of the
deletions varied from 254 bases to a premature termina-
tion of the transcript, resulting in the truncation of the
Whether or not these transcripts are translated into
active receptors remain to be established. The lack of
regulatory and inhibitory domains could easily result
in the generation of constitutively active receptors. A
EGFR that is not internalised would very quickly be
overexpressed on the cell surface, without the ability to
be down-regulated. The loss of inhibitory domains
would leave the substrate binding sites of the tyrosine
kinase available for interaction with its substrates, even
in an non-ligand-bound state.
Amongst the ligands that bind to the EGFR, a general-
ised motif containing six conserved cysteines is found,
which via disulphide bonds creates three peptide loops
. This similar folding of the mature peptides ensures
a common conformational structure.
AR, HB-EGF and BTC have, in addition to the
conserved motif, extended N-termini that confers fur-
ther specificity for target cells. AR and HB-EGF both
possess a highly basic N-terminus that enables these
growth factors to bind to heparin or heparan sulphate
proteoglycans expressed on the cell surface [34, 35]. This
can increase the activating potential of AR and HB-EGF
by localising the growth factors on the cell surface, close
to the EGFR.
All the ligands are synthesised as large membrane-
bound, glycosylated precursors which, at least in the
case of EGF and TGF-a, have been shown to possess
biological activity , suggesting that the growth fac-
tors might be able to activate EGFR via an auto- or
juxtacrine mechanism, while they still remain bound to
Coexpression of EGFR and one or more of its
ligands might result in an autocrine loop, resulting in
a constant activation of the EGFR tyrosine kinase
domain, leading to uncontrolled growth.
Ligand binding to the extracellular domain causes allos-
teric changes in the intracellular part of the receptor
resulting in the activation of the intracellular tyrosine
kinase. The autophosphorylation of the C-terminal end
removes an alternate substrate/competitive inhibitor
conformation, permitting access of cellular substrates
to the tyrosine kinase domain.
EGFR is activated by a three step mechanism. The
by guest on July 14, 2011
binding of any of the specific ligands to the receptor
induces dimerisation of the ligand-binding receptors.
The EGFR may dimerise with another EGFR, or it can
form heterodimers with other members of the EGFR
receptor family . The dimerisation results in the
autophosphorylation of five specific tyrosine (Tyr) resi-
dues (Tyr 1173, 1148, 1086, 1068, and 992) in the car-
boxy-terminal end of the intracellular part of EGFR,
with Tyr-1173 as the major autophosphorylation site .
The autophosphorylation of these five tyrosines re-
sults in the formation of binding sites for the substrates
of the tyrosine kinase needed for signal transduction.
Generally receptor motifs containing phosphotyrosyl
residues are recognised by intracellular proteins con-
taining src homology 2 motifs, (SH2). This recognition
is an important part of the signal transduction pathway.
SH2-containing signal proteins that directly or indirectly
interacts with the autophosphorylated EGFR include
enzymes such as PLC-yl, GAP and the syp phospho-
tyrosine phophatase, as well as non-enzymatic adapter
molecules such as the p85 subunit of phosphatidylinosi-
tol 3-kinase, the src homology and collagen (She) pro-
tein, Grb-2  and Nek .
Mutational analysis have shown that the removal of
the autophosphorylation sites has a severe effect on
substrate binding if all five sites are removed . How-
ever, if only one site is altered, the other autophos-
phorylation sites appear to be able to compensate for
the loss of one site. A progressive reduction in EGFR
affinity for PLC-yl is recorded with the loss of Tyr-1173
(27%), Tyr-1173 and Tyr-1148 (65%), and Tyr-1173, Tyr-
1148 and Tyr-1068 (82%) .
The recognition by PLC-yl of the tyrosine kinase
domain of the EGFR seems to be independent of the
recognised autophosphorylation site, which explains the
gradual loss of EGFR affinity for PLC-yl.
In the case of EGFRvIII, which is poorly autophos-
phorylated, the mutation of a single autophosphoryla-
tion site abolishes tyrosine kinase activity .
The down-regulation of EGFR is partly accomplished
by internalisation of the activated EGFR, followed by
degradation in the lysosomes, and partly by the de-
sensitisation induced by phosphorylation of serine and
threonine residues in the intracellular domain [39-41].
EGFR's are normally diffusely distributed on the sur-
face of the cell. Upon ligand binding they cluster in
coated pits and are endocytosed in vesicles that ulti-
mately fuses with lysosomes . Both the receptor and
the ligand are then degraded in the lysosomes. A domain
in the regulatory C-terminal end of the EGFR (from
aminoacids 957 to 1022), has been shown to be required
for endocytosis, and the deletion of the entire tyrosine
kinase domain has no influence on the endocytosis of
the EGFR [8, 43]. A kinase deficient receptor generated
by introducing point mutations eliminates ligand-induced
endocytosis . These data show that internalisation is
dependent on tyrosine kinase activity in the EGFR, at
least if the tyrosine kinase domain is present. This might
suggest the occurrence of conformational changes in the
intracellular domain upon ligand-binding, involving
both the tyrosine kinase domain and the regulatory
C-terminus. These conformational changes would then
expose sequences in the C-terminus that dictate interac-
tion with coated pits and subsequent internalisation. It is
likely that the lack of internalisation seen in EGFRvIII
is caused by the inability to adapt to the endocytotic
The phosphorylation of serine and threonine residues
has been shown to desensitise the EGFR. Desensitisa-
tion refers to a reduced ability of EGFR mediated signal
transduction, despite an unchanged number of receptors
at the cell surface. The phosphorylation of threonine 654
and serines 1002, 1046 and 1047 has been associated
with a decrease in the ability of EGF to stimulate
receptor dimerisation, tyrosine kinase activity, phospha-
tidylinositol turnover and receptor internalisation .
EGFR mediated signal transduction
Tyrosine phosphorylation is a key element in the signal
transduction mediated by EGFR. The stimulation of
PLC-y by the EGFR mediated tyrosine phosphorylation
causes the release of Ca2+ from intracellular compart-
ments and the generation of diacylglycerol, the activator
of protein kinase C (PKC) . PKC is a serine/threo-
nine kinase  that possibly is responsible for the phos-
phorylation of the serine/threonine residues involved in
the desensitisation of EGFR.
Another protein that is activated by the EGFR tyro-
sine kinase domain is Ras, which leads to DNA syn-
thesis and cell proliferation, through a pathway leading
from the cell surface to the nucleus [48, 49]. This path-
way involves a large number of protein factors besides
Ras, including Raf, MAPK , cytosolic kinases and
nuclear transcription factors .
The SH2 adaptor protein Grb-2 recruits the Ras
GDP/GTP exchange factor, Sos, to the plasma mem-
brane upon binding to activated EGFR. This activates
the MAP kinase pathway, one of the most important
membrane-to-nucleus signalling pathways in eukaryotes
. As constitutive activation of MAP kinase-mediated
mitogenic signalling pathways elicits transformation
, the constitutive signalling by EGFR/EGFRvIII
overexpression may have a significant influence on the
acquirement of the maligne phenotype.
EGFR tyrosine kinase is also involved in the pro-
gression of cells through G] phase and into S phase.
This progression is mediated by a family of protein
kinases, the cyclin dependent kinases, (CDK) and their
corresponding activating partners, the cyclins . Pro-
gression through Gi phase requires activation of the
various cyclin-CDK kinase complexes. One of the crit-
ical substrates of Gi CDKs is the retinoblastoma pro-
tein, (RB), whose phosphorylation and subsequent re-
lease of RB-bound transcription factors are required for
by guest on July 14, 2011
Gi to S phase transition. The blocking of EGFR ligand
binding by a monoclonal antibody has been shown to
reduce Gi phase CDK activities, causing G] cell cycle
The identification of the substrates for the EGFR
tyrosine kinase is far from complete. Transfection studies
with the cell adhesion molecule epithelial cadherin
(E-cadherin), has led to the hypothesis that the tyrosine
phosphorylation of a E-cadherin associated protein,
P-catenin, might lead to the loss of cell-cell adhesion,
resulting in a more metastatic phenotype . p-Catenin
might be a substrate for EGFR tyrosine kinase, thereby
connecting EGFR activity and metastatic potential.
The level of E-cadherin has influence on the EGFR
level in the cell. Ca2+ mediated down-regulation of
E-cadherin expression resulted in a strong up-regulation
of EGFR in keratinocytes, whereas E-cadherin trans-
fection reversed this effect .
It has been shown that internalised activated EGFRs
are still autophosphorylated and catalytically active .
If the internalised EGFR continues to trigger signal
transduction pathways after internalisation, the proteins
interacting with the EGFR are likely to associate with
the internalised EGFRs. This implies that the signalling
role of EGFR continues after internalisation, and is
only down-regulated when the receptor is degraded in
the lysosomes. The prolonged activation period would
enhance the signal transduced into the cell by the acti-
EGFR and its role in the development of the malignant
In a large number of tumours EGFR status is altered, due
to overexpression and/or mutations. Amplified EGFR
signalling might induce uncontrolled cell growth and
a malignant phenotype. Apart from EGFR mutations,
overexpression of EGFR or its ligands, or coexpression
of ligands and receptor might lead to an abnormal
EGFR mediated signal transduction.
Gene amplification of the EGFR gene has been
observed in a number of different tumours, and found
to be present in approx. 40% of glioblastoma multi-
forme . In an fluorescence in situ hybridisation
(FISH) assay, 18 out of 29 grade 3 and 4 gliomas
displayed EGFR gene amplification . Overexpres-
sion of EGFR were also frequently observed in breast,
bladder, cervix, kidney, and ovarian tumours , as
well as in lung cancer and various squamous carcino-
Treatment with EGF of a oesophageal cancer cell line
expressing E-cadherin and EGFR induced changes in
the cellular morphology and phosphorylation of P-
In some colon cancer cell lines treatment with EGF,
or TGF-a, caused a reduction of E-cadherin, and an
increase of a2-integrin, carcinoembryonic antigen (CEA)
and CD44 . These changes might increase the meta-
static potential of the cells. Hypothetically, the reduc-
tion in E-cadherin level leads to decreased cell-cell
adhesion enabling initial detachment of cells from the
primary tumour. The increased integrin and CEA ex-
pression might enhance attachment and spreading of
cells through the extracellular matrix, while increased
expression of CD44 could enable attachment of tumour
cells to endothelial cells and facilitate access to the
Possible roles of EGFR in research and cancer
The EGFR has been used as a prognostic marker for a
number of years, as the overexpression of EGFR was
correlated to a poor prognosis in a number of cancer
forms, including breast cancer , gliomas , squa-
mous carcinoma  and laryngeal cancer . In other
cases, e.g., non-small-cell lung cancer, there is contro-
versy whether or not EGFR overexpression can be used
as a prognostic marker [67-71].
More recently the EGFR has been studied intensively
as a target for monoclonal antibodies. The overexpres-
sion of EGFR in many tumours compared to normal
tissue, makes it possible to use EGFR as a target for the
delivery of cytotoxins or radioactive isotopes preferen-
tially to the tumour cells, or to use EGFR as a target for
A number of studies have been published using
EGFR specific antibodies to modulate cell growth on
cell lines of various cancer forms. A EGFR-blocking
monoclonal antibody has been shown to up-regulate
p27Km and to inhibit proliferation by arresting cell
cycle progression in Gj, when administered to a pro-
static cancer cell line .
The use of EGFR-blocking antibodies has been inves-
tigated using xenografts of a squamous cell carcinoma
cell line overexpressing EGFR . The blocking anti-
bodies were found uniformly localised on tumour mem-
branes, and induced almost complete regression. In vitro
studies using the same cell line and antibodies revealed
that the treatment with EGFR blocking antibodies in-
duced terminal differentiation.
Another way to inhibit EGFR activity is to prevent
translation of EGFR mRNA by the use of antisense
oligonucleotides. These antisense oligonucleotides can
be synthesised by a oligosynthesiser as phosphothiorate
oligos (S-oligos), and administered to the cells in solu-
tion, or the antisense sequence can be antisense mRNA,
obtained by transfecting the antisense EGFR sequence
into the relevant cell lines. The use of S-oligos specific to
the EGFR ligands AR and TGFa, has been used in
combination with EGFR-blocking antibodies to inhibit
growth of a human colon cancer cell line which coex-
presses both EGFR and its ligands . These experi-
ments showed an additive inhibitory effect when using
the combination of blocking antibodies and antisense
by guest on July 14, 2011
The stable transfection of EGFR specific antisense
mRNA constructs into a human epidermoid carcinoma
cell line that overexpresses EGFR, considerably reduced
the level of EGFR expressed in the cells and restored
serum dependent growth .
Another way to target EGFR overexpressing cells is
to use the ligands as carriers of e.g. cytotoxins. A fusion
protein consisting of TGFa and Pseudonomas exotoxin
have been examined for cytotoxic effects on normal and
tumourigenic breast cancer cells both in vitro and in vivo
. The toxin inhibited cell growth in vitro, as well as
xenografts of breast cancer cell lines expressing EGFR
in nude mice. Cell lines that did not express EGFR were
unaffected by the toxin both in vitro and in vivo. This
suggests that the use of cytotoxic fusion proteins might
be a possible way to inhibit tumour growth of EGFR
EGFR function can be suppressed by construction
of dominant negative receptors, that inhibit EGFR
signalling by heterodimerisation [77-79]. Introduction
of dominant negative receptors through gene therapy
might prove to be a powerful tool in the inhibition of
EGFR overexpressing tumours. The transfer of the Neu
ectodomain to EGFR expressing human glioma cells
inhibits the transformed phenotype and can revert cell
growth and proliferation to a quiescent normal level
. A Neu ectodomain form (N691stop) leads to in-
hibition of EGF-induced DNA synthesis, less efficient
EGF-induced internalisation and down-regulation of
EGFR activity, thereby reducing the oncogenic poten-
tial of EGFR-N691stop coexpressing fibroblasts .
The Neu ectodomain has a high affinity for the EGFR,
which makes it a viable biologic construct for gene
therapy of human glioblastoma. A similar Neu mutant,
T691stop is able to form heterodimers with EGFRvIII,
and might be a way to revert the mitogenic potential of
this EGFR mutant .
As the EGFR tyrosine kinase is necessary for signal
transduction it is a good target for therapy, using tyro-
sine kinase inhibitors . A number of studies have
investigated the effect of tyrosine kinase inhibitors on
cancer cell lines, in vitro [82-86] and in vivo [87-89].
Two classes of tyrosine kinase inhibitors are the tyr-
phostins [90-92] and the dianilinophtalimides (DAPH)
[93, 94]. The use of a tyrphostin, RG-13022 has been
shown to inhibit TGF-a-induced growth and EGFR-
phosphorylation in vitro . DAPH has been shown to
inhibit EGFR tyrosine kinase activity in vitro, and oral
administration of a DAPH, CGP 54211 selectively in-
hibited the level of EGFR phosphorylation in a im-
planted human cell line in nude mice. As a consequence
of the inhibited EGFR activity necrosis and inhibition
of tumour growth was observed . Therefore tyrosine
kinase inhibitors might be a powerful therapeutic tool
against EGFR overexpressing cancers.
EGFR blocking antibodies, and radiolabelled EGF
or antibodies have been tested in a number of phase I
trials in patients with squamous lung carcinoma [73, 95]
and gliomas [72, 96, 97]. These studies have shown that
the antibodies bind specifically to the tumour and that
EGFR blocking antibodies can be administered safely
to patients having tumours overexpressing EGFR. The
doses administered were sufficient to inhibit tumour
growth. A phase II study using 125I-labeled EGFR spe-
cific antibodies has been conducted on patients with
malignant astrocytoma, astrocytoma with anaplastic
foci, and glioblastoma multiforme . These studies
revealed encouraging results with a one-year survival of
60%, and a median survival of 15.6 months for patients
with glioblastoma multiforme, compared to a 50% death
rate within six months when the patients are treated by
The antiproliferative effect of anti-EGFR monoclo-
nal antibodies is enlarged by combination with other
agents such as the cyclic adenosine monophosphate
(cAMP) analogue 8-chloro-cAMP, which inhibits a
cAMP dependent serine-threonine kinase which is over-
expressed in many human cancers. This combination
treatment delayed tumour growth significantly in mice
when compared to anti-EGFR monoclonal antibody
treatment alone .
The generation of EGFRvIII specific monoclonal
antibodies have been used to detect EGFRvIII in glio-
blastomas . Using this approach, 8 of 11 tumours
previously found EGFRvIII negative using polyvalent
anti-EGFRvIII sera, were shown to be EGFRvIII pos-
itive. To be able to detect EGFRvIII in cancer cells,
biopsies or xenografts must be tested, as the mutation
tend to disappear when cells are cultured in vitro .
The reason for this phenomenon is not known.
Anti-EGFRvIII specific antibodies might prove to
be superior to anti-EGFR antibodies with respect to
antibody mediated treatment, as the EGFRvIII mutant
is expressed exclusively on tumour cells and not on
normal tissue. This could possibly reduce the toxicity
after administration of isotopes or cytotoxins, coupled
to specific antibodies.
Recently it has been shown that the tyrosine kinase
inhibitor tyrphostin AG 1478 preferentially inhibits tyro-
sine kinase activity in EGFRvIII transfected cell lines,
when compared to the same cell lines transfected with
wtEGFR . This might be used for specific targeting
EGFRvIII expressing cells in cancer therapy.
In a large number of tumours, EGFR is mutated or
overexpressed. The EGFR gene is often amplified and
deletion mutations found in cancer cells have been
shown to have an constitutive active tyrosine kinase.
This suggests that the EGFR plays an important role in
the development of the malignant phenotype of many
A number of deletion mutants have been found,
mainly in glioblastomas, but lately also in other malig-
nancies (Table 1). The development of new detection
methods such as reverse transcriptase polymerase chain
by guest on July 14, 2011
Table 1. Occurrence of EGFR deletion mutations in human neoplasia.
Human glioma xenograft
Figure 2. a) Schematic presentation of the use of a EGFRvIII specific
antibody as a tumour specific carrier for a radioactive isotope or a
cytotoxin. The antibody will only target cells expressing EGFRvIII;
b) and the radioactive isotope might then kill targeted, as well as
neighbouring cells, thereby targeting radiation therapy to the tumour.
Cytotoxins might get transferred from the targeted cell to neighbour-
ing cells through intracellular transport mechanisms.
reaction (RT-PCR), and the use of EGFR-mutant spe-
cific antibodies, might prove that EGFR mutations are
even more common than observed hitherto.
So far, EGFRvIII has been found in: high- and low-
grade gliomas, paediatric gliomas, medulloblastomas,
breast and ovarian carcinomas as well as non-small-cell
lung cancer. EGFRvIII has not been found or has not
been tested in other cancer forms. The list of cancers
where EGFRvIII is found will probably grow after
implementation of more sensitive methods such as RT-
PCR, RNase protection assays and specific monoclonal
The use of EGFR specific antibodies used for cancer
therapy is promising, but serious problems can be envi-
sioned, due to heterogeneity within the tumour, and the
fact that normal cells also express EGFR. Therefore, it
will be preferable to target the cancer cells with tumour
specific antibodies to avoid toxicity in normal cells,
when coupling an radioactive isotope, or a cytotoxin to
In this respect EGFRvIII is particularly interesting
as the deletion in the extracellular domain creates a
tumour specific epitope, that can be used as the target
for monoclonal antibodies (Figure 2a). This approach
could have significant therapeutic value by coupling
radioactive isotopes, or cytotoxins to the antibody. As
EGFRvIII is a ligand independent constitutive active
receptor the use of a blocking antibody is not possible.
As the EGFRvIII constantly stimulates cell growth it is
likely that the tumour cells expressing the EGFRvIII
mutant are the most proliferative. The coupling of radio-
active isotopes to a EGFRvIII specific monoclonal anti-
body would then target the fastest proliferating cells. A
radioactive isotope coupled to a specific monoclonal
antibody could kill neighbouring cancer cells not ex-
pressing EGFRvIII, as well as the cell expressing the
antigen, thereby overcoming the problems arising from
tumour heterogeneity (Figure 2b).
The use of EGFRvIII specific antibodies and trans-
fected cell lines expressing EGFR or EGFRvIII, could
be very useful tools to investigate the role of EGFR and
EGFRvIII, in the development of the malignant pheno-
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Received 26 June 1997; accepted 1 September 1997
Hans Skovgaard Poulsen
Section for Radiation Biology 5074
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