Review
`
`
`
`Annals 0f0ncology 8: ll97—l206, 1997.
`O 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-Thomsenz & H. Skovgaard Poulsenl
`‘Sectionfor Radiation Biology, The Finsen Centre, Rigshaspitalet, 2 Tumourpatholagical laboratory, Institute ofMolecular Pathology, University of
`Copenhagen, Copenhagen, Denmark
`
`Summary
`
`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 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.
`
`(I) the structure and
`In this review we will focus on:
`function of EGFR, (II) implications of receptor/ligand coex-
`pression and EGFR mutations or overexpression,
`(111)
`its
`effect on cancer cells, (IV) the development of the malignant
`
`phenotype and (V) the clinical aspects of therapeutic targeting
`of EGFR.
`
`Key words: cancer, epidermal growth factor receptor, signal-
`ling, tyrosine kinase
`
`Abbreviations: AR — arnphiregulin; hp — basepairs; BTC -
`betacellulin; cAMP — cyclic adenosine monophosphate; CDK
`— cyclin dependent
`ltinase; DAPH - dianilinophtalimides;
`E-cadherin — epithelial cadherin; EGF — epidermal growth
`factor; EGFR — epidermal growth factor receptor; GAP -
`GTPase-activating protein; HB-EGF — Heparin-binding
`EGF-like growth factor; MAPK — mitogen-activated protein
`kinase; PKC — protein kinase C; PLC-7 ~ phospholipase C-y;
`RB — retinoblastoma protein; RT-PCR — reverse transcriptase
`polymerase chain reaction; Shc — src homology and collagen
`protein; S-oligos — phosphothiorate oligos; TGF-at — trans-
`forming growth factor-oz; wtEGFR — wildtype EGFR
`
`Introduction
`
`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) [1]. 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 [2].
`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 GT1-"ase—activating protein
`(GAP) [5, 6], which leads to an increase in catalytical
`activity [7].
`The activated receptor/ ligand complex is endocytosed
`and either degraded within the lysosomes [8], or recycled
`
`to the plasma—membrane [9]. Endocytosis and degra-
`dation induces down—regulation of the growth factor
`induced signal.
`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
`H86 aminoacids, M, 170 kDaltons (kDa) {IO}, 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 l).
`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-oz (TGF-at) [11], am-
`phiregulin (AR) [12], Heparin-binding EGF—like growth
`factor (HB-EGF) [5], and betacellulin (BTC) [13].
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`1198
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`EGF receptor
`
`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
`
`is
`and resembles the v-erb-B oncoprotein [19, 20]. It
`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 [20].
`EGFRVII, found in gliomas with amplified rearranged
`EGFR genes [23], 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 [23], and it
`responds very similar to the wildtype EGFR (wtEGFR)
`to growth factors [23]. EGFRVII does not seem to have
`any influence on the malignant phenotype of the g1io-
`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
`WIEGFR. 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 [26], in 5 of 32 lung carcino-
`mas [IS], in 21 of 27 breast carcinomas [16, l7], in 4 of6
`paediatric gliomas, in 6 of 7 medulloblastomas, and in
`24 of 32 ovarian carcinomas [17] (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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`Figure 1. Schematic structure of EGFR, with the extra- and intra-
`cellular domains.
`
`The cytoplasmic carboxy—terrnina1 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
`and differentiation.
`
`EGFR deletion mutations
`
`A large number of deletions of the EGFR mRNA has
`been observed in ‘a number of neoplasia, first in gho-
`blastorna [14], but recently also in non~small-cell lung
`carcinomas [15], breast cancer [16], paediatric gliomas,
`medulloblastomas, and ovarian carcinomas [17].
`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 [18].
`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 lcinasc.
`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
`
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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 [27] and enhances
`the tumourigenicity of transfected human glioma cells in
`nude mice [28].
`The tyrosine kinase of EGFRVIII is much less auto-
`phosphorylated when compared to wtEGFR [29]. 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 [29], 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
`tissues.
`
`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 SV4O based expression
`vector carrying the EGFRVIII CDNA in chinese hamster
`ovary cells, revealed an altered subcellular location of
`EGFRVIII, when compared to wtEGFR [32]. EGFRVIII
`was found primarily in the endoplasrnatic reticulum,
`whereas wtEGFR was expressed on the cell surface.
`This intracellular localisation has not been observed in
`
`1199
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`tory/internalisation domains of the EGFR [14]. 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
`mRNA.
`
`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.
`
`EGFR ligands
`
`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
`[33]. This similar folding of the mature peptides ensures
`a common conformational structure.
`
`in addition to the
`AR, HB-EGF and BTC have,
`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-at, have been shown to possess
`biological activity [36], suggesting that the growth fac-
`tors might be able to activate EGFR via an auto- or
`juxtacrine mechanism, while they still remain bound to
`the membrane.
`
`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.
`
`human tumours, frozen sections of xenografts or other
`EGFRVIII transfected cell lines and might be restricted
`to these particular transfected cells.
`
`EGFR signalling
`
`EGFR activation
`
`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 Ca“ regula-
`
`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
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`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 [37]. The dimerisation results in the
`autophosphorylation of five specific tyrosine (Tyr) resi-
`dues (Tyr 1173, 1148, 1086, 1068, and 992) in ‘the car-
`boxy-termjnal end of the intracellular part of EGFR,
`with Tyr-1173 as the major autophosphorylation site [3].
`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.
`SI-I2-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 [38] and Nck [4].
`Mutational analysis have shown that the removal of
`the autophosphorylation sites has a severe effect on
`substrate binding if all five sites are removed [4]. 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%) [4].
`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 [29].
`
`EGFR inactivation
`
`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—4l].
`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 [42]. 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 [44]. 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
`conformation.
`
`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 [45].
`
`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) [46]. PKC is a serine/threo-
`nine kinase [47] 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 [50], cytosolic kinases and
`nuclear transcription factors [51].
`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
`[52]. As constitutive activation of MAP kinase-mediated
`mitogenic signalling pathways elicits transformation
`[52],
`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 G1 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 [53]. Pro-
`gression through G1 phase requires activation of the
`various cyclin-CDK kinase complexes. One of the crit-
`ical substrates of G, CDKs is the retinoblastoma pro-
`tein, (RB), whose phosphorylation and subsequent re-
`lease of RB-bound transcription factors are required for
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`G, to S phase transition. The blocking of EGFR ligand
`binding by a monoclonal antibody has been shown to
`reduce G1 phase CDK activities, causing G1 cell cycle
`arrest [54].
`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,
`[3-catenin, might lead to the loss of cell-cell adhesion,
`resulting in a more metastatic phenotype [55]. B-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. Ca“ 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 [56].
`It has been shown that internalised activated EGFRS
`
`are still autophosphorylated and catalytically active [57].
`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-
`vated EGFR.
`
`EGFR and its role in the development of the malignant
`phenotype
`
`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 [58].
`In an fluorescence in situ hybridisation
`(FISH) assay, 18 out of 29 grade 3 and 4 gliomas
`displayed EGFR gene amplification [59]. Overexpres-
`sion of EGFR were also frequently observed in breast,
`bladder, cervix, kidney, and ovarian tumours [60], as
`well as in lung cancer and various squamous carcino-
`mas [61].
`Treatment with EGF of a oesophageal cancer cell line
`expressing E-cadherin and EGFR induced changes in
`the cellular morphology and phosphorylation of B-
`catenin [62].
`In some colon cancer cell lines treatment with EGF,
`or TGF-oz, caused a reduction of E-cadherin, and an
`increase of at;-integrin, carcinoembryonic antigen (CEA)
`and CD44 [63]. These changes might increase the meta-
`
`1201
`
`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
`circulation.
`
`Possible roles of EGFR in research and cancer
`treatment
`
`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 [61], gliomas [64], squa-
`mous carcinoma [65] and laryngeal cancer [66]. 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—7l].
`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
`gene therapy.
`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
`p27 Kw‘ and to inhibit proliferation by arresting cell
`cycle progression in G1, when administered to a pro-
`static cancer cell line [72].
`The use of EGFR-blocking antibodies has been inves-
`tigated using xenografts of a squamous cell carcinoma
`cell line overexpressing EGFR [73]. 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 TGFav, 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 [74]. These experi-
`ments showed an additive inhibitory effect when using
`the combination of blocking antibodies and antisense
`S-oligos.
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`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 [75].
`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
`[76]. 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 viva. This
`suggests that the use of cytotoxic fusion proteins might
`be a possible way to inhibit tumour growth of EGFR
`overexpressing tumours.
`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
`[80]. A Neu ectodomain form (N69lstop) 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-N69lstop coexpressing fibroblasts [77].
`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,
`T69lstop is able to form heterodimers with EGFRVIII,
`and might be a way to revert the mitogenic potential of
`this EGFR mutant [80].
`As the EGFR tyrosine kinase is necessary for signal
`transduction it is a good target for therapy, using tyro-
`sine kinase inhibitors [81]. 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-at-induced growth and EGFR—
`phosphorylation in vitro [82]. 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 [87]. 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 1251-labeled EGFR spe-
`cific antibodies has been conducted on patients with
`malignant astrocytoma, astrocytoma with anaplastic
`foci, and glioblastoma multiforme [98]. These studies
`revealed encouraging results with a one—year survival of
`60%, and a median survival of 15.6 months for patients
`with glioblastoma rnultiforme, compared to a 50% death
`rate within six months when the patients are treated by
`surgical resection.
`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 [99].
`The generation of EGFRVIII specific monoclonal
`antibodies have been used to detect EGFRVIII in glio-
`blastomas [16]. Using this approach, 8 of ll tumours
`previously found EGFRVIII negative using polyvalent
`anti-EGFRVIII sera, were shown to be EGFRVIII pos-
`itive. To be able to detect EGFRvIIl in cancer cells,
`biopsies or xenografts must be tested, as the mutation
`tend to disappear when cells are cultured in vitro [25].
`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 [100]. This might be used for specific targeting
`EGFRVIII expressing cells in cancer therapy.
`
`Future perspectives
`
`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
`cancers.
`
`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
`
`
`
`
`
`§.I9qIl.I’.'-3/\()j\]U01S1"-K13/3J0'S[”2{l.lYlOi‘[).lO}X0'3lIO{l{l‘2//I(lllqIUOJJ'[)Qp'20[UA(\0(_[
`
`
`
`
`
`APOTEX EX. 1019-006
`
`

`
`Table I. Occurrence of EGFR deletion mutations in human neoplasia.
`
`E