Epidermal Growth Factor Receptor Tyrosine Kinase ...selected as a potential target for chemoprevention. Because growth factor networks are redundant, selective inhibition of signaling

Vol. 5, 657-666, August /996 Cancer Epidemiology, Biomarkers & Prevention 657

Review

Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors as

Potential Cancer Chemopreventives

Gary J. Kelloff,’ Judith R. Fay, Vernon E. Steele,Ronald A. Lubet, Charles W. Boone, James A. Crowell,and Caroline C. Sigman

Chemoprevention Branch. Division of Cancer Prevention and Control,

National Cancer Institute, Bethesda, Maryland 20892 [G. J. K., V. E. S.,

R. A. L.. C. W. B., J. A. Cl. and CCS Associates, Mountain View, California94043 [J. R. F., C. C. 5.1

Abstract

Among the most important targets for chemopreventiveintervention and drug development are deregulated signaltransduction pathways, and protein tyrosine kinases arekey components of these pathways. Loss of tyrosinekinase regulatory mechanisms has been implicated inneoplastic growth; indeed, many oncogenes code foreither receptor or cellular tyrosine kinases. Because of itsderegulation in many cancers (bladder, breast, cervix,colon, esophagus, head and neck, lung, and prostate), theepidermal growth factor receptor (EGFR) has beenselected as a potential target for chemoprevention.Because growth factor networks are redundant, selectiveinhibition of signaling pathways activated in precancerousand cancerous cells should be possible. Requirements forspecific EGFR inhibitors include specificity for EGFR,high potency, activity in intact cells, and activity in vivo.

Inhibition of autophosphorylation is preferred, because it

should result in total blockade of the signaling pathway.Inhibitors that compete with substrate rather than at theATP-binding site are also preferable, because they arenot as likely to inhibit other ATP-using cellular enzymes.

Several classes of specific EGFR inhibitors have beensynthesized recently, including structures such asbenzylidene malononitriles, dianilinophthalimides,quinazolines, pyrimidines, [(alkylamino)methyl]-acrybophenones, enollactones, dihydroxybenzyl-aminosalicylates, 2-thioindoles, aminoflavones, andtyrosine analogue-containing peptides. A possible testingstrategy for the development of these and other EGFRinhibitors as chemopreventive agents includes thefollowing steps: (a) determine EGFR tyrosine kinaseinhibitory activity in vitro; (b) evaluate EGFR specificityand selectivity (relative to other tyrosine kinases andother protein kinases); (c) determine inhibition of EGFR-mediated effects in intact cells; (d) determine inhibition ofEGFR-mediated effects in vivo (e.g., in nude mouse tumor

xenografts); and (e) determine chemopreventive efficacyin vivo (e.g., in the hamster buccal pouch or mouse or ratbladder).

Mechanism-based Strategies in the Development ofCancer Chemopreventive Agents: Signal TransductionPathways

A successful and comprehensive strategy to address the cancerproblem encompasses both preventive and therapeutic ap-

proaches. Several types of prevention programs can be envi-

sioned; one is chemoprevention, which involves prophylacticintervention with drugs or micronutrients to inhibit, delay, or

reverse the carcinogenic process before malignancy. Develop-ment of agents that can be used for this type of proscriptiveintervention involves numerous research avenues, including

identification of chemicals that prevent tumorigenesis in animalmodels or in relevant in vitro screening assays. Another ap-proach is to target specific mechanisms involved in the carci-nogenic process. Such a rational, targeted strategy toward de-velopment of chemopreventive drugs has been facilitated by

increased understanding of the biochemical and genetic abnor-malities involved in carcinogenesis. Discovery of agents withnovel mechanisms of action that counter these abnormalitiesand have chemopreventive potential is a major focus of theNational Cancer Institute Chemoprevention Branch drug devel-

opment program (1-3).

One of the most significant advances in recent years isincreased understanding of the biochemical control mecha-nisms involved in regulating cell growth and development.

Cells respond to signals from extracellular stimuli via a com-plicated network of highly regulated events, collectively re-ferred to as signal transduction pathways. Stimulation of thesepathways results in changes in transcriptional activity (re-viewed in Refs. 4 and 5). Although normal cells respond

appropriately to extracellular stimuli, many precancerous and

cancerous cells have lost this ability and display aberrant sig-

naling (reviewed in Ref. 6).Numerous places to interfere with deregulated signaling

pathways can be targeted as potential sites for chemopreventiveintervention. Key components of these pathways are the PTKs,2which catalyze the transfer of the ‘y-phosphate of ATP to thehydroxyl group of tyrosine on numerous proteins (7). Loss ofPTK regulatory mechanisms has been implicated in neoplasticgrowth; indeed, many oncogenes code for PTKs (reviewed inRefs. 8 and 9).

Received 2/2/96; revised 5/7/96; accepted 5/8/96.

The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in

accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

I To whom requests for reprints should be addressed. at ChemopreventionBranch, National Cancer Institute, Executive Plaza North, Suite 201, 6130

Executive Boulevard, Bethesda, MD 20892.

2 The abbreviations used are: PTK. protein tyrosine kinase; AR, amphiregulin;

CThJ, cervical intraepithelial neoplasia; CIS, carcinoma in situ; CSF-lr, colony-

stimulating factor I receptor; DMBA, 7, 1 2-dimethylbenz(a)anthracene; EGFR.epidermal growth factor receptor; ER, estrogen receptor; HPV, human papillo-

mavirus; IHC, immunohistochemical; tNSr, insulin receptor; PDGFR, platelet-

derived growth factor receptor; PK, protein kinase; 5CC, squamous cell carci-

noma; TGF-a, transforming growth factor a; DAPHI, 4.5-dianilinophthalimide;

DAPH2, 4,5-bis(4-fluoroanilino)phthalimide.

on February 13, 2020. © 1996 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from

http://cebp.aacrjournals.org/

Plasma

Membrane

TGFct, Other Ligands

658 Review: EGFR Inhibitors as Cancer Chemopreventives

Two general classes of PTKs are currently recognized:receptor tyrosine kinases, which receive signals directly

through their extracellular domains; and cellular tyrosine ki-nases, which are signal transducers (reviewed in Ref. 10).

Specific PTKs activated during the development of many hu-man neoplasias have been identified. Examples of aberrant

expression of receptor PTKs are EGFR in head and neckcancers ( I I ), p 1 85�-erhB.2 in breast and ovarian cancers, andPDGFR in glioblastomas and breast cancers (reviewed in Ref.

12).

Targeting Specific PTKs: EGFR

Based on the redundancy of growth factor networks, selective

inhibition of signaling pathways activated in precancerous andcancerous cells should be possible. Proliferation of normal cellsis dependent on more than one growth factor, and one growthfactor activates multiple intracellular signaling pathways. Nu-merous genetic knockout experiments have established that if a

particular growth factor-signaling pathway is inactivated, analternative pathway takes over (reviewed in Refs. 8 and 12).

Because overexpression or mutation of an oncogene can lead toconstitutive activation of a single signaling pathway, inhibitionof this specific pathway should not disturb other pathways

necessary for normal cell function. Thus. inhibiting specificPTKs activated in target tissues should result in fewer effects onthe growth of normal compared with precancerous or cancerouscells (reviewed in Refs. 8 and 12).

As noted above, many specific PTKs associated with can-cers of various organs have been identified. One of the mostextensively studied is the product of the c-erbB-1 proto-onco-

gene. EGFR (reviewed in Refs. 13 and 14). Disrupting theEGFR-mediated signaling pathway represents a novel approach

for cancer prevention. The rationale for using EGFR antago-nists as chemopreventive agents, analysis of potential clinicallyrelevant target organs, and identification of inhibitors withspecificity toward intrinsic EGFR tyrosine kinase activity arethe subjects of this review.

EGFR and Signal Transduction

EGFR is a I 70-kilodalton transmembrane protein that is amember of a family of related receptor PTKs that also includesthe proteins encoded by the c-erbB-2 (neu), c-erbB-3, andc-erhB-4 genes (reviewed in Ref. 15). Presently recognized

EGFR ligands are EGF, TGF-a, AR, betacellulin, and heparin-binding EGF (reviewed in Ref. 16). Some ligands includingAR, EGF, and TGF-a are capable of acting as inhibitors, as

well as stimulators, of cell proliferation, depending on thespecific circumstances (reviewed in Ref. 17). The importance

of EGF and TGF-a in controlling cell growth has been wellestablished (reviewed in Refs. 14 and 16); the importance ofother ligands is only beginning to be explored.

Activation of EGFR can occur via autocrine (ligands se-creted by the same cell), paracrine (ligands secreted by othercells), or juxtacrine (membrane-bound forms of growth factorsactivate the receptor on adjacent cells) mechanisms (reviewed

in Ref. 15). On ligand binding, EGFR dimerizes with neigh-boring receptors and is autophosphorylated at three major ty-rosine residues (reviewed in Ref. I 8). Subsequently, the recep-

tor interacts with a number of proteins that are elements ofsignal transduction pathways, including phospholipase Cy,

phosphatidylinositol-3’-kinase, growth factor receptor-binding

protein 2, Src family kinases, and components of the Jak/STATpathway (Fig. I; reviewed in Refs. 19 and 20).

Nuclear

Membrane

Fig. 1. Signal transduction via the EGFR pathway. On ligand binding. EGFR

dimerizes with neighboring receptors and is autophosphorylated at three major

tyrosine residues. Subsequently. the receptor interacts with several proteins,

which are elements of signal transduction pathways. including phospholipase C’y

(PLC’y), phophatidylinositol-3’-kinase (P13-Kinase), growth factor receptor bind-

ing protein 2 (Grb2, Src family kinases, and components of the Jak./STAT

pathway. MAPK, mitogen-activated protein kinase.

Association of EGFR with Carcinogenesis

General Considerations

Deregulated expression of EGFR and its ligands has beenassociated with the development of neoplasia in both animalsand humans. Aberrant EGFR signaling can occur with receptoroverexpression or with mutated forms of the receptor. Bothmethods of activation have been associated with human can-

cers, although overexpression is much more common. The mostfrequently identified receptor mutant, EGFRvIII, has lost amino

acids 6-273. This truncation results in ligand-independent ty-

rosine kinase activity, altered subcellular location, increasedstability (21, 22), and enhanced tumorigenicity (23). Until re-cently, this mutation had only been identified in a limitednumber of gliomas. However, using more sensitive methods, ithas been detected in up to 57% of high-grade and 86% oflow-grade glial tumors, 78% of breast cancers, 73% of ovariancancers, and 16% of non-small cell lung cancers, but not in anynormal tissues examined to date (24-26). EGFR overexpres-sion generally occurs in the absence of detectable alterations in

the gene, although some cases of amplification and rearrange-ment have been observed (reviewed in Ref. 27). Recently, Soberet a!. (28) reported that the elements of the signal transduction

pathway activated by EGFR are determined by the level ofreceptor expressed. At physiological receptor levels, EGF bind-

ing results in phosphorylation of Src homology 2 domain-containing proteins, which is sufficient to induce a normal

mitogenic response. However, when expression of EGFR isincreased, other substrates, such as phospholipase Cy and Ras-GAP, become phosphorylated. This may explain, at least in

part, how EGFR overexpression contributes to carcinogenesis.Increased expression of the EGFR ligands, particularly

TGF-a, has also been linked to tumorigenesis. Transgenic miceoverexpressing TGF-a develop benign skin lesions and cancersof the liver and mammary gland (29, 30). TGF-a can cooperatewith both viral and cellular oncogenes in the induction ofneoplasia in multiple tissues (31, 32), and evidence to support

a role for TGF-a in transformation independent of its stimula-tory effects on cell growth has been presented (33). EGF has

also been associated with carcinogenesis both in vitro and in

vivo (reviewed in Ref. 34). Additionally, the oncogenic action

of ras genes may be mediated in part by the EGFR-signalingpathway; both TGF-a (35) and other ligands (36) have beenimplicated.



Cancer Epidemiology, Biomarkers & Prevention 659

Role of Aberrant EGFR Signaling in the Development of

Specific Human Cancers

Abnormalities in the EGFR-signaling pathway have been

associated with the development of many human cancers, al-though in most tissues, the precise role remains unclear. Muchof the available clinical data have been generated from studies

of established cancers. These investigations have shown that in

some organs, EGFR is overexpressed in a subset of cancers;

overexpression may or may not be associated with poor prog-nosis (reviewed in Ref. 14).

Targets for chemopreventive intervention are tissues inwhich aberrant EGFR-mediated signal transduction occurs at a

relatively early time point during tumor development: bladder,

breast, cervix, colon, esophagus, head and neck, lung, andprostate. Examination of changes in EGFR signaling during

premalignant stages of carcinogenesis has been less extensively

studied than in fully transformed tissues. However, two general

patterns have emerged (discussed in detail below). The first

pattern is a trend toward increased expression of EGFR during

the early stages of carcinogenesis, followed by receptor down-regulation. often subsequent to increased ligand production.

This pattern has been observed, for example, in the lung, cervix,and prostate.

The second recurring pattern observed is expansion ofreceptor and/or ligand expression from a subset of cells innormal tissue (usually in the basal layer) to extended cellular

layers during neoplastic progression (e.g. , head and neck, blad-der, and cervix). These changes in cellular distribution are

usually detected using IHC techniques, which allow the main-

tenance of tissue architecture. However, it should be noted thatchanges in receptor distribution can be obscured by the use of

ligand-binding assays, which, despite their increased sensitivityrelative to immunohistochemistry, generally necessitate the use

of tissue homogenates.

Furthermore, interpretation of clinical studies has beenclouded by the use of “normal-appearing” tissue from cancer

patients to determine normal receptor and ligand expressionpatterns and levels. It is well known that field-wide damageoccurs in many tissues exposed to a common carcinogen orharboring a genetic defect. This helps explain the increased risk

that persons who have been treated for a primary tumor exhibitfor the development of second primary tumors (37); indeed,such “cured” cancer patients are important cohorts for chemo-

prevention clinical trials. Because changes in expression may

occur during the very early stages of tumorigenesis, they wouldlikely be overlooked if histologically normal tissue from cancer

patients were used to assess normal tissue levels. For example,such early changes have been noted in expression of EGFR

during the development of oral cancers (see below).

Head and Neck. Increased expression of EGFR protein hasbeen found in histologically normal tissue adjacent to head and

neck SCC compared with cancer-free, non-smoking controls(I 1). Levels of EGFR remained elevated but did not increase in

hyperplastic and dysplastic tissue; a second large increase in

receptor levels was noted in carcinomas. Additionally, althoughonly the basal layer expressed EGFR in the normal epithelium,

expression expanded to all layers, including the superficiallayer, during neoplastic progression. Increased levels of EGFR

and TGF-a mRNA have also been found in histologically

normal mucosa isolated from head and neck SCC patientscompared with noncancer controls (38).

Deregulated signaling through the EGFR pathway has alsobeen examined at specific head and neck cancer sites. Tumorand normal-appearing mucosal samples from patients with oral

SCC, as well as the normal-appearing oral mucosa of heavydrinkers and smokers, displayed increased EGFR expression

compared with levels detected in healthy, non-drinking, non-smoking controls (39). Shirasuna et a!. (40) reported EGFRexpression limited to the basal cells in oral mucosa from

healthy normal controls but extending to other cell layers in oralleukoplakia. Connective tissue from normal controls stainedweakly for EGF, and the intensity increased as lesions pro-

gressed to higher degrees of dysplasia, with the strongest stain-ing in carcinomas. Taken together, these observations suggest

that deregulated expression of EGFR and ligands is an earlyevent during the development of head and neck cancers.

Retinoids prevent the development of second primary tu-mors in cured head and neck cancer patients (41), and these

effects have been suggested to result from down-regulation ofEGFR and TGF-a (38). Some (42) but not all evidence supports

a role for modulation of the EGFR-signaling pathway in the

a.ntiproliferative effects of retinoids in head and neck cancers(43).

Lung. In biopsy specimens from heavy smokers, the percent-

age of cells expressing EGFR increased as the degree of bron-chial epithelial changes progressed from normal appearing tosquamous metaplasia to dysplastic squamous metaplasia to CIS

(44). However, the percentage of positively staining cells wasdecreased in SCC relative to CIS. A shift in the distribution ofcells expressing EGFR was also noted. In the normal epithe-

hum, EGFR was only expressed in the basal layer, but asneoplasia progressed, EGFR extended upward through the ep-

ithelial layer to occupy the full thickness of the epithelium.

It should be noted that abnormal EGFR distribution has

been observed in reactive (squamous metaplasia and inflam-matory atypia) as well as precancerous (dysplasia and CIS)

bronchial lesions associated with both non-small cell lung can-

cer adenocarcinomas and SCC; however, staining was moreintense and involved more of the superficial layer of the epi-

thelium in precancerous compared with reactive lesions. Simul-taneous aberrant expression of EGFR and p53 occurred in SCCand associated preinvasive lesions but not adenocarcinomas

(45).

Esophagus. Deregulated EGFR-mediated signaling has beenlinked to the development of both esophageal adenocarcinomas

and SCC. In Barrett’s mucosa, a premalignant lesion associatedwith the development of esophageal adenocarcinoma, EGFR

and TGF-a expression increased with higher degrees of dys-plasia. Both were also increased in high-risk intestinal meta-plasia compared with lower-risk gastric cardiac metaplasia

(46, 47).

In squamous esophageal lesions, stronger IHC staining forEGFR was observed in dysplasias and CIS than in surroundingnormal mucosa; in invasive cancers, variations in intensity andhomogeneity of receptor expression were reported (48).

Bladder. The distribution of EGFR differs between normal

and neoplastic urothelia. In the normal bladder, EGFR is onlyexpressed on cells of the basal layer, whereas in malignant,dysplastic, and histologically normal tissue from bladder cancerpatients, EGFR is equally expressed on cells of the urothelial

layers, including those directly in contact with urine (49). Raoet a!. (50) observed a progressive increase in the percentage of

EGFR-expressing cells obtained from tissue distant from thecarcinoma, to tissue adjacent to the tumor, to tumor tissue.

Furthermore, patients whose non-muscle-invasive tumors (PTa

and pT3) expressed EGFR were significantly more likely toprogress toward invasive disease (5 1 ). Because high levels ofEGF are excreted in the urine, expression of EGFR in the




premalignant and malignant bladder transitional epithelium

could have a significant impact on proliferation of these cells(49). Messing and Resnikoff (49) is currently evaluating

EGFR as a biomarker for bladder neoplasia in a Phase II

clinical chemoprevention trial of the antiproliferative2-difluoromethylornithine.

Prostate. Strong positive IHC staining for EGFR has consis-tently been reported in all basal cells from normal and benignprostate glands. Although reports of EGFR-positive prostatecancers vary, results of seven different studies found dimin-

ished EGFR expression in basal cells of tumors when comparedwith benign glands (reviewed in Ref. 52). Both high-gradeprostatic intraepithelial neoplasia and malignant lesions express

significantly less EGFR protein compared with normal,

atrophic, hyperplastic, and low-grade prostatic intraepithelialneoplasia (53). In one study in which reduced EGFR levels

were noted in malignant compared with normal or benign

prostatic hyperplastic cells, expression of the EGFR ligand

TGF-a was only observed in malignant cells (54).

Colon. Salomon et a!. ( I 7) collated data on 559 colon tumorsfrom 12 studies and concluded that 25-77% expressed EGFR.In general, no significant differences between EGFR levels inadjacent. noninvolved cobonic mucosa and tumor tissue wereobserved. Koenders ci a!. (55) reported higher EGFR expres-

sion in the proximal compared with the distal colon in normal-appearing, but not carcinomatous, tissue. They suggested thatthese regional differences might account, at least in part, for thelack of quantitative differences in EGFR bevels between nor-

mal-appearing and cancerous tissue observed in other studies.

Indeed, this group also reported significantly higher levels of

EGFR in normal-appearing tissue than in carcinomas, suggest-ing that aberrant EGFR expression may be an early event

during colon cancer development.A number of observations suggest that EGFR ligands can

influence the growth and development of precancerous andcancerous colon cells. TGF-a levels were higher in coloncarcinomas compared with normal-appearing mucosa (56), and

a progressive increase in the percentage of lesions expressingTGF-cs was observed during the progression from cobonic ad-enomas to CIS to invasive cancers (57). TGF-cs enhanced thegrowth of an adenoma cell line, and growth was completely

inhibited by anti-EGFR antibodies (58). Deoxycholate, an en-dogenous colon tumor promoter, induces TGF-a mRNA invitro, further supporting a role for this growth factor in the

development of colorectab cancer (59). Additionally, studies

suggest that TGF-a expression can contribute to the transfor-mation of colon cells independent of proliferative effects (33).

Other EGFR bigands have also been implicated in cob-rectal cancer development. Saeki ci a!. (60) detected AR im-munoreactivity in approximately 100% of normal colon sam-pIes, 15% of noninvolved adjacent mucosab samples, 64% of

adenomas, and 50% of colon carcinomas. These results suggestthat changes in AR expression occur in normal-appearing,high-risk coborectal mucosa relative to normal tissue.

Cervix. Most studies agree that in the normal cervical epithe-

hum, expression of EGFR is limited to the basal and parabasalcells, whereas in CIN, expression expands throughout the full

thickness of the epithebium (61-64). Most (61, 62, 65) but notall (66) studies have also shown that expression of EGFR in

cancerous tissue decreases relative to intraepithelial lesions.Indeed, based on preliminary findings that EGFR levels in-

creased in low- and medium-grade CIN but decreased in high-grade CIN and carcinoma, Mitchell and coworkers (65) are

currently evaluating EGFR as a biomarker in chemopreventive

efficacy trials of 2-difluoromethybornithine and the retinoid

alb-trans-N-(4-hydroxyphenyl)retinamide.

In support of the importance of deregulated signaling via

the EGFR pathway during the development of cervical cancer,

immortalization of normal human ectocervical epitheliab cellswith HPV- 16, a known risk factor for cervical cancer, results in

increased EGFR bevels. Retinoic acid reduces EGFR protein

levels and EGF binding in HPV-16-immortalized human ecto-

cervical cells but not in normal cells (67). This is interesting in

light of the ability of retinoic acid to cause regression of CIN

lesions (68).

However, other groups have found no correlation between

expression of EGFR and the presence of HPV or between

EGFR and low- versus high-risk HPV types when stratified by

grade of CIN (63, 64). Because the malignant potential of CIN

is highly correlated to HPV type, these results suggest that

EGFR expression may be a marker of proliferation rather than

an integral component of the cervical transformation process.

Breast. The results of studies examining expression of EGFR

and its ligands in breast cancers vary significantly. Klijn et a!.

(69) compiled data from 40 different series of patients (n =

5232) and calculated that, on average, 45% (range, 14-91%) of

human breast cancers express EGFR. A much higher percent-

age (about 80%) express one or more EGFR bigands. Cancer

cells backing EGFR that continue to express EGFR-activating

ligands may do so to stimulate stromab cells needed to support

tumor growth (70).

Although the actual percentages of tumors expressing

EGFR differ among studies, a consistent inverse correlation

between expression of EGFR and ER in breast cancers has been

reported (69, 70). Some cancers express both receptors; how-

ever, evidence suggests that even when both receptors arepresent, they are not expressed in the same cancer cells. Co-

expression in tumor tissue was only observed in a subset of

cells from a mixed invasive ductal CIS. In contrast, EGFR and

ER are frequently coexpressed in normal-appearing cells ob-

tamed from breast cancer patients (7 1). One explanation for

these changes in expression patterns is that malignant conver-

sion may be associated with attainment of independence for at

least one of these growth factors. Furthermore, this suggests

that premalignant cells unresponsive to antiestrogens may still

be responsive to growth inhibition via the EGFR-signaling

pathway.

Particularly relevant for the purposes of chemoprevention,

expression of EGFR in ductal cells of needle aspirates from

women at high risk for breast cancer development (first-degree

relative with breast cancer, prior breast cancer, or precancerous

mastopathy) was significantly higher than in bow-risk controls

(72).

Taken together, these observations suggest that normal

breast cells are dependent on both EGFR and ER for growth.

Cells at high risk for tumor development may overexpress

EGFR, leading to increased proliferative activity. As they pro-

gress toward malignancy, these cells may lose their dependence

on one or both of these growth factors. However, the subset of

transformed cells that no longer express cellular receptors may

continue to express EGFR bigands to stimulate the growth of

stromal cells needed to support tumor cell growth.

Thus, it is clear that EGFR signaling is deregulated during

the carcinogenic process in a number of tissues, making selec-

tive inhibition of this pathway a viable chemopreventive

approach.



Benzylidene Malononitriles Dianhlinophthalimides

H

0 N o

X�N�,N�X

DAPH1, X=HDAPH2, X=F

RG 14620

Quinazolines I(All�amino)methYllaaYloPhen0nes

i�) � N(CH�)2R,”�”NH ..-

�: Ph-CH2-0 � �

CAQ: R,=Cl; R�,R�=H EGFR Inhibitor 18AG 1478: R,=Cl; R,,R�=OCH,PD 153035: R,=Br; R,,R�=0CH,

Enollactones Dthydroxybenzylaminosalicylates

OH OH C0,(CH,)3-Ph

�

BE.23372M 2-Hydroxy-5-[N-[(2,5-dihydroxy-phenyl)methyl]amino]benzoicAcid 3-Phenylpropyl Ester

2-Thioindoles Aminoflavones

COOH NH�

c�’SJ$31� ::c#{231}r6

HOOC NH2 0

PD 146568 6-Hydroxy-3’,5,7-triaminoflavone

where X is one ofthe following heterocyclic tyrosine analogs:

Cancer Epidemioloaj�, Biomarkers & Prevention 661

Fig. 2. Structures of EGFR-specific tyrosine kinase inhibitors.

Inhibiting the EGFR-signaling Pathway

Although the effects of retinoids on EGFR function, such asthose described above for head and neck and cervix, may beinvolved in its observed chemopreventive action, these com-

pounds are well known to have numerous other biologicalactivities (reviewed in Ref. 73). More definitive conclusionsregarding the effects of modulation of EGFR function can bedrawn by examining the consequences of specifically blockingEGFR activity. Antitumor activity against xenografts of EGFR-dependent cell lines has been demonstrated with EGFR-specificmonocbonal antibodies (e.g., Ref. 74). Another approach is toblock EGFR function by inhibiting the receptor’s intrinsic

tyrosine kinase activity.Numerous naturally occurring compounds inhibit PTK

activity. Quercetin, a flavonoid and well-known chemopreven-

tive agent (reviewed in Ref. 75), was the first PTK inhibitordiscovered. However, quercetin also inhibits several other en-zymes including cAMP-dependent PKA, PKC, and other ATP-

using enzymes (reviewed in Ref. 76). The related isoflavonegenistein is more selective toward PTKs than quercetin, but ittoo inhibits other enzymes, such as histidine protein kinases(reviewed in Ref. 77) and topoisomerases (reviewed in Ref.78). Other natural products, such as erbstatin, herbimycin A,bavendustin A, and (+)-aeroplysinin-l, are also potent PTKinhibitors with increased selectivity toward PTKs and are thus

potential lead compounds for the design of novel antisignaling

drugs. Numerous synthetic tyrosine phosphorybation inhibitors,

collectively known as tyrphostins, have been developed (re-viewed in Ref. 76); some specifically inhibit EGFR tyrosine

kinase activity and are discussed below.

Requirements for Specific PTK Inhibitors

Several authors have elaborated the requirements for de-velopment of specific PTK inhibitors (e.g., 10, 12, 76, 77, 79).

Requirements include: specificity among the kinases [e.g. , ser-me and threonine protein kinases (such as PKC) and histidineprotein kinases]; specificity toward the target PTK, in this case,

EGFR (numerous other PTKs, such as PDGFR, CSF-lr, andINSr, may also be inhibited); high potency; activity in intact

cells; and activity in vivo. Inhibitors can act at the level ofautophosphorybation or on downstream targets; autophospho-rylation is the preferred site, because it should result in total

blockade ofthe signaling pathway. Inhibitors that compete withsubstrate rather than at the ATP-binding site are also preferable,because they are not as likely to inhibit other AlP-using ceb-lular enzymes. Additionally, the high ATP concentrations in thecell may render inhibitors that are quite effective against theisolated enzymes inactive in intact cells (80).

Tyrosine Analog Containing Peptides

EGFR Kinase Inhibitors

The following discussion presents an overview of the structural H-Ar Arg-GIu.GIu-Leu-Gin-Aap-Aap-X-GIu-OH

classes of drugs that have been developed as EGFR inhibitors

(see Fig. 2). Based on their activity in in vitro and in vivoscreening assays, some of these drugs may have chemopreven-

tive potential. It should be noted that PTK inhibitors that do notmeet all the requirements set forth above may still be viablechemopreventive drugs under appropriate circumstances.

Also, most currently available animal data have been ob-tamed in models relevant for establishing chemotherapeuticeffectiveness, i.e. , inhibition of growth of transplantable tumors

or tumor cells in vivo. Because changes in EGFR-mediated Decapepades

signaling occur during the process of carcinogenesis, effective-ness or ineffectiveness toward established tumors may not




accurately predict activity or inactivity toward precancerouslesion growth and development. This differential effectivenesshas been demonstrated, for example, with retinoids, which arewell-established chemopreventive agents but have generallybeen ineffectual against established tumors in animal models(reviewed in Ref. 81).

Benzylidene Malononitriles

Extensive work on structures based on erbstatin and tyro-

sine incorporating the benzylidene malononitrile nucleus has

been carried out by Levitzki ( 12, 76) and Gazit et a!. (82). Someof these compounds can discriminate between PTKs; some caneven discriminate between EGFR and the closely relatedpl85’�”�2, which have 80% homologous kinase domains(12, 80). Based on its inhibitory activity toward EGFR kinase

activity, Rh#{244}ne-Poubenc Rorer is developing the tyrphostin RG14620 as a topical antipsoriatic. Abnormalities in EGFR regu-bation are implicated in the etiology of this disease (83). RG14620 inhibits EGF-stimulated cancer cell proliferation in vitro

and EGF-dependent tumor growth in nude mice (84). Topicaland i.p. pharmacokinetic data on RG 14620 have been pub-lished (85). RG-l3022 may also be undergoing developmentfor treatment of EGFR-associated cancers (10, 84); however,

studies have shown that this drug shows limited specificity for

EGFR (86).

Dianilinophthalimides

The class of DAPH1 compounds differs by only two

carbon-carbon bonds from the PKC inhibitor staurosporineagbycone; however, they have very different physicochemical

and biochemical profiles. DAPHI (CGP 5241 1) is specific forEGFR relative to PKC. casein kinases, and several other PTKs.In cells, DAPH1 inhibited autophosphorylation of EGFR andthe closely related p1 85cerbB2 but not PDGFR. Most impor-tantly, DAPHI displayed antitumor activity in a nude mousexenograft model against tumors overexpressing EGFR or

pl85c��2 but had no effect on PDGF-dependent tumors. The

compound did not cause gross toxicity after 15 days of treat-ment, although it did not have absolute specificity for EGFR(87).

To enhance the stability of DAPH1 in vivo, blockade of

metabolic hydroxylation was achieved by synthesis of afluorine-substituted derivative, DAPH2. In cells, DAPH2selectively inhibited signals mediated via the EGF andpl85��2 pathways, but not other tyrosine kinases; selective

antitumor activity was also demonstrated for EGF-dependentrelative to PDGF-dependent xenografts in mice. However,DAPH2 was not as selective toward specific PKC isozymes asthe parent compound; in particular, it inhibited the PKC-f32isozyme with potency equal to that of EGFR. Pharmacokineticsof DAPH2 in rats and mice has been determined (88).

Quinazolines

Fry et a!. (89) have developed quinazobine derivatives, asexemplified by the highly potent and specific EGFR kinaseinhibitor PD 153035 [4-(3-bromoanilino)-6,7-dimethoxyquina-zoline]. It is competitive with respect to AlP, rapidly sup-presses autophosphorylation of EGFR, with a bow picomolarinhibition constant, and selectively blocks EGF-mediated cel-lubar processes, including mitogenesis, early gene expression,and oncogenic transformation. However, PD 153035 was re-cently reported to be inactive in vito against the growth ofEGFR-dependent tumors (90). As noted above, this result does

not necessarily preclude its having chemopreventive activity.

Further structure-activity studies on quinazoline and relatedheterocyclic derivatives have been carried out by this Parke-Davis research group (91); however, PD 153035 remains themost potent compound identified.

The closely related quinazolines CAQ (92) and AG 1478(93) have also been synthesized. AG 1478 has been reported to

be selective for EGFR and to block autophosphorylation in

intact cells. It is being evaluated in vivo (cited in Ref. 12).

Pyrimidines

A study of 7-aminopyrido[4,3-d]pyrimidines containing

aromatic side chains revealed that this series displayed EGFRinhibitory activity similar to the corresponding quinazobines(94); however, as for the related quinazolines, none was aspotent as PD 153035.

[(Alkylamino)methyljacrylophenones

This group of compounds was designed to act as multi-substrate complex inhibitors based on the transition state pro-posed for tyrosine kinases. In vitro, they selectively inhibitedEGFR relative to v-ab!, c-src, or PKC. i.p. administration of theEGFR inhibitor 18 (1 -[4-(benzyboxy)phenyl]-2-[(dimethyl-amino)methyl]-3-[3,4-dimethybphenyl)-thio]propan-1-one) at

0.1 x the maximum tolerated dose significantly inhibited thegrowth of human breast carcinoma cells in nude mice (95).However, based on the differences between inhibition of EGF-dependent tyrosine phosphorylation and antiproliferative activ-

ity, the authors suggested that other mechanisms of action are

also likely (95).

Enollactones

Tanaka et a!. (96) isolated and synthesized the fungalmetabolite BE-23372M, which consists of an enoblactone ring

and two catechob rings. It appears to be specific for EGFR andis competitive with respect to substrate peptide and to ATP.Autophosphorylation of solubilized EGFR and of EGFR inintact cells was also inhibited.

Dihydroxybenzylaminosalicylates

A series of 5-[(2,5-dihydroxybenzyl)amino]salicybate

compounds have been tested against EGFR kinase activity (97).The 5-[(2,5-dihydroxybenzyl)-amino]salicybate moiety waschosen as a simplified model for lavendustin A, one of the mostpotent in vitro natural product PTK inhibitors identified to date.Some of these compounds were competitive with respect to

ATP; others were noncompetitive. All the inhibitors were non-competitive with respect to the peptide substrate. Based onselectivity toward EGFR relative to PKC and PKA and the

ability to inhibit EGFR autophosphorylation, promising com-pounds were identified (e.g., 2-hydroxy-5-[N-[(2,5-dihy-

droxyphenyl)methyb]amino]benzoic acid 3-phenylpropyl es-ter). No results from studies of the inhibitory activity of thesecompounds toward the growth of tumors bearing amplifiedEGFR are yet available.

2-Thioindoles

A group at Parke-Davis (98), in collaboration with re-searchers from the University of Auckland School of Medicine(99, 100), screened approximately 100,000 compounds from

their chemical library for inhibitory activity against a variety of

tyrosine kinases. Using this approach, 2-thioindobes were found




Table I Steps in preclinical evaluation of EGFR inhibitors as

chemopreventive drugs

1 . in vitro inhibition of EGFR tyrosine kinase activity (including inhibition of

EGFR autophosphorylation)

2. In vitro assays with a battery of other tyrosine kinases (e.g., p185’ ��hB�2

p60�_.”#{149},PDGFR. CSF-lr, and INS-r) and other protein kinases to

determine specificity and selectivity

3. Inhibition of autophosphorylation and EGFR-mediated proliferation and

transformation in intact cells (e.g., A43l carcinoma cells)

4. In SitS) inhibition of EGFR in tumor system dependent on EGFR pathway

(e.g., xenografts of A43l cells in nude mice)

5. In l’fl’() demonstration of chemopreventive activity in animal models of

carcinogenesis dependent on or strongly associated with EGFR pathway

(e.g., possibly DMBA-induced hamster buccal pouch, N-butyl-N-(4-

hydroxybuty)nitrosamine-induced rat or mouse bladder, DMBA- or N-

methyl-N’-nitrosourea-induced rat mammary gland. and azoxymethane-

induced rat colon)

to be inhibitors of EGFR. Some compounds in this series, suchas PD 146568, have shown selectivity toward EGFR.

Aminoflavones

Some members of a series of synthetic aminoflavones

(e.g., 6-hydroxy-3’,5,7-triaminoflavone) were effective in vitro

against EGFR kinase (101).

Tyrosine Analogue-containing Peptides

Two decapeptides containing the fluorenylmethoxycar-

bonyb derivatives of two homochirab tyrosine analogues, a pyr-idine N-oxide and a pyridone, were synthesized by Andrews etal. (102). Both peptides inhibited autophosphorylation in vitro,

but relatively high concentrations were required. Although theinhibitory mechanism was not determined, the authors sug-

gested that these peptides have the potential to serve as mech-anism-based (irreversible) inhibitors based on their similarity tothe enzyme substrate.

Approach to Developing EGFR Kinase Inhibitors asChemopreventive Agents

A number of in vitro and in vivo tests may be used in the

development of EGFR kinase inhibitors as potential chemopre-ventive agents. A possible testing strategy is outlined below(Table 1). Priorities for further development would be based onresults at each of the following steps (in order).

Determine EGFR Tyrosine Kinase Inhibitory Activity

Inhibition of EGFR-catalyzed incorporation of [32P]ATP

into various substrate peptides can be assessed (e.g., Ref. 89).Sources of EGFR include A43 1 human epidermoid carcinomacells (e.g., Refs. 89 and 92) and the purified recombinant EGFRintracellular domain (e.g., Ref. 87). It is important to demon-strate inhibition of EGFR autophosphorylation.

Evaluate EGFR Specificity and Selectivity

The specificity (e.g. , serine and threonine protein kinaseversus PTK) and selectivity (various PTKs versus EGFR)

should be determined. Fry et a!. (98) have described an exten-

sive screening battery examining the effects of potential inhib-itors on several PTKs in addition to EGFR, includingp185��”�2, p6o%.sr( PDGFR, CSF-Ir, and INSr. Barret et a!.

(79) have recently described a simple method to ascertain theability of a compound to inhibit the activity of a panel of protein

kinases using a single substrate to facilitate targeting of specificPTKs and cell types.

Determine Inhibition of EGFR-mediated Effects in Intact

Cells

Selective inhibition of autophosphorylation and effects onEGFR-mediated proliferation and transformation can be exam-med in whole cells. A43l carcinomas cells, which express highbevels of EGFR, have been extensively used for this purpose(e.g., Refs. 89 and 98). Swiss 3T3 fibroblasts stimulated with

various growth factors can be used to determine inhibitor se-

bectivity on phosphorylation reactions (e.g., Refs. 87, 89, and

98).

Determine Inhibition of EGFR-mediated Effects in Vivo

Several authors (e.g. , Refs. 8 and 98) have discussedrequirements for testing PTK inhibitors in vivo. Minimal re-quirements include deregulation of the specific PTK in thetarget tissue and demonstration that tumor growth depends onthe targeted signaling pathway. In this case, EGFR should beknown to be overexpressed in the tumor, and tumor growth

should be dependent on its expression. Xenografts of A43 Icells in nude mice overexpress EGFR, and their growth can beinhibited by treatment with anti-EGFR monocbonal antibodies

(reviewed in Refs. 87 and 98). This animal model has been usedto demonstrate activity of EGFR kinase inhibitors in a chemo-therapeutic setting (e.g., Ref. 87).

Determine Chemopreventive Efficacy in Vivo

DMBA-induced cancer in the hamster buccal pouch has

been suggested as a model for human head and neck cancers(103). Overexpression of EGFR in this model has been ob-served in both dysplastic lesions and SCCs ( 104). Pendingvalidation of the dependence of these lesions on EGFR fortumor growth, this model may be especially useful to assess the

chemopreventive effects of EGFR kinase inhibitors. Tests for

chemopreventive efficacy in other established animal tumormodels in which high bevels of proliferation are particularlyimportant to carcinogenesis (e.g. , rat bladder and colon and

mammary and mouse bladder) may logically follow. See thearticle by Steele et a!. (105) for a description of the animalmodels currently used in the National Cancer Institute Chemo-prevention Branch drug development program.

Discussion and Conclusions

The use of specific and selective PTK inhibitors represents a

rational, targeted approach toward development of chemopre-ventive drugs. Because of the redundancy of cellular signaling

pathways, inhibition of a specific deregulated pathway in can-cerous or precancerous lesions offers the possibility of minimaleffects on normal cell function.

Deregulated signaling via the EGFR-mediated pathwayhas been linked to carcinogenesis in several target organs of

high interest to the Chemoprevention Branch, as reviewedabove. However, the precise role of aberrant EGFR signalingduring neoplastic development in most tissues remains unclear.Interpretation of results has been further obfuscated by the useof normal-appearing tissue from cancer patients to determinereceptor and bigand expression in normal tissue, which could

obscure potential field effects. However, in many tissues, in-creased expression of EGFR occurs early during neoplastic

progression, followed by down-regulation, often subsequent to




increased ligand production. This suggests that inhibition ofEGFR tyrosine kinase activity may be a viable chemopreven-

tive strategy.Targeting EGFR activity may afford some unique chemo-

preventive opportunities. Based on the inverse correlation be-

tween expression of ER and EGFR observed during breastcancer development, premalignant cells unresponsive to anties-

trogens may still be responsive to growth inhibition via theEGFR signaling pathway. In the bladder, expression of EGFRin a normal-appearing, high-risk urothelium but not in normal

tissue exposed to urine may provide a means to selectivelytarget precancerous cells.

Several authors have elaborated on problems with the

current methods available for establishing the effectiveness ofspecific PTK inhibitors (e.g., Refs. 7-9, 77, 79, and 98). Theserange from appropriate methods for in vitro testing to identifi-

cation of relevant animal models. Establishing selectivity andspecificity toward PTKs may be quite difficult because of the

numerous protein kinases currently known. Additionally, manyof the compounds specific for a particular PTK inhibit other

protein kinases at higher doses; thus, high doses could lead toback of selectivity and specificity accompanied by toxicity.

Careful dose-response studies will be necessary to establishtherapeutic windows.

Furthermore, effects on unknown enzymes may occur. For

example, Osherov ci’ a!. (80) developed two tyrphostins, both ofwhich are competitive with respect to ATP but specific in vitro

for EGFR or p1 85c-erbB-2 despite the 80% homology betweenthe kinase domains ofthe two receptors. In intact NIH/3T3 cellstransfected with either EGFR or pl85c�1��l (possessing the

pl85�r�a kinase domain), autophosphorylation and endoge-nous substrate phosphorylation were not inhibited by either

compound, despite the observation that mitogenic signaling

induced by EGF in both transfected cells was blocked by thesetwo agents. The authors suggested that the high intracellularATP levels prevented binding of the inhibitors to their receptors

and that the antiproliferative effects of the compounds weremediated by an element downstream, presumably a tyrosine

kinase.Unfortunately, the design of EGFR inhibitors directed at

the catalytic site is hindered by the lack of a three-dimensionalstructure for the enzyme (106). Many EGFR kinase inhibitorsidentified to date are competitive with ATP, increasing the

likelihood of interacting with other ATP-using enzymes. Asdiscussed above, such compounds may be inactive or active

only at much higher concentrations in intact cells comparedwith the isolated enzyme because of high intracellular ATP

bevels. Despite these hurdles, progress has clearly been made indeveloping candidate drugs with apparent specificity toward

EGFR kinase activity, some of which are active in vivo. Recent

publication of the structure of the INSr kinase domain (107),which shares extensive homology to other PTKs (106), as well

molecular modeling of the EGFR catalytic site (108), should

enhance drug discovery efforts (10).

Acknowledgments

We gratefully acknowledge Mary K. Doeltz for her review of and contributions

to the manuscript.

References

I . Kelloff, G. J., Boone, C. W., Crowell, J. A., Steele, V. E., Lubet, R., and

Sigman. C. C. Chemopreventive drug development: perspectives and progress.

Cancer Epidemiol.. Biomarkers & Prey.. 3: 85-98.

2. Kelloff, G. I., Boone, C. W., Steele, V. E., Fay, J. R., Lubet, R. A., Crowell,

I. A.. and Sigman. C. C. Mechanistic considerations in chemopreventive drugdevelopment. J. Cell. Biochem., Suppl.. 20: 1-24. 1994.

3. Kelloff, G. J., Boone, C. W., Steele, V. E., Fay. J. R.. and Sigman. C. C.

Inhibition ofchemical carcinogenesis. In: J. Arcos, M. Argus, and Y. Woo (eds.).

Chemical Induction of Cancer: Modulation and Combination Effects, pp. 73-122.

Boston: Birkh#{228}user Boston, Inc., 1995.

4. Karin, M., and Hunter, T. Transcriptional control by protein phosphorylation:

signal transmission from the cell surface to the nucleus. Curt’. Biol.. 5: 747-757.

1995.

5. Hill, C. S., and Treisman, R. Transcriptional regulation by extracellular sig-

nals: mechanisms and specificity. Cell. 80: 199-21 1, 1995.

6. Cooper. G. M. Oncogenes and growth factors. In: Oncogenes, Chap. 1 1, pp.

163-173, Boston: Jones and Bartlett Publishers, 1990.

7. Chang, C-i., and Geahlen, R. L. Protein-tyrosine kinase inhibition: mecha-

nism-based discovery of antitumor agents. J. Nat. Prod. (Lloydia), 55: 1529-

1560. 1992.

8. Powis, G. Signalling pathways as targets for anticancer drug development.

Pharmacol. & Ther., 62: 57-95, 1994.

9. Powis, G., and Workman, P. Signalling targets for the development of cancerdrugs. Anti-Cancer Drug Des., 9: 263-277. 1994.

10. Levitzki, A.. and Gazit, A. Tyrosine kinase inhibition: an approach to drug

development. Science (Washington DC), 267: 1782-1788, 1995.

I I . Shin, D. M., Ro. J. Y., Hong. W. K.. and Hittelman, W. N. Dysregulation of

epidermal growth factor receptor expression in premalignant lesions during head

and neck tumorigenesis. Cancer Res., 54: 3153-3159. 1994.

12. Levitzki, A. Signal-transduction therapy. A novel approach to disease man-

agement. Eur. J. Biochem.. 226: 1-13. 1994.

I 3. Gullick, W. J. Prevalence of aberrant expression of the epidermal growthfactor receptors in human cancers. Br. Med. Bull., 47: 87-98, 1991.

14. Salomon, D. S., Brandt. R., Ciardiello, F., and Normanno, N. Epidermal

growth factor-related peptides and their receptors in human malignancies. Cnt.

Rev. Oncol. Hematol., /9: 183-232, 1995.

15. Kumar, V., Bustin, S. A., and McKay, I. A. Transforming growth factor a.

Cell Biol. Int., /9: 373-388, 1995.

16. Lee, D. C., Fenton. S. E., Berkowitz, E. A., and Hissong. M. A. Transforming

growth factor a: expression. regulation. and biological activities. Pharmacol.

Rev., 47: 51-85, 1995.

17. Salomon, D. S., Normanno, N.. Ciardiello, F., Brandt, R., Shoyab. M., and

Todaro, G. I. The role of amphiregulin in breast cancer. Breast Cancer Res. Treat..

33: 103-114, 1995.

18. Boonstra. J.. Rijken. P.. Humbel, B.. Cremers, F.. Verkleij. A., and van

Bergen-Henegouwen. P. The epidermal growth factor. Cell Biol. mt., 19: 413-

430, 1995.

19. Malarkey, K., Belham. C. M., Paul, A., Graham. A.. McLees, A., Scott, P. H..

and Plevin, R. The regulation of tyrosine kinase signalling pathways by growthfactor and G-protein-coupled receptors. Biochem. I.. 309: 361-375, 1995.

20. Briscoe, J., Guschin. D., and Muller, M. Just another signalling pathway.

Corn Biol., 4: 1033-1035, 1994.

21. Ekstrand, A. I., Longo, N., Hamid, M. L., Olson, I. L., Liu, L., Collins, V.P., and James, C. D. Functional characterization of an EGF receptor with atruncated extracellular domain expressed in glioblastomas with EGFR gene

amplification. Oncogene, 9: 2313-2320, 1994.

22. Ekstrand, A. J., Liu, L., He, I., Hamid, M. L., Longo. N.. Collins. V. P.. and

James, C. D. Altered subcellular location of an activated and tumour-associated

epidermal growth factor receptor. Oncogene. /0: 1455-1460, 1995.

23. Nishikawa, R., ii, X-D., Harmon, R. C., Lazar, C. S., Gill, G. N., Cavenee,W. K., and Huang. H-J. S. A mutant epidermal growth factor receptor common

in human glioma confers enhanced tumongenicity. Proc. Natl. Acad. Sci. USA.

91: 7727-7731.

24. Garcia de Palazzo, I. E., Adams. G. P., Sundareshan, P., Wong. A. I., Testa,J. R.. Bigner. D. D.. and Weiner. L. M. Expression of mutated epidermal growth

factor receptor by non-small cell lung carcinomas. Cancer Res., 53: 3217-3220,

1993.

25. Wikstrand, C. I., Hale, L. P., Batra, S. K., Hill, M. L., Humphrey, P. A.,Kurpad, S. N., McLendon, R. E., Mostacello, D., Pegram. C. N., Reist, C. I.,

Traweek, S. T., Wong, A. I., Zalutsky, M. R., and Bigner, D. D. Monoclonalantibodies agalnst EGFRvIII are tumor specific and react with breast and lung

carcinomas and malignant gliomas. Cancer Res., 55: 3140-3148, 1995.

26. Moscatello, D. K., Holgado-Madruga, M., Godwin, A. K., Ramirez, 0.,

Gunn, 0., Zoltick, P. W., Biegel, I. A., Hayes. R. L.. and Wang. A. J. Frequent

expression of a mutant epidermal growth factor receptor in multiple humantumors. Cancer Res., 55: 5536-5539, 1995.




27. Carter, T. H., and Kung, H. I. Tissue-specific transformation by oncogenicmutants of epidermal growth factor receptor. Crit. Rev. Oncog., 5: 389-428,

I 994.

28. Soler, C., Alvarez, C. V., Beguinot. L.. and Carpenter. 0. Potent SHCtyrosine phosphorylation by epidernal growth factor at low receptor density or inthe absence of receptor autophosphorylation sites. Oncogene. 9: 2207-2215,

1994.

29. Jhappan. C., Stahle. C.. Harkins, R. N.. Fausto, N., Smith, G. H., and Merlino,

0. T. TGFa overexpression in transgenic mice induces liver neoplasia and

abnormal developmentofthe mammary gland and pancreas. Cell. 61: 1137-1 146,1990.

30. Sandgren. E. P., Luetteke, N. C.. Palmiter, R. D., Brinster, R. L., and Lee. D.

C. Overexpression of TGFa in transgenic mice: induction of epithelial hyperpla-

sia, pancreatic metaplasia. and carcinoma of the breast. Cell. 61: 1 121. 1990.

31. Murakami, H., Sanderson, N. D., Nagy, P., Marina, P. A., Merlino, 0., and

Thorgiersson. S. S. Transgenic mouse model for synergistic effects of nuclear

oncogenes and growth factors in tumorigenesis: interaction of c-myc and trans-

forming growth factor a in hepatic oncogenesis. Cancer Res., 53: 1719-1723,

1993.

32. Sandgren. E. P., Luetteke. N. C.. Qiu. T. H.. Palmiter. R. D.. Bnnster, R. L..and Lee, D. C. Transforming growth factor a dramatically enhances oncogene-

induced carcinogenesis in transgenic mouse pancreas and liver. Mol. Cell. Biol.,

13: 320-330, 1993.

33. Ziober, B. L., Willson, J. K. V., Hymphrey. L. E., Childress-Fields. K., and

Brattain, M. 0. Autocrine transforming growth factor a is associated with

progression of transformed properties in human colon cancer cells. J. Biol. Chem.,

268: 691-698, 1993.

34. Stoscheck. C. M., and King. L. E., Jr. Role of epidermal growth factor incarcinogenesis. Cancer Res.. 46: 1030-1037, 1986.

35. Glick, A. B., Spom, M. B.. and Yuspa. S. H. Altered regulation of TGFfJI

and TGFa in primary keratinocytes and papillomas expressing v-Ha-ras. Mol.Carcinog.. 4: 210-219, 1991.

36. Dlugosz, A. A., Cheng. C., Williams, E. K., Darwiche, N., Dempsey, P. J.,

Mann, B., Dunn, A. R., Coffey. R. J.. Jr., and Yuspa. S. H. Autocrine transforming

growth factor a is dispensable for v-rasHa-induced epidermal neoplasia: potential

involvement of alternate epidermal growth factor receptor ligands. Cancer Res.,

55: 1883-1893, 1995.

37. Lippman. S. M., Lee. I. S., Lotan. R., Hittelman, W., Wargovich. M. J.. and

Hong. W. K. Biomarkers as intermediate end points in chemoprevention trials.

I. NatI. Cancer Inst.. 82: 555-560, 1990.

38. Grandis. J. R.. and Tweardy. D. J. TGFa and EGFR in head and neck cancer.

J. Cell. Biochem., 17F: 188-191, 1993.

39. Bergler, W., Bier, H.. and Ganzer. U. The expression of epidermal growth

factor receptors in the oral mucosa of patients with oral cancer. Acta Oto-Rhino-

Laryngol. BeIg., 246: 121-125, 1989.

40. Shirasuna, K., Hayashido, Y., Sugiyama, M., Yoshioka. H., and Matsuya, T.Immunohistochemical localization of epidermal growth factor (EGF) and EGF

receptor in human oral mucosa and its malignancy. Virchows Arch. A Pathol.

Anal. Histopathol., 418: 349-353. 1991.

41 . Hong, W. K., Lippman. S. M.. and Wolf, 0. T. Recent advances in head andneck cancer-larynx preservation and cancer chemoprevention: the Seventeenth

Annual Richard and Hinda Rosenthal Foundation Award Lecture. Cancer Res.,

53: 5113-5120, 1993.

42. Lippman, S. M., Garewal, H. S., and Meyskens. F. L. Retinoids as potential

chemopreventive agents in squamous cell carcinoma of the head and neck. Prey.

Med.. 18: 740-748, 1989.

43. Beenken, S. W., Huang. P., Sellers, M., Peters, 0., Listinsky. C., Stockard,

C.. Hubbard. W., Wheeler, R.. and Grizzle, W. Retinoid modulation of biomar-kers in oral leukoplakia/dysplasia. J. Cell. Biochem., 19 (Suppl.): 270-277, 1994.

44. Yoneda, K. Distribution of proliferating-cell nuclear antigen and epidermalgrowth factor receptor in intraepithelial squamous cell lesion of human bronchus.

Mod. Pathol., 7: 480-486, 1994.

45. Rusch, V., Klimstra, D., Linkov, I., and Dmitrovsky, E. Aberrant expression

of p53 or the epidermal growth factor receptor is frequent in early bronchial

neoplasia. and coexpression precedes squamous cell carcinoma development.

Cancer Res., 55: 1365-1372, 1995.

46. Jankowski, J., Hopwood. D.. and Wormsley, K. 0. Flow-cytometric analysis

of growth-regulatory peptides and their receptors in Barrett’s oesophagus and

ocsophageal adenocarcinoma. Scand. J. Gastroenterol., 27: 147-154, 1992.

47. Jankowski, I., McMenemin, R., Yu, C., Hopwood, D., and Wormsley. K. G.

Proliferating cell nuclear antigen in oesophageal diseases. Correlation with trans-

forming growth factor a expression. Gut. 33: 587-591, 1992.

48. Stemmermann, G.. Heffelfinger. S. C.. Noffsinger. A., Hui. Y. Z.. Miller. M.

A., and Fenoglio-Preiser. C. M. The molecular biology of esophageal and gastric

cancer and their precursors: oncogenes, tumor suppressor genes, and growth

factors. Hum. Pathol., 25: 968-981, 1994.

49. Messing, E. M.. and Reznikoff, C. A. Epidermal growth factor and its

receptor: marker of-and targets for-chemoprevention of bladder cancer. J.

Cell. Biochem., 161: 56-62, 1992.

50. Rao, J. Y., Hemstreet, 0. P., III, Hurst, R. E., Banner. R. B., Jones, P. L., Mm,K. W., and Fradet, Y. Alterations in phenotypic biochemical markers in bladder

epithelium during tumongenesis. Proc. Nstl. Acad. Sci. USA. 90: 8287-8291, 1993.

51. Neal. D. E., Sharples, L., Smith, K., Fennelly, J., Hall, R. R., and Harris, A.

L. The epidermal growth factor receptor and the prognosis of bladder cancer.Cancer (Phila.), 65: 1619-1625, 1990.

52. Ware, J. L. Prostate cancer progression. Implications of histopathology.Am. J. Pathol., 145: 983-993, 1994.

53. Turkeri, L. N., Sake, W. A., Wykes, S. M.. Grignon. D. J.. Pontes. J. E.. and

Macoska, J. A. Comparative analysis of epidermal growth factor receptor gene

expression and protein product in benign. premalignant, and malignant prostatetissue. Prostate, 25: 199-205, 1994.

54. Myers. R. B., Kudlow, J. E., and Grizzle. W. E. Expression of transforminggrowth factor-a, epidermal growth factor and the epidermal growth factor recep-

tar in adenocarcinoma of the prostate and benign prastatic hyperplasia. Mod.

Pathol., 6: 733-737, 1993.

55. Koenders, P. 0., Peters, W. H. M., Wobbes, T., Beex. L. V. A. M.,

Nagengast, F. M., and Bendrad, T. J. Epidermal growth factor receptor levels are

lower in carcinomatous than in normal colorectal tissue. Br. J. Cancer, 65:

189-192. 1992.

56. Liu, C., Woo, A., and Tsao. M-S. Expression oftransforming growth factor-ain primary human colon and lung carcinomas. Br. J. Cancer, 6: 425-429. 1990.

57. Tanaka, S., Imanishi, K., Haruma, K., Tsuda, T., Yoshihara, M., Sumii, K..

and Kajiyama, 0. Immunoreactive transforming growth factor-a and epidermalgrowth factor in colorectal adenomas and carcinomas. Oncology (Basel), 49:

381-385. 1992.

58. Markowitz, S. D., Molkentin, K., Gerbic, C., Jackson, J., Stellato. T.. andWilson, J. K. V. Growth stimulation by coexpression of transforming growth

factor-a and epidermal growth factor-receptor in normal and adenomatous human

colon epithelium. J. Clin. Inyest,, 86: 356-362. 1990.

59. Anderson. B. D., Karnes, W. E., and Pearson, R. K. Bile acids regulate the

growth rate of transformed colonic epithelium and increase TGFa mRNA levels

in vitro (Abstract). Gastroenterology. 104: A384, 1993.

60. Saeki. T., Stromberg, K.. Qi. C-F., Gullick, W. J., Tahara, E., Normanno, N.,

Ciardiello, F., Kenney. N., Johnson, 0. R.. and Salomon, D. S. Differentialimmunohistochemical detection of amphiregulin and cripto in human normal

colon and colorectal tumors. Cancer Res., 52: 3467-3473, 1992.

61. Goppinger, A.. Wittmaack, F. M.. Wintzer, H. 0.. Lkenbcrg. H., and

Bauknecht, T. Localization of human epidermal growth factor receptor in cervical

intraepithelial neoplasias. J. Cancer Res. Clin. Oncol., 1 15: 259-263, 1989.

62. Mittal. K., Pearson, J., and Demopoulos. R. Patterns of mRNA for epidermalgrowth factor receptor and keratin B-2 in normal cervical epithelium and in

cervical intraepithelial neoplasia. Gynecol. Oncol.. 38: 224-229, 1990.

63. Chapman, W. B., Lorincz. A. T., Willett, 0. D.. Wright. v. C., and Kurman.R. J. Epidermal growth factor receptor expression and the presence of human

papilloma virus in cervical squamous intraepithelial lesions. Int. J. Gynecol.Pathol., 11: 221-226, 1992.

64. Tervahauta, A., Syijanen, S., and Syijanen, K. Epidermal growth factor receptor.

c-erbB-2 proto-oncogene and estrogen receptor expression in human papillomavirus

lesions of the uterine cervix. tnt. I. Gynecol. Pathol.. 13: 234-240, 1994.

65. Mitchell, M. F., Hittelman. W. N., Lotasi, R., Nishioka, K., Tortolero-Luna,

0.. Richards-Kortum, R., and Hong, W. K. Chemoprevention trials in the cervix:

design, feasibility. and recruitment. J. Cell. Biochem., 23: 104-1 12, 1995.

66. McGlennen, R. C., Ostrow, R. S., Carson, L F., Stanley. M. S.. and Faras, A. J.Expression of cytokine receptors and markers of differentiation in human papillo-

mavinis-infected cervical tissues. Am. J. Obstet. Gynecol., 165: 696-705, 1991.

67. Sizemore, N., and Rorke, H. A. Human papillomavinss 16 immortalization ofnormal ectocervical epithelial cells alters retinoic acid regulation of cell growth and

epidermal growth factor receptor expression. Cancer Ret.. 53: 451 1-4517, 1993.

68. Meyskens, F. L., Jr., Surwit, E., Moon. T. E., Childers. J. M., Davis, J. R.,

Dorr, R. T., Johnson, C. S., and Alberts, D. S. Enhancement of regression ofcervical intraepithelial neoplasia II (moderated dysplasia) with topically applied

all-trans-retinoic acid: a randomized trial. J. NatI. Cancer Inst., 86: 539-543,

1994.

69. Klijn, J. 0. M., Berns, P. M. J. J.. Schmitz, P. I. M., and Foekens. J. A. The

clinical significance of epidermal growth factor receptor (EGF-R) in humanbreast cancer: a review on 5232 patients. Endocr. Rev., 13: 3-17. 1992.

70. Ethier, S. P. Growth factor synthesis and human breast cancer progression.J. Nail. Cancer Inst.. 87: 964-973, 1995.




7 1 . van Agthoven, T., Timmermans, M.. Fockens, J. A.. Dorssers, L. C. I., and

Henzen-Logmans. S. C. Differential expression of estrogen. progesterone, andepidermal growth factor receptors in normal. benign. and malignant human breast

tissues using dual staining immunohistochemistry. Am. I. Pathol.. 144: 1238-

1246, 1994.

72. Fabian, C. J.. Zalles, C., Kamel, S., Kinder, B. F., McKittrick, R., Tranin, A. S.,

Zeiger. S.. Moore, W. P.. Hassanein. R. S.. Simon, C., Johnson, N., Vergara. G.,iewell, W. R.. Lin, F., Bhatia, P., and Chin, T. Prevalence of aneuploidy. overex-pressed ER. and overexpressed EGFR in random breast aspirates of women at high

and low risk for breast cancer. Breast Cancer Res. Treat.. 30: 263-274, 1994.

73. Gudas, L. I., Spom. M. B., and Roberts, A. B. Cellular biology and bia-

chemistry of the retinoids. In: M. B. Spom. A. B. Roberts, and D. S. Goodman(eds.), The Retinoids: Biology, Chemistry, and Medicine. Ed. 2. Chap. 11. pp.

443-520. New York: Raven Press, Ltd., 1994.

74. Masui. H., Kawamoto, T., Sato, I. D., Wolf, B., Sato. G., and Mendelsohn,

J. Growth inhibition of human tumor cells in athymic mice by anti-epidermal

growth factor receptor monoclonal antibodies. Cancer Res.. 44: 1002-1007, 1984.

75. Stavric, B. Quercetin in our diet: from potent mutagen to probable anticar-

cinagen. Clin. Biochem.. 27: 245-248, 1994.

76. Levitzki. A. Tyrphostins: tyrosine kinase blockers as novel antiproliferative

agents and dissectors of signal transduction. FASEB I., 6: 3275-3282. 1992.

77. Boutin, J. A. Tyrosine protein kinase inhibition and cancer. Int. I. Biochem..

26: 1203-1226, 1994.

78. Barnes, S., and Peterson, T. G. Biochemical targets of the isoflavone genis-

tein in tumor cell lines. Proc. Soc. Exp. Biol. Med.. 208: 103-108, 1995.

79. Barret, I. M., Emould, A. P., Ferry, G., Genton, A.. and Boutin, J. A.

Integrated system for the screening of the specificity of protein kinase inhibitors.

Biochem. Pharmacol., 46: 439-448, 1993.

80. Osherov, N., Gazit, A.. Gilon, C., and Levitzki, A. Selective inhibition of the

epidermal growth factor and HER2/neu receptors by tyrphostins. J. Biol. Chem.,

268: 11134-11142, 1993.

81. Moon, R. C., Mehta, R. G., and Rao, K. V. N. Retinoids and cancer in

experimental animals. In: M. B. Spom, A. B. Roberts, and D. S. Goodman (edt.),

The Retinoids: Biology. Chemistry, and Medicine, Ed. 2, Chap. 14, pp. 573-595.

New York: Raven Press, Ltd., 1994.

82. Gazit, A., Osherov, N., Posner, I., Bar-Sinai, A., Gilon, C., and Levitzki, A.

Tyrphostins. 3. Structure-activity relationship studies of a-substituted benzyli-

denemalononitrile 5-S-aryltyrphostins. I. Med. Chem.. 36: 3556-3564, 1993.

83. Ben-Bassat, H., Vardi, D. V., Gazit, A., Klaus, S. N., Chaouat, M.,

Hartzstark, Z., and Levitzki, A. Tyrphostins suppress the growth of psoriatickeratinocytes. Exp. Dermatol.. 4: 82-88, 1995.

84. Yoneda, T., Lyall. R. M., Alsina, M. M., Persons. P. E., Spada, A. P.,Levitzki, A., Zilberstein, A., and Mundy, G. R. The antiproliferative effects of

tyrosine kinase inhibitors tyrphostins on a human squamous cell carcinoma in

s’itro and in nude mice. Cancer Res., Si: 4430-4435, 1991.

85. Khetarpal, V. K.. Markham, P. M., and Ziemniak, I. A. Dispositional char-

acteristics of a tyrosine kinase inhibitor (RG 14620) in rats and rabbits following

intravenous administration of dermal application. Drug Metab. Dispos.. 22:

216-223, 1994.

86. Reddy, K. B.. Mangold. G. L., Tandon. A. K.. Yoneda, T., Mundy. G. R..

Zilberstein, A., and Osborne, C. K. Inhibition of breast cancer cell growth in vitro

by a tyrasine kinase inhibitor. Cancer Res., 52: 3636-3641, 1992.

87. Buchdunger. E., Trinks, U., Mett, H., Regenass, U., Muller, M., Meyer. T.,

McGlynn, E., Pinna, L. A., Traxler, P., and Lydon, N. B. 4,5-Dianilinophthalim-

ide: a protein-tyrosine kinase inhibitor with selectivity for the epidermal growth

factor receptor signal transduction pathway and potent in rico antitumar activity.Proc. NatI. Acad. Sci. USA, 9!: 2334-2338, 1994.

88. Buchdunger. E., Mett, H., Trinks. U., Regenass, U., Muller, M.. Meyer. T..

Beilstein, P., Wirz, B., Schneider, P., Traxler, P., and Lydon, N. B. 4.5-bis(Flu-oroanilino)phthalimide: a selective inhibitor of the epidermal growth factor re-

ceptor signal transduction pathway with potent in vito antitumor activity. Clin.

Cancer Ret., 1: 813-821, 1995.

89. Fry. D. W., Kraker. A. I., McMichael, A., Ambroso. L. A.. Nelson, I. M.,Leopold. W. R., Connors, R. W.. and Bridges. A. J. A specific inhibitor of the

epidermal growth factor receptor tyrosine kinase. Science (Washington DC), 265:

1093-1095, 1994.

90. Hook, K. E., Kunkel, M. W., Elliot, W. L., Howard, C. T., and Leopold, W.

R. Epidermal growth factor receptor tyrosine kinase in A43 I xenografts: inhibi-

tion by PD 153035 14-(3.bromoanilina)-6,7-dimethaxyquinazolinel. Proc. Am.

Assoc. Cancer Res., 36: 434, 1995.

91. Rewcastle, G. W., Denny, W. A., Bridges. A. J., Zhou. H., Cody. D. R..

McMichael, A., and Fry. D. W. Tyrosine kinase inhibitors. 5. Synthesis and

structure-activity relationships for 4-[(phenylmethyl)aminoj- and 4-(phenylami-

no)quinazolines as potent adenosine 5’-triphosphate binding site inhibitors of the

tyrasine kinase domain of the epidermal growth factor receptor. J. Med. Chem.,38: 3482-3487, 1995.

92. Ward, W. H. i.. Cook, P. N., SIster, A. M., Davies, D. H., Holdgate, 0. A.,

and Green, L. R. Epidermal growth factor receptor tyrosine kinase. Investigationof catalytic mechanism, structure-based searching and discovery of a potentinhibitor. Biochem. Pharmacol., 48: 659-666, 1994.

93. Barker, A. I.. Davies, D. H. (inventors), and Imperial Chemical Industries.

PLC (assignee). Pharmaceutical composition comprising quinazoline derivatives

inhibit receptor tyrosine kinase(s) and are used for treating cancers e.g., Ieuke-

mia(s) and tumors of breast, lung, colon, and rectum. European Patent 520,722.

December 30. 1992.

94. Thompson. A. M.. Bridges, A. J.. Fry, D. W., Kraker, A. J., and Denny, W.

A. Tyrosine kinase inhibitors. 7. 7-Amino-4-(phenylamino)- and 7-amino-4-[(phenylmethyl)aminojpyrido[4.3-iflpydmidines: a new class of inhibitors of the

tyrasine kinase activity of the epidermal growth factor receptor. J. Med. Chem.,38: 3780-3786, 1995.

95. Traxler, P., TrinkS, U., Buchdunger, E., Mett. H., Meyer. T., Muller. M.,

Regenass, U., Rosel, J., and Lydon, N. l(Alkylamino)methyljacrylophenones:

potent and selective inhibitors of the epidermal growth factor receptor protein

tyrosine kinase. J. Med. Chem., 38: 2441-2448. 1995.

96. Tanaka, S., Okabe, T., Chieda, S., Endo, K., Kanoh, T., Okura, A., andYoshida, E. BE-23372M, a novel and specific inhibitor for epidermal growth

factor receptor kinase. Jpn. J. Cancer Res,, 85: 253-259, 1994.

97. Chen, H., Boiziau, J., Parker, F., Mailliet, P., Commercon, A., Tocque. B.. Ic

Pecq, i-B., Roques, B-P., and Garbay, C. Structure-activity relationships in aseries of 5-[(2.5-dihydroxybenzyl)aminolsalicylatc inhibitors of EGF-receptor-

associated tyrosine kinase: importance of additional hydrophobic aromatic inter-actions. J. Med. Chem., 37: 845-859, 1994.

98. Fry. D. W., Kraker, A. I., Connors, R. C.. Elliott, W. I., Nelson. J. M.,

Showalter, H. D. H., and Leopold, W. R. Strategies for the discovery of noveltyrosine kinase inhibitors with anticancer activity. Anti-Cancer Drug Des., 9:

331-351, 1994.

99. Thompson. A. M., Rewcastle, G. W., Tercel, M., Dobrusin, E. M., Fry, D.

W., Kraker, A. J., and Denny, W. A. Tyrosine kinase inhibitors. I . Structure-

activity relationships for inhibition of epidermal growth factor receptor tyrosinekinase activity by 2.3-dihydro-2-thioxo-lH-indole-3-alkanoic acids and 2,2’-

dithiobis(lH-indole-3-alkanoic acids). J. Med. Chem., 36: 2459-2469, 1993.

100. Palmer, B. D., Rewcastle, G. W., Thompson. A. M., Boyd. M.. Showalter,

H. D. H., Serccl, A. D., Fry, D. W., Risker, A. J.. and Denny, W. A. Tyrosinekinase inhibitors. 4. Structure-activity relationships among N- and 3-substituted2,2’-dithiobis(lH-indoles) for in vitro inhibition of receptor and nonreceptor

protein tyrosine kinascs. J. Med. Chem., 38: 58-67, 1995.

101. Cushman, M., Thu. H., Geahien, R. L. and Kraker, A. J. Synthesis andbiochemical evaluation of a series of aminoflavones as potential inhibitors of protein-

tyrosine kinases pS6lck, EGFr, and p6Ov-src. I. Med. Chem., 37: 3353-3362, 1994.

102. Andrews, D. M., Gregoriou. M., Page. T. C. M., Peach, J. M., and PraU, A.

J. Potential mechanism-based tyrasine kinase inhibitors. Part 2. Design and

synthesis of peptides containing heterocyclic tyrosine analogues. J. Chem. Soc.

Perkin Trans. I, 11: 1335-1340, 1995.

103. Gimenez-Conti, I. B., and Slaga. T. I. The hamster cheek pouch carcino-

genesis model. I. Cell. Biochem., 17F: 83-90. 1993.

104. Shin, D. M., Gimenez, I. B., Lee, J. S., Nishioka, K., Wargovich, M. J.,

Thacher, S., Lotan, R., Slaga. T. I.. and Hong, W. K. Expression of epidermal

growth factor receptor, polyamine levels, ornithine decarboxyase activity, micro-

nuclei, and transglutaminase I in a 7.12-dimethylbenz(a)anthracene-induced ham-

ster buccal pouch carcinogenesis model. Cancer Res.. 50: 2505-2510. 1990.

105. Steele, V. E., Moon, R. C., Lubet, R. A., Grubbs, C. I., Reddy. B. S.,

Wargovich, M., McCormick, D. I., Pereira, M. A., Crowell, J. A., Bagheri. D..Sigman, C. C., Boone, C. W., and Kelloff, G. J. Preclinical efficacy evaluation of

potential chemopreventive agents in animal carcinogenesis models: methods andresults from the NCI chemoprevention testing program. J. Cell. Biochem. Suppl.,

20: 32-54, 1994.

106. Burke, T. R.. Jr. Protein-tyrosine kinases: potential targets for anticancer

drug development. Stem Cells, 12: 1-6, 1994.

107. Hubbard, S. R., Wei, I., Ellis, I., and Hendrickson, W. A. Crystal structureof the tyrosine kinase domain of the human insulin receptor. Nature (Land.), 372:

746-754, 1994.

108. Knighton. D. R.. Cadena. D. I., Zheng, J., Ten Eyck. I. F.. Taylor, S. S.,

Sowadski, J. M., and Gill, G. N. Structural features that specify tyrosine kinase

activity deduced from homology modeling of the epidermal growth factor recep-tar. Proc. Natl. Acad. Sci. USA, 90: 5001-5005, 1993.



1996;5:657-666. Cancer Epidemiol Biomarkers Prev G J Kelloff, J R Fay, V E Steele, et al. potential cancer chemopreventives.Epidermal growth factor receptor tyrosine kinase inhibitors as

Updated version

http://cebp.aacrjournals.org/content/5/8/657

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cebp.aacrjournals.org/content/5/8/657To request permission to re-use all or part of this article, use this link



http://cebp.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

Epidermal Growth Factor Receptor Tyrosine Kinase ...selected as a potential target for chemoprevention. Because growth factor networks are redundant, selective inhibition of signaling