PD153035, a Tyrosine Kinase Inhibitor, Prevents …...PD153035, a Tyrosine Kinase Inhibitor,...

Vol. 3, 2099-2/06, November /997 Clinical Cancer Research 2099

3 The abbreviations used are: EGF. epidermal growth factor: MAb.

monoclonal antibody: FBS, fetal bovine serum.

PD153035, a Tyrosine Kinase Inhibitor, Prevents Epidermal Growth

Factor Receptor Activation and Inhibits Growth of Cancer Cells in

a Receptor Number-dependent Manner’

Monique Bos, John Mendelsohn,

Young-Mee Kim, Joan Albanell, David W. Fry,

and Jose Baselga2Laboratory of Receptor Biology and Department of Medicine,

Memorial Sloan-Kettering Cancer Center. New York, New York10021 [M. B., J. M., Y-M. K., J. B.]: Department of MedicalOncology. Vall d’Hebron University Hospital, 08035 Barcelona.Spain [J. A., J. B.]; and Parke-Davis Pharmaceutical Research,

Division of Warner-Lambert Company, Ann Arbor, Michigan 48105

[D. W. F.]

ABSTRACT

PD153035 is reported to be a specific and potent inhib-

itor of the epidermal growth factor (EGF) receptor tyrosine

kinase and, to a lesser degree, of the closely related HER2/

neu receptor. We show that PD153035 inhibits EGF-de-

pendent EGF receptor phosphorylation and suppresses the

proliferation and cbonogenicity of a wide panel of EGF

receptor-overexpressing human cancer cell lines. EGF re-

ceptor autophosphorylation in response to exogenous EGF

was completely inhibited at PD153035 concentrations of

>75 n�i in cells overexpressing the EGF receptor. In con-

trast, PD153035 only reduced heregulin-dependent tyrosine

phosphorylation in HER2/neu-overexpressing cell lines at

significantly higher concentrations (1400-2800 nr�i).

PD153035 exposure did not affect the expression of either

EGF receptors or HER2/neu. PD153035 caused a dose-de-

pendent growth inhibition of EGF receptor-overexpressing

cell lines at low micromolar concentrations, and the IC50 in

monolayer cultures was less than 1 p.i�i in most cell lines

tested. At doses of up to 2.5 �M, the IC50 for HER2/neu-

overexpressing cells was not reached. In colony-forming

assays, the PD153035 growth-inhibitory activity in cultures

driven by endogenous (autocrine) ligand was correlated with

EGF receptor number, with higher activity in cells express-

ing higher numbers of EGF receptors and only minimal

activity in cells expressing normal numbers of EGF recep-

tors but high HER2/neu levels. PD153053 also abolished all

Received 5/2 1/97; accepted 7/31/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with I 8 U.S.C. Section 1734 solely to

indicate this fact.

I This work was supported by an American Society of Clinical Oncol-

ogy Career Development Award (to J. B.) and by NIH Grants CA65746and CA37641.2 To whom requests for reprints should be addressed. at Department ofMedical Oncology, Vall d’Hcbron University Hospital. Paseo VaIld’Hcbron 1 19-129, 08035 Barcelona, Spain. Fax: 34-3-2746059; E-mail: [email protected].

growth effects mediated by the addition of exogenous EGF;

this condition could be reversed upon removal of the com-

pound. Cotreatment with C225, an anti-EGF receptor-

blocking monoclonal antibody, further enhanced the antitu-

mor activity of PD153035, suggesting mechanisms of action

for C225 other than competition with ligand binding. This

batter finding also suggests that combined anti-EGF receptor

strategies may be of enhanced benefit against tumors with

high levels of EGF receptor expression.

INTRODUCTION

The EGF3 receptor is a Mr 170,000 plasma membrane

glycoprotein composed of an extracellubar ligand-binding do-

main, a transmembrane lipophilic segment, and an intracellular

protein tyrosine kinase domain with a regulatory COOH-termi-

nab segment ( 1 ). Following bigand binding, EGF receptor dimer-

ization occurs, which results in high-affinity ligand binding,

activation of the intrinsic protein tyrosine kinase activity, and

tyrosine autophosphorylation ( I ). These events lead to activa-

tion of a cascade of biochemical and physiological responses

that are involved in the mitogenic signal transduction of cells

(2).

The EGF receptor has been proposed as a potential target

for new anticancer agents for a variety of reasons: coexpression

of high levels of EGF receptor and its ligands leads to a

transformed cellular phenotype (3, 4); the expression of EGF

receptors is elevated in many epithelial tumors and tumor-

derived cell lines (4, 5); and this overexpression correlates with

a poor clinical outcome in a number of malignancies (6). Fur-

thermore, MAbs that block bigand-induced activation of the

EGF receptor tyrosine kinase inhibit the growth of human tumor

xenografts (7-9).

Recently, the quinazobones have been identified as a new

class of tyrosine kinase inhibitors (10). One of these com-

pounds, PD153035, is a reversible inhibitor of the EGF receptor

tyrosine kinase and, to a lesser degree, of the closely related

HER2/neu receptor ( 1 1 ). Prior studies with cell lysates had

shown that PD153035 inhibited the isolated tyrosine kinase

activity of the EGF receptor at picomolar concentrations but had

little effect against a panel of non-EGF receptor tyrosine kinase

receptors, except for the closely related HER2/neu receptor.

PD 1 53035 inhibited EGF receptor autophosphorylation in

mouse fibroblasts and A43 1 human epidermoid cells at nano-

molar concentrations (1 1). PD153035 was also capable of

blocking EGF-mediated mitogenesis and oncogenic transforma-

tion in fibroblasts overexpressing EGF receptors ( 1 1).

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2100 PD153035 Treatment of Cancer Cells

Here, we have studied the capacity of PD 153035 to inhibit

ligand-induced EGF receptor phosphorylation and cell growth

in a panel of human carcinoma cell lines with various levels of

EGF receptor and/or HER2/neu receptor overexpression. We

report that PD153035-mediated inhibition is dependent upon the

level of expressed EGF receptor numbers, with less activity

against cells that have lower receptor number and only HER2/

izeu receptor overexpression. Interestingly. cotreatment with

C225. an anti-EGF receptor MAb, further enhances the antitu-

mor activity of PD153035, suggesting that combined anti-EGF

receptor strategies may be of benefit against tumors with high

levels of EGF receptor expression.

MATERIALS AND METHODSCompounds and Antibodies. Tyrosine kinase inhibitor

PD153035 and anti-EGF receptor MAbs 225 and 528 have been

described previously (7, 8. 1 1 ). A human-murine chimeric ver-

sion of MAt, 225 (C225) was used for these experiments (12).

Rabbit polyclonal antibody RK-2, against the COOH-terminal

portion of the EGF receptor, was a gift from Dr. J. Schlessinger

(New York University Medical Center, New York, NY). Anti-

HER2/neu Ab2 and Ab3 MAbs were from Oncogene Science

(Cambridge, MA). EGF was from Collaborative Research (Wal-

tham, MA). Antiphosphotyrosine antibody 4GlO was from Up-

state Biotechnology Inc. (Lake Placid, NY). Heregulin, a bigand

that phosphorylates HER2/neu, was a gift from Dr. M.

Slikowski (Genentech Inc., South San Francisco, CA).

Cell Lines and Culture Media. For the present studies,

cell lines with various levels of either EGF receptor or HER2/

neti expression were chosen. Vulvar squamous carcinoma cells

(A431), breast adenocarcinoma cells (BT474, MDA-MB-231,

MDA-MB-453, MDA-MB-468, and SK-BR-3), colon adeno-

carcinoma cells (SW620), prostate adenocarcinoma cells

(DU145), and cervical squamous carcinoma cell lines (C4i,

ME18O, and SiHa) were obtained from the American Type

Culture Collection (Rockville, MD). Difi colon adenocarcinoma

cells were kindly provided by Dr. B. Boman (Creighton Uni-

versity, Omaha, NE). The breast adenocarcinoma cell line

MCF7/HER-2, transfected with HER2/neu, was a gift of Dr. C.

Benz (University of California, San Francisco, CA). The num-

bers of EGF receptors per cell in the studied cell lines were as

follows: Difi, 4.8-5 x 10� receptors/cell ( 13); A43 1 , 2 X I0�

receptors/cell ( 14); MDA-MB-468, 2 X 10� receptors/cell (15);

DUI45, 2.5 x l0� receptors/cell (16); MDA-MB-23l, 2.5 X

l0� receptors/cell ( 17): BT-474, I .4 X 10� receptors/cell (18);

SK-BR-3, I X l0� receptors/cell (17); ME-l80, 8.6 X l0�

receptors/cell ( 19); SiHa, 5 X l0� receptors/cell ( 19); and C4i,

2.2 X � receptors/cell ( 19). SW620, MDA-MB-453, and

MCF7IHER-2 cells were considered to have low EGF receptor

levels because the receptor could not be detected by Western

blot analysis, a method that correlates well with receptor number

(17). HER2/neu-overexpressing cell lines MD-MB-453, BT474,

SK-BR-3. SW620, and MCF7IHER-2 all had HER2/neu levels

of >40 ng/mg protein (Ref. 20 and data not shown). The CMK

megakaryoblastic leukemia cell line, which lacks EGF receptors

and HER2/neu, was kindly provided by Dr. H. Avraham (Beth

Israel Deaconess Medical Center, Boston, MA).

A43l, MDA-MB-231, MDA-MB-453, MDA-MB-468,

SK-BR-3, DU145, and Difi cells were grown in monolayer

culture with DMEM and Ham’s F-l2 medium (1 : 1), with 10%

FBS. BT474 cells were grown in DMEMIHam’s F-l2 medium

(1: 1) with 10% FBS, 300 mg/liter L-glutamine, and 10 p.g/ml

human insulin. SW620, C4i, SiHa, and ME18O cells were grown

in RPMI 1640 with 10% FBS, 300 mg/liter L-glutamine, 100

units/ml penicillin, and 100 �ig/ml streptomycin. MCF7IHER-2

cells were cultured in DMEM/Hl6 medium (1 g/Iiter glucose),

with 10% FBS, 100 units/mi penicillin, 100 units/mi streptomy-

cm, and 400 �g/ml G-418. CMK cells were cultured in Iscove’s

modified Dulbecco’s medium with 10% FBS, 300 �ig/m1 L-

glutamine, 100 p.g/ml ampicillin, and 50 p.g/ml kanamycin. All

cells were grown at 37#{176}Cand 5% CO,.

Western Immunobbotting. Selected cell lines were

grown in 100-mm dishes until subconflucnce and then incubated

in serum-free medium for 18 h. The monolayers were exposed

to various concentrations of PD153035 for 2 h and then treated

with either EGF (20 nM) for 10 mm or heregulin (20 nM) for 15

mm. Then, the medium was removed, the cells were washed

twice with cold PBS, and the monolayer was scraped into 1 ml

of ice-cold lysis buffer [50 mtvi Hepes (pH = 7), 150 msi NaCI,

10% glycerol, 1% Triton X-lOO, 5 m�vt EDTA, 1 mM MgC1,, 25

mM NaF, 1 mM sodium orthovanadate, I mr�i phenylmethylsul-

fonyl fluoride, 25 �g/ml aprotinin, 50 jig/mi leupeptin, and 1%

bovine albumin]. The lysate was transferred to a microfuge tube,

placed on ice for 15 mm, and centrifuged S mm at 10,000 X g.

The supernatant was transferred to a clean microfuge tube,

followed by immunoprecipitation with either anti-EGF receptor

MAb 528 (5 rig) or anti-HER2/neu MAb Ab2 (30 p.g) for 2 h at

4#{176}C.Immunocomplexes were harvested by the addition of 50 p.1

of protein A-Sepharose in PBS and washed 4 times with the

buffer by centrifugation. Precipitates were resuspended in 30 p.1

of Laemmli buffer, heated to 100#{176}Cfor S mm, and centrifuged

to obtain the supernatant. Samples were loaded onto a 6% SDS

polyacrylamide gel, followed by overnight transfer to polyvi-

nylidene difluoride membranes. Membranes were blocked with

a solution containing 150 mM NaCI, 1% BSA, 0.1% Tween 20,

and 20 nM Tris (pH 7.4) for I h, then incubated for an additional

hour with either the antiphosphotyrosine antibody (1:15,000),

anti-EGF receptor RK-2 antibody (1 :2,000), or anti-HER2/neu

Ab3 ( I :7,500). Membranes were washed four times for S mm

each in the same solution and incubated for 45-60 mm with a

1: 10,000 dilution of horseradish peroxidase-conjugated second-

ary antibody. Subsequently, membranes were vigorously

washed four times for 5 mm each in the same solution, followed

by a 1-mm incubation with a luminol-based solution and chemi-

luminescent detection.

Monolayer Growth Assay. Cells were seeded in six-

well plates (model 3046; Falcon, Lincoln Park, NJ) at l0�

cells/well. The next day, cells were changed to medium con-

taming 0.5% FBS for 18 h, and then PD153035 was added at

various concentrations to the cultures. After 72 h of treatment,

cells were washed once with PBS, harvested with 0.1% human

trypsin-l mM EDTA in PBS, and counted with a Coulter coun-

ter. The CMK cells grow in suspension and, therefore, did not

require trypsmnization.

Soft Agar Colony-forming Assay. To perform soft agar

assays, a bottom layer of 1 ml of the corresponding culture

medium containing 0.7% agar (Difco Laboratories, Detroit, MI)



PD153035 (nM)A I

U- Ig�

e, �n in �,j � ig) 0

Difi � C’) r-

A431 #{149}s

MDA-MB468 �

PD153035 (nM)

p170

..-p170

�- p170

.�-p185

.�-p185

-.w � � � � � I-

PD153035 (nM)

Difi anti-P-tyr

Difianti.EGFr

. �, ww

PD153035 (nM)

II) �0 0 0 0

�3 It) � I�l it) 0 �C) I c� �- L!::J �“ �‘- �

�I

� “1 csj It) It) 0 �

C) I c’j ‘- tV-. � �

SKbr3 ‘� � �,.

SW620 #{149}�S� �

MCF7/HER-2 __

B I

SKbr3 anti-P-tyr

SKbr3 � -anti-HER-2 � -- �- p185

Fig. I Inhibitory capacity of PD153035 on EGF receptor tyrosine

kinase and HER2/neu tyrosine kinase was evaluated by tyrosine kinase

phosphorybation assays. A. EGF receptor-overexpressing cell lines Difi.A43l. and MDA-MB-468 were treated with PD153035 at various con-centrations for 2 h and then with 20 nM EGF for 10 mm (top). PD 153035

treatment at concentrations of >75 nM resulted in complete inhibition ofEGF receptor phosphorylation. Bottom. HER2/neu-overexpressing cell

lines SK-BR-3, SW620, and MCF7/HER-2 were treated with PD 153035

at various concentrations for 2 h and then with 20 nM heregulin for 15

mm. PD153035 only inhibited HER2/neu phosphorylation at the highestconcentrations tested ( 1400-2800 nM). B. increasing concentrations of

PD153035 did not affect the expression of EGF receptor protein in Dificells, as shown in Western blotting assays with antibody RK2 (top).

Likewise, PD153035 did not affect HER2/neu protein levels in 5K-

BR-3 cells, as determined by Western blotting with antibody Ab3(bottom).

and 10% FBS was prepared in 35-mm six-well plates (model

3046; Falcon). After the bottom layer was solidified, 20,000

cells per dish were added in 1 .5 ml of culture medium contain-

ing the sample, 0.35% agar, and 10% FBS. The additions also

contained PD153035 at different concentrations. Triplicates

were performed for every condition. Cells were incubated for

1 1-14 days at 37#{176}Cin 5% CO, atmosphere. Colonies with more

Clinical Cancer Research 2101

0U. Lfl � � 0C.,, to to � E�J � �

C) Woc� �- C’) N.

Table I Inhibitory effects of PD153035 in cultures of EGF receptor

versus HER-2/nezi-overexpressing human cancer cell lines

Relative cell count (%)“IC3, �-____

Cell line (tiM) 0.125 P.M 0.25 tiM 0.5 � 2.5 tiM

EGF receptorA431 0.22 84 51 18 3

Difi 0.3 68 54 27 22

DU145 0.4 68 54 38 22MDA-MB-468 0.68 77 63 51 36

ME18O 0.95 87 60 53 38

C4i 2.5 77 65 60 52

SiHa NR” 78 78 73 66

HER-2/neu

SK-BR-3 NR 102 105 90 51MDA-MB-453 NR 99 96 96 81MCF7IHER-2 NR 86 84 79 67

SW620 NR 100 98 95 91Control’

CMK NR 104 95 1(X) 84

�- p185 “ Results are expressed as a percentage of the cell

untreated cell cultures from the same cell line.

1’ NR. not reached.

( The non-EGF receptor/non-HER-2/neu-bearing leukemia cell lineCMK was used as a control.

.u.-p170 than 25 cells were then counted manually. The experiments

were repeated, and the results were comparable.

.*- p1 70

RESULTS

Inhibition of Ligand-induced Receptor Phosphoryla-

tion. First, the inhibitory potency of PD 153035 was tested by

analysis of receptor phosphorylation against a panel of cancer

cell lines overexpressing various levels of EGF receptor and

�-. p185 HER2/neu. With EGF receptor-overexpressing cell lines,

PD 153035 caused a dose-dependent decrease in ligand-induced

EGF receptor phosphorylation. In cell lines with the highest

level of EGF receptor overexpression, Difi, A43 1 , and MDA-

MB-468, drug concentrations of �75 nM resulted in complete

inhibition of receptor autophosphorylation (Fig. 1A, top). In all

other treated EGF receptor-expressing cell lines, receptor phos-

phorylation was completely inhibited after treatment with

PD153035 at dose levels of 350 nM or higher. Because EGF

receptor phosphorylation may be decreased indirectly by down-

regulation of the receptor, it was important to assess whether the

effect of PD153035 was caused by decreased EGF receptor

protein levels. This proved not to be the case because increasing

doses of PD153035 did not influence the expression of the EGF

receptor protein despite complete inhibition of receptor phos-

phorylation. as shown by Western blotting with anti-EGF re-

ceptor antibody RK2 (Fig. lB. to/, and data not shown). Pro-

longed exposure to PD153035 (72 h) did not result in receptor

down-regulation (data not shown).

The effect ofPDlS3O3S on HER2/neu was evaluated in the

HER2/neu-overexpressing cell lines SK-BR-3, SW620 and

MCF7IHER-2. In these cell lines, PD153035 inhibited (incom-

pletely) HER2/neu phosphorylation, which was induced by the

addition of heregubin, only at the highest concentrations tested

(1400-2800 nM; Fig. 1A, bottom). This occurred in the absence

of an effect on HER2/neu protein levels, as shown by Western

number in



A B

EGFr+

. CMK (Control)

U C4i

0 ME18O

+ SIHa

#{163}DU 145

0 MDA-MB-231

x DIF1

� MDA-MB-468

1 00

0

0 80

�60

C 400

(320

0

0.1

HER-2lneu+

. CMK (Control)

. SW620

0 SK-BR-3

� MCF7/HER-2

0 MDA.MB-453

0.1 1 3

PD153035 (I.LM)1 3

PD153035 (�.tM)

Fig. 2 Growth-inhibitory effects of PD153035 in colony-forming assays of EGF receptor- or HER2/neu-exprcssing cancer cell lines. Mean colony

number of triplicate experiments was calculated for every treatment point. Data points, percentages of the colony number observed when the samecell line was not exposed to PD153035. The leukemia cell line CMK, which lacks EGF receptors and HER2/neu, was used as a control. A, PD 153035

markedly inhibited the cbonogenic growth of EGF receptor-overexpressing cell lines in a dose-dependent manner. B, PD153035’s effects on

cbonogenic growth of HER2/neu-overcxprcssing cell lines were minor.

41S SK-BR-32.5

0 2a)U

C1.5�

0C0U �.

� 0.5�

ME18O

BT-474

MDA-MB.231

MDA-MB-468


blotting with anti-HER2/neu antibody Ab3 (Fig. 1B, bottom).

Hence, PD153035 was more potent by a factor of one to two

orders of magnitude in inhibiting EGF receptor tyrosmne kinase

compared with HER2/neu tyrosine kinase.

Inhibition of Cell Proliferation in Monolayers. To as-

sess whether this specific EGF receptor tyrosine kinase inhibi-

tory effect resulted in growth inhibition in monolayer cell cub-

ture, different EGF receptor-overexprcssing cell lines (A43 1,

Difi, MDA-MB-468, MDA-MB-231, DU145, SiHa, C4i, and

MEl 80) were treated with PD153035 at increasing concentra-

tions of 0. 125-2.5 p.M. as described in the “Materials and

Methods.” Leukemia cell line CMK, which lacks EGF receptors

and HER2/neu, was used as a control. PD153035 caused a

dose-dependent growth inhibition of EGF receptor-positive cell

lines, beginning at less than micromolar concentrations, and the

IC50 was less than 1 p.M in most cases (Table 1 ). In contrast, in

the HER2/neu-overexpressing cell lines (SK-BR-3, MDA-MB-

453, and MCF7/HER-2), the growth-inhibitory effect of

PD 153035 was very modest and almost comparable with its

effect on the CMK control cells, except at the highest concen-

tration (Table I).

Inhibition of Growth in Soft Agar. To further charac-

terize the growth-inhibitory capacity of PD153035 on the panel

of EGF receptor- and HER2/neu-expressing human cancer cell

lines, a series of cbonogenic growth assays was conducted. Cells

were plated and treated with PD I 53035 at concentrations from

0. 125 to 2.5 p.M. Fig. 14 shows the dose-dependent, growth-

inhibitory effect of PD153035 on the cbonogenic growth of EGF

receptor-overexpressing cell lines. A43 1 cells were not tested

because they do not grow well in soft agar. The inhibition of

clonal growth was clearly more pronounced in EGF receptor-

overexpressing cancer cell lines than it was in HER2/neu-

overexpressing cell lines (Fig. 2B). The relationship between

EGF receptor number and response to PD153035 was further

10 100 1000

Number EGF receptors (xl 0�)

Fig. 3 Relationship between the level of EGF receptor expression and

growth inhibition by PD153035. PD153035 concentrations that resultedin a 50% inhibition of clonogenic growth were plotted versus thenumber of EGF receptors for each cell line tested. As shown, thereappeared to be an exponential relation between the level of EGF recep-tor overexpression and the growth-inhibitory effects of PD153035 forthe different cell lines tested.

analyzed by plotting the IC50 of PD153035 for cell lines versus

their EGF receptor numbers (Fig. 3). We found an exponential

relationship between the number of EGF receptor and growth-

inhibitory effects of PD153035, with increasing PD153035 sen-

sitivity for cell lines expressing higher numbers of receptors

(Fig. 3). The only exception was the HER2/neu-overexpressing

cell line SK-BR-3, which also expresses a high number of EGF

receptors (1 X i05 receptors/cell). In SK-BR-3 cells, PD153035



A. B... : �# ..

,.

a )L ,.

.. a...

. .. . ,- . % . .. S:.. ‘ ‘. I � �

.. . I ‘ I� #{149}:#{149}�,� , ,.

‘ . . . -

:� � �#{149} ‘:“ #{149}

. . . . . . . ft..

V � � _ �,.#{149} . �


Fig. 4 Concentration-depend-

ent effects of PD153035 on

the clonogenic growth of Difi

cells grown in the absence of

PD153035. Size and number of

colonies ofDifi cells when grown

in the absence of PD153035 (A)

and when increasing concentra-

tions of PD153035 were added:

0.125 p.M (B): 0.25 p.M (C); and

0.5 p.M (D). Both the numberand size of the colonies were

inversely related to the dose of

PDI 53035.

induced only a modest inhibition of growth both in monolayer

as well as in soft agar (Figs. 2 and 3). Fig. 4 illustrates the

morphological changes of Difi colonies when treated with

PD153035. Higher drug concentrations resulted in a decrease in

the number as well as the size of the colonies.

Abrogation of EGF-mediated Growth Effects and Re-

versibility. In the cell cultures studied above, proliferation

was driven by exogenous (autocrine) ligand. We evaluated

whether PD153035 was also capable of preventing the growth

effects of exogenous EGF. A43 1 cells are growth stimulated by

EGF at picomolar concentrations but inhibited at higher con-

centrations (21 ), whereas SiHa cells are growth stimulated by all

concentrations of EGF (19). These cells were grown in mono-

layer culture in the absence or presence of PD153035 at con-

centrations of 0.25 or 1 p.M. After 24 h of exposure to

PD153035, 200 pM-l00 nM EGF was added. Treatment with

PD153035 resulted in a complete abolition of EGF-induced

growth effects upon A43l cells (Fig. SA) and SiHa cells (Fig.

SB). The data suggest that inhibition of EGF receptor tyrosine

kinase by PD153053 results in unresponsiveness to the growth-

regulatory activity of exogenous EGF and that this effect is

complete and cannot be reversed by high EGF concentrations.

The growth-inhibitory capacity and the insensitivity to

EGF-induced growth-regulation observed with PD153035 is

reversible upon removal of the compound. In the experiment

shown in Fig. 6, PD153035-containing medium of the EGF-

treated SiHa cells was removed and replaced with fresh me-

dium, in the absence of EGF or containing 2 or 20 nM EGF.

Upon removal of PD153035. cells regained their capacity to

grow and to respond to exogenous EGF. Similar data were

obtained with other cell lines (data not shown).

Combined Therapy with PD153035 and the Anti-EGF

Receptor-blocking Antibody MAb C225. To investigate

whether a combined anti-EGF receptor strategy would be more

efficacious than PD153035 alone, we performed a series of

studies with PD153035 given in combination with the EGF

receptor-blocking MAb C225. This human-murine chimeric

MAb, currently under clinical evaluation (22), is derived from

our well-characterized murine MAb 225, which binds to the

receptor with an affinity comparable to that of the natural ligand

(Kd 2 nM), competes with EGF binding, blocks the activation

of receptor tyrosine kinase by EGF or transforming growth

‘p.’ #{149}#{149}

#{149}1

� � .

S � i’..

Ito � � #{149}#{149} I

e � � .�

.. I . ‘ . _&f �. . . . .�..p.. .‘? -‘.‘-�#{149}�. . ,.‘

I :� . � #{149} ..�.. .. a.... �#{149} , � :.4’:#{149}

. ,.... . �‘L.. �

factor a, and inhibits the growth of cell lines expressing high

level of EGF receptors (7-9, 12).

PD153035 was added to A43 1 cell monolayer cultures, and

MAb C225 was added 24 h later. After 72 h cells were counted.

an additive growth inhibitory effect was found for every treat-

ment point (Fig. 7). This additive effect was observed with all

concentrations ofPDl53O3S, including concentrations (0.25 and

0.5 p.M) higher than those required to achieve complete tyrosine

kinase inhibition in our assay (see Fig. 1A). Thus, even in a

situation of complete inhibition of EGF receptor tyrosine kinase

by PD153035, C225 can further inhibit cell growth, suggesting

the possibility of an alternative mechanism of action.

DISCUSSION

Here, using a large panel of human cancer cell lines ex-

pressing various levels of the EGF receptor and/or the closely

related HER2/neu receptor, we have shown that the quinazolone

PD153035 is a highly specific and potent inhibitor of ligand-

dependent EGF receptor phosphorylation. In addition,

PD153035 suppresses proliferation and cbonogenicity with

greater efficacy in cell lines expressing greater numbers of

receptors. Our studies also show that concentrations that were

active against cells expressing high levels of EGF receptors did

not significantly affect proliferation of cells expressing high

levels of HER2/neu and normal levels of EGF receptors.

Fry et al. (1 1) have previously shown that PD 153035

inhibited isolated EGF receptor tyrosine kinase at picomolar

concentrations and HER2/neu receptor at micromolar concen-

trations. At concentrations as high as 50 p.M, PD153035 had

little effect against isolated recombinant platelet-derived growth

factor receptors, fibroblastic growth factor receptors, colony-

stimulating factor- 1 receptors, insulin receptors, or src tyrosine

kinases. At nanomolar concentrations, PD153035 inhibited

EGF- but not basic fibroblastic growth factor- or platelet-de-

rived growth factor-induced receptor phosphorylation in Swiss

3T3 fibroblasts and EGF receptor autophosphorylation in hu-

man A43 1 epidermoid carcinoma cells. In our studies with a

panel of EGF receptor-expressing cell lines, we have observed

that autophosphorylation was completely inhibited in overex-

pressing cancer cells at concentrations of �75 nM. We also

found that PD153035 did not alter the expression of the EGF



A B

SiHa300

250

‘:‘ 200’

C).0

E 150

C

� 100.

50

0

a-C)

.0

E

C

C)C.)

300

250

200

150

100

50

0�

O-o---o--o-O

A 431

I I I I

0 1 2 3

Days after PD153035 wash-out


0 0.2 2 20 100 0 0.2 2 20 100

EGF(nM) EGF(nM)

Fig. 5 PD153035 prevents EGF-mediated growth effects. A, SiHa cells, which are growth stimulated by EGF, were grown in monolayer culture in

the presence or absence of various concentrations of PD153035 (#{149},0 p.tvi; #{149},0.25 p.M: 0. 1 p.M). After 24 h, EGF at 200 pM, 2 nst, 20 nM, and 100flM was added. Mean cell numbers of triplicate experiments were assayed after 72 h. Data points, percentages of cell numbers compared with the same

cell line grown in the absence of both PD153035 and EGF. B, A431 cells, which are growth stimulated by picomolar concentrations of EGF but

inhibited by higher doses. were cultured under the same conditions as SiHa cells, as described above. PD153035 addition resulted in a complete

inhibition of both growth-stimulatory and -inhibitory effects of EGF in A43l cells.

I

Fig. 6 Reversibility of PD153035 growth inhibition. After 72 h of

treatment with PD153035 at 1 p.M. medium from SiHa cells was

removed and replaced with fresh medium containing EGF at 2 nM (�)or 20 nsi (0) or media alone (#{149},control). Cells were counted daily for

3 days. Data points, percentages of the cell number observed at the time

PD153035 was removed.

receptor, indicating that its effect on EGF receptor tyrosine

kinase was not due to decreased EGF receptor protein levels. In

contrast, PD153035 only reduced tyrosine phosphorylation in

HER2/neu-overexpressing cell lines at concentrations in the

range of 1400-2800 nM. These results are in line with the data

published by Fry et a!. (1 1) on activity against isolated HER2/

neu tyrosine kinase compared with EGF receptor tyrosine

kinase.

PD153035 induced a marked inhibition of growth both in

monolayer and soft agar with cells expressing high levels of

EGF receptors but not with cells bearing normal levels of EGF

receptors and high levels of HER2/neu. The only exception was

observed in SK-BR-3 cells, which overexpress both EGF recep-

tor and HER2/neu and were not significantly growth inhibited

by PD153035 despite inhibition of EGF receptor phosphoryla-

tion (data not shown). These findings are in contrast with the

inhibition of growth observed in BT474 cells, which express

levels of both receptors similar to those expressed by SK-BR-3

cells (Fig. 3). A possible explanation for this discrepancy is that

SK-BR-3 cells have a potential but not obligatory EGF receptor

autocrine loop because they are also resistant to anti-EGF re-

ceptor blocking MAbs (23) and other EGF receptor-targeted

treatment strategies (17).

The degree of growth inhibition observed with PD 153035

was dependent on the number of EGF receptors per cell, with

increased efficacy in cells with higher receptor numbers. This

dependency was better observed in soft agar assays, a more

stringent test of mitogenic capacity because several cycles of

cell division are required to form a detectable colony (Fig. 3).

Prior studies with EGF-receptor-blocking antibodies have

shown that they inhibit the growth of nearly all cells that have

an active EGF receptor autocrine pathway, independent of the

number of receptors (6). Although all of the cells that were

inhibited by PD153035 are known to produce ligand (19, 24),

the clear relationship between increased receptor number and

improved response to treatment with PD153035 may be an

indication that an increased receptor number could result in a

higher capacity for receptor autophosphorylation by additional

mechanisms of receptor activation, such as spontaneous dimer-

ization of receptors.

The observation that PD153035 completely prevented ef-



A 431

0a-

C0U

0

a-C)

.0E

C

C)C.)

100

80 -

60

40

20

0’

C225 (nM) � �‘ �. ‘�� ,�,. ‘4) c� c� 4� ��#{149}�. ‘V��)4) ,�Y�O

PD153035 (p.M) 0 0.1 0.25 0.5

�[1


Fig. 7 Inhibitory effects ofPD153035 in combination withanti-EGF receptor MAb C225in monolayer cultures of A43 Icells. Cell number was deter-mined after 72 h. Columns, cell

growth expressed as percent-ages of the cell number in un-treated cultures of A431 cells(control).

fects of exogenous EGF upon SiHa and A43 1 cells is important

because it shows that the antigrowth effects of this compound

cannot be rescued by supraphysiological amounts of ligand.

Presumably, this is a consequence of the location of the receptor

kinase downstream from the ligand-binding site. As expected,

this effect is different from our observations with anti-EGF

receptor antibodies because carefully titrated amounts of the

ligand can reverse the growth-inhibitory capacity of the MAb

(25). Our studies also show that PD153035 is cytostatic and that

cells regain their growth capacity and ligand sensitivity upon

drug removal. These findings suggest that continuous drug

exposure will be required to achieve clinical efficacy and that

further preclinical studies should take this fact into consider-

ation. In vivo studies with another tyrosine kinase inhibitor have

also shown lack of antitumor activity when drug concentrations

fell bellow the optimal in vitro dose, further supporting the need

for continuous drug exposure (26).

Combined treatment of A431 cells with PD153035 and

antireceptor MAb C225 caused an additive inhibitory effect,

despite the fact that EGF receptor tyrosine activity was com-

pletely blocked with PD153035 concentrations higher than 75

flM (Fig. 1) and could not be rescued with high concentrations of

EGF (Fig. SA). Furthermore, treatment of cells with normal

number of EGF receptors with MAb 225 could inhibit cell

growth in our earlier studies (for review see Ref. 6). This

suggests that MAb C225 might have effects other than compet-

ing with ligand binding and, thereby, preventing tyrosine kinase

activation. Studies by Fan et a!. (27) have shown that MAb 225

produces antibody-mediated EGF receptor dimerization, result-

ing in receptor down-regulation. Down-regulation of receptors

by the antibody appears to be an important factor in its growth-

inhibitory capacity because a monovalent 225 Fab’ fragment,

which retains capacity to block binding and tyrosine kinase

activation of receptors by exogenous ligand, was unable to

induce receptor dimerization and down-regulation and had

weaker antiproliferative capacity (27). In contrast, we have not

observed EGF receptor down-regulation with PD153035 (Fig.

1B). Thus, combined antireceptor treatment strategies with a

tyrosine kinase inhibitor and agents that down-regulate the

receptor may offer a therapeutic advantage.

In conclusion, the data presented here demonstrates the

efficacy and specificity of PD153035 as an anticancer agent

against EGF receptor-overexpressing human tumor cell lines.

Further in vivo studies with this class of specific EGF tyrosine

kinase inhibitors arejustified and may provide the basis for their

future clinical development.

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1997;3:2099-2106. Clin Cancer Res M Bos, J Mendelsohn, Y M Kim, et al. cells in a receptor number-dependent manner.growth factor receptor activation and inhibits growth of cancer PD153035, a tyrosine kinase inhibitor, prevents epidermal

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