Download Identification of Epidermal Growth Factor

Identification of Epidermal Growth Factor Receptor
and c-erbB2 Pathway Inhibitors by Correlation With
Gene Expression Patterns
Katja Wosikowski, Danita Schuurhuis, Kathryn Johnson, Kenneth D. Paull,
Timothy G. Myers, John N. Weinstein, Susan E. Bates*
Background: Growth factor receptor-signaling pathways are
potentially important targets for anticancer therapy. The
interaction of anticancer agents with specific molecular targets can be identified by correlating target expression patterns with cytotoxicity patterns. We sought to identify new
agents that target and inhibit the activity of the epidermal
growth factor (EGF) receptor and of c-erbB2 (also called
HER2 or neu), by correlating EGF receptor, transforming
growth factor (TGF)-a (a ligand for EGF receptor), and
c-erbB2 messenger RNA (mRNA) expression levels with the
results of cytotoxicity assays of the 49 000 compounds in the
National Cancer Institute (NCI) drug screen database. Methods: The levels of mRNAs were measured and used to generate a molecular target database for the 60 cell lines of the
NCI anticancer drug screen. The computer analysis program, COMPARE, was used to search for cytotoxicity patterns in the NCI drug screen database that were highly correlated with EGF receptor, TGF-a, or c-erbB2 mRNA
expression patterns. The putative EGF receptor-inhibiting
compounds were tested for effects on basal tyrosine phosphorylation, in vitro EGF receptor tyrosine kinase activity,
and EGF-dependent growth. Putative ErbB2-inhibiting
compounds were tested for effects on antibody-induced
ErbB2 tyrosine kinase activity. Results: EGF receptor
mRNA and TGF-a mRNA levels were highest in cell lines
derived from renal cancers, and c-erbB2 mRNA levels were
highest in cells derived from breast, ovarian, and colon cancers. Twenty-five compounds with high correlation coefficients (for cytotoxicity and levels of the measured mRNAs)
were tested as inhibitors of the EGF receptor or c-erbB2
signaling pathways; 14 compounds were identified as inhibitors of these pathways. The most potent compound, B4, inhibited autophosphorylation (which occurs following activation) of ErbB2 by 50% in whole cells at 7.7 µM. Conclusions:
Novel EGF receptor or c-erbB2 pathway inhibitors can be
identified in the NCI drug screen by correlation of cytotoxicity patterns with EGF receptor or c-erbB2 mRNA expression levels. [J Natl Cancer Inst 1997;89:1505–15]
Receptor tyrosine kinases contain an extracellular ligandbinding domain, a membrane-spanning domain, and an intracellular domain with tyrosine kinase activity. The type I family of
growth factor receptors includes the epidermal growth factor
(EGF) receptor, and the tyrosine kinases c-erbB2, c-erbB3, and
c-erbB4 (1,2). Growth factors interact with their specific receptors at the cell surface, activating the receptor tyrosine kinase. A
cascade of downstream signaling proteins are activated, includ-
ing Ras, Raf, mitogen-activated protein kinase (MAPK) kinase,
and MAPK, eventually leading to altered gene expression and
increased growth rate (3–6).
The EGF receptor is a 170-kd transmembrane glycoprotein
that is found on many epithelial cell types. It is activated by at
least three ligands, EGF, transforming growth factor-a (TGF-a),
and amphiregulin (7,8). An autocrine growth pathway has been
proposed for TGF-a in normal and malignant human breast
epithelium (9). ErbB2 (also called HER2 and neu) is a 185-kd
transmembrane tyrosine kinase with considerable homology to
the EGF receptor, although a ligand for ErbB2 has not yet been
clearly identified. EGF receptor and c-erbB2 have 43% sequence
homology between their extracellular domains and 82% between
their tyrosine kinase domains. Both EGF receptor and c-erbB2
are overexpressed in certain types of tumors, notably, breast,
ovary, bladder, colon, kidney, and head and neck cancers as
well as squamous carcinomas of the lung (10–13). In addition, a
relationship between overexpression of c-erbB2 or EGF receptor
and poor prognosis in patients with breast cancer has been reported by several groups (12,14–17). Therefore, the EGF receptor and c-erbB2 signaling pathways are potentially important
targets for anticancer therapy.
Chemotherapy has proven to be successful in selected cancers, stimulating the search for other effective antineoplastic
drugs. To identify new agents with potential antitumor activity,
the National Cancer Institute (NCI) established a panel of 60
human tumor cell lines organized into subpanels representing
leukemia, melanoma, and cancers of the lung, breast, colon,
kidney, ovary, prostate, and central nervous system (CNS). The
cytotoxic effects of natural products, synthetic compounds, and
semisynthetic compounds are being determined using these 60
cell lines, and the data are stored in a database that now contains
cytotoxicity information from assays with more than 49 000
compounds (18–20). The patterns of cytotoxicity among the cell
lines in the screen are evaluated using the computer analysis
program COMPARE. This program calculates the degree of
similarity (Pearson correlation coefficient, or ‘‘r’’ value) between the pattern of cytotoxicity of an index compound and all
other compounds in the database. Similar patterns of cytotoxic*Affiliations of authors: K. Wosikowski, D. Schuurhuis, K. Johnson, S. E.
Bates (Medicine Branch, Division of Clinical Sciences), K. D. Paull (Information Technology Branch, Development Therapeutics Program, Division of Cancer Treatment, Diagnosis, and Centers), T. G. Myers, J. N. Weinstein (Laboratory of Basic Sciences), National Cancer Institute, Bethesda, MD.
Correspondence to: Susan E. Bates, M.D., National Institutes of Health, Bldg.
10, Rm. 12N226, Bethesda, MD 20892. E-mail: [email protected]
See ‘‘Notes’’ following ‘‘References.’’
© Oxford University Press
Journal of the National Cancer Institute, Vol. 89, No. 20, October 15, 1997
ARTICLES 1505
ity identify compounds with similar targets and mechanisms
of action (19,20). For example, agents that bind tubulin are
highly correlated despite diversity in structure (21). Measurement of various molecular markers has been undertaken in the
60 cell lines, using COMPARE to correlate the patterns of expression with the cytotoxicity patterns for the 49 000 compounds
in the NCI database (22,23).
This last approach was used to identify unknown compounds
in the drug screen database that may target the EGF receptor, the
c-erbB2 signaling pathway, or the action of TGF-a. The success
of this approach relies on three prerequisites: that the level of a
target is quantitatively related to its activity in the cell, that
inhibition of that target will have an impact on cellular growth,
and that compounds exist for which inhibition of that target is a
predominant mechanism of action. We determined the levels of
messenger RNAs (mRNAs) encoding the EGF receptor, ErbB2,
and TGF-a in the 60 cell lines comprising the NCI anticancer drug screen, generated expression patterns, and used
COMPARE to find compounds with correlated patterns of sensitivity. The putative EGF receptor compounds were tested for
effects on basal tyrosine phosphorylation, in vitro EGF receptor
tyrosine kinase activity, and EGF-dependent growth. Putative
ErbB2 compounds were tested for effects on ErbB2 tyrosine
kinase activity in biochemical assays and in whole cells.
Methods
Cell Culture
The cell lines comprising the NCI anticancer drug screen panel were obtained
and processed as previously described (18,24,25). A number of additional cell
lines were used as positive controls and as tools in the assays testing activity of
identified compounds. SK-BR-3 and MDA-MB-453 cells were obtained from
the American Type Culture Collection (Rockville, MD) and A431 cells were
provided by Ira Pastan (NCI, Bethesda, MD). The EGF receptor overexpressing
MCF-7 TH cells are a drug-resistant subline isolated from parental MCF-7 cells
by intermittent exposure to 1 mM doxorubicin. The cells were a gift from Tom
Hamilton (Fox Chase Cancer Center, Philadelphia, PA). Cells were grown as
monolayer cultures in Iscove’s minimal essential medium (IMEM) (Biofluids
Inc., Rockville, MD) supplemented with 10% fetal calf serum (FCS) (Life Technologies, Inc. [GIBCO-BRL], Gaithersburg, MD), 2 mM glutamine, 15 mM
HEPES, and 25 mg/mL gentamicin (all from Biofluids Inc.) in a humidified 5%
CO2 atmosphere at 37 °C. The MCF-10A immortalized human breast epithelial
cells were provided by David Salomon (NCI, Bethesda, MD) and were cultured
in Dulbecco’s minimal essential medium (DMEM)–Ham’s F12 (1:1) medium
containing 5% horse serum, 100 U/mL penicillin, and 100 mg/mL streptomycin
(all from Biofluids Inc.) plus 20 mM HEPES, 4 mg/mL insulin, 500 ng/mL
hydrocortisone, and 20 ng/mL EGF (all from Collaborative Biomedical Products, Bedford, MA) (26).
RNA Extraction and Electrophoresis
Total RNA was extracted by homogenization of cells in guanidine isothiocyanate buffer followed by centrifugation over a cesium chloride cushion (27).
To compare the RNA quantities and quality, RNA concentration was measured
by spectrophotometry and 5 mg total RNA was electrophoretically separated in
1% agarose–6% formaldehyde gels. Gels were stained with ethidium bromide
and examined to confirm regular loading. RNA that was not comparably loaded
was re-extracted, remeasured, and re-evaluated.
RNase Protection Assay
A 141-base pair (bp) fragment of the EGF receptor complementary DNA
(cDNA), a 470-bp fragment of c-erbB2 cDNA, and a 603-bp fragment of TGF-a
DNA were used to generate 32P-labeled antisense riboprobes by SP6, T7, and T7
polymerase transcription (Promega Corp., Madison, WI), respectively, in the
presence of [a-32P]uridine triphosphate (specific activity, 3000 Ci/mmol) (Du
1506 ARTICLES
Pont NEN, Boston, MA) as described previously (28). For the ribonuclease
(RNase) protection assay, 30 mg of total RNA was hybridized with 3 × 105
cpm-labeled riboprobe and then digested for 30 minutes at 25 °C with 40 mg/mL
RNase A and 28 mg/mL RNase T1. Following extraction, samples were separated on a 6% polyacrylamide gel and autoradiography was performed. The
relative amount of mRNA was quantified using a Phosphor Imager, coupled to
Image Quant software (Molecular Dynamics, Sunnyvale, CA), or a Fotoeclipse
densitometer (Fotodyne Inc., Hartland, WI).
COMPARE Analysis and Determination of Pearson
Correlation Coefficients
Three separate COMPARE analyses were performed using the relative expression of the EGF receptor, c-erbB2, and TGF-a mRNAs in the 60 cell lines
of the NCI drug screen. Pearson correlation coefficients, or ‘‘r’’ values, were
obtained for each expression pattern, with the cytotoxicity pattern of compounds
in the NCI drug screen database. The database stores the bioactivity data of
compounds as the −log10 GI50 (e.g., a GI50 of 10−4 M is stored as +4 and a GI50
of 10−9 is stored as +9) (24). GI50 is the designation for a time zero-corrected
IC50, the concentration of a compound causing 50% growth inhibition. Thus, cell
lines represented by larger values indicate sensitivity to the tested compound,
whereas cell lines represented by smaller numbers indicate resistance. When
+log10 values of the EGF receptor, c-erbB2, and TGF-a mRNA levels were used
for the COMPARE analysis, then greater mRNA expression correlated with
enhanced sensitivity of the cell to the database compound. A list of compounds
having the highest correlation coefficients was sorted in order of descending
correlation coefficients. Compounds at the top of the list are those that correlate
best with the expression pattern being analyzed. Compounds were obtained from
the Drug Screen repository (Drug Synthesis and Chemistry Branch, NCI,
Bethesda, MD), having been contributed from diverse sources, including academia, industry, and government.
For the purpose of this article, compounds chosen from the NCI database and
used in further experiments were assigned the E, B, or EB numbers shown in
Table 1. This nomenclature allows rapid identification of the COMPARE analysis from which the compound was obtained: E, EGF receptor; B, c-erbB2; or
EB, both EGF receptor and c-erbB2. Compounds are originally contributed to
the drug screen as discreet (indicating that the structure is to be held confidential)
or nondiscreet). Both discreet and nondiscreet compounds were used in the
study.
Cluster Analysis
Patterns of averaged data GI50 data were obtained as previously described
(29). The pattern for each drug was centered by subtracting from each value the
pattern mean and dividing by the pattern standard deviation. The Euclidean
distance between patterns normalized in this fashion approximate the additive
inverse of the respective Pearson correlation coefficients (Distance 4 1 − Pearson correlation coefficient) and can thus be compared with the Pearson correlation coefficient calculation used by the COMPARE program. The JMP computer software for Macintosh (version 3.1.5, SAS Institute, Cary, NC) was used
to generate the dendogram by the average-linkage clustering method. Reference
compounds selected from a group of 216 standard agents on the basis of their
mechanism of action were added to the group before clustering. Average linkage
is a commonly used technique, which is agglomerative, starting with individual
compounds and ending with a single cluster containing all of the compounds.
Each join of individual compounds or previously joined clusters represents a
node on the tree that is plotted at a particular similarity value (mean Pearson
correlation coefficient). When the node joins two compounds, the Pearson correlation coefficient is simply the average of the two. When two previously
formed clusters are joined, the mean Pearson correlation coefficient is calculated
by considering all possible intercluster compound pairings. For example, the
topoisomerase II inhibitors, labeled with a m in Fig. 3, are all joined into a single
cluster whose node has a mean Pearson correlation coefficient of .55. Thus, the
topoisomerase II inhibitors in the NCI screen have cytotoxicity patterns that are
correlated on average at the .55 level.
EGF-Dependent Growth Assay
The inhibitory effect of compounds (E1 through E11 and EB1 through EB5)
on EGF-dependent growth of MCF-10A breast epithelial cells was determined in
Journal of the National Cancer Institute, Vol. 89, No. 20, October 15, 1997
Table 1. Thirty compounds among the top 56 identified by COMPARE, using the EGF receptor, c-erbB2, and TGF-a expression profiles*
EGF receptor
c-erbB2
No.
PCC
Rank
Name
No.
PCC
Rank
676495
676497
623436
671526
676498
646148
Discreet
616511
656128
Discreet
643315
682027
669365
672768
615554
646019
680581
680625
Discreet
675223
656125
656126
631229
676496
646015
669083
650706
651295
654970
668885
0.726
0.715
0.704
0.658
0.631
0.626
0.611
0.599
0.596
0.593
0.579
0.579
0.574
0.554
0.553
0.548
0.545
0.544
0.543
0.541
0.540
0.537
0.536
0.534
0.528
0.523
0.523
0.518
0.516
0.508
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
37
TP38-001
TP4EK
TGFa.-PE40
Toxin.delta.53L
TP4EK-K6
EB1
643315
646019
655128
Discreet
683039
648202
646148
611388
656125
657871
646081
640490
668937
640820
Discreet
668885
674987
655439
673189
669714
Discreet
682883
10121
682879
671373
Discreet
669627
178249
668419
669164
0.614
0.566
0.560
0.558
0.554
0.552
0.542
0.532
0.504
0.504
0.499
0.494
0.491
0.485
0.482
0.480
0.477
0.471
0.469
0.469
0.468
0.465
0.461
0.458
0.457
0.453
0.449
0.447
0.435
0.423
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
24
27
30
33
34
38
46
56
E1
EB2
PD153717
E2
E3
EB3
EB4
E4
TP38-279
E5
E6
E7
EB5
TGF-a
Name
EB2
EB3
e23(dsFV)PE38
EB1
B1
EB4
B2
B3
B4
EB5
B5
B6
B7
B8
B9
No.
PCC
Rank
Name
671526
Discreet
643892
Discreet
643438
Discreet
682765
648200
636467
Discreet
311046
650426
Discreet
648202
643315
644673
668456
616511
676497
634308
655128
Discreet
669365
644674
641886
642550
642195
675223
673999
676495
0.451
0.451
0.443
0.426
0.424
0.422
0.421
0.417
0.415
0.4122
0.408
0.406
0.406
0.404
0.402
0.401
0.401
0.400
0.399
0.399
0.398
0.396
0.395
0.394
0.391
0.389
0.384
0.383
0.381
0.377
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
36
Toxin.delta.53L
TP4EK
PD153717
TP38-001
*EGF 4 epidermal growth factor; TGF-a 4 transforming growth factor; PCC 4 Pearson correlation coefficient.
96-well plates. Eight hundred MCF-10A cells were plated in the absence and
presence of 20 ng/mL EGF in DMEM–Ham’s F12 (1:1) medium containing
2.5% horse serum and supplemented as described above. Cells were treated with
increasing concentration of the compound and fixed and stained after 5 days in
culture as described. The results were expressed as the difference (in %) from the
control value (untreated wells without EGF or compound) that was plotted as
0%. Positive values indicate more growth than in control untreated cells, and
negative values indicate inhibition of growth. Experiments were performed in
quadruplicate.
Cell Membrane Kinase Assay
Cells were plated in 10-cm Petri dishes and were allowed to attach overnight.
To avoid interference of EGF receptor or ErbB2 ligands possibly present in the
serum, the medium was replaced with serum-free medium composed of phenol–
red free IMEM supplemented with 2 mM glutamine and 15 mM HEPES (Biofluids Inc.) plus 1 mg/mL essentially fatty acid-free bovine serum albumin
[BSA], 5 mg/mL insulin, 5 mg/mL transferrin, and 5 ng/mL selenium (all from
Sigma Chemical Co., St. Louis, MO) for 24–48 hours.
For the EGF receptor kinase assay, membrane extracts were prepared. A431
cells were washed two times in ice-cold PBS and lysed in hypotonic lysis buffer
(10 mM NaCl, 15 mM EDTA, 8.5 mM NaH2PO4, 11.5 mM Na2HPO4, 20 mg/mL
aprotinin [ICN Biomedicals, Aurora, OH], 20 mg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1 mM sodium orthovanadate [all from
Sigma Chemical Co.]). After sonication, lysates were cleared by centrifugation
(200g, 5 °C, 15 minutes). Supernatants were centrifuged (100 000g, 5 °C, 60
minutes) and membranes were resuspended in 50 mM HEPES, pH 7.4, 125 mM
NaCl, 10% (vol/vol) glycerol. Two micrograms of membrane preparation was
preincubated with 120 mM compound or genistein (LC Laboratories, Woburn,
MA) in the presence or absence of 2 × 10−8 M EGF for 10 minutes at room
temperature. The kinase reaction was initiated by the addition of an equal volume (20 mL) of 50 mM HEPES, 125 mM NaCl, 4 mM MnCl2, 24 mM MgCl2,
and 4 mM adenosine 58-triphosphate (ATP) with 0.8 mCi [g-32P]ATP (specific
activity, 3000 Ci/mmol; Du Pont, NEN) per reaction for 4 minutes at 4 °C as
described by Osherov (30). The reaction was stopped by the addition of sodium
dodecyl sulfate (SDS)-sample buffer.
For the ErbB2 assay, serum-starved cells were stimulated with 10–25 mg/mL
anti-ErbB2 recombinant human monoclonal antibody (HER2, provided by Genentech, Inc., South San Francisco, CA) (31) for 5 minutes at 37 °C and harvested in TNESV (50 mM Tris, pH 7.6, 1% [vol/vol] Nonidet P-40 [NP-40], 2
mM EDTA, 100 mM NaCl, 1 mM vanadate, 20 mg/mL aprotinin, 20 mg/mL
leupeptin, and 1 mM PMSF) as described (32). ErbB2 was immunoprecipitated
from 1 mg of protein using anti-ErbB2 monoclonal antibody (MAb), recognizing
a cell surface epitope (c-Neu; Ab-5; Oncogene Science, Inc., Manhasset, NY)
and protein A-sepharose beads (Pharmacia Biotech, Inc., Piscataway, NJ). Immunoprecipitates were preincubated with 120-mM compounds or the erbstatin
analog (methyl 2,5-dihydroxycinnamate, LC Laboratories) in buffer (50 mM
HEPES, pH 7.4, 125 mM NaCl) for 15 minutes at 4 °C, followed by incubation
with an equal volume (30 mL) of reaction mix (50 mM HEPES, 125 mM NaCl,
4 mM MnCl2, 24 mM MgCl2, and 2 mM ATP with 15 mCi [g-32P]ATP per
reaction) for 5 minutes at 4 °C. The reaction was stopped by the addition of hot
SDS-sample buffer.
In both assays, proteins were separated by 7% SDS–polyacrylamide gel elec
trophoresis (SDS–PAGE). Gels were fixed in 10% (vol/vol) acetic acid, 30% (vol/
vol) methanol, and 10% (vol/vol) glycerol, then autoradiography was performed.
ErbB2 Autophosphorylation Assay
In 60-mm Petri dishes, 106 MDA-MB-453 cells were plated in IMEM containing 10% FCS and allowed to attach. Cells were serum starved for 24 hours
in serum-free IMEM medium before the addition of 200 mM test compound for
1 hour, followed by stimulation with 25 mg/mL anti-ErbB2 antibody (rhuMAb
HER2, Genentech, Inc.) for 5 minutes and then lysis in TNESV. Twenty to 70
microgram protein extracts were separated by 7% SDS–PAGE. The proteins
were transferred to Immobilon-P (Millipore Corp., Bedford, MA) membranes,
blocked with 5% BSA, and probed with antiphosphotyrosine antibody (RC20H;
Transduction Lab., Cincinnati, OH) to detect tyrosine phosphorylated ErbB2 or
an antibody to detect actin (Ab-1; Oncogene Research Products, Cambridge,
Journal of the National Cancer Institute, Vol. 89, No. 20, October 15, 1997
ARTICLES 1507
MA) to confirm regular loading of the gel as previously described (32). IC50
determinations were made by plotting the level of phosphorylated ErbB2 obtained at four concentrations of test compound (range, 4–200 mM).
whereas those to the left of the mean indicate the lowest
levels.
Results
Selection of Compounds Based on the Correlation of
Cytotoxicity With High mRNA Expression
Expression of EGF Receptor, c-erbB2, and TGF-a mRNAs
in the 60 Cell Lines of the NCI Anticancer Drug Screen
To identify compounds that target the EGF receptor, c-erbB2,
and the TGF-a pathway, the levels of the respective mRNAs in
the 60 cell lines in the drug screen were determined by RNase
protection assays. Fig. 1 shows representative mRNA results for
kidney, colon, breast, and ovarian cell lines, along with photographs of the ethidium bromide-stained gels demonstrating RNA
integrity and the comparability of quantities of RNA used in the
RNase protection assays. The levels of EGF receptor, c-erbB2,
and TGF-a mRNAs in all cell lines were determined in at least
two independent experiments. A cell line known to have high
levels of expression of these mRNAs was included in each experiment as a positive control (MCF-7 TH, MDA-MB-453, or
MDA-MB-231). The expression levels were quantitated by use
of a fotoeclipse densitometer or a Phosphor Imager and were
related to the levels of expression in the control MCF-7 TH,
MDA-MB-453, and MDA-MB-231 cells, which were assigned a
value of 1000 for EGF receptor, c-erbB2, and TGF-a mRNAs,
respectively. Levels for EGF receptor in the 60 cell lines ranged
from 0 to 3763; for c-erbB2, from 0 to 27 973; and for TGF-a,
from 0 to 10 091. EGF receptor expression was highest in renal
cell lines. Highest levels of c-erbB2 expression were observed in
ovarian, breast, and colon cells. TGF-a mRNA levels were highest in renal cell lines. Levels of all three mRNAs were lowest in
leukemia cells.
Generation of EGF Receptor, c-erbB2, and TGF-a
Expression Patterns
As shown previously, the cytotoxicity pattern of a given drug
can predict its mechanism of action using the COMPARE program (19,21). A variety of molecular targets have been measured in the 60 cell lines and expression patterns have been generated to compare them to the cytotoxicity patterns in
the database (22,23). As shown in Fig. 2, expression patterns
for EGF receptor, c-erbB2, and TGF-a mRNAs in the 60
cell lines were generated using the +log10 of the quantitative
values of EGF receptor, c-erbB2, and TGF-a mRNAs. In
the mean graphs shown in Fig. 2, the bars pointing to the right
of the mean (the vertical line in the center) represent the
highest levels of EGF receptor, c-erbB2, and TGF-a mRNAs,
To identify compounds to which cells expressing high levels
of EGF receptor, c-erbB2, or TGF-a might be more sensitive,
the COMPARE program was entered, using the +log10 values of
the EGF receptor, c-erbB2, or TGF-a mRNA levels. EGF receptor, c-erbB2, and TGF-a mRNA expression in the 60 cell
lines was correlated by COMPARE analysis with the sensitivity of these cell lines to the more than 49 000 drugs in the
NCI database. Table 1 shows 30 compounds among the top 56
identified by COMPARE as having the highest Pearson correlation coefficients. Larger values represent a better positive correlation between high levels of EGF receptor, c-erbB2, or
TGF-a mRNAs and sensitivity of the cells to the listed compound.
The TGF-a-PE40 chimeric toxin, consisting of TGF-a
coupled to Pseudomonas exotoxin (PE) (33) and four of its
derivatives displayed the highest Pearson correlation coefficients (r 4 .726–.631) with EGF receptor expression pattern. As
shown in Table 1, a total of six TGF-a-toxin chimeras were
found among the top 24 compounds. TGF-a-toxin has previously been demonstrated to have increased cytotoxicity in cells
that express high levels of EGF receptor on the cell surface (33).
Also high on the EGF receptor COMPARE list is the tyrosine
kinase inhibitor NSC-669365 (PD153717, Parke-Davis, Ann Arbor, MI), which has been shown to be a specific inhibitor of the
EGF receptor tyrosine kinase. PD153717 inhibits EGF receptor
autophosphorylation in A431 cells with an IC50 of 100 nM and
specifically inhibits EGF-mediated mitogenesis in fibroblasts,
but has no activity against PDGF- or FGF-mediated events (Fry
DW: unpublished observations). Another specific EGF receptor
kinase inhibitor, NSC-669364 [PD153035, Parke-Davis (34)]
was identified in the top 100 compounds from the EGF receptor
COMPARE analysis (r 4 .436).
The highest correlation coefficient for compounds identified by the c-erbB2 COMPARE analysis was .614. A recombinant anti-ErbB2 immunotoxin (e23(dsFv)PE) (35) was fifth
on the c-erbB2 COMPARE list. Recombinant immunotoxins selectively bind to and kill cells that are recognized by
the antigen-binding domain. Two of the TGF-a-toxin chimeras
and OVB3-PE (36), an antibody that recognizes an ovarian cellspecific antigen coupled to PE toxin, were also among the top
100 compounds in the c-erbB2 COMPARE analysis.
Fig. 1. Expression of epidermal growth factor (EGF) receptor, transforming
growth factor (TGF)-a, and c-erbB2 messenger RNAs in cell lines from the
National Cancer Institute anticancer drug screen. Total RNA was isolated from
the 60 cell lines comprising the anticancer drug screen and 30 mg were analyzed
using a ribonuclease (RNase) protection assay. 32P-labeled antisense fragments
from EGF receptor, TGF-a, and c-erbB2 were protected against RNase degradation by hybridization to total RNA. The protected bands were separated on a 6%
polyacrylamide gel and visualized by autoradiography. Shown are autoradiograms of the same exposure time for EGF receptor, TGF-a, or c-erbB2 in
ovarian, breast, renal, and colon cell lines. Ethidium bromide (EthBr)-stained
agarose gels as shown were used to confirm the integrity of the RNA and to
confirm that comparable amounts of total RNA were used in the assays. Both the
28S and the 18S ribosomal RNA bands are shown in the photograph.
1508 ARTICLES
Journal of the National Cancer Institute, Vol. 89, No. 20, October 15, 1997
Fig. 2. Expression patterns of epidermal
growth factor (EGF) receptor, c-erbB2, and
transforming growth factor (TGF)-a messenger RNA (mRNA) levels in the cell lines of
the National Cancer Institute anticancer drug
screen. Expression patterns were obtained by
plotting positive and negative values (deltas)
generated from a set of mRNA expression
levels determined in each cell line. The mean
+log10 of EGF receptor, TGF-a, or c-erbB2
mRNA expression level was obtained for the
panel of 60 cell lines. The individual value
for each cell line then was subtracted from
the mean expression level for the panel to
create the corresponding delta. Bars to the
right identify cell lines that express high levels of EGF receptor, c-erbB2, and TGF-a
mRNAs.
As in the EGF receptor COMPARE analysis, the compound
with the highest Pearson correlation coefficient (r 4 .451) in the
TGF-a analysis was a TGF-a-chimeric toxin derivative (Toxin.delta.53L) (37). Two more TGF-a-toxin chimeras were among
the top 36 compounds. The tyrosine kinase inhibitor PD153717
identified in the EGF receptor COMPARE analysis was also
identified in the TGF-a COMPARE analysis (r 4 .395).
Highlighted compounds were chosen, based on correlation
coefficients and availability, for use in further experiments.
Compounds with an E designation were derived from the EGF
receptor COMPARE analysis, B compounds were from the
c-erbB2 analysis, and EB compounds were found both in the
EGF receptor and c-erbB2 analysis. Compounds E8 through E11
were derived from an early COMPARE analysis, performed using a preliminary dataset.
Activity Patterns of Putative Kinase Inhibitors Compared
With Those of Standard Anticancer Agents
To evaluate whether the putative EGF receptor and ErbB2
kinase inhibitors have distinctive cytotoxicity patterns (indicative of distinct mechanisms of action), a cluster analysis was
performed. The 25 compounds that were identified in Table 1
plus six toxin chimeras and two known EGF receptor tyrosine
kinase inhibitors (Parke-Davis) were analyzed together with 24
standard antineoplastic agents of known mechanism of action.
Much as a phylogenetic tree is used to indicate genetic sequence similarities, a cluster tree such as the one shown in Fig.
3 can be employed to indicate similarities in the cytotoxicity
patterns of compounds tested in the screen. In the process of the
clustering, compounds with similar cytotoxicity patterns are
Journal of the National Cancer Institute, Vol. 89, No. 20, October 15, 1997
ARTICLES 1509
correlation coefficient 4 .47), as were the group of toxin chimeras (labeled ‘‘■’’) (average correlation coefficient 4 .34).
Only at correlation coefficient values of .20 to .17 do the known
TGF-a-toxin chimeras join other compounds. For reference, this
is about the value at which the antitubulin standard agents join
the other standard agents of vastly different mechanisms of action (.14). Furthermore, the putative tyrosine kinases as a group
join the standard agents at a value of −.06.
If the hypothesis that differences in activity pattern indicate
differences in mechanisms of action is correct, then the clustering reveals that distinct mechanisms of cytotoxicity prevail for
each of the two groups: the erbB2 kinase inhibitors (●) and the
TGF-a toxin chimeras (■). Furthermore, these mechanisms are
different from those of the standard agents. The most distinctive
difference between the standard agents and our selected compounds is that the leukemia cells were insensitive to our selected
compounds. This is in contrast to most conventional cytotoxic
(standard) agents, which are highly cytotoxic in leukemic cells.
Identification of Compounds E1, E10, and E11 as Putative
EGF Receptor Tyrosine Kinase Inhibitors
Initial experiments demonstrated inhibition of overall tyrosine phosphorylation in A431 cell membranes by compounds E1
and E11, and to a lesser extent, E6, E7, and E10 (data not
shown). Since basal levels of tyrosine phosphorylation reflect
not only the EGF receptor pathway but also other tyrosine kinases, we directly assessed whether the compounds could inhibit
autophosphorylation of the EGF receptor following stimulation
by EGF using an in vitro kinase assay. Fig. 4 shows the results
for EGF-induced EGF receptor autophosphorylation in membranes prepared from A431 cells. Genistein, a nonspecific tyrosine kinase inhibitor, was used as a control. Compounds E1 and
E11 and, to a lesser extent, E9 and E10 inhibited EGF-induced
EGF receptor autophosphorylation. Among the 15 compounds
evaluated, three compounds (E1, E10, and E11) inhibited both
basal tyrosine phosphorylation in A431 membranes and the
basal and EGF-induced EGF receptor autophosphorylation.
Fig. 3. Dendogram result of cluster analysis of compounds based on their cytotoxicity patterns in the cell lines of the drug screen. Members of clusters of
particular interest were labeled m (topoisomerase inhibitors), ● (putative epidermal growth factor [EGF] receptor or ErbB2 kinase inhibitor), and ■ (transforming growth factor [TGF-a] toxin chimeras). PCC-Pearson correlation coefficient. The cluster analysis was performed as described in the ‘‘Methods’’
section.
grouped together in a cluster. The compounds in each cluster are
joined at a node from which branches leading to the cluster
emerge. The height of the node indicates the degree of similarity
between the compounds in the clusters. This is expressed in
terms of a mean Pearson correlation coefficient. For example,
topoisomerase II inhibitors, including the anthracyclines, mitoxantrone, and amsacrine (mAMSA) (labeled with a bold ‘‘m’’),
form a cluster; the node from which the cluster emerges is shown
at a correlation level of .55. Similarly, the tubulin-active agents,
including the colchicines, vincas, and taxanes, form a separate
cluster with a node at 0.5.
A subset of the putative ErbB2 kinase inhibitors (labeled with
a bold ‘‘●’’) were very similar in pattern to each other (average
1510 ARTICLES
EGF-Dependent Growth Inhibition in MCF-10A
Mammary Epithelial Cells
To confirm the functional inhibition of the EGF receptor
pathway, the compounds E1 through E11 and EB1 through EB5
and genistein were individually added to MCF-10A cells cultured in media with and without the presence of added EGF.
MCF-10A cells are immortalized, nontransformed human mammary epithelial cells that display increased growth on the addition of exogenous EGF (26). As shown in Fig. 5, the addition of
EGF (shaded bars) to control MCF-10A cells stimulated their
growth 50%–75% above basal levels of growth (0% on the Y
axis). Addition of compounds E1, E2, E8, E10, EB2, EB3, EB5,
and genistein inhibited the EGF-stimulated growth without inhibiting basal growth. At higher concentrations, both basal and
EGF-stimulated growth are inhibited, suggesting nonspecific cytotoxicity. The remaining eight compounds not shown had no
specific effect on EGF-stimulated growth, since inhibition of
EGF-dependent growth occurred concurrently with inhibition of
basal growth. These results confirm the ability of the compounds
to functionally inhibit the EGF receptor pathway-dependent
growth of MCF-10A cells.
Journal of the National Cancer Institute, Vol. 89, No. 20, October 15, 1997
Fig. 4. In vitro effect of selected
compounds on basal and epidermal
growth factor (EGF)-induced EGF
receptor autophosphorylation. Membrane preparations from A431 cells
were preincubated with compound
in the presence or absence of 2 ×
10−8 M EGF (10 minutes, room temperature), followed by incubation for
4 minutes at 4 °C with reaction mix
(see ‘‘Methods’’ section). Sodium
dodecyl sulfate (SDS)-sample buffer
was added and samples were heated
and proteins were separated by gelelectrophoresis. Incorporation of
[32P] ATP was visualized by autoradiography. Shown is the 170-kd
band that represents the EGF receptor.
Identification of B4 as the Most Potent Putative ErbB2
Tyrosine Kinase Inhibitor
An anti-ErbB2 monoclonal antibody, rhuMAb HER2, was
used to stimulate ErbB2 autophosphorylation in MDA-MB-453
human breast cancer cells prior to cell lysis and incubation with
the test compounds or with the erbstatin analog positive control.
Four of the 14 compounds evaluated (B1, B2, B3, and B5)
inhibited ErbB2 autophosphorylation in membrane preparations
following stimulation with rhuMAb HER2 antibody (data not
shown).
This assay was repeated in intact MDA-MB-453 cells that
were pretreated with the test compounds and then stimulated
with the rhuMAb HER2 antibody prior to cell lysis and examination for ErbB2 autophosphorylation. In this type of assay, the
intracellular part of ErbB2 is not as freely accessible as in the in
vitro tyrosine kinase assay. Seven compounds (EB2, EB5, B1,
B2, B3, B4, and B9) at 200 mM and the erbstatin analog at 500
Fig. 5. Effect of selected compounds on epidermal growth factor (EGF)dependent growth in MCF-10A cells. MCF-10A cells were plated in medium
without (dotted bars) or with (shaded bars) 20 ng/mL EGF. Cells were treated
with increasing concentrations of the test compound and fixed and stained after
5 days in culture as described in the ‘‘Methods’’ section. The results were expressed
as the difference (in %) from the control value (untreated wells without EGF or
compound) that was plotted as 0%. Positive values indicate more growth than in
control untreated cells and negative values indicate inhibition of growth. The
experiment was performed in quadruplicate (mean ± standard deviation). The
experiment shown is representative of three independent experiments.
Journal of the National Cancer Institute, Vol. 89, No. 20, October 15, 1997
ARTICLES 1511
mM inhibited basal and antibody-induced autophosphorylation
of ErbB2, as shown in Fig. 6.
We performed dose–response experiments with these compounds and identified B4 as the most potent agent. As a control
for the nonspecific effect of cytotoxic agents, we examined the
effect of doxorubicin, dactinomycin, or cisplatin on induced autophosphorylation of ErbB2. No inhibition was observed, even
when 200 mM drug concentrations were used.
Diverse Chemical Structures of the Putative EGF Receptor
or c-erbB2 Pathway Inhibitors
Fig. 7 presents the chemical structures of 11 of the 14 compounds that displayed an inhibitory effect in at least one of the
functional assays performed (in vitro kinase, whole cell kinase,
and EGF-dependent growth assay). The diversity of their chemical structures is apparent. No two compounds shown here belong
to similar chemical classes. B4 has a structure that resembles the
tyrphostin class of compounds.
Discussion
In this study, EGF receptor, c-erbB2, and TGF-a mRNA
expression levels in the 60 cell lines comprising the NCI anticancer drug screen were analyzed and correlated by COMPARE
analysis with sensitivity to the more than 49 000 compounds in
the NCI database. High correlation coefficients, which should
indicate compounds most effective in cell lines containing high
levels of these mRNAs, were sought to identify potential inhibi-
tors of growth factor signaling. Six compounds found among the
top 30 correlated with EGF receptor mRNA level also were
found in the top 30 correlated with c-erbB2 mRNA level. The
putative growth factor signaling pathway inhibitors cluster in a
dendogram as if they represent a mechanism of drug action
distinct from those of the well-known cytotoxic antitumor
agents. Three (E1, E10, and E11) of 15 compounds tested inhibited both basal tyrosine phosphorylation in A431 membranes
and basal and EGF-induced EGF receptor autophosphorylation.
Seven (E1, E2, E8, E10, EB2, EB3, and EB5) of 16 compounds
inhibited EGF-dependent growth. Four (B1, B2, B3, and B5)
and seven (B1, B2, B3, B4, B9, EB2, and EB5) of 14 compounds
tested inhibited ErbB2 autophosphorylation in cell membrane
preparations and in intact MDA-MB-453 cells, respectively. The
most effective of these compounds (B4) inhibited antibodyinduced ErbB2 autophosphorylation, with an IC50 of 7.7 mM in
the intact cell assay. These results confirmed the hypothesis that,
by correlating expression patterns with cytotoxicity patterns,
COMPARE analysis could identify compounds that inhibit EGF
receptor and c-erbB2 pathways.
Underlying this hypothesis is the prerequisite that the level of
EGF receptor or c-erbB2 is quantitatively related to its activity
or importance, and clinical studies have suggested this would be
true. High levels of EGF receptor and c-erbB2 mRNA and protein expression have been observed in a variety of solid human
cancers and have been associated with the presence of metastatic
disease and an increased probability of tumor recurrence and
poor patient survival (10,12–17). High levels of expression of
EGF receptor or c-erbB2 mRNA or protein has also been correlated with resistance to chemotherapy (including doxorubicin,
Fig. 6. Effect of selected compounds on
basal and antibody-induced ErbB2 autophosphorylation in intact MDA-MB-453
cells. Cells were preincubated with 200
mM compound for 1 hour, then stimulated
with rhuMAb HER2 for 5 minutes and
harvested. Protein samples were run on
sodium dodecyl sulfate–polyacrylamide
gel electrophoresis gels and electrophoretically transferred to Immobilon membrane. The membrane was hybridized with
anti-phosphotyrosine or anti-actin antibody. The 185-kd bands reflect the tyrosine phosphorylated state of the ErbB2
protein.
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Journal of the National Cancer Institute, Vol. 89, No. 20, October 15, 1997
Fig. 7. Chemical structures of 11 from among the 14 compounds that displayed inhibitory effect in at least one of the functional assays performed. The E, B, and
EB numbers assigned can be identified in Table 1. Ph 4 phenyl.
tamoxifen, cisplatin, and vinblastine) in various cancer cell lines
and tumors (14,38,39).
Another prerequisite for identification of EGF receptor or
c-erbB2 pathway inhibitors in the drug screen is that compounds
exist that have inhibition of the EGF receptor or c-erbB2 pathways as the major mechanism of action. Numerous agents have
been described that intercept the EGF receptor or c-erbB2 signaling pathway at binding of the ligand to its receptor or at
tyrosine kinase activation. Natural tyrosine kinase inhibitors
have been described, such as genistein, lavendustin A, erbstatin,
and herbimycin A, which exhibit a rather broad specificity (40–
43). Since the naturally occurring tyrosine kinase inhibitors frequently require micromolar concentrations for kinase inhibition,
100–200 mM was selected as the concentration range to screen
the compounds identified by COMPARE. Dose–response studies with the compounds from the c-erbB2 COMPARE analysis
confirmed relatively high IC50s, ranging from 7.7 to 277 mM.
More selective tyrosine kinase inhibitors have been reported,
including NSC-669364 [PD153035, Parke-Davis (34)], which
was identified in the EGF receptor COMPARE analysis,
and numerous tyrphostins (44). These more selective inhibitors
have also been shown to be very potent, inhibiting kinase
activity in the nanomolar range. The compounds identified by
COMPARE may be viewed as lead compounds for which structure-activity studies designed to increase potency could be conducted.
New cancer drugs can be identified by looking for active
chemical compounds through use of high throughput screening
or can be designed by use of a rational and specific concept of
drug–target interaction (30,34,44–48). The NCI drug screen has
attempted to combine screening and molecular target strategies
simultaneously. The chief advantage of such an approach is that
compounds that interact with a target may be identified on the
basis of cytotoxicity pattern, thus obviating the need to directly
screen 49 000 compounds for their effect on that target. However, the target must be important, its activity in or importance
to the cell must be reflected quantitatively in its level, and inhibition must have an impact on cell growth within the 48-hour
assay. Thus, there will be targets for which this approach will not
be satisfactory.
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ARTICLES 1513
The COMPARE analysis has the ability to be extremely sensitive and specific, depending on the ‘‘seed’’ pattern used. For
example, using paclitaxel (Taxol) cytotoxicity as a seed,
approximately 50 of the top 55 compounds identified by
COMPARE are taxanes (data not shown). Reduced sensitivity,
specificity, and accuracy can be expected from seeds built of
laboratory-measured quantities representing RNA or protein
amounts or even from functional assays. The percentage of compounds that have tyrosine kinase inhibitory effects in a randomly
selected group is much less than the percentage of inhibitory compounds we obtained by selecting compounds using
COMPARE. It was reported that in a screen of 150 000 compounds, less than 1.8% of the compounds inhibited v-scr tyrosine kinase activity (49). From our selected compounds, four
(28%) of 15 inhibited EGF receptor autophosphorylation and
four (29%) of 14 inhibited ErbB2 autophosphorylation in cell
membrane preparations. Fourteen of 25 compounds examined
were active in at least one of the assays performed. In contrast,
nonspecific tyrosine kinase inhibitors, such as genestein and
herbimycin, give low correlation coefficients with the EGF receptor mRNA expression pattern, which can be attributed to
their lack of specificity (r 4 .077 and −.573, respectively).
On the other hand, whether the compounds that were negative
in the in vitro assays represent false-positive correlations is not
clear. Our confirmatory studies concentrated on the inhibitory
effect of identified compounds on early events in the signal
transduction pathway. By testing for inhibition of autophosphorylation, we restricted our investigation to the identification
of compounds with a mechanism of action at the tyrosine kinase
level or upstream. A downstream mechanism of action would
not be detected by the in vitro assays and other assays would be
needed.
Although compounds that inhibit EGF receptor kinase have
been under development by multiple investigators, the potential
contribution of such an agent to anticancer therapy has not been
clearly defined. The results of the studies presented here, in
which the EGF receptor COMPARE analysis ranks TGF-a toxin
chimeras at the top followed by compounds that include new and
known EGF receptor kinase inhibitors, suggest that this pathway
is of importance in cell growth. Thus, as a class of compounds
that are distinct from the standard cytotoxic agents, these inhibitors have the potential to be important in anticancer therapy.
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Notes
Supported in part by the International Medical Student Exchange Program,
The Netherlands (D. Schuurhuis).
We thank Ed Sausville for his critical reading of the manuscript, Curtis Hose
and Anne Monks for providing the cell lines, and Bob Schultz and Suzanne
Radtke for their help in the development of this project.
Permission to publish information relating to the following compounds is
appreciated: NSC-682027 and NSC-680625, Peakdale Fine Chemicals Limited,
Glossop, Derbyshire, U.K.; NSC-643892, Bionet Research Ltd, Camelford,
U.K.; NSC-680581, D. Heber, Pharmaceutical Institute, Kiel, Germany; NSC636467; NSC-636467, LIPHA SODEPHARM, Chilly-Mazarin, France; and
NSC-640820 and NSC-642550, Bayer AG, Wuppertal, Germany.
Presented in preliminary form at the 1996 meeting of the American Association for Cancer Research, Washington, DC.
Manuscript received December 6, 1996; revised June 10, 1997; accepted July
10, 1997.
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