Vol. 5, 3653–3660, November 1999
Clinical Cancer Research 3653
The Outcome of Heregulin-induced Activation of Ovarian Cancer
Cells Depends on the Relative Levels of HER-2 and
HER-3 Expression1
Fengji Xu, Yinhua Yu, Xiao-Feng Le,
Cinda Boyer, Gordon B. Mills, and
Robert C. Bast, Jr.2
Division of Medicine, University of Texas M. D. Anderson Cancer
Center, Houston, Texas 77030 [F. X., Y. Y., X-F. L., G. B. M.,
R. C. B.], and Duke University Medical Center, Durham, North
Carolina 27710 [C. B.]
ABSTRACT
Members of the epidermal growth factor receptor family of tyrosine kinases, including epidermal growth factor
receptor, c-erbB-2 (HER-2), c-erbB-3 (HER-3), and
c-erbB-4 (HER-4), can be coexpressed at different levels in
nonhematopoietic tissues. Amplification and overexpression
of HER-2 is found in approximately one-third of cancers
that arise in the breast and ovary. In our previous studies,
heregulin (HRG) and anti-HER-2 antibodies inhibited proliferation, increased invasiveness, and enhanced tyrosine autophosphorylation of SKBr3 breast cancer cells that overexpressed HER-2. In the present report, the effects of HRG
and anti-HER-2 antibody have been compared in six ovarian cancer cell lines. HRG inhibited anchorage-independent
growth of SKOv3 cells that overexpressed HER-2 (105 receptors/cell) but stimulated the growth of OVCA420,
OVCA429, OVCA432, OVCA433, and OVCAR-3 cells that
expressed lower levels of the receptor (104 receptors/cell).
Thus, cell lines with a high level of HER-2 relative to HER-3
or HER-4 were growth inhibited, whereas cell lines with
lower levels of HER-2 were growth stimulated by HRG.
Stimulation or inhibition of clonogenic growth did not correlate with endogenous expression of HRG or with the impact of exogenous HRG on phosphorylation of HER-2,
HER-3, or HER-4. Anti-HER-2 antibodies inhibited the
growth of SKOv3 cells but failed to affect the growth of the
other cell lines. In OVCAR-3 cells that had been transfected
with HER-2 cDNA to increase expression to 105 receptors/
cell, HRG inhibited rather than stimulated growth. Conversely, when HER-2 expression by SKOv3 cells was downregulated by transfection of the viral E1A gene, HRG
Received 1/28/99; revised 8/10/99; accepted 8/17/99.
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.
1
Supported by Grant RO1 CA 39930 from the NIH, Department of
Health and Human Services.
2
To whom requests for reprints should be addressed, at Division of
Medicine, University of Texas M. D. Anderson Cancer Center, Box 092,
1515 Holcombe Boulevard, Houston, TX 77030.
stimulated rather than inhibited growth. To evaluate the
relative importance of HER-3 and HER-4, NIH 3T3 cells
were cotransfected with HER-2 and HER-3 or with HER-2
and HER-4. HRG inhibited the growth of cells with a high
ratio of HER-2:HER-3, whereas HRG stimulated the growth
of cells with low levels of the two receptors. In cells that
express only HER-2 and HER-4, HRG stimulated the
growth of cells that expressed HER-4 independent of HER-2
levels. Anti-HER-2 antibodies inhibited the growth of transfectants with high levels of HER-2 expression independent of
HER-3 or HER-4 expression. In ovarian cancer cells that
express all three receptors, the relative levels of HER-2 and
HER-3 appear to determine the response to HRG. Taken
together, these studies support the concept that the level of
HER-2 expression can modulate response to HRG, determining whether the response is stimulatory or inhibitory. In
contrast, agonistic antibodies that bind to HER-2 alone inhibit anchorage-independent growth but fail to mimic
HRG’s ability to stimulate growth of cells with low HER-2:
HER-3 ratios.
INTRODUCTION
Four members of the EGFR3 family of tyrosine kinase
growth factor receptors have been identified: (a) EGFR; (b)
c-erbB-2 (HER-2); (c) c-erbB-3 (HER-3); and (d) c-erbB-4
(HER-4). These four receptor proteins are normally coexpressed
at different levels in diverse tissues, excluding the hematopoietic
system. Aberrant expression of EGFR has been observed in a
variety of human tumors. In breast and ovarian cancer, increased
or persistent expression of the EGFR has been associated with a
poor prognosis (1, 2). HER-2 gene amplification and or overexpression is found in approximately one-third of breast and
ovarian cancers. Overexpression of HER-2 has been associated
with a poor prognosis in node-positive breast cancers and in
many, but not all, studies of advanced ovarian cancer (3, 4). The
prognostic significance of levels of HER-3 and HER-4 expression are less well defined.
HRG was initially isolated during the search for a ligand
that binds to the HER-2 receptor. The several isoforms of HRG
bind to cells that express HER-3 or HER-4 alone, but not to cells
that express only HER-2 (5–7). However, HRG can bind to
heterodimers of HER-2 and HER-3 or HER-2 and HER-4 (8 –
10). Interactions between HER-2 and HER-3 may be important
for HRG-induced signaling in that HER-3 lacks tyrosine kinase
activity but contains multiple SH2 binding sites. HER-2 and
3
The abbreviations used are: EGFR, epidermal growth factor receptor;
HRG, heregulin; FBS, fetal bovine serum; TCM, tissue culture medium.
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3654 Outcome of HRG-induced Activation of Ovarian Cancer Cells
Fig. 1 HER family (HER-1,
HER-2, HER-3, HER-4) and
HRG receptor expression in
ovarian cancer cell lines.
HER-4 are capable of phosphorylating multiple substrates but
contain a limited repertoire of SH2 binding sites.
Our previous studies have demonstrated that HRG and
anti-HER-2 antibodies inhibit proliferation, increase invasiveness, and enhance tyrosine autophosphorylation of breast cancer
cells that overexpress HER-2, such as SKBr3 cells (11, 12).
Other investigators have reported that HRG stimulates the
growth of breast cancer cells that express low levels of the
HER-2 receptor (13), but that ovarian cancer cells may be
refractory to growth stimulation by the ligand (14). The present
study documents that HRG can, in fact, stimulate anchorageindependent growth of ovarian cancer cells. The ratio of HER2:HER-3 appears to be important in determining whether cells
that express both receptors are stimulated or inhibited by HRG.
In cells that express only HER-2 and HER-4, HRG stimulates
cell proliferation independent of HER-2 levels. When all three
receptors are present, the ratio of HER-2:HER-3 appears to be a
critical determinant in regulating clonogenic growth.
MATERIALS AND METHODS
Cell Lines, Antibodies, and HRG. Human epithelial
ovarian cancer cell lines OVCA420, OVCA429, OVCA432, and
OVCA433 were maintained in MEM supplemented with 10%
FBS, 2 mM L-glutamine, nonessential amino acids, 1 mM sodium
pyruvate, 100 units/ml penicillin, and 100 mg/ml streptomycin
(TCM). OVCAR-3 was maintained in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. SKOv3 was maintained in
McCoy 5A medium containing 10% FBS, 2 mM L-glutamine,
100 units/ml penicillin, and 100 mg/ml streptomycin. The NIH
3T3 murine fibroblast cell line and the 17313 cell line (15) were
kindly provided by Dr. S. Mckenzie (Applied BioTechnology/
Oncogene Science, Cambridge, MA). The 17313 cell line was
produced by transfection of the full-length human HER-2 gene
into NIH 3T3 cells. Both murine cell lines were cultured in
DMEM supplemented with 10% FBS and 2 mM L-glutamine.
Medium for 17313 was additionally supplemented with 400
mg/ml G418 (Life Technologies, Inc., Grand Island, NY).
NIH3T3/H4 13-2-3 cell line, transfected with HER-4, and the
parental NIH3T3-7 cell line were generously provided by Dr.
B. D. Cohen (Bristol-Myers Squibb, Seattle, WA). Both cell
lines were cultured in NIH 3T3 medium. For growth of 3T3/H4
13-2-3, the medium was supplemented with 250 mg/ml G418
and 2.5 mM histidinol (Sigma, St. Louis, MO). For all experiments, cells were detached with 0.25% trypsin-0.02% EDTA
and washed once in complete medium before use.
Murine monoclonal antibodies to the extracellular domain
of p185 were obtained from Applied BioTechnology/Oncogene
Science (TA1, ID5, RC1, RC6, NB3, BD5, PB3, and OD3) and
from Chiron, Inc. (Emeryville, CA; 454C11, 741F8, and 736
G9). All antibodies were of the IgG1 isotype except PB3
(IgG2a) and OD3 (IgM). MOPC21 (IgG1) was used as an
isotype-matched control that did not bind to p185. The H3.105
anti-HER-3 antibody was purchased from NeoMarker (Fremont,
CA) and the 10-4 and 6-4-11 anti-HER-4 antibodies were generously provided by Dr. B. D. Cohen. Antibodies against HRG
(including a and b types) and phosphotyrosine were purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and
Upstate Biotechnology (Lake Placid, NY), respectively.
Recombinant HRG b was obtained from Genentech (South
San Francisco, CA).
Antibody and HRG Binding to EGFR and HER-2,
HER-3, and HER-4 Receptors. A live cell radioimmunoassay was used to determine the binding of monoclonal antibodies
and HRG to different cell lines and transfectants. Cells were
trypsinized and seeded at a density of 2 3 104 cells/well in
96-well removable plates. After overnight incubation, monolayers were washed with 1% FBS in TCM supplemented with 0.1%
sodium azide. For indirect binding assays, different monoclonal
antibodies (10 mg/ml) were added in volumes of 50 ml to cell
monolayers. After incubation at 4°C for 2 h, 125I-labeled sheep
antimouse antibody F(ab9)2 fragment (200,000 cpm/well) was
added in a volume of 100 ml, and incubation was continued for
another 2 h. Nonspecific binding was determined by adding
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Clinical Cancer Research 3655
Fig. 2 Effect of HRG and anti-p185 antibody on anchorageindependent growth of different
ovarian cancer cell lines (#,
P , 0.005; *, P , 0.001).
Fig. 3 Effect of HRG (10 ng/
ml) on anchorage-independent
growth of ovarian cancer cell
lines that express different
number receptors of HER family and HRG (#, P , 0.005; *,
P , 0.001).
125
I-labeled sheep antimouse antibody F(ab9)2 fragment (50 ml)
to cells without monoclonal antibodies or to empty wells. After
incubation on ice for 4 h, unbound antibodies were removed by
washing the wells four times with ice-cold TCM containing 5%
FBS with 0.1% sodium azide. Individual wells were then detached, and radioactivity was determined in a Packard gamma
counter. For direct binding assays, TA1, H3-105, 10-4 monoclonal antibodies, and HRG were directly labeled with 125I using
the Iodogen technique and incubated with different cell lines.
The EBDA program was used to calculate the number of binding sites/cell (16, 17).
Transfection of HER-2, HER-3, and HER-4 cDNA.
Plasmid 9002, containing the full-length human HER-2 gene,
was obtained from Applied BioTechnology/Oncogene Science,
Inc. Plasmid CHER-3x, containing the full-length human
HER-3 gene, was a gift from Dr. Greg Plowman (SUGEN, Inc.,
Redwood City, CA). Plasmid H4y, containing the full-length
human HER-4 gene, was provided by Dr. B. D. Cohen. NIH
3T3 cells and OVCAR-3 cells were transfected by Lipofectamin
as directed by the manufacturer (Life Technologies, Inc.). At
least seven independent transfectants were cloned and cultured
continuously with G418 or hygromycin. Transfected cells were
selected for their ability to bind antibodies directed against
HER-2, HER-3, or HER-4.
Assays of Anchorage-independent Growth. Anchorage-independent cell growth was measured in 35-mm tissue
culture dishes (Nunc, Inc., Naperville, IL). A 1-ml layer of 0.6%
agar (Difco, Detroit, MI) in TCM was solidified in the bottom of
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3656 Outcome of HRG-induced Activation of Ovarian Cancer Cells
mg of normal mouse or rabbit IgG (Santa Cruz Biotechnology)
together with 20 ml of protein A/G-agarose conjugate. In this
study, 250, 500, and 750 mg of protein from total cell lysates of
the six ovarian cancer cell lines were used for immunoprecipitation. Lysates were then immunoprecipitated overnight at 4°C
with 3 mg each of antibodies reactive with HER-2, HER-3, and
HER-4 and 20 ml of protein A/G-agarose conjugate.
Statistical Analysis. Statistical analysis was performed
using the two-sided Student’s t test. WinSTAT 3.1 software was
used for this statistical analysis.
RESULTS
Fig. 4 HRG expression in different ovarian cancer cell lines. A, total
lysates (60 mg/lane) from six ovarian cancer cell lines were separated on
8% SDS-PAGE. Western blotting was performed to determine the
relative level of HRG expression with an anti-HRG antibody. The same
membrane used for HRG detection was stripped and reprobed with an
anti-b-actin antibody to normalize differences in protein loading. A
breast cancer cell line, MDA-MB-231, was used as a known positive
control for HRG. B, The HRG expression of MDA-MB-231 cells was
set as 1. After normalization of protein loading, relative HRG expression
was calculated.
each dish. Cells to be assayed were suspended in 1 ml of 0.3%
agar in TCM supplemented with antibody, control medium, or
different concentrations of HRG or diluant. Cells (2 3 104) were
seeded in each dish. MOPC21, a monoclonal antibody that did
not bind to p185HER-2 or to other cell surface determinants, was
used as a control. Cells were incubated for 10 –14 days at 37°C
in 5% CO2 and 95% humidified air. Colonies containing more
than 30 cells were counted using inverted phase microscopy.
Preparation of Total Cell Lysate and Western Immunoblot Analysis. Cells were treated with or without 10 ng/ml
HRG for different intervals and solubilized in lysis buffer. The
lysates were cleared by centrifugation at 14,000 rpm for 10 min.
Protein concentration of the lysates was measured by the BCA
assay (Pierce Chemical Company, Rockford, IL). Equal
amounts of protein were boiled in Laemmli SDS sample buffer
and resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with specific antibodies. The
signals were visualized with peroxidase-conjugated second antibodies and the enhanced chemiluminescence system (Amersham, Arlington Heights, IL).
Immunoprecipitation. Aliquots of total cell lysates containing equal amounts of protein in lysis buffer [137 mM NaCl,
20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1 mM DTT, 1% NP40,
10% glycerol, and protease inhibitors] were precleared with 2
Expression of EGFR, HER-2, HER-3, and HER-4 in
Ovarian Cancer Cell Lines. Six ovarian cancer cell lines
were characterized for expression of EGFR, HER-2, HER-3,
and HER-4. The 225, TA1, H3-105, and 10-4 antibodies that
recognized each of these receptors were directly labeled with
125
I and incubated with each cell line, and the number of binding
sites was estimated (Fig. 1). The number of binding sites/cell
was taken as an estimate of the receptor level. All six cell lines
express intermediate levels of EGFR (104–105 receptors/cell)
and low levels of HER-3 and HER-4 (103–104 receptors/cell).
One of the six cell lines (OVCAR-3) had low levels of HER-2,
with ,103 receptors/cell; four of the six cell lines (OVCA420,
OVCA429, OVCA432, and OVCA433) had intermediate expression of HER-2, with 104–105 receptors/cell; whereas
SKOv3 had 10 –100-fold higher levels of HER-2, with 105–106
receptors/cell (Fig. 1).
Binding of HRG to Ovarian Cancer Cells. Using a
direct radioimmunoassay, we estimated the number of HRG
binding sites/cell associated with different ovarian cancer cell
lines (Fig. 1). Similar binding of HRG was observed in each of
the six cell lines, although p185HER-2 levels varied from 104–
106 receptors/cell, consistent with the possibility that HER-2 did
not contribute substantially to HRG binding.
Reactivity of Monoclonal Antibodies with EGFR and
HER-2, HER-3, and HER-4 Receptors. Our previous studies defined the reactivity of 11 monoclonal antibodies with the
extracellular domain of p185HER-2 and measured their ability to
inhibit the growth of SKBr3 breast cancer cells (11). To further
investigate all four EGFR family members, these 11 monoclonal
antibodies and one additional anti-HER-2 antibody, 736G9,
were incubated with NIH 3T3 cell lines that had been transfected individually with EGFR, HER-2, HER-3, or HER-4.
Binding of antibodies was measured indirectly with 125I-labeled
sheep antimouse F(ab9)2. Each of these 12 monoclonal antibodies bound strongly to the HER-2 receptor, but not to the EGF,
HER-3, or HER-4 receptors. The 225 anti-EGFR, H3.105 antiHER-3, and 10-4 anti-HER-4 antibodies bound strongly to the
relevant receptors, but not to other receptors in this family. HRG
bound to cells that expressed HER-3 or HER-4, but not to cells
that expressed EGFR or HER-2 alone, consistent with previous
reports (18).
Stimulation or Inhibition and Anchorage-independent
Growth with HRG. The effect of HRG on anchorage-independent growth was measured in each of the six ovarian cancer
cell lines. HRG stimulated growth in five of the six cell lines but
inhibited the growth of SKOv3 cells that overexpressed HER-2
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Clinical Cancer Research 3657
Fig. 5 Phosphorylation of HER-2, HER-3, and HER-4 before and after HRG treatment. All six ovarian cancer cell lines at 50% confluence were
treated with 10 ng/ml HRG for 20 min. After treatment with lysis buffer [137 mM NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 1 mM DTT, 1%
NP40, 10% glycerol, and protease inhibitors], 250, 500, and 750 mg of protein from total cell lysates of these cell lines were used to immunoprecipitate
HER-2, HER-3, and HER-4 with individual monoclonal antibodies, respectively. Western blot analysis with an anti-phosphotryrosine antibody was
used to determine the phosphorylation status of HER-2, HER-3, and HER-4 in each cell line before and after HRG treatment.
Fig. 6 Effect of HRG (10 ng/ml) on anchorage-independent growth of
OVCAR-3 and HER-2-transfected OVCAR-3 ovarian cancer cells (#,
P , 0.05; *, P , 0.001).
(Figs. 2 and 3). Stimulation or inhibition was dependent on the
concentration of HRG. As much as 10 ng/ml HRG was required
to produce significant growth inhibition in SKOv3 cells. To
evaluate the possible contribution of endogenous HRG expression to growth regulation, we have measured relative HRG
expression in six ovarian cancer cell lines by Western blotting
(Fig. 4). Each of the six cell lines expressed the 44-kDa HRG
protein. OVCAR-3 cells exhibited the highest level of HRG
expression, and OVCA432 had the lowest level among these six
cell lines. Consequently, no correlation was found between the
response to HRG treatment and the endogenous expression of
HRG protein.
Interaction with ligand and with antibody has been shown
to activate the HER-2 kinase (11). HER-4 can also be activated
by ligand, whereas HER-3 lacks kinase activity but can be
phosphorylated by interaction with other members of the HER
family (18). To explore the possibility that differences in phos-
phorylation of receptors might correlate with growth stimulation
or inhibition, the phosphorylation status of HER-2, HER-3, and
HER-4 was measured after treatment with HRG. As shown in
Fig. 5, increased phosphorylation of HER-2 after HRG treatment was observed in every cell line. Increased phosphorylation
of HER-3 after HRG treatment was observed in OVCAR-3,
OVCA420, OVCA429, and OVCA432, whereas little, if any,
phosphorylation of HER-3 was observed in SKOv3 and
OVCA433. Increased phosphorylation of HER-4 after HRG
treatment was observed only in OVCAR-3 cells (Fig. 5). Thus,
no clear correlation was observed between the impact of HRG
on clonogenic growth and tyrosine phosphorylation of HER-2,
HER-3, and HER-4.
The monoclonal antibody ID5, which binds to the extracellular domain of p185HER-2, inhibits the anchorage-independent growth of cells that overexpress HER-2 (19). Incubation
with ID5 failed to inhibit or to stimulate growth in the five
ovarian cancer cell lines that exhibited low levels of p185HER-2
but did inhibit the anchorage-independent growth of SKOv3
cells that expressed 105–106 receptors/cells (Fig. 2). Incubation
with HRG and ID5 did not prevent HRG-induced stimulation of
anchorage-independent growth of the five cell lines with low
levels of HER-2 expression (Figs. 2 and 3), and the ID5 antibody did not block HRG binding (data not shown).
Effects of HRG on Transfected Ovarian Cancer Cells
with Different Levels of HER-2 Expression. Because the
ovarian cancer cell lines were derived from different patients, an
apparent correlation between p185HER-2 expression and the effect of HRG on anchorage-independent growth might relate to
other abnormalities in the cells. Consequently, we have examined ovarian cancer cells and transfectants in which HER-2
expression could be up-regulated or down-regulated on the same
background. OVCAR-3 cells express 103 HER-2 receptors/cell
and are stimulated by HRG. Transfection of OVCAR-3 cells
with full-length HER-2 cDNA produced the OVU15 clone that
expressed 105 p185HER-2 sites/cell. Expression of HER-3 and
HER-4 was similar in the transfectants and in parental cells.
HRG stimulated the growth of the parental OVCAR-3 cells
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3658 Outcome of HRG-induced Activation of Ovarian Cancer Cells
Fig. 7 Effect of HRG and anti-p185 antibodies on anchorage-independent growth of
SKOv3 and E1A-transfected
SKOv3 ovarian cancer cells (*,
P , 0.01; **, P , 0.001).
Fig. 8 Effect of HRG and anti-p185 antibody on anchorageindependent growth of NIH
3T3 transfectants with HER-2
and different levels of HER-4
(*, P , 0.05; **, P , 0.001).
ND, not detected.
2– 4-fold but inhibited the growth of the OVU15 cells with
greater p185HER-2 expression (Fig. 6).
Introduction of E1A into cells that overexpress HER-2 can
lower HER-2 expression and inhibit tumor cell growth (20). The
SKOv3 cell line that overexpressed HER-2 was transfected with
E1A to produce E1A-SKOv3 cells that had substantially lower
levels of p185HER-2 (Fig. 7). Similar levels of HER-3 and
HER-4 were found once again in the parental and transfected
cells. HRG at high concentrations inhibited the growth of parental SKOv3 cells but stimulated the anchorage-independent
growth of transfectants with decreased HER-2 expression.
Effect of HRG on NIH 3T3 Cells Transfected with
HER-2, HER-3, and/or HER-4. Ovarian carcinoma cell lines
express different levels of EGFR, HER-2, HER-3, and HER-4.
HRG can interact with multiple receptors and permit crosstalk to
occur (9, 21, 22). Consequently, we have studied interactions of
HRG with NIH 3T3 cells that have been transfected with
HER-2, HER-3, or HER-4 cells, alone or in combination. HRG
failed to stimulate NIH 3T3 cells that had been transfected with
HER-2 or HER-3 alone (data not shown). By contrast, a 5–7fold stimulation of colony formation in anchorage-independent
assays was observed when cells transfected with HER-4 were
treated with HRG (Fig. 8).
In transfectants that expressed HER-2 and HER-3, the ratio
of the two receptors proved to be important. With high levels of
HER-2 ($105) and low levels of HER-3 (clone E21), HRG and
ID5 inhibited anchorage-independent growth. When similar levels of HER-2 and HER-3 were expressed (clone E22), HRG, but
not ID5, stimulated cell growth (Fig. 9). Transfectants with
intermediate levels of HER-2 (clone E6) exhibited a lower level
of growth stimulation by HRG (Fig. 9). Consequently, the
inhibition or stimulation of anchorage-independent growth by
HRG appeared to depend on the ratio of HER-2:HER-3 expression in cells that bore only these two receptors. Transfectants
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Clinical Cancer Research 3659
Fig. 9 Effect of HRG and anti-p185 antibodies on anchorage-independent growth of NIH
3T3 cell transfectants with different levels of HER-2 and
HER-3 (#, P , 0.01; *, P ,
0.001).
that contained combinations of HER-2 and HER-4 were stimulated regardless of the ratio of HER-2:HER-4 (Fig. 8). The
anti-HER-2 ID5 antibody regularly inhibited clonogenic growth
in cells that expressed 106 HER-2 receptors/cell.
DISCUSSION
In our earlier studies, HRG and certain anti-HER-2 antibodies inhibited clonogenic growth of the SKBr3 breast cancer
cell line that overexpressed HER-2 (19). In the present study,
clonogenic growth of SKOv3, an ovarian cancer cell line that
overexpressed HER-2, was similarly inhibited by HRG and an
anti-HER-2 antibody. Conversely, HRG stimulated anchorageindependent growth of five ovarian cancer cell lines that expressed lower levels of HER-2. All six cell lines expressed
modest but demonstrable levels of HER-3 and HER-4. All six
cell lines bound HRG and exhibited similar densities of binding
sites for this ligand, consistent with the previously reported
importance of HER-3 and HER-4 for binding HRG (18). In
contrast to a previous report (14), it appears that HRG is capable
of regulating the growth of ovarian cancer cell lines. The apparent difference between reports may relate to the different
assays used to monitor growth of cancer cells.
In the clinic, the adverse prognostic significance of HER-2
overexpression appears greatest in node-positive breast cancer
(1, 3) and in late-stage ovarian cancer (4). Although it may seem
paradoxical that clonogenic growth can be inhibited by ligand
and by agonistic anti-HER antibodies in cells that overexpress
the receptor, recent data suggest that agonists increase the invasiveness of cells that overexpress HER-2 (12). Consequently,
enhanced potential for invasion and metastasis, rather than an
increased rate of growth or clonogenicity, may be associated
with HER-2 overexpression. However, HRG might exert autocrine growth stimulation in cells with lower levels of HER-2,
and the prognostic significance of HRG expression in cells with
normal HER-2 levels deserves further study.
Overexpression of HER-2 has been found in approximately
30% of ovarian cancer (4), whereas overexpression of HER-3
and HER-4 has not been reported to date. Consistent with these
studies of cancer tissues, HER-2 levels in the six ovarian cancer
cell lines varied over 3 orders of magnitude, whereas the levels
of EGFR, HER-3, and HER-4 varied by only 1 order of magnitude. Thus, marked heterogeneity of expression was observed
with only one of the four members of the HER family of
receptors. A corollary of this observation is that different levels
of HER-2 expression will produce markedly different ratios of
HER-2:HER-3 and HER-2:HER-4. When HRG was incubated
with each of the six cell lines, growth inhibition appeared to
correlate with high levels of HER-2 that produced high HER2:HER-3 and HER-2:HER-4 ratios. Inhibition or stimulation of
growth with HRG did not correlate with endogenous HRG
expression or with HRG-induced phosphorylation of HER-2,
HER-3, or HER-4. Consequently, downstream effects of HER-2
overexpression must contribute to the growth inhibition observed.
The effect of HRG in NIH 3T3 cells transfected with
HER-2 and HER-3 or HER-2 and HER-4 suggest that the ratio
of HER-2:HER-3 is important for cell growth regulation,
whereas the ratio of HER-2:HER-4 is not. In OVCAR-3 cells
and SKOV-3 cells that express all three receptors, modulation of
HER-2 levels altered the response to HRG and to agonistic
anti-HER-2 antibody. Our data suggest that the interaction of
HER-2 and HER-3 may be particularly important for HRG or
antibody-induced growth inhibition, overriding the stimulation
produced by HER-4 homodimers or HER-2/HER-4 heterodimers. However, our data do not permit us to distinguish
between the importance of the ratio of HER-2:HER-3 and the
absolute level of HER-2 in determining response to HRG.
Anti-HER-2 antibodies inhibited the growth of cells with
high levels of HER-2, regardless of HER-3 or HER-4 expression. Antibody-mediated growth stimulation was not observed.
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3660 Outcome of HRG-induced Activation of Ovarian Cancer Cells
The ID5 antibody binds to HER-2 alone and presumably signals
predominantly though this receptor. HRG can signal through
HER-4 homodimers, through HER-2/HER-3 heterodimers, or
through HER-2/HER-4 heterodimers. Our results pose the interesting possibility that signaling through HER-2 alone inhibits
clonogenic growth, and that this is recognized most readily in
cells that overexpress HER-2. Additional studies are underway
to delineate signaling pathways in cells treated with HRG or
with agonistic HER-2 antibodies.4 Both agents can induce autophosphorylation of HER-2 and activate mitogen-activated
protein kinase. The ID5 anti-HER-2 antibody, but not HRG,
increases activity of phospholipase-C g and perturbs diacylglycerol levels. HRG, but not ID5, increases phosphatidylinositol 39-kinase activity, AKT kinase activity, p70S6 kinase activity, and c-jun-NH2-kinase activity as well as inducing
differentiation. Thus, HRG and ID5 signal by different pathways.
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Downloaded from clincancerres.aacrjournals.org on July 12, 2017. © 1999 American Association for Cancer
Research.
The Outcome of Heregulin-induced Activation of Ovarian
Cancer Cells Depends on the Relative Levels of HER-2 and
HER-3 Expression
Fengji Xu, Yinhua Yu, Xiao-Feng Le, et al.
Clin Cancer Res 1999;5:3653-3660.
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