(Received for publication, June 26, 1995)
From the
HER2, the erbB-2/neu proto-oncogene product,
is a 185-kDa transmembrane glycoprotein related to the epidermal growth
factor receptor. Overexpression of HER2 was reported in several human
adenocarcinomas, including mammary and ovarian carcinomas. A family of
glycoproteins, the heregulin/neu differentiation factors, was
characterized and implicated as the ligands for HER2. Recently, it has
been shown that HER2 alone is not sufficient to reconstitute high
affinity heregulin receptors and that HER3 or HER4 may be the required
components of the heregulin receptors on mammary carcinoma cells
(Sliwkowski, M. X., Schaefer, G., Akita, R. W., Lofgren, J. A.,
Fitzpatrick, V. D., Nuijens, A., Fendly, B. M., Cerione, R. A.,
Vandlen, R. L., and Carraway, K. L., III(1994) J. Biol. Chem. 269, 14661-14665; Plowman, G. D., Green, J. M., Culouscou,
J.-M., Carlton, G. W., Rothwell, V. M., and Buckley, W.(1993) Nature 366, 473-475). Using the Cytosensor
to measure the extracellular acidification rate, we have examined
the effects of recombinant human heregulin-
on three mammary
carcinoma cell lines expressing HER2 (MDA-MB-453, SK-BR-3, and MCF-7),
an ovarian carcinoma cell line expressing HER2 (SK-OV-3), and CHO-K1
and 293-EBNA cells stably transfected with HER2. By reverse
transcription polymerase chain reaction and Western blotting, we found
that the breast cells also express HER3 and that the ovarian line
co-expresses the HER4 message. A dramatic increase in the acidification
rate was observed for the mammary carcinoma cells co-expressing high
levels of HER2 and HER3. In contrast, the ovarian cells expressing high
levels of HER2 and low levels of HER4 or CHO-K1 and 293-EBNA cells
expressing HER2 alone were not responsive to heregulin. When these same
transfected cells were exposed to monoclonal anti-HER2 antibody
followed by anti-IgG to cause aggregation of the HER2 molecules, an
increase in the acidification rate was observed, indicating coupling of
transfected HER2 to the signal transduction pathway. Transfection of HER2 into MCF-7 cells, on the other hand, gave 4-fold enhanced
acidification responses. These data, together with the previously
reported high affinity heregulin binding and activation of tyrosine
phosphorylation in HER2 and HER3 co-transfected cells support the role
of HER2 and HER3 as components of the heregulin receptor in breast
cells.
A family of EGF ()receptor-related proteins has
recently been described that includes the proto-oncogene products of erbB-2/neu (HER2), erbB-3 (HER3), and erbB-4 (HER4)(1, 2) , which have a high
degree of sequence homology (40-50%) and similar molecular
structure, containing a glycosylated extracellular domain, a
transmembrane domain, and a conserved tyrosine kinase domain. The HER2
receptor was the first of these discovered by two groups who screened
human genomic libraries at low stringency using EGF receptor-based
probes(3, 4) . Subsequent to this, the HER3 (5) and HER4 (6) receptors were identified using
similar techniques. Despite the structural similarity between these
receptors, they have no binding affinity for any of the EGF ligands,
which leaves open the possibility that ligands exist for each of these
receptors.
The identity of the ligands for these receptors has been the object of much investigation resulting in the identification of several candidate ligands for HER2. A 30-kDa protein (7) and heregulin (8) were purified from the conditioned medium of MDA-MB-231 mammary carcinoma cells. Neu differentiating factor was purified from the conditioned medium of ras-transformed fibroblasts(9) , and Neu activating factor was purified from the conditioned medium of human ATL-2 T cells(10) . Each of these putative ligands was shown to activate phosphorylation of the HER2 protein, p185. Subsequent to the cloning of Neu differentiating factor (11) and heregulin(8) , peptide ligands with a high degree of sequence homology with heregulin/Neu differentiating factor, the glial growth factors-I, -II, and -III (12) and a protein that stimulates muscle acetylcholine receptor synthesis (ARIA) (13) were identified. These findings indicate that a family of heregulin-like ligands exist that are important in the development and regeneration of the nervous system and are products of the same gene, produced by alternatively spliced mRNA(12, 13) .
Whether the heregulin/Neu differentiating factor is truly a ligand
for HER2 was recently disputed by the finding that heregulin did not
activate phosphorylation of p185 in ovarian cell lines expressing HER2
and in ovarian cells transfected with HER2(14) . These results
suggest that another component in addition to HER2 is required for the
heregulin receptor. More recently, both HER4 (16) and
HER3(17, 18) have been separately reported to be the
additional component required in combination with HER2 to reconstitute
heregulin high affinity binding and heregulin-mediated p185
phosphorylation. We have recently found using a sensor-based
microphysiometer, Cytosensor(19, 20) , that heregulin-
stimulates an
increase in the extracellular acidification rate in SK-BR-3 cells,
which express high levels of HER2(21) . Using this technique to
monitor cellular metabolism, we have measured the cellular response of
a series of carcinoma cell lines expressing HER2, HER3, and HER4 and
cells transfected with HER2 for their metabolic response to
heregulin-
. Our results indicate that cells expressing both HER2
and HER3 respond to heregulin, and those expressing HER2 and low levels
of HER4 or HER2 alone do not.
Figure 1:
Acidification response of human mammary
and ovarian carcinoma cells to heregulin-. Carcinoma cells were
prepared and loaded into Cytosensor
capsules as
described under ``Materials and Methods.'' The cells were
monitored in the Cytosensor
until acidification rates
stabilized, and then media containing various concentrations of
heregulin-
were switched into the fluid stream for 15 min. A, SK-BR-3 cells were exposed to 100 (2.2 nM)
(
), 10 (
), 1 (
), or 0 (
) ng/ml
heregulin-
. B, MDA-MB-453 cells were exposed to 80 (1.8
nM) (
), 20 (
), 5 (
), or 0 (
) ng/ml
heregulin-
. C, MCF-7 cells were exposed to 100 (
)
or 0 (
) ng/ml heregulin-
. D, SK-OV-3 cells were
exposed to 100 (
) or 0 (
) ng/ml
heregulin-
.
Figure 2: Expression of HER2 and HER3 in human mammary and ovarian carcinoma cells. Human mammary and ovarian carcinoma cell lines were tested for the presence of HER2 mRNA by Northern blot analysis and p185 by Western blot analysis. A, total RNA (20 µg) isolated from human mammary carcinoma cells (MDA-MB-453, MCF-7, and SK-BR-3), human ovarian tumor cells (SK-OV-3), and human kidney cells (293) was fractionated in a 1% agarose-formaldehyde gel. After transferring to a nylon membrane, the blot was hybridized with a biotinylated HER2 cDNA probe. The hybridized blot was incubated with a streptavidin-alkaline phosphatase conjugate followed by chemiluminescent detection as described under ``Materials and Methods.'' B, the membrane fraction isolated from the same breast and ovarian cell lines were solublized and electrophoresed in SDS-polyacrylamide gels for Western blot analysis as described under ``Materials and Methods.'' The p185 bands were detected using monoclonal anti-HER2 antibody followed by horseradish peroxidase-labeled anti-IgG and chemiluminescent detection. The faint p185 band for MCF-7 is invisible in the photograph, but it is present on the original film. C, Western blot analysis of the mammary and ovarian cell lines for HER3 using anti-HER3 peptide rabbit antibody was conducted as described for B. The p160 bands corresponding to HER3 were detected by chemiluminescent detection.
Figure 3:
RT-PCR detection of HER2, HER3, and HER4
mRNA expression in human mammary and ovarian carcinoma cells. Total RNA
was isolated and RT-PCR was performed as described under
``Materials and Methods.'' A, the ethidium
bromide-agarose gels of the PCR products using primers for HER2, HER3,
and HER4 are shown here. The DNA size standard
X174/HaeIII is also included. B, PCR products
from A were blotted to a nylon membrane, probed with the
corresponding biotinylated probes, and detected as described under
``Materials and Methods.''
Figure 4: Overexpression of HER2 RNA in CHO-K1, 293-EBNA, and MCF-7 stably transfected with pCEP/HER2 expression plasmid. Clonal cell lines stably transfected with pCEP/HER2 plasmid was isolated as described under ``Materials and Methods.'' A, Northern blot analysis of total RNA was performed using a biotinylated HER2 cDNA probe. SK-BR-3 RNA was included as a positive control for HER2 mRNA expression. B, Western blot analysis of membrane fractions of CHO-K1, 293-EBNA, and MCF-7 cells transfected with pCEP/HER2 was performed as described under ``Materials and Methods.'' The p185 bands were detected using monoclonal anti-HER2 antibody followed by horseradish peroxidase-labeled anti-IgG and chemiluminescent detection. SK-BR-3 was included as a positive control.
Figure 5:
Lack of acidification response by
HER2-transfected CHO-K1 and 293 cells to heregulin-.
HER2-transfected CHO-K1 and 293 EBNA cells were prepared and loaded
into Cytosensor
capsules as described under
``Materials and Methods.'' The cells were monitored in the
Cytosensor
until acidification rates stabilized, and
then media containing 100 ng/ml heregulin-
or 2 µg/ml
anti-HER2 monoclonal antibody were switched into the fluid stream for
15 min. The cells treated with anti-HER2 were immediately exposed to 4
µg/ml anti-mouse IgG for an additional 15 min. A,
CHO/HER2-H cells were exposed to heregulin (
) or media alone
(
). B, CHO/HER2-H cells were exposed to anti-HER2
(
) or control IgG1 (
) followed by anti-IgG. Parental CHO-K1
cells were also exposed to anti-HER2 (
) or control IgG1
(
) followed by anti-IgG. C, 293/HER2-O cells were
exposed to heregulin (
) or media alone (
). D,
293/HER2-O cells were exposed to anti-HER2 (
) or control IgG1
(
) followed by anti-IgG. Parental CHO-K1 cells were also
exposed to anti-HER2 (
) or control IgG1 (
) followed by
anti-IgG. Due to the presence of a high concentration of buffer salts
in the anti-HER2 antibody preparation, the apparent acidification rates
were depressed by approximately 12% any time the preparation was
present in the chamber.
Figure 6:
Increased acidification response to
heregulin- in HER2-transfected MCF-7 cells. Cells were prepared
and loaded into Cytosensor
capsules as described under
``Materials and Methods.'' A, the MCF/HER2-A cells
were monitored in the Cytosensor
until acidification
rates stabilized, and then media containing 50 ng/ml heregulin-
(
) or media alone (
) were switched into the fluid stream for
15 min. B, parental MCF-7 cells were also tested for response
to 50 ng/ml heregulin-
(
) or media alone (
) under the
same conditions as in A.
Ligand binding/cross-linking and tyrosine phosphorylation
assays have been used widely to study the interaction between heregulin
and HER2 in breast cancer cells(1, 2, 18) .
In the present study, we have employed extracellular acidification
measurements to quantitate the specific response of HER2-expressing
mammalian cells to recombinant heregulin-. It has been
demonstrated that ligand-receptor interactions generally result in the
activation of second messenger pathways that trigger increases in
extracellular acidification as measured by a
microphysiometer(19, 20) . Earlier studies showed that
tyrosine kinase-dependent EGF receptor signaling activates increased
extracellular acidification(19, 23) . Our results here
show that human mammary carcinoma cells MDA-MB-453 and SK-BR-3 respond
strongly and rapidly to heregulin stimulation by increasing their
extracellular acidification rate, whereas MCF-7 cells, another mammary
carcinoma cell line, only give a very weak response. The heregulin
responsiveness of these mammary carcinoma cells correlates well with
HER2 expression levels as determined by immunohistochemical staining
and Northern blot and Western blot analyses. This correlation does not,
however, apply to CHO-K1 and 293-EBNA cells stably transfected with HER2 cDNA or to an ovarian carcinoma cell line SK-OV-3, which
naturally expresses high levels of HER2. These cells fail to respond to
heregulin at all. In concordance with our results, a previous
biochemical study demonstrated heregulin cross-linking to HER2 and
heregulin-activated tyrosine phosphorylation of p185 in human tumor
cells of breast, colon, and neuronal origin but not in ovarian cells
that express the receptor in natural abundance nor in transfected
ovarian cells and fibroblasts overexpressing the
receptor.(14) . Taken together, these results indicate that
heregulin activation of cells is dependent on the presence of a
tissue-specific component in addition to HER2, which is absent in
ovarian cells. This hypothesis is further supported by our finding that
the mammary carcinoma cell line MCF-7 that is transfected with HER2 cDNA and selected for overexpression gives an enhanced
acidification response to heregulin.
It is conceivable that the additional component required for heregulin responsiveness may be a tyrosine kinase receptor closely related to HER2, such as HER3 or HER4, and that the heregulin pathway may be mediated by receptor cross-talk or heterodimerization. This possibility is suggested by several studies that have shown EGF receptor-mediated tyrosine phosphorylation of HER2 (26, 27, 28, 29) in transformed rodent and human cell lines. EGF receptor and HER2 have also been shown to act synergistically to trigger transformation of rodent fibroblasts (30) by a mechanism that may involve EGF-induced heterodimerization of EGF receptor and HER2(31) . Using the sensitive RT-PCR method and Western blotting, we detected expression of HER3 at much greater levels in mammary carcinoma cells than in ovarian cells. In contrast, the ovarian cell line SK-OV-3 was the only one tested that expressed significant levels of HER4 message, although the HER4 protein was not detectable by Western blotting. The correlation between extracellular acidification and other data suggests that cells expressing both HER2 and HER3 respond to heregulin stimulation, whereas those expressing HER2 and low levels of HER4 do not. This is in agreement with a recent study in which co-expression of HER2 and HER3 in COS-7 cells reconstituted high affinity heregulin binding and heregulin-induced tyrosine phosphorylation(17) . Our findings, on the other hand, are in contrast to an earlier study that indicated that HER4 mRNA was undetectable in ovarian cells but was expressed in mammary carcinoma cells(6) . The same group also reported data indicating that HER4 is a co-receptor required for heregulin-mediated signal transduction (15) using receptor-transfected cells. This may be the case for cells expressing high levels of the HER4 protein.
In summary, our results indicate that HER2 expression alone does not
impart heregulin responsiveness. Moreover, based on our evidence, cells
naturally co-expressing HER2 and HER3 respond to heregulin; we
speculate that HER3 is the receptor for heregulin, which is in
agreement with the results of Carraway et al.(16) and
Kita et al.(32) . Ligand-driven heterodimerization of
HER3 and HER2 may form a ligand-receptor complex that in turn triggers
the heregulin signal transduction pathway as foreseen by Peles et
al.(14) and proposed by Sliwkowski et
al.(17) . In subsequent experiments, we have
co-transfected HER2 and HER3 cDNA into CHO cells and
reconstituted the acidification response to heregulin in these cells, ()which validates our results with the mammary carcinoma
cells. Our present study also indicates that HER4 does not play a role
in heregulin-mediated signal transduction in the mammary carcinoma
cells tested.