From the
We investigated the mechanism of action of the human prolactin
(hPRL) receptor on four different breast cancer cell lines, T-47D,
MCF-7, BT-474, and SK-BR3, that express elevated levels of the receptor
compared with normal cells. Cells treated with human growth hormone
(hGH), which binds and activates the hPRL receptor, exhibited
bell-shaped dose-response growth curves consistent with the sequential
dimerization mechanism proposed for the hPRL receptor (Fuh, G., Colosi,
P., Wood, W.I., and Wells, J.A.(1993) J. Biol. Chem. 268,
5376-5381). Growth stimulation was enhanced by
Zn
Breast cancer affects one in every eight women in the United
States and is a significant cause of death (Kelsey and Gammon, 1991).
It has been shown that hPRL
The hPRL receptor
has been cloned from mammary cancer cells (Boutin et al.,
1989). It contains a single extracellular domain, a transmembrane, and
an intracellular domain. Ligand-induced dimerization is believed to be
responsible for signaling. Mutational studies have shown that hormones
that can activate the receptor contain two binding sites for the
receptor, Site 1 and Site 2 (Fuh et al. (1993); for review,
see Wells and de Vos(1993)). These sites bind in a sequential fashion
where the hormone binds first through Site 1 and then Site 2
(Fig. 1). Furthermore, monoclonal antibodies to the PRL receptor
can cause weak signaling (Djiane et al.,1985).
Here,
we investigate the mechanism of action of the hPRL receptor on four
different human breast cancer cell lines. We find these cells can be
stimulated to grow by addition of hGH, and the growth activation
pattern fits the sequential dimerization model for the hPRL receptor.
Furthermore, antagonists to the hPRL receptor inhibit the growth of
these cells. Surprisingly, not all breast cancer cell lines respond
equivalently to these antagonists. In particular, one cell line
(BT-474) is more potently inhibited by an hPL (not an hGH) antagonist
and produced transcripts that coded for a novel form of the
extracellular domain of the prolactin receptor. These studies provide
further support for the importance of the hPRL receptor in breast
cancer and suggest that antagonists to it may help to treat the
disease.
T-47D cells were from the American Type Culture Collection.
The other breast cancer cell lines, BT-474, MCF-7, and SK-BR3, were
provided by G. Lewis at Genentech. hGH was from Genentech, and variants
of hGH (G120R, K168A/E174A, and D171A/R64A) were provided by B.
Cunningham at Genentech. hPL and hPRL were from B. DeVos at Genentech.
The G120R-hPL mutant was made by site-directed mutagenesis (Kunkel
et al., 1987) and purified as described (Lowman et
al., 1991). The hPRLbp was expressed in Escherichia coli and purified as described (Cunningham et al., 1990). The
monoclonal antibody
(405) to the HER-2
(405) was provided
by P. Carter at Genentech. Polyclonal antibodies to the hPRLbp (PC4)
were generated in rabbits, using the hPRLbp as an immunogen, and
purified on an hPRLbp affinity column as described (Cunningham et
al., 1990).
Both T-47D cells (see
Fig. 2A) and SK-BR3 cells (see Fig. 2B)
showed a bell shape when treated with increasing concentrations of hGH.
The bell shape reflects the sequential dimerization mechanism for
activation (Fig. 1) seen when hGH is added to FDC-P1 cells
transfected with either the hGH (Fuh et al., 1992) or hPRL
receptor (Fuh et al., 1993). The maximal enhancement in cell
growth observed in these and other studies on MCF-7 and BT-474 cells
(not shown), varied from 110 to 160% of the untreated control depending
on the cell line. While the maximal response may vary from experiment
to experiment, each dose-response curve showed the characteristic bell
shape. The modest enhancement in hGH-simulated growth suggests that
these cells are growing at near maximal rate in the assays. This is not
surprising since there are a wide variety of growth stimulants in serum
used in the assay media (Biswas et al., 1987), and breast
cancer cells have been shown to produce their own growth factors (Huff
et al., 1988).
Growth of MCF-7 and BT-474 cells
is only marginally stimulated by additional hGH. We hypothesized that
sufficient amounts of a lactogen (or other growth factors) may be
present in serum or produced by MCF-7 cells and BT-474 to maximally
stimulate them to grow. We therefore tested whether growth of these
cells in serum could be inhibited by addition of either the hGH or hPL
G120R analog. Indeed, in the case of MCF-7 cells, both analogs could
inhibit growth, and the inhibition by G120R-hGH was nearly 90%
(Fig. 3C). The G120R-hPL was about 10-fold less potent
than the G120R-hGH analog, consistent with its weaker binding to the
hPRL receptor.
These results were in contrast to those found in
BT-474 cells (Fig. 3D). Here, the G120R-hPL analog was
considerably more potent than the G120R-hGH analog in inhibiting
serum-stimulated growth of BT-474 cells. The inhibition by both analogs
was more pronounced in the presence of added Zn
T-47D cells were more resistant to the inhibition by
either antagonist. The greatest inhibition was only 12% that of cells
not treated with the antagonists (Fig. 3A). T-47D cells
have more hPRL receptors than other cell lines (), and to
antagonize their growth would require that the antagonist occupy many
more receptors at a time. Previous experiments suggest that only a
small portion of the hPRL receptors need to signal to exert a maximal
effect (Ashkenazi et al., 1987; Fuh et al., 1993).
For example, the K
Data are reproduced from Shiu (1979).
We thank Dr. Tim Clackson for advice in PCR cloning
and general encouragement, Gail Lewis for advice in breast cancer cell
assays, and Wayne Anstine for graphics.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, which preferentially increases the affinity of
hGH for the hPRL receptor. Furthermore, receptor-selective variants of
hGH that bind the hPRL receptor but not the hGH receptor were
agonistic, providing additional support that specific binding to the
hPRL receptor can stimulate these breast cancer cells to grow. On this
basis we produced variants of hGH and human placental lactogen (hPL)
that were potential antagonists because they bind but do not dimerize
the hPRL receptor. The hPL-based antagonist was less potent than the
hGH-based antagonist toward the growth of MCF-7 cells, consistent with
the lower affinity of hPL for hPRL receptor than for hGH. However, the
hPL-based antagonist was more potent than the hGH antagonist for BT-474
cells. Antibodies to the hPRL receptor inhibited growth of FDC-P1 cells
transfected with the hPRL receptor; these also inhibited MCF-7 cells
and T47D cells but not BT-474 cells. A unique feature of BT-474 cells
was found when screening its cDNA revealed the presence of a novel
alternative splice of the hPRL receptor that codes for the soluble
extracellular domain; this may explain these differential inhibitory
effects. These studies provide further molecular insight into the
potential role of the hPRL receptor in breast cancer and demonstrate
that hPRL receptor antagonists can inhibit the growth of breast cancer
cells.
(
)
receptors are
present in 40-70% of tumor biopsies (Bonneterre et al.,
1990; Murphy et al., 1984). Furthermore, many breast cancer
cell lines express 2-10 times more hPRL receptors than normal
cells (Shiu, 1979) (). Lactogenic hormones such as hPRL or
hPL that bind specifically to the hPRL receptor can stimulate the
growth of human breast cancer cell lines (Shiu, 1985; Manni et
al., 1986; Biswas et al., 1987). Although it is believed
that lactogenic hormones may play a role in the growth and development
of breast cancer in animal models (Phares, 1986; Platica et
al., 1991; Tornell et al., 1991), the importance of these
hormones in the human disease is still unclear. For example, higher
circulating levels of lactogenic hormones have been found in breast
cancer patients by some investigators (Maddox et al., 1992)
but not by others (Love et al., 1991).
Figure 1:
Sequential dimerization model for
activation of the hPRL receptor by hGH, hPL, or hPRL. These hormones
have two sites for binding the hPRL receptor. Binding occurs first
through Site 1 (middlepanel) and then Site 2
(lowerpanel). At very high concentrations the
hormone can antagonize itself by blocking receptors using Site 1 and
thus prevent dimerization (bottompanel). Figure
taken from Fuh et al. (1993) and published with
permission.
A
homolog of hGH, hPL, can also activate the hPRL receptor. Unlike hPRL,
hPL and hGH require Zn to bind the hPRL receptor via
Site 1 (Cunningham et al., 1990; Lowman et al.,1991). The structural basis for this was elaborated in a recent
x-ray crystal structure of the complex between hGH and the
extracellular domain of the hPRL receptor (Somers et al.,
1994). Based upon the sequential dimerization mechanism it has been
possible to build hPRL receptor antagonists that bind to the receptor
via a normal Site 1 but do not allow efficient dimerization because of
a mutation introduced into Site 2 (Fuh et al., 1993).
Cell Proliferation Assay for Testing Agonists and
Antagonists
Breast cancer cells were cultured in a 1:1 mixture
of Dulbecco's modified Eagle's medium and Ham nutrient
mixture F-12 medium supplemented with 2 mM glutamine, 100
units of penicillin/ml, 100 µg of streptomycin/ml, and 10% FBS. For
assays, 1 10
trypsinized cells were added to each
well of a 96-well plate and allowed to incubate at 37 °C for
6-18 h in media with 1% diafiltered FBS before hormones were
added. The final assay medium had 15 µM of ZnSO
and either 1% diafiltered FBS for assaying agonists or 3% FBS for
assaying antagonists. We used diafiltered FBS to remove potential small
molecule growth agonists (like steroids) and to better control the
amount of zinc salts present. Trypsinization followed by incubation in
low serum media served to fast the cells before hormone treatments.
When assaying MCF-7 cells, no zinc was added because additional zinc
increased the background growth, making the antagonists less effective.
After incubating cells with the hormones for 3 or 4 days, cells were
pulsed with 1 µCi/well of [
H]thymidine
(Amersham Corp.) for 6 h. The cells were harvested onto the glass
filters, and radioactivity was measured in a scintillation counter (Fuh
et al., 1993). Standard errors were calculated from three to
five replicate samples at each hormone concentration. The proliferation
assay of FDC-P1 cells transfected with the hPRL receptor was performed
as described (Fuh et al., 1993). Briefly, cells were grown in
suspension with hGH, and the hGH was removed 24 h prior to each assay.
About 4
10
cells were added to each well and
incubated with the indicated hormone for 18 h. Cells were pulsed with
[
H]thymidine for 4 h before harvesting. The assay
medium was supplemented with 1% horse serum to reduce the background
growth. Competitive displacement of [
I]hGH was
used to determine the affinity of hGH analogs for hPRLbp as described
(Cunningham et al., 1990).
Cloning of a Variant Form of the hPRL
Receptor
mRNA was extracted from BT-474 and T-47D cells and
primed with random hexameric oligonucleotides. Degenerate PCR primer
pairs were designed from the most conserved regions of the hPRL
receptors and hGH receptors. PCR was performed with Taq polymerase (Perkin-Elmer) at an annealing temperature of 55 °C
on cDNA derived from about 0.13 µg of mRNA. The PCR fragments were
then cloned into the pCRII vector (Invitrogen). The PCR
fragments from individual clones were digested with two restriction
enzymes, and the fragments were compared with those of the hGH or hPRL
receptor by agarose gel electrophoresis. The clones having a
restriction digest pattern different from that of the hGH or hPRL
receptor were sequenced. The presence of the transcript for the
alternatively spliced form of the hPRL receptor was confirmed by PCR of
the cDNA with variant specific primers at a higher annealing
temperature (72 °C).
Activation of the hPRL Receptor Can Stimulate Breast
Cancer Cells To Grow
Four breast cancer cell lines (T-47D,
MCF-7, BT-474, and SK-BR3) were chosen for study based on the fact that
they have different numbers of hPRL receptors/cell (). We
first wished to establish whether hGH could stimulate these cells to
proliferate in our system, and if so determine the characteristics of
the activated receptor.
Figure 2:
Growth stimulation of breast cancer cells
T-47D (panel A) or SK-BR3 (panel B) upon addition of
hGH or hGH analogs. Cells were treated with increasing concentrations
of hormone as indicated for 4 days. DNA synthesis was measured by
[H]thymidine incorporation as described under
``Materials and Methods.'' A, hGH plus (
) or
minus (
) 15 µM of ZnSO
. B, wild
type hGH (
), K168A/E174A (
), or R64A/D171A (
), all
with 15 µM ZnSO
.
Previous studies have shown that
Zn enhances binding of hGH and activation of the hPRL
receptor, but it does not affect binding of hGH to the hGH receptor
(Cunningham et al., 1990; Fuh et al., 1993). The
concentration of hGH required for half-maximal stimulation of T-47D
cells (EC
) was shifted from
1 nM to
0.1
nM when additional Zn
was provided to the
cells (Fig. 2A). Receptor-selective variants of hGH have
been made that bind specifically to either the hGH or hPRL receptors
(Cunningham and Wells, 1991). The thymidine incorporation of SK-BR3
cells was stimulated by a variant that binds selectively to the hPRL
receptor (R64A/D171A) but not by one that binds selectively to the hGH
receptor (K168A/E174A) (Fig. 2B). Similar results were
found for T-47D cells (not shown). hPRL is known to bind only the hPRL
and not hGH receptor. Treatment of T-47D cells with 2 and 20
nM hPRL resulted in thymidine incorporation that was 108
± 18 and 138 ± 7% of untreated cells, which was similar
to the DNA synthesis in cells treated with hGH. These studies show that
hGH and hPRL can modestly stimulate these cells to grow and suggest
that these hormones exert their effect through binding and dimerization
of an hPRL-like receptor.
Antagonists to the hPRL Receptor Inhibit hGH-stimulated
Growth
FDC-P1 cells transfected with the full-length hPRL
receptor can be stimulated to grow with either hGH or hPRL (Fuh et
al., 1993). An hGH analog, G120R, antagonizes hGH-stimulation of
these cells because it blocks binding at Site 2 and does not allow
receptor dimerization (Fig. 1). When T-47D cells were stimulated
to grow with 1 nM hGH, growth could be inhibited by the G120R
analog in a dose-dependent manner (Fig. 3A).
Figure 3:
Antagonism of T-47D cells with G120R-hGH
() (panel A), FDC-P1 cells transfected with the hPRL
receptor by G120R-hGH (
) or G120R-hPL (
) (panel B),
MCF-7 cells with G120R-hGH (
) or G120R-hPL (
) (panel
C), and BT474 cells by G120R-hGH with ZnSO
(
) or
without ZnSO
(
) or by G120R-hPL with ZnSO
(
) or without ZnSO
(
) (panel D).
T-47D cells and transfected FDC-P1 cells were incubated with 1
nM hGH plus the serial dilutions of antagonists. MCF-7 cells
and BT-474 cells did not require additional hGH for maximal stimulation
and were treated with increasing concentrations of the indicated
antagonists. The 100% value for all panels represents data
from control cells treated with assay media alone. See ``Materials
and Methods'' for details.
hPL
binds selectively to the hPRL receptor, albeit about 10-fold weaker
than hGH. Like hGH, it requires Zn for binding the
hPRL receptor (Lowman et al., 1991). We produced the G120R
variant of hPL and found that it inhibits hGH-stimulated growth of the
FDC-P1 cells transfected with the hPRL receptor
(Fig. 3B). The IC
for G120R-hPL was about
8-fold higher than for G120R-hGH.
,
consistent with binding to an hPRL-like receptor. However, it is
possible that the receptors on BT-474 cells are different from hPRL
receptor since they are inhibited more by the hPL antagonist than the
hGH antagonist.
for binding the
prolactin receptor on either Nb2 cells or FDC-P1 cells transfected with
the hPRL receptor is about 10 times higher than the EC
for
stimulation of growth by hGH (Fuh et al., 1993).
BT-474 Cells Produce a Transcript for a Soluble Form of
the hPRL Receptor
Polyclonal antibodies to the hPRLbp
(designated PC4) inhibited hPRL-stimulated growth of FDC-P1 cells
transfected with the hPRL receptor (Fig. 4A). Previous
studies have shown that the PC4 antibody does not compete with hormone
binding to the hPRL receptor (Cunningham et al., 1990). It is
possible that the antibody interferes with dimerization of the hPRL
receptor by reacting at a site where the receptors come together as
seen when monoclonal antibody 5 binds to the hGH receptor (Cunningham
et al., 1991). The PC4 antibody inhibits growth of
serum-stimulated MCF-7 cells (Fig. 4B) and hGH-induced
growth of T-47D cells (data not shown). However, the PC4 antibody was
ineffective at inhibiting the growth of BT-474 cells even when present
at a concentration of 2 µM (Fig. 4C). This
compares with the inhibition of BT-474 cells by the G120R-hPL analog,
which at 1 µM caused about 40% inhibition. This suggested
the possibility that BT-474 cells could produce a variant form of hPRL
receptor and thus may not bind the antagonistic antibody to hPRL
receptor.
Figure 4:
Inhibition of the growth of FDC-P1 cells
transfected with the hPRL receptor (panel A) or of breast
cancer cells MCF-7 (panel B) or BT-474 (panel C) with
polyclonal antibodies to the extracellular domain of the hPRL receptor
(hPRLbp). In panelA, FDC-P1 cells were incubated
with increasing concentrations of hPRL alone (), polyclonal
antibodies (PC4) to the hPRLbp alone (
), or PC4 in the presence
of 2 nM hPRL (
). MCF-7 cells (panelB)
or BT474 cells (panelC) were treated with PC4
(
) or G120R-hPL (
). For panelsB and
C, the 100% value represents data from cells treated with
assay media alone. DNA synthesis was measured as described under
``Materials and Methods.''
To better understand the difference in hormone specificity
on BT-474 cells, we screened for cDNAs that were homologous to the hGH
and/or hPRL with the receptor using degenerate PCR (Fig. 5). We
isolated two different clones. One of these isolated from both T-47D
and BT-474 cells was identical to the hPRL receptor previously reported
(Boutin et al., 1989). The second one, isolated from only
BT-474 cells, had the transmembrane domain exon deleted and
consequently a frameshift after Ser. This produced a stop
codon after two additional residues, Ala
and
Trp
. This novel transcript would code for a soluble
extracellular domain of the hPRL receptor (Fig. 5). A similar
construct has been generated by recombinant methods and expressed in
E. coli (Cunningham et al., 1990). The protein is
soluble and binds hPRL with virtually the same affinity as the
full-length receptor (Cunningham et al., 1990). The cDNA for
the extracellular domain transcript represented about 5-10% of
the total cDNA coding for the hPRL receptor in BT-474 cells and was not
found in the other breast cancer cells we have studied (data not
shown). We were able to amplify this transcript from BT-474 cells by
PCR with primers that are specific for the alternatively spliced form
(data not shown).
Figure 5:
DNA and deduced protein sequence of an
alternatively spliced transcript of the hPRL receptor that codes for
the hPRLbp from BT-474 cells. The arrow points to the deleted
portion, which includes transmembrane domain in the shadedbox. The degenerate PCR primers I and III (underlined in the sequence) were first used to clone the hPRL receptors, and
primers I and II were used to prime the variant specific
transcript.
The significance of the transcript for a soluble
form of the hPRL receptor has yet to be determined, and further work is
required to show that this transcript is translated and expressed. Such
a form is reminiscent of the soluble form of hGH receptor (Baumann
et al., 1986) thought to be shed by proteolysis of the
full-length receptor (Leung et al., 1987). There are many
other examples of soluble cytokine receptors for which various
biological functions have been implicated including extending the
circulating half-life of the hormone and antagonizing the action of
certain cytokines (for review, see Rose-John and Heinrich(1994)). The
production of the soluble extracellular domain could potentially
protect the BT-474 cells from inhibition by the PC4 polyclonal antibody
and could explain the reversal in apparent inhibition by the
antagonists if it bound more tightly to G120R-hGH than to G120R-hPL.
Prospects for Use of hPRL Receptor Antagonists in Breast
Cancer
These studies survey the possibilities for using hPRL
receptor antagonists in breast cancer. In most cases the hPRL receptor
present on these four breast cancer cell lines was able to stimulate
growth and did so in a dimerization-dependent manner. We find that
receptor number as well as the presence or absence of the soluble hPRL
receptor can have profound effects on the effectiveness of agonists or
antagonists to the receptor. Very high levels of current antagonist
were required for substantial inhibition of growth, and this would
likely prohibit their clinical use. However, it is possible to increase
the affinity of Site 1 in the G120R-hGH analog and make it a more
potent antagonist to the hGH receptor (Fuh et al., 1992;
Lowman et al., 1991; Lowman and Wells, 1993). Such a strategy
could be applied here to improve either the hGH or hPL antagonists
toward the hPRL receptor. Our finding that BT-474 cells produce a
unique transcript coding for the extracellular domain could represent a
breast cancer-specific form of the receptor. Although antagonists to
the hPRL receptor alone were not dramatically effective at inhibiting
the growth for most of these cell lines, perhaps the analogs may be
more effective when used in conjuction with other growth inhibitors. In
fact we find that the G120R hGH antagonist was more effective at
inhibiting the growth of SK-BR3 cells when added in the presence of an
inhibitory antibody to the HER-2 receptor than for just the antibody
alone (data not shown). Thus, an antagonist to the hPRL receptor may be
potentially important in the treatment of this complex disease.
Table:
hPRL receptors are abundant in various human
breast cancer cell lines
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.