©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Prolactin Receptor Antagonists That Inhibit the Growth of Breast Cancer Cell Lines (*)

Germaine Fuh , James A. Wells (§)

From the (1) Department of Protein Engineering, Genentech, Inc., 460 Pt. San Bruno Blvd., South San Francisco, California 94080 and the Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, California 94143

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, 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.


INTRODUCTION

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() 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).

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).


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).

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.


MATERIALS AND METHODS

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).

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).


RESULTS AND DISCUSSION

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.

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).


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.

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, 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.

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 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

Data are reproduced from Shiu (1979).



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: hPRL, human prolactin; hGH, human growth hormone; hPL, human placental lactogen; hPRLbp, the extracellular domain of the hPRL receptor; FBS, fetal bovine serum; PCR, polymerase chain reaction; mutants are designated by the wild-type residue (in single-letter code) followed by its position and the mutant residue; multiple mutants are indicated by a series of single mutants separated by slashes.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.