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Endocrinology Vol. 144, No. 12 5300-5307
Copyright © 2003 by The Endocrine Society

Inhibition of Prolactin (PRL)-Induced Proliferative Signals in Breast Cancer Cells by a Molecular Mimic of Phosphorylated PRL, S179D-PRL

Matthew D. Schroeder, Jennifer L. Brockman, Ameae M. Walker and Linda A. Schuler

Department of Comparative Biosciences (M.D.S., J.L.B., L.A.S.), University of Wisconsin, Madison, Wisconsin 53706; and Division of Biomedical Sciences (A.M.W.), University of California, Riverside, California 92521

Address all correspondence and requests for reprints to: Dr. Linda A. Schuler, Department of Comparative Biosciences, University of Wisconsin, 2015 Linden Drive, Madison, Wisconsin 53706. E-mail: schulerl{at}svm.vetmed.wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Posttranslational modifications of prolactin (PRL), including phosphorylation, vary with physiologic state and alter biologic activity. In light of the growing evidence for a role for PRL in proliferation in mammary cancer, we examined the ability of a mimic of phosphorylated human PRL, S179D-PRL, to initiate signals to several pathways in mammary tumor cells alone and in combination with unmodified PRL. Unmodified PRL employed multiple pathways to increase cellular proliferation and cyclin D1 levels in PRL-deficient MCF-7 cells. S179D-PRL was a weak agonist compared with unmodified PRL with regard to cellular proliferation, cyclin D1 levels, and phosphorylation of signal transducer and activator of transcription 5 and ERKs. However, S179D-PRL was a potent antagonist of unmodified PRL to these endpoints. In contrast to the reduced levels of the long isoform of the PRL receptor observed in response to a 3-d incubation with unmodified PRL, S179D-PRL up-regulated expression of this isoform, 4-fold. These studies support the utility of this mutant as a PRL antagonist to proliferative signals in mammary epithelial cells, including a potential role in breast cancer therapeutics.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALTHOUGH PROLACTIN (PRL) exerts effects on many diverse physiological processes, one of the best studied targets is the mammary gland (for review, see Refs. 1 and 2). PRL is critical for both mammogenesis and lactation, and accumulating evidence indicates that it may play a role in mammary carcinogenesis as well (for review see Refs. 3, 4, 5). In rodent models, systemic PRL treatment or pituitary transplants increased development of spontaneous, chemically induced, and oncogene-initiated mammary tumors (6, 7, 8), and mammary transgenic PRL induced tumors in virgin females (9, 10). Although the mechanism(s) whereby this occurs are not well understood, PRL increased bromodeoxyuridine incorporation in morphologically normal structures as well as epithelial hyperplasias and adenocarcinomas in the neu-related lipocalin-PRL transgenic and pituitary isograft models (10, 11), suggesting that augmented proliferation contributes to the disease process. The role of PRL in human breast cancer has been controversial because correlations between breast cancer development and progression, and circulating levels of PRL have been conflicting; and inhibition of pituitary PRL synthesis with bromocriptine did not alter the disease course (for review see Ref. 3). A more recent large prospective study by Hankinson et al. (12), however, shows a correlation between PRL levels in the high normal range and increased risk of breast cancer. Furthermore, production of PRL within primate mammary epithelial cells themselves indicates that the pituitary may not be the only relevant source of this hormone (for review see Refs. 3 and 4). The high levels of PRL receptors (PRLRs) in a large portion of tumors (13, 14, 15) suggest that the PRL signaling pathway may be a useful therapeutic target in human disease.

PRL can be modified posttranslationally by various processes including phosphorylation and glycosylation, as well as proteolytic cleavage (for review, see Refs. 16 and 17). The variability of these modifications with physiologic context prompted the hypothesis that they may contribute to the diverse effects of PRL at its targets, including the mammary gland (18, 19). Phosphorylation, in particular, has been shown to decrease its mitogenic action in the PRL-dependent rat lymphoma cell line, Nb2 (18, 20). To more easily study the activity of phosphorylated PRL, Walker and colleagues (21, 22) designed a phosphomimic, substituting an aspartate for serine at amino acid 179 of human PRL (hPRL) (S179D-PRL), a major site of phosphorylation. In their hands, this mutant was an effective antagonist for unmodified PRL-induced proliferation of Nb2 cells (22, 23). However, Goffin and co-workers (24) observed no evidence for antagonism and only weak agonistic activity using their own preparations of this mutant hormone in the same system. Recent studies analyzing development of the mammary gland during pregnancy and responses of mammary epithelial cells in vitro have demonstrated a complex spectrum of activity for S179D-PRL. S179D-PRL treatment during pregnancy inhibited murine alveolar growth (25, 26), while concomitantly increasing ß-casein transcripts (25). In the normal murine mammary cell line, HC11, which differentiates in response to PRL, Walker and colleagues (27, 28) found that S179D-PRL increased transcripts for ß-casein relative to unmodified PRL, and that this was blocked by selective inhibitors of ERKs.

The effect of PRL on mammary cells that have already undergone neoplastic changes may be distinct from normal cells. Study of PRL actions in human mammary tumor cells has been hampered by the endogenous PRL production, which makes experimental manipulation of PRL availability difficult. To provide a model to examine PRL signaling pathways and target genes in human tumor cells, we have derived PRL-deficient MCF-7 cells that demonstrate increased sensitivity to exogenous hormone (29). PRL increases proliferation of these cells by facilitating the G1/S transition, in part by increasing cyclin D1 expression and activity and decreasing p21 levels (29, 30). Using this system and homologous hormone, here we have examined the signals transmitted by S179D-hPRL to several well-characterized pathways, and its effect on the actions of unmodified PRL. Our data demonstrate that S179D-PRL is a weak agonist that is able to antagonize proliferative signals initiated by unmodified PRL in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Antibodies used in Western analyses were as follows: cyclin D1 (MS-210-P1) from NeoMarkers (Fremont, CA); signal transducer and activator of transcription (STAT) 5 pTyr694 (71–6900), PRLR long form (lPRLR) (35–9200) and PRLR intermediate form (iPRLR) (34–4800) from Zymed Labs, Inc. (San Francisco, CA); STAT 5 (sc-835) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and phospho-p44/42 ERK (Thr202/Tyr204) (9101) and p44/42 ERK (9102) from NEB Cell Signaling (Beverly, MA). The enhanced chemiluminescence kit was purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). All of the remaining reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Recombinant wild-type (WT)-PRL and S179D-PRL were prepared as previously described (22, 24). Data presented are from experiments performed with S179D-PRL synthesized in the Walker laboratory unless otherwise stated. Effects on cyclin D1 levels in the MCF-7-derived cells, and STAT5 activity in Chinese hamster ovary (CHO) cells were confirmed with a preparation from the Goffin laboratory. Purified natural human PRL [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)-PRL; lot AFP9042] was obtained through the National Hormone and Pituitary Program and Dr. Parlow. Selective chemical inhibitors were purchased from the following vendors: AG490, EMD Biosciences, Inc. (San Diego, CA); PP1, Biomol Research Lab (Plymouth Meeting, PA); LY294002, Sigma-Aldrich Corp.; PD98059, Calbiochem (La Jolla, CA); U0126, Promega (Madison, WI); ICI182,780, Tocris Cookson, Inc. (Ellisville, MO).

PRL-deficient MCF-7 cell culture
The MCF-7 derived subline (29) was grown in RPMI 1640 medium containing 10% horse serum and 50 µM ganciclovir. The cells were transferred to the above medium minus the ganciclovir 4 d before use. For growth assays, cells were plated at 4 x 105 cells/60-mm tissue culture dish. After seeding, the cells were washed once with serum-free RPMI 1640 and cultured in serum-free RPMI 1640 for 48 h before treatment with vehicle, NIDDK-PRL, WT-PRL (unmodified PRL), or S179D-PRL (all at 100 ng/ml). For some experiments, selective chemical inhibitors or vehicle was added as noted. The cells were harvested with trypsin at the indicated times and viable cells counted using a hemocytometer.

Western analyses
PRL-deficient MCF-7 cells were plated as described for the growth assays before treatments. Cells were harvested into 75 µl lysis buffer [25 mM Tris (pH 8.0), 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM sodium orthovanadate, and 20 mM sodium fluoride]. The cellular debris was removed by centrifugation at 10,000 rpm at 4 C for 10 min, and the protein concentration in the supernatant was determined using the bicinchoninic acid kit (Pierce Chemical Co., Rockford, IL). Lysates (30 µg protein) were electrophoresed through standard Laemmli SDS-polyacrylamide gels (12%), transferred to polyvinylidene fluoride membranes, and then probed with the appropriate antibodies. Membranes were blocked 4 h in 0.25% gelatin in 100 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, and 0.1% Tween 20; washed once with 100 mM Tris-HCl (pH 7.5), 150 mM sodium chloride and 0.1% Tween 20; and incubated in primary antibody overnight at 4 C (cyclin D1, 1:500; STAT 5 pTyr694, 1:1000; STAT 5, 1:5000; Phospho-p44/42 ERK, 1:5000; p44/42 ERK, 1:1000; lPRLR, 1:2000; iPRLR, 1:6250). Proteins were visualized using enhanced chemiluminescence as previously described (31). Quantification of the signals was performed using a Molecular Dynamics Personal SI densitometer and ImageQuant (version 4.2a) software (Molecular Dynamics, Inc., Sunnyvale, CA). For some experiments, the parental MCF-7 cell line and T47D line were similarly examined.

CHO-K1 cell culture, transient transfection and reporter gene assays
CHO-K1 cells were maintained in DMEM/F12 containing 5% FBS and penicillin/streptomycin (Life Technologies, Inc., Gaithersburg, MD), and were transiently transfected using Superfect (QIAGEN Inc., Valencia, CA) as described (30). The PRE3 plasmid consists of three copies of the consensus sequence for the STAT 5 binding site (TTCTTGGAA) from the ß-casein promoter (PRL response element, PRE), upstream of a luciferase reporter (29). The human lPRLR construct was graciously provided by Dr. C. Clevenger (32), and the cytomegalovirus -ß-galactosidase construct by Dr. C. Caskey (33).

Luciferase activity of cell lysates was determined as described (30). Luciferase values were corrected for transfection efficiency by determining the ratio of luciferase activity to ß-galactosidase activity and expressed as relative luciferase units.

Statistical analyses
Statistical analyses were performed using Prism version 3.02 (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
S179D-PRL blocks WT-PRL-induced increase in numbers of PRL-deficient MCF-7 cells
To determine if the different forms of PRL elicited different responses in our PRL-deficient mammary tumor cells, we evaluated the change in cell number following incubation with purified natural PRL (NIDDK-PRL; a mixture of unmodified and posttranslationally modified hormone) (22), PRL synthesized in Escherichia coli and therefore without posttranslational modifications (WT-PRL), or S179D-PRL similarly prepared (Fig. 1AGo). At 48 h, treatment with 100 ng/ml of either NIDDK-PRL or WT-PRL doubled the number of cells, whereas the same concentration of S179D-PRL had no effect. Preparations of S179D-PRL from both the Walker and Goffin laboratories yielded similar results. Furthermore, S179D-PRL was able to block the response to WT-PRL, with 10 ng/ml inhibiting WT-PRL (100 ng/ml) action about 50% (Fig. 1BGo). Because the PRL-deficient MCF-7 cells have a low rate of apoptosis under these conditions (unpublished observations), the higher number of cells that resulted from treatment with either NIDDK-PRL or WT-PRL primarily reflects an increase in proliferation.



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  		FIG. 1. S179D-PRL inhibits WT-PRL-induced growth of PRL-deficient MCF-7 cells. A, Cells were plated at equal densities, cultured in serum-free medium for 48 h, and then treated with the appropriate ligand (NIDDK-PRL, WT-PRL or S179D-PRL, Walker (W) or Goffin (G) preparations, all 100 ng/ml). The number of viable cells was counted at each time point as described in Materials and Methods. Results are expressed as the mean ± SE of triplicate plates. B, Cells were plated at equal densities, cultured in serum-free medium for 48 h, and then treated with vehicle, WT-PRL (100 ng/ml) alone or WT-PRL (100 ng/ml) + S179D-PRL (10 ng/ml or 100 ng/ml). The number of live cells was counted 48 h after treatment. The number of cells in the vehicle control was subtracted from the experimental plates and the increases in response to hormone treatments are shown. Both A and B are representative experiments of at least three experiments. Different lower case letters indicate significant differences among treatments using ANOVA followed by Student-Newman-Keuls posttest (P < 0.05).

 
S179D-PRL blocks WT-PRL-induced increase in cyclin D1
Because of the key role of cyclin D1 in regulating the G1/S transition and mediating the proliferative response to PRL, we examined the effect of S179D-PRL on cyclin D1 levels. WT-PRL increased cyclin D1 levels in a dose-dependent manner as we observed previously with NIDDK-PRL (29) (Fig. 2AGo). WT-PRL (100 ng/ml) yielded maximal stimulation, and was used for all subsequent studies. S179D-PRL had no detectable effect on cyclin D1 unless high concentrations (1000 ng/ml) were employed, which only resulted in a modest change (Fig. 2BGo). Again, S179D-PRL was able to compete with WT-PRL to decrease cyclin D1 to the level of the untreated control (Fig. 2CGo). To determine if S179D-PRL was able to inhibit PRL action in other mammary tumor cell lines, we examined the parental MCF-7 cells as well as T47D cells, both of which express endogenous PRL (29, 34, 35). As expected, WT-PRL only marginally increased cyclin D1 levels consistent with activity of the endogenous hormone, whereas S179D-PRL inhibited basal expression (Fig. 2DGo).



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  		FIG. 2. S179D-PRL blocks WT-PRL induction of cyclin D1 protein. A–C, After 48 h in serum-free media, PRL-deficient MCF-7 cells were treated for 6 h with vehicle or hormones as shown, and cyclin D1 levels in lysates were determined by Western analysis. Blots shown are representative of at least three experiments. D, Representative Western analysis of cyclin D1 in lysates from parental MCF 7 and T47D cells at 6 h after treatment.

 
PRL increased cell numbers and cyclin D1 levels by multiple pathways
PRL has been shown to activate multiple signaling cascades in many experimental systems, including mammary epithelial cells (for review see Refs. 1 and 3). To investigate the effect of S179D-PRL on pathways leading to increased cell number and cyclin D1 levels, we employed selective chemical inhibitors to ascertain the role of these signaling cascades in WT-PRL action on these endpoints. As shown in Fig. 3AGo, JAK2, Src family kinases, and phosphatidylinositol 3'-kinase are essential mediators of the PRL-induced increase in cell number, as are the ERKs (Fig. 3AGo). JAK2, phosphatidylinositol 3'-kinase, and ERKs are also critical for the PRL-induced increase in cyclin D1, with Src family kinases playing less critical roles (Fig. 3BGo). In contrast, inhibition of estrogen action with ICI182,780 did not alter either the proliferative response to PRL, nor the relative increase in cyclin D1 levels.



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  		FIG. 3. Multiple signaling pathways mediate PRL-induced increases in cell number and cyclin D1 in PRL-deficient MCF-7 cells. A, MCF-7 cells were plated and treated with vehicle (open bars) or WT-PRL (100 ng/ml; solid bars) as in Fig. 1AGo in the presence of vehicle or chemical inhibitors selective for kinase intermediates (PD78059, 20 µM; U0126, 10 µM; AG490, 25 µM; PP1, 20 µM; LY294002, 20 µM; ICI182780, 100 nM), and the net change in cell number determined as described for Fig. 1Go. B, MCF-7 cells were plated and treated with vehicle or WT-PRL (100 ng/ml) as in Fig. 2Go in the presence of vehicle or chemical inhibitors selective for kinase intermediates as for Fig. 3AGo. Cyclin D1 levels were determined by Western analysis. Blots shown are representative of at least three experiments.

 
S179D-PRL decreased WT-PRL-induced phosphorylation of STAT 5
STAT 5 is a key mediator of PRL-induced increases in cyclin D1 transcription (30), as well as a major downstream molecule of JAK2 to many other targets of PRL (36). Therefore we examined STAT 5 phosphorylation at Y694 after stimulation with WT-PRL or S179D-PRL using an antibody that recognizes this modification in both STATs 5a and 5b (Fig. 4AGo). WT-PRL elicits a robust response, but as for cyclin D1 levels, S179D-PRL displayed only very weak activity at the highest concentration (1000 ng/ml). Tyrosine phosphorylation of STAT 5 in response to WT-PRL at this site was inhibited by increasing concentrations of S179D-PRL (Fig. 4BGo). However, the level of phosphorylation was never completely reduced to control levels even at 10,000 ng/ml (data not shown). To investigate the net effect on STAT 5 activity, a simple STAT 5 responsive enhancer linked to a reporter gene (PRE3-luciferase) was employed. The PRL-deficient MCF-7 cells exhibited only a weak activation (30%) of this promoter in response to PRL (data not shown). Therefore, we used CHO cells, which have been a useful model to examine PRL signaling, to quantitatively evaluate effects of S179D-PRL on STAT 5 activation. These cells produce negligible levels of PRL and only low levels of PRLR, so PRLR isoform complement can be controlled. In CHO cells cotransfected with the human lPRLR and the strongly STAT 5-responsive PRE3-luciferase construct, WT-PRL increased luciferase expression 6-fold (Fig. 4CGo). S179D-PRL, prepared by both the Walker and Goffin laboratories, had no activity at 100 ng/ml, and only very low activity at 1000 ng/ml, similar to endpoints in the MCF7-derived cells. When both WT-PRL and S179D-PRL were added together, S179D-PRL significantly but incompletely inhibited the WT-PRL-enhanced promoter activity (Fig. 4CGo), similar to the effect on phosphorylation of STAT 5 at Y694 in the MCF-7-derived cells.



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  		FIG. 4. S179D-PRL reduces WT-PRL-induced phosphorylation of STAT5 at Y694 in PRL-deficient MCF-7 cells, and PRE-driven luciferase activity in transfected CHO cells. A and B, After 48 h in serum-free media, PRL-deficient MCF-7 cells were treated for 15 min with vehicle or hormones as shown and levels of STAT 5 phosphorylated Y694 or total STAT 5 in lysates determined by Western analysis using antibodies that recognize both STATs 5a and 5b under these conditions. Blots shown are representative of at least three experiments. C, CHO cells were transiently transfected with the PRE3-luciferase reporter construct, lPRLR, and ß-galactosidase constructs as described in Materials and Methods, and treated as indicated with WT-PRL, S179D-PRL, or a combination of the two forms, using preparations from the Walker (solid bars) and Goffin (open bars) laboratories. After 24 h, samples were assayed for luciferase activity and ß-galactosidase activity was used to correct for transfection efficiency. Activity is presented relative to untreated PRE3-transfected cells, and data represent the mean of five separate experiments, ±SEM. Different letters indicate significant differences between treatment groups (Student’s t test, P < 0.05).

 
WT-PRL-stimulated ERK activation is attenuated by S179D-PRL in the MCF-7-derived cells
PRL also uses other signaling pathways, including ERKs (see Refs. 1 and 3). WT-PRL stimulated ERK 1 and ERK 2 phosphorylation maximally at 15 min in this system (Fig. 5CGo). As was the case for STAT 5 phosphorylation, S179D-PRL demonstrated only slight activity toward ERKs at 1000 ng/ml (Fig. 5AGo). In contrast to the ability of S179D-PRL to inhibit PRL-induced increases in cyclin D1 and STAT 5 phosphorylation, it was unable to decrease WT-PRL stimulated ERK 1 and ERK 2 phosphorylation at this time, even at concentrations of 10 µg/ml (Fig. 5BGo). However, S179D-PRL shortened the duration of WT-PRL stimulated ERK phosphorylation (Fig. 5CGo). Whereas WT-PRL initiated a biphasic response, falling after a peak at 15 min and then rising slightly again at 135 min, equal concentrations of S179D-PRL brought levels to basal by 90 min and prevented the second rise.



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  		FIG. 5. S179D-PRL inhibits the second phase of ERK phosphorylation by WT-PRL. A and B, After 48 h in serum-free media, PRL-deficient MCF-7 cells were treated for 15 min with vehicle or hormones as shown and phospho-ERK or ERK levels in lysates determined by Western analysis. Blots shown are representative of at least three experiments. C, After 48 h in serum-free media, cells were treated for the indicated times with vehicle or hormones as shown and phospho-ERK and ERK levels in lysates determined by Western analysis.

 
S179D-PRL increased lPRLR levels
The PRLR gene is alternatively spliced to yield multiple receptor isoforms with different signaling capacities (for review see Ref. 3). Target cells express more than one isoform, with relative and absolute levels varying with target and physiologic state. Exposure to ligand has been reported to augment or down-regulate receptors in different experimental systems (for reviews, see Refs. 37, 38, 39). MCF-7 cells express primarily the long PRLR isoform (lPRLR), although low levels of other isoforms are present (Brockman, J. L., unpublished observations). In the PRL-deficient MCF-7 cells, incubation with WT-PRL modestly reduced levels of the lPRLR (Fig. 6Go, A and B). In marked contrast, S179D-PRL increased lPRLR levels by 4-fold over long incubation periods (72 h). However, this increase was not apparent at earlier times (24 or 48 h; data not shown). Levels of the iPRLR isoform, which are present at much lower levels, did not change with treatment (Fig. 6AGo, lower blot). In the reciprocal of the experiments shown in Figs. 2CGo, 4BGo, and 5BGo, increasing concentrations of WT-PRL were employed to inhibit the effects of S179D-PRL. As shown in Fig. 6CGo, WT-PRL competes effectively for S179D-PRL signaling to this endpoint as well.



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  		FIG. 6. S179D-PRL increases levels of lPRLR in PRL-deficient MCF-7 cells. Cells were cultured for 24 h in serum-free media, treated with vehicle or hormone as shown, and levels of PRLR determined by Western analyses. A, Representative Western analysis of lPRLR and iPRLR following hormone treatment (100 ng/ml) at 0 and 72 h after treatment as shown. B, Change in lPRLR levels compared with control (0 h) from three independent experiments (mean ± SE), vehicle (black bars), WT-PRL (gray bar), and S179D-PRL (white bar). Different letters indicate significant differences among treatments using ANOVA followed by Student-Newman-Keuls posttest (P < 0.05). C, Representative Western analysis of lPRLR at 72 h after treatment. Blot is representative of at least three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mammary tumor cells, like many other extrapituitary cells, synthesize PRL (for review, see Refs. 3 and 40). We know little about this PRL: the relatively low levels of expression compared with pituitary lactotrophs have limited our knowledge of posttranslational modifications and how they may be altered by environmental context. Here we assessed the relative activities of unmodified hPRL and a molecular mimic of phosphorylated hPRL, S179D-PRL, in human mammary tumor cells in the absence of confounding endogenous PRL production. In this system, S179D-PRL was a weak PRL agonist for proliferation. However, it was an effective antagonist of unmodified PRL for this activity at low concentrations (0.4–4.0 nM). Our data demonstrate that PRL, like EGF and estrogens (41, 42), employs a complex signaling network to signal to proliferation in breast cancer cells. Low concentrations of S179D-PRL antagonized the effect of unmodified PRL on the endpoints examined, including cyclin D1 levels, phosphorylation of STAT 5 at Y694, and the duration of ERK phosphorylation, which has been linked to cell cycle progression in other systems (43). Although S179D-PRL at 4 nM reduced WT-PRL activity to unstimulated levels for some of these endpoints, including cell number and cyclin D1 levels, for others, including phosphorylation of Y694 of STAT5, acute ERK phosphorylation, and activation of a PRL-responsive enhancer, this antagonism was incomplete. Antagonistic activity was evident not only in this cell line, but also in the parental MCF-7 and T47D mammary tumor cell lines, where S179D-PRL reduced cyclin D1 levels below those of the unstimulated cells, arguably by opposing endogenously synthesized PRL.

These generally antagonistic activities of S179D-PRL at low concentrations observed in our MCF-7-derived cells contrast with more complex effects observed in other systems by both the Walker and Goffin laboratories. In rat Nb2 cells, which display high levels of a mutant form of the lPRLR with a partial deletion in the cytoplasmic domain (39), S179D-PRL effectively induced tyrosine phosphorylation of STAT 5a, and at least in the Walker laboratory, but not Goffin laboratory, also antagonized cell proliferation (23, 24). In murine HC11 cells, S179D-PRL initiated only low levels of tyrosine phosphorylation but higher levels of serine phosphorylation of STAT 5a compared with unmodified hormone (27). However, S179D-PRL strongly activated ERKs in these cells, and selective inhibitors linked this pathway to the increase in ß-casein transcripts. In contrast, in our studies using the MCF-7-derived cells, which express predominantly the lPRLR but also low levels of other PRLR isoforms, S179D-PRL did not inhibit the first phase of ERK activation, but did attenuate the second phase. Interestingly, Goffin and colleagues (24) observed signaling to ERKs after 15 min of exposure to S179D-PRL using conditions similar to those examined here in another human mammary tumor cell line, T47D). Although differences in the hormone preparations from the Walker and Goffin laboratories have been cited as a reason for some of their apparently conflicting observations (24), this does not account for our findings in the MCF7 subline described here, since protein prepared in these laboratories had the same effects. Clearly, species differences in hormone and receptors (44), endogenous production of PRL, tissue origin and oncogenic mutations, as well as PRLR isoform complement, can confound results and will take some time to unravel. In the current study, we have eliminated the complication of endogenous PRL production and have used species appropriate hormone preparations.

The low concentrations of S179D-PRL necessary to exert antagonistic effects in the PRL-deficient MCF-7 cells as well as some studies of Nb2 cells (22) are consistent with a high affinity for the PRLR. However, Goffin and colleagues (24) found that this mutant had about a 10-fold lower affinity for the human lPRLR stably overexpressed in 293 cells than WT-PRL, assessed by competition for binding with radiolabeled WT-hPRL. In the present studies in the PRL-deficient MCF-7 cells, similar concentrations of S179D-PRL were required to inhibit responses to WT-PRL, and of WT-PRL to block the S179D-PRL-induced increase in lPRLR levels. These data are consistent with the altered structure of this mutant modifying interactions with the PRLR, thereby affecting conformational changes in the receptor and consequently activation of downstream signaling pathways.

Factors governing total PRLR expression and relative levels of PRLR isoforms are not well understood (for reviews see Refs. 37, 38, 39). Depending on the model system, concentration of PRL, and time course, PRL has been observed to both up-regulate and down-regulate membrane binding proteins. In contrast to down-regulation of the lPRLR in the MCF-7-derived cells in response to WT-PRL, S179D-PRL treatment markedly increased lPRLR levels above those in cells cultured in serum-free media. These data suggest that at least some target cells may demonstrate increased sensitivity to WT-PRL signals mediated by this isoform following long term exposure to S179D-PRL. Levels of iPRLR, in contrast, were not affected by S179D-PRL; other PRLR isoforms (45, 46) were not examined. Whether the S179D-PRL-induced increase in lPRLR represents reduced degradation and/or increased synthesis was not ascertained in these studies. The requirement for longer incubations suggests that indirect mechanisms may play a role. The interactions of S179D-PRL with the PRLR and the underlying mechanisms and trafficking of the PRLR isoforms subsequent to S179D-PRL binding are under investigation.

These studies demonstrate that low concentrations of S179D-PRL antagonize WT-PRL signaling to several pathways in breast cancer cells that may impact on carcinogenic behavior, particularly proliferation. These observations suggest that this compound may prove useful in understanding the actions of PRL in mammary tumorigenesis and progression, as well as in development of therapeutic approaches.


   Acknowledgments
 
We are grateful to Jennifer Gutzman, Sophie Bernichtein, and Dr. Vincent Goffin for reagents and helpful discussions.


   Footnotes
 
This work was supported by NIH Grant R01-CA-78312 and the University of Wisconsin Center for Women’s Health and Women’s Health Research (to L.A.S.) and by grant DAMD17-00-1-0180 (to A.M.W.).

Abbreviations: CHO, Chinese hamster ovary; hPRL, human PRL; iPRLR, intermediate form of the PRLR; lPRLR, long form of the PRLR; NRL-PRL, PRE, PRL response element; PRL, prolactin; PRLR, PRL receptor; STAT, signal transducer and activator of transcription; WT, wild-type.

Received July 2, 2003.

Accepted for publication September 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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