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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 11 3692-3699
Copyright © 1997 by The Endocrine Society

Original Studies

Coexpression and Cross-Regulation of the Prolactin Receptor and Sex Steroid Hormone Receptors in Breast Cancer1

Christopher J. Ormandy2, Rosemary E. Hall, David L. Manning, John F. R. Robertson, Roger W. Blamey, Paul A. Kelly, Robert I. Nicholson and Robert L. Sutherland

Cancer Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia; Breast Cancer Laboratory, Tenovus Cancer Research Center, University of Wales College of Medicine (D.L.M., R.I.N.), Cardiff CF-4XX, Wales, United Kingdom; the Department of Surgery, City Hospital (J.F.R.R., R.W.B.), Nottingham, United Kingdom NG5 1PB; and INSERM U-344 Endocrinologie Moléculaire, Faculté de Médecine Necker-Enfants Malades (P.A.K.), 75743 Paris Cédex 15, France

Address all correspondence and requests for reprints to: Dr. C. J. Ormandy, Cancer Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia. E-mail: c.ormandy{at}garvan.unsw.edu.au


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The sex steroid hormones and PRL interact synergistically to control the neoplastic growth of the mammary gland. The basis for this hormonal synergy is unknown, but may involve cellular coexpression of the sex steroid and PRL receptors, coupled with receptor cross-regulation. To examine this hypothesis the expression of the sex steroid and PRL receptors was examined in 20 human breast cancer cell lines and 123 primary breast cancers. Regulation of sex steroid receptors by PRL and of the PRL receptor by sex steroids was examined in T-47D and MCF-7 breast cancer cells. Northern analysis of the breast cancer cell lines and tumors indicated that the PRL receptor and the sex steroid receptors were coexpressed. The level of PRL receptor expression in the breast cancer cell lines was linearly related to that of the estrogen and progesterone receptors, but not to that of the androgen receptor. In MCF-7 and T-47D cells, acute treatment with progestins and androgens and long term treatment with estrogens increased PRL receptor levels. Analysis of sex steroid receptor messenger ribonucleic acid and binding activity showed that acute PRL treatment produced a time- and concentration-dependent increase in progesterone receptor and a decrease in androgen receptor. These results indicate that receptors for sex steroids and PRL are coexpressed and are cross-regulated, providing a potential mechanism for the observed synergy among estrogen, progesterone, and PRL in the control of tumor growth.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
THE PRL receptor (PRLR) binds a group of hormones that comprise PRL, the placental lactogens, and primate GHs. It is a member of the cytokine receptor superfamily and exerts a mitogenic effect in a variety of tissues (1, 2, 3, 4). In the mammary gland, treatment with a combination of estrogen, progesterone, and PRL stimulates the proliferation of mammary epithelial cells to produce lobuloalveoli in vivo (5) and in vitro (6, 7). The individual roles of these hormones in mammary development have been recently confirmed by knockout mouse studies, in which an absence of the progesterone or PRLR produced defects in mammary gland development after puberty, the latter apparent in heterozygous animals (8, 9), whereas the absence of an estrogen receptor (ER) resulted in the attainment of prepubertal mammary development only (10).

The PRLR also provides a major mitogenic signal in rodents (11, 12); raised PRL levels accelerate the growth of mammary tumors induced by 7,12 dimethylbenz({alpha})anthracene in rats, whereas PRL ablation reduces their growth (13). Recent transgenic animal models have shown dramatic increases in mammary tumors in mice overexpressing human GH (hGH), which binds to both PRL and GH receptors, but not in those that overexpress bovine GH, which binds only to the GH receptor (14).

The role of PRL in human breast cancer is less clear. A large literature comparing serum PRL levels and the incidence of human breast cancer is difficult to interpret due to the problems associated with measurements of serum PRL (15). Of these, the largest prospective study concluded that PRL is a weak tumor promoter for human breast cancer (16). Experimentally, PRL is a mitogen for human breast cancer tissue and cells in culture, and human breast cancer cells express both the PRLR and PRL, raising the possibility of an autocrine/paracrine system of growth regulation (17, 18, 19, 20, 21, 22, 23).

In normal mammary gland development (15, 24, 25) and experimental mammary tumors (26, 27, 28), PRL and the sex steroids act synergistically to exert their mitogenic effects; however, the mechanism behind this synergy remains to be defined. A possible scenario is that mammary epithelial cells coexpress receptors for the sex steroid and lactogenic hormones, and that the lactogens and the sex steroids cross-regulate the levels of their receptors, resulting in a dramatic increase in the number of receptor molecules available for signaling. To examine this hypothesis we used Northern analysis of 20 breast cancer cell lines and 123 primary breast cancers to determine whether the PRLR is coexpressed with the sex steroid receptors, and using MCF-7 and T-47D breast cancer cell lines have investigated the regulation of sex steroid receptors by PRL and the regulation of the PRLR by sex steroid hormones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell lines, primary cancers, and reagents

Cell culture reagents were obtained from Cytosystems (Sydney, Australia), C.S.L.-Novo (Sydney, Australia), and Corning (Sydney, Australia). Cells were obtained from American Type Culture Collection (Rockville, MD), except for MCF-7, MDA-MB-157, MDA-MB-231, MDA-MB-330, T-47D, and HBL-100, which were from E. G and G. Mason Research Institute (Worcester, MA). Cells were cultured in HEPES-buffered (pH 7.4) RPMI 1640 supplemented with 6 mmol/L L-glutamine, 10 µg/mL porcine insulin, 20 µg/mL gentamicin sulfate (GM) with 5% FCS. Steroid treatments were carried out in GM with 1% dextran-charcoal-treated FCS. DNA fingerprinting used probes for human polymorphic epithelial mucin and {alpha}-globin 3'-hypervariable region. Mycoplasma testing used the Gen-Probe T. C. rapid detection system supplied by BioMediq (Doncaster, Australia). Biopsies of primary, pretherapy breast cancers were obtained at surgery and collected into liquid nitrogen. Probes were fragments from human complementary DNAs (cDNAs) encoding the ER (29), the progesterone receptor (PgR) (30), the androgen receptor (AR) (31), and the PRLR (32). The 18S oligonucleotide was synthesized to bases 151–180 of the rat sequence (33). Steroid hormones were purchased from Sigma Chemical Co. (St. Louis, MO) and Amersham (Sydney, Australia). ICI 164348 was a gift from Dr. Alan Wakeling, Zeneca Pharmaceuticals (Macclesfield, UK). hGH was donated by Dr. G. E. Chapman and was iodinated (34) to a specific activity of 50–70 Ci/g.

Assay of cell monolayer PRLR-binding activity

Triplicate, confluent, 12-well plates received 500 µL GM-1% BSA containing either 60,000 cpm [125I]hGH (~0.03 nmol/L) or 60,000 cpm [125I]hGH plus 50 nmol/L unlabeled hGH overnight at 20 C. The wells were then emptied, washed twice with PBS, and solubilized with 1 mL 0.1 mol/L NaOH-1% (vol/vol) Triton X-100. Wells were sampled, and the radioactivity was measured for 5 min. Specific binding was calculated by subtracting the average nonspecific binding from the average total binding. The specific binding SE was calculated by taking the square root of the sum of the squares of the total and nonspecific binding SEs. Cells were counted using a hemocytometer. This method has previously been shown to detect only PRLR (35).

Steroid binding assays

MCF-7 cells were harvested during log phase growth. Cells (4 x 105) were added to a series of 1.5-mL Eppendorf tubes and pelleted, the supernatant was aspirated, and the pellet was resuspended by brief vortex mixing in 200 µL GM containing [3H]ORG 2058 (10 nmol/L), [3H]estradiol (4 nmol/L), or [3H]dihydrotestosterone (20 nmol/L) in the presence or absence of a 100-fold molar excess of unlabeled steroid for 1 h at 37 C. The cells were pelleted, then resuspended in 200 µL ice-cold 5% BSA-phosphate-buffered saline and placed on ice for 20 min, followed by centrifugation and aspiration of the wash solution. The Eppendorf lid was removed, and the tube was placed in 7 mL ACS scintillation fluid, shaken to resuspend the cell pellet, and counted for 5 min using a Beckman liquid scintillation counter (Beckman, Palo Alto, CA).

Northern and Southern analysis

Cells were trypsinized and counted using a hemocytometer. Ribonucleic acid (RNA) from breast tumors and cell pellets (up to 108 cells) was prepared using standard methods and subjected to Northern analysis (36). Blots were washed in 0.2 x SSC (standard saline citrate)-1% SDS for 30 min at 65 C before autoradiography at -70 C with two intensifying screens. Variation in RNA content per lane was measured by reprobing with an oligonucleotide for the 18S ribosomal subunit. Autoradiographs were quantified by longitudinal densitometric scans and were analyzed using the Bio-Rad 1D analyst computer program (Bio-Rad Laboratories, Richmond, CA). Results were corrected for 18S signal intensity. DNA was prepared by SDS-proteinase K digestion of 1 x 108 breast cancer cells overnight at 37 C followed by phenol-chloroform extraction and ethanol precipitation as detailed previously (36). Genomic DNA (20 µg) was digested overnight with 5 U EcoRI and fractionated followed by Southern capillary transfer to Zetaprobe (Bio-Rad) nylon membrane, hybridized, and washed as before.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
PRLR expression in human breast cancer cell lines

PRLR gene expression was examined in 20 human breast cancer cell lines (Fig. 1Go, top panel). Major PRLR messenger RNA (mRNA) transcripts of 13.7, 3.4, and 2.6 kb were detected in the breast cancer cell lines. Two additional minor species of 11.6 and 10.5 kb were seen using polyadenylated mRNA blots (data not shown). The large bands may be produced by a combination of partially spliced heteronuclear RNA transcripts, 5'-untranslated regions of differing lengths (37), differing 3'-untranslated regions due to alternate polyadenylation site usage (38), and differing degrees of polyadenylation (38). Longitudinal densitometric measurements of Northern blots from up to four independent RNA preparations were made. Optical density values were normalized against MCF-7 signal intensity and the mean ± SE or range was calculated (Fig. 2Go). Five cell lines expressed high PRLR mRNA levels (MDA-MB-134, BT 483, T-47D, BT 474, and MDA-MB-361), and eight additional lines expressed moderate to low PRLR levels. The remainder expressed no detectable PRLR mRNA. Analysis by reverse transcription-PCR (RT-PCR) of some of the negative lines (HBL-100 and MDA-MB-231) showed that faint PRLR signals could be produced, whereas others (BT-20) produced no signal (39). The relationship between PRLR mRNA level and PRLR-binding activity was also investigated in some of the PRLR-positive cell lines (Fig. 3Go). The PRLR mRNA level was positively correlated (r2 = 0.925) with PRLR-binding activity, indicating that potential posttranscriptional mechanisms of PRLR regulation are not likely to be of significant effect in the maintenance of steady state PRLR levels, although acute effects of steroids on PRLR protein half-life may still occur (40).



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  		Figure 1. Northern analysis of PRLR and ER gene expression in total RNA from human breast cancer cell lines. Total RNA (20 µg/lane) was hybridized with [{alpha}-32P]deoxy-CTP-labeled human PRLR cDNA (top panel), human ER cDNA (middle panel), and an oligonucleotide directed against the 18S ribosomal subunit (bottom panel). The autoradiographs of the cDNAs were exposed for 48 h without intensifying screens.

 


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  		Figure 2. Level of PRLR gene expression in human breast cancer cell lines. PRLR mRNA levels were measured by Northern analysis in up to four separate RNA preparations from each cell line. Multiple measurements were standardized using the level found in MCF-7 cells, and error bars represent either the SE of three or more separate determinations or the range of duplicate determinations. Histograms without error bars represent single determinations.

 


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  		Figure 3. Relationship between the level of PRLR gene expression and PRLR-binding activity. PRLR mRNA levels shown in Fig. 2Go were plotted against PRLR-binding activity measured in confluent monolayer cultures. The line of best fit and the r2 value calculated by linear regression are indicated, and this analysis excludes the cell lines shown by open symbols, which expressed high levels of PRLR mRNA, but undetectable (MDA-MB-134) or barely detectable (MDA-MB-361) levels of PRLR-binding activity.

 
Two cell lines were identified that expressed high levels of PRLR mRNA but levels of PRLR-binding activity that were at the level of detection (MDA-MB-361) or undetectable (MDA-MB-134). RT-PCR analysis of PRLR mRNA indicated no differences in the size of the major PCR products of the extracellular and intracellular regions of the receptor among T-47D, MCF-7, MDA-MB-361, and MDA-MB-134 cells. The MDA-MB-361 and MDA-MB-134 cell lines may carry an undetected small mutation that results in a loss of receptor binding or may produce large quantities of PRL that prevent iodinated tracer binding through receptor saturation.

To determine whether the high expression of the PRLR observed in some cell lines was due to gene amplification, the signal intensity obtained from Southern analysis (Fig. 4AGo) was compared to the level of PRLR mRNA obtained by Northern blot (Fig. 4BGo). This analysis showed that Southern and Northern blot signal intensities were unrelated (r2 = 0.009; P = 0.68). Thus, gene amplification cannot account for the high level of PRLR observed in some cell lines, indicating instead that mechanisms regulating gene expression must be responsible.



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  		Figure 4. Relationship between PRLR gene amplification and mRNA levels. A, Genomic DNA (10 µg) was digested with EcoRI, size fractionated, and Southern blotted before hybridization with 32P-labeled PRLR cDNA. B, The PRLR signal intensity in A was quantified by densitometric scans and is compared to the level of PRLR mRNA from Fig. 2Go. Linear regression analysis gave an r2 of 0.009 (P = 0.68), indicating no correlation between the gene copy number and the level of gene expression.

 
Coexpression of PRLR and sex steroid receptors in breast cancer cells

The Northern blots used for the measurement of PRLR were reprobed with cDNAs for ER (Fig. 1Go, middle panel), PgR, AR, pS2, and the epidermal growth factor receptor (blots not shown), and an oligonucleotide to the 18S ribosomal subunit to control for loading (Fig. 1Go, lower panel). The signal intensities obtained for all genes were compared using Kendall’s rank correlation test (Table 1Go). PRLR mRNA expression was significantly correlated with the expression of ER and PgR, and with AR and pS2 with lesser significance. When the level of PRLR gene expression was plotted against the level of ER, PgR, or AR gene expression, PRLR was positively correlated with the signal intensity for ER (r2 = 0.8; P = 0.0001) and PgR (r2 = 0.3; P = 0.02), but was not related to the level of AR mRNA (r2 = 0.1; P = 0.3; Fig. 5Go). The only ER negative cell lines that expressed moderate levels of PRLR mRNA were BT-549 and MDA-MB-453. BT-549 expressed both the PgR and the AR, whereas MDA-MB-453, which is also PgR negative, overexpresses the AR (41) and shows elevated PRLR-binding activity in response to androgens (42), indicating that these two cell lines retain responsiveness to some of the sex steroid hormones. These results clearly demonstrate that the PRLR is coexpressed with the sex steroid hormone receptors. The relationships between expression of the steroid hormone receptors, glucocorticoid receptor, vitamin D receptor, and the epidermal growth factor receptor in a subset of these breast cancer cell lines are described in an earlier report from this laboratory (43).


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  		Table 1. Kendall’s rank correlation test in breast cancer cell lines

 


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  		Figure 5. Comparison between the level of PRLR gene expression and AR, PgR, and ER gene expression. The Northern blot shown in Fig. 1Go was reprobed with the human AR cDNA and human PgR cDNA. Levels of gene expression were quantified by densitometry and are plotted against the level of PRLR gene expression. Linear regression analysis indicated that the PRLR mRNA level was correlated to the ER mRNA level (r2 = 0.8; P = 0.0001) and the PgR mRNA level (r2 = 0.3; P = 0.02), but was not correlated to the level of AR mRNA (r2 = 0.1; P = 0.3).

 
Coexpression of PRLR and ER in human primary breast cancers

PRLR, ER, PgR, and AR gene expression was examined by Northern analysis of RNA from 186 breast cancers. A representative Northern blot of 13 tumors is shown in Fig. 6Go. As expected for RNA derived from surgical specimens, some samples of mRNA were degraded and were excluded from the analysis (46 samples). Additional samples that showed mostly normal histology were also excluded (17 samples), leaving 123 samples available for analysis.



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  		Figure 6. AR, ER, PgR, and PRLR gene expression in primary breast cancers. Primary human breast tumors were placed into liquid nitrogen after surgery. Total mRNA was prepared by the guanidinium-cesium chloride method, and 10 µg RNA were run per lane. Northern analysis was carried out using 32P-labeled cDNAs. Loading was controlled by hybridization with an oligonucleotide to the 18S ribosomal subunit. A representative blot of 13 samples is shown, which was sequentially probed with the indicated cDNAs. Lanes 1 and 4 show examples of samples that were excluded from the analysis due to low 18S signal.

 
Filters were quantitated by longitudinal densitometric scans and corrected for 18S signal intensity. Frequency distribution analysis of the level of PRLR expression (data not shown) indicated a normal distribution skewed toward low expression, with a separate small group of tumors that expressed very high levels of the PRLR. The correlation between the expression of the PRLR and the sex steroid hormone receptors was analyzed in two ways; positive or negative values were assigned by visual examination of the autoradiographs (Table 2Go), which allowed {chi}2 analysis (Table 3Go). Optical densities were compared using Kendall’s rank correlation test (Table 3Go). Both statistical methods confirmed that the sex steroid and PRLRs were coexpressed.


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  		Table 2. Tumor frequency distribution according to receptor status

 

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  		Table 3. Tumor frequency distribution statistical analysis

 
Previous examination of the coexpression of the PRLR with the ER by RRa have produced conflicting results, with reports indicating both coexpression (44, 45, 46, 47, 48, 49, 50) and independent expression (51, 52, 53, 54). A range from 2–72% of PRLR-positive tumors was reported in these studies due to differences in assay type (55), the radioligand used (56), the quality of the membrane preparation (57), and whether endogenous lactogens are dissociated from the PRLR (51). Thus, studies that report low numbers of PRLR-positive tumors generally fail to find a correlation with ER. We show here by Northern analysis that the PRLR is coexpressed with the ER. Reports using other alternatives to RRa, such as immunohistochemistry in 25 breast tumor samples (17) or RT-PCR in 12 breast cancer cell lines (39), also support the conclusion that the PRLR and the ER are coexpressed.

In a number of tumor samples, normal tissue was also taken adjacent to the tumor, and in six cases, histological examination showed that the normal tissue contained a significant epithelial component. RNA was prepared from normal and tumor tissue from each of these samples and was analyzed for PRLR mRNA levels. The PRLR and sex steroid receptors were also coexpressed in these samples. In all cases, the level of PRLR mRNA per µg total RNA found in the tumor was higher than that found in the surrounding normal tissue (data not shown). Although differences in the relative epithelial to stroma ratio between normal and tumor portions cannot be dismissed, these observations raise the possibility that tumors express higher levels of PRLR than normal epithelial tissue.

Regulation of PRLR by sex steroids and regulation of sex steroid receptors by PRL

The ability of the sex steroids to modulate PRLR levels is summarized in Fig. 7Go. In MCF-7 cells estrogen was unable to regulate PRLR mRNA or binding levels under acute exposure conditions (10 nmol/L for 24 h) despite being able to induce the expression of retinoic acid receptor-{alpha} under identical experimental conditions (58). After long term estrogen depletion followed by exposure to estrogen for 3 days, PRLR mRNA levels increased in both MCF-7 and T-47D cells, and this effect was abrogated by a 10-fold molar excess of the antiestrogen ICI 164384. A number of studies (42, 59, 60) confirm no effect of acute estrogen on PRLR levels, whereas one using EFM-19 human breast cancer cells reported an increase in PRLR after short term estrogen exposure (61). In contrast acute exposure to both dihydrotestosterone and progesterone was able to increase PRLR mRNA and PRLR ligand-binding activity in MCF-7 cells (Fig. 7Go) and T-47D cells (not shown), as previously reported by this laboratory and others (40). These data suggest an indirect mechanism of estrogen action on PRLR, and the well characterized positive regulation of PgR by estrogen provides a possible intermediary.



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  		Figure 7. Regulation of PRLR gene expression by steroid hormones. MCF-7 cells were grown in RPMI 1640–5% FCS and were changed to RPMI 1640–1% dextran-charcoal stripped FCS (SFCS) 24 h before exposure to vehicle or 10 nmol/L estradiol (E), dihydrotestosterone (DHT), or Organon 2058 (ORG) for 24 h. Levels of PRLR gene expression (light gray bars) were measured by Northern analysis of 20 µg total RNA, whereas levels of PRLR protein (dark gray bars) were measured by ligand-binding activity. Both are expressed as a percentage of the vehicle-treated control value. MCF-7 cells were also grown in 5% SFCS for three passages before exposure to vehicle, 10 nmol/L E, or 10 nmol/L E plus 100 nmol/L of the pure antiestrogen ICI 164384 (ICI) for 3 days (3 d) before Northern analysis of PRLR gene expression. Experiments shown are representative of multiple replicates.

 
MCF-7 cells were also treated with various concentrations of human PRL, and the effects on sex steroid receptor mRNA level and binding activity were examined (Fig. 8Go). PRL produced a concentration-dependent increase in PgR mRNA and receptor-binding activity. In contrast PRL treatment caused a concentration-dependent decrease in AR mRNA levels and AR-binding activity. No effect of PRL on ER was seen at 24 h, although PRL has been reported to increase ER-binding activity in MCF-7 cells after 36–60 h of incubation (62), indicating that, like the effect of estrogen on the PRLR, long term, rather than acute, treatment may be required. Where positive receptor cross-regulation occurs (PRLR and PgR, PRLR and ER), the levels of mRNA for these receptors tend to be positively correlated (Fig. 5Go), but where both negative and positive cross-regulations occur (PRLR and AR), this effect is lost.



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  		Figure 8. Effect of PRL on steroid hormone receptor-binding activity and mRNA levels. MCF-7 cells were grown in RPMI 1640–5% FCS and were changed to RPMI 1640–1% SFCS 24 h before addition of the indicated concentrations of human PRL for 24 h. Cells were then either assayed for steroid hormone-binding activity (upper panel) or steroid hormone receptor mRNA levels (lower panel). Error bars represent the range of two experiments and are smaller than the symbol where not indicated. Circles, PgR; triangles, AR; squares, ER.

 
We have shown that human breast cancer cells and tumors coexpress the PRL and sex steroid receptors and that their expression levels are cross-regulated. This receptor cross-regulation provides a potential mechanism to explain the observed synergy between PRL and the sex steroid hormones during mammary development and in neoplastic growth, especially that between progesterone and PRL.

It is becoming apparent that in many circumstances the steroids and PRL do not act alone, but require the presence of each other for full activity. For example, the expression of the rat casein gene requires both PRL and glucocorticoid (63); the prolactin-inducible protein (PIP) from breast cancer cells is regulated by a combination of androgen and PRL (64); expression of the uteroferrin gene requires estrogen, progesterone, and PRL (65); and the expression of uteroglobin by the uterus requires progesterone and PRL (66). In the later example, where PRL and progesterone act synergistically (67), PRL increased uterine PgR levels, and progesterone increased uterine PRLR levels, identical to the situation defined here in breast cancer cells and indicating that receptor cross-regulation may provide a general mechanism allowing synergy among estrogen, progesterone, and PRL in many target tissues. These results suggest that the use of antisteroid hormone therapy in breast cancer may be attacking only half of the synergistic equation, which underpins the hormonal control of cancer cell proliferation.


   Acknowledgments
 
The authors thank Christine Lee for the cell line RNA preparation, and Anna deFazio and Rebbeca Sini for the cell line ER and 18S Northern analysis.


   Footnotes
 
1 This work was supported by grants from the Kathleen Cuningham Foundation, Australia; the New South Wales State Cancer Council; and the National Health and Medical Research Council of Australia. Back

2 C. J. Martin Fellow of the National Health and Medical Research Council of Australia. Back

Received May 7, 1997.

Revised July 2, 1997.

Accepted August 6, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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