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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 2 667-674
Copyright © 1998 by The Endocrine Society

Original Studies

Increased Expression of Prolactin Receptor Gene Assessed by Quantitative Polymerase Chain Reaction in Human Breast Tumors Versus Normal Breast Tissues

Philippe Touraine, Jean-François Martini, Brigitte Zafrani, Jean-Claude Durand, Françoise Labaille, Catherine Malet, André Nicolas, Christine Trivin, Marie-Catherine Postel-Vinay, Frédérique Kuttenn and Paul A. Kelly

INSERM Unité 344, Faculté Medecine Necker (Ph.T., J-F.M., F.L., M-C.P.V., P.A.K.), 75730 Paris Cedex 15, France; Institut Curie (B.Z., A.N.), 75005 Paris, France; Clinique St. Jean de Dieu (J-C.D.), 75007 Paris, France; Département d’Endocrinologie, Hôpital Necker (C.M., F.K.), Paris, France; Laboratoire d’Explorations Hormonales, Hôpital Necker (C.T.), 75015 Paris, France

Address all correspondence and requests for reprints to: Philippe Touraine, INSERM Unité 344, Faculté Medecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The role of PRL in human breast tumorigenesis is not well understood. One of the limitations is the difficulty of accurately measuring PRL receptors (PRLR) in human tissues. We established a quantitative PCR method (Q-PCR) in T-47D human breast cancer cells and applied it to 29 patients, 25 of whom presented with either cancer or fibroadenoma. Four patients underwent a mammoplasty, and normal epithelial cells were cultured before Q-PCR. In T-47D cells, 31 x 106 messenger RNA molecules were detected per microgram of total RNA. In all patients, expression of the PRLR gene was detected, varying from 1500 to 1 x 106 molecules/µg of RNA in normal tissues and from 4500 to 34.7 x 106 molecules/µg of RNA in tumors. PRLR expression was always greater in tumor than in normal contiguous tissue and similar in cultured mammary epithelial cells and normal breast tissues. Estradiol and progesterone receptor-negative tumors expressed low levels of PRLR transcripts, similar to normal breast tissue from menopausal women. Immunocytochemical analysis of PRLR confirmed stronger staining in almost all tumor samples compared with normal tissues. A messenger RNA encoding locally produced human PRL was also identified by RT-PCR in every sample tested. Our results confirm PRLR gene expression in all tissues studied, and moreover, indicate that this expression is increased in human breast tumors vs. normal contiguous tissues.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE ROLE of lactogenic hormones in the induction and growth of rodent mammary tumors is well known (1). However, it is not established whether lactogenic hormones such as PRL have specific effects on the development of human breast cancer (2). Because PRL actions are mediated by a specific membrane receptor, studies on the expression of PRL receptor (PRLR) in human breast tissues are of relevance to better understand the role of PRL in breast tumorigenesis. PRLR has been identified in human breast cancer cell lines in culture (3, 4) and in human breast biopsies (5, 6). Detection of PRLR by hormone binding or immunocytochemistry (ICC) showed the presence of PRLR in 20–80% of the breast samples (7, 8, 9). Furthermore, this presence seems to be correlated with the overall survival without recurrence of these patients (10). It is important to ascertain whether the above-mentioned methods are sufficiently sensitive to allow association of PRLR expression with a specific pattern of progression of the disease. It is also important to better understand the putative role of PRL and its receptor in breast tumorigenesis; to date, no quantitative data are available concerning their expression in normal breast tissue (11), which is necessary for such comprehension.

RT-PCR is an established method to detect gene expression and has been widely used in mammary tissues (11, 12). However, this method is qualitative and does not provide precise levels of gene expression in normal vs. tumor tissues. Quantitative PCR (Q-PCR), in contrast, provides a rapid and reliable way to quantitate the amount of a given messenger RNA (mRNA) in samples, even with very low levels of gene (13, 14, 15, 16, 17).

In the present study, we assessed PRLR expression by Q-PCR and ICC in normal human breast tissues and tumors. A higher expression was found in all tumors as compared with contiguous normal tissues, suggesting an increased pattern of expression of PRLR in breast tumors.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Twenty-nine patients were studied. Their age, histological diagnosis, hormonal status, estradiol receptor (ER) and progesterone receptor (PR) status, and the eventual hormonal treatments are indicated in Table 1Go.


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  		Table 1. Main clinical characteristics of patients

 
Twenty-five patients underwent surgery for a breast tumor. Seven patients had fibroadenoma and 1 patient had a radial scar with no sign of malignancy. One patient had liver metastasis of a breast cancer diagnosed 3 yr before. The remaining 16 patients had a histological diagnosis of cancer, all invasive except for 1 patient. For each patient, tumor tissue and contiguous normal tissue were obtained, after informed consent.

Four patients had a surgical reductive mammoplasty. These patients were studied specifically for the measure of PRLR gene expression in normal breast epithelial cells and for comparison with values from in vivo normal tissue.

Primary cell culture of human epithelial cells

Primary cultures of normal human breast epithelial cells were obtained as previously described elsewhere (18). Neither PRL nor GH was added to the culture medium. In brief, the normal breast tissue was first enzymatically digested with collagenase (0.15%) and hyaluronidase (0.05%) in Ham’s F10 and then filtered consecutively through 500-, 300-, and 150-µm sieves to eliminate undigested tissue. The cell material retained on a final 60-µm sieve was used for epithelial cell culture.

Construction of internal control and synthesis of internal control RNA

The plasmid for the preparation of synthetic internal control RNA (cRNA) was constructed by inserting a 50-bp fragment of the human GH receptor complementary DNA (cDNA) into a portion of the human PRLR (hPRLR) cDNA (Fig. 1Go). The 50-bp fragment was generated by PCR using a forward primer with a constitutive NcoI restriction site (5'-TCTACTTTCCATGGCTCTTAAT-3' and a reverse primer with an additional NcoI restriction site (5'-TACAAATACCATGGCTGTTAGC-3'). This fragment was digested with NcoI and introduced into the hPRLR cDNA previously digested with NcoI and treated with alkaline phosphatase. The subsequent chimaeric hPRLR plasmid was cleaved with EcoRI, and the 2773-bp fragment was subcloned into the EcoRI site of Bluescript vector (Stratagene Cloning System, San Diego, CA) containing an oligo d(A) tail inserted at the HindIII site.



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  		Figure 1. Schematic representation of PRLR and plasmid construction for generation of cRNA. A, hPRLR cDNA with shaded rectangle representing mature receptor; transmembrane domain is shown black. On left is position of first and last amino acid of mature receptor, whereas on right, first and last nucleotides of cDNA and primer positions (PRLR41 and PRLR50). Size of amplicon (360 bp) is indicated. B, Plasmid construction for internal control: pBluescript vector with T7 promoter and an oligo d(A) tail are represented. Position of SalI site used for plasmid linearization is also indicated. C, Structure of modified insert containing additional 50 bp (cross-hatched area) is shown, with primer positions. Size of amplicon of cRNA (410 bp) is also indicated.

 
After digestion by SalI, the chimaeric PRLR construct served as a template for in vitro transcription by T7 RNA polymerase to generate internal cRNA, using the protocol supplied by the manufacturer (Boehringer Mannheim, Mannheim, Germany). The cRNA was purified with phenol-chloroform extraction and subsequently by oligo(dT) chromatography. The purified cRNA was resuspended in diethylprocarbonate-treated water. The absolute number of cRNA molecules was calculated using spectrophotometric absorbency at 260 nm (Beckman DU-64, Fullerton, CA), the molecular weight of the cRNA molecule (976,260 g/mol) and Avogadro’s number. The cRNA solution was diluted to 1 x 106 molecules/µL in the presence of RNAsin (Promega, Madison, WI), aliquoted, and stored at -80 C.

Oligonucleotide primers used for amplification of PRLR messenger

Oligonucleotide primers designed for the amplification of the hPRLR transcript were purchased from Genosys (Cambridge, England). The forward primer (PRLR-41) has the following sequence 5'-CTGTGGATTAAATGGTCTCC-3' and the reverse primer (PRLR-50), the sequence 5'-TGCAGGTCACCATGCTATA-3' (nucleotides 723-1083). These primers amplify the transmembrane domain and do not distinguish the PRLR long form from the eventual intermediate form also described (11). For PCR, PRLR-41 was also used as 5'-end labeled with [{gamma}-32P]ATP (>5000Ci/mmol; Amersham, Arlington Heights, IL) using T4 polynucleotide kinase, and unincorporated nucleotides were removed using a Biogel P-60 column (Bio-Rad Labs., Richmond, CA).

cDNA synthesis and establishment of Q-PCR

Total RNA was extracted according to the procedure of Chomcsynski and Sacchi (19). Then RNA preparations were divided into aliquots of 1 µg/tube containing 1 U/µL of RNAsin (Promega) and stored at -80 C.

Establishment of the Q-PCR was based on experiments previously performed in our laboratory (17, 18). The first step consisted of titration to evaluate the number of molecules of mRNA per microgram of total RNA. Titration consisted of RT of both total RNA (1 µg) and cRNA at various concentrations, from 10,000 to 40 x 106 molecules for T-47D cells and from 1000 to 4 x 106 for the tissue samples. Once titration was performed, RT was performed again using 1 µg total RNA and the number of cRNA molecules previously determined. Three-fold serial dilutions of the cDNA mixture were amplified by PCR. The reaction was carried out using 10 µL of each diluted RT mixture in PCR buffer (50 mM KCl, 1.5 mM MgCl2, and 10 mM Tris-HCl, pH 8.3), 200 µM deoxynucleotide triphosphates, 12.5 pmol forward and 25 pmol reverse primers, 1.8 x 106 cpm 32P-end-labeled PRLR 41-oligo, and 1.5 U Taq polymerase (Perkin-Elmer/Cetus, Norwalk, CT) in a final volume of 50 µL. After an initial denaturation at 95 C for 5 min, the amplification profile was 30 sec of denaturation at 94 C, 1 min of annealing at 52 C, and 1 min 30 sec of extension at 72 C, for 25 cycles. Two kinds of negative control were prepared: 1) total RNA was omitted in RT (RNA control), and 2) cDNA product was omitted in the PCR reaction (DNA control).

Analysis of PCR-amplified cDNA products

PCR products (20 µL) were separated on 5% nondenaturing polyacrylamide gel stained with ethidium bromide. The bands corresponding to each specific PCR product were excised from gels, and the amount of incorporated radioactivity (counts per min) was plotted against the amount of template (cRNA or total RNA). Radioactivity from negative controls served as background. The linear regression of each dilution curve was calculated, and the absolute number of target mRNA molecules was estimated by extrapolating the value of 1 µg total RNA to the internal control. Results were expressed as the number of molecules per microgram total RNA.

Analysis of human PRL mRNA expression

Four micrograms of total RNA were used for RT using the conditions described for PRLR. PCR was performed with the following set of primers, purchased from Genosys: forward 5'-TGCCAGGTGACCCTTCGAGACCTG-3' and reverse 5'-GACTATCAGCTCCATGCCCTCTAG-3'-primers (nucleotides 31–404). The reaction was carried out using 10 µL of each diluted RT mixture in PCR buffer (see above), 200 µM deoxynucleotide triphosphates, 25 pmol forward and reverse primers, and 1.5 U Taq polymerase in a final volume of 50 µL. The amplification profile was 30 sec of denaturation at 94 C, 1 min of annealing at 69 C, and 1 min 30 sec of extension at 72 C during 32 cycles, after an initial denaturation at 95 C for 5 min.

Immunohistochemical study of PRLR expression

Immunochemistry was performed on all samples. The sections were stained using a streptavidin biotin peroxydase method. The immunoperoxydase reaction was visualized by the use of diaminobenzidine as a chromogen. Negative controls were performed by omitting the primary antibody. Immunostaining for PRLR was performed using a monoclonal antibody (U5) that has been shown to cross-react with the human PRLR (20) and has been used at a concentration of 5 µg/mL.

Staining for PRLR was recorded in tumors and surrounding normal glandular tissue. The intensity of staining was noted as absent, weak, moderate, or strong.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Q-PCR in T-47D cells

The synthetic internal control was designed in such a manner that amplicons of cRNA could be easily distinguished from those of the target mRNA by size (410 bp for cRNA and 360 bp for target mRNA), but sufficiently close to permit a comparison (Fig. 2Go).



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  		Figure 2. Ethidium bromide staining of PCR products of total T-47D RNA (lane 1), control RNA (lane 2), and T-47D + cRNA (lane 3). M, Molecular size markers.

 
Q-PCR was performed initially on T-47D cells. An initial value of approximately 25 x 106 molecules of mRNA per microgram of total RNA was observed after two titrations. In a second step, dilution curves were performed. They were parallel and exponential for both cRNA and PRLR mRNA, indicating that Q-PCR was equally efficient for both control and target RNA without any competition that could interfere with reliable quantitation. Three different experiments were performed on T-47D cells: a value of 31 x 106 mRNA molecules encoding PRLR per microgram of total RNA was calculated. Different concentrations of primers and a varying number of cycles were also tested, indicating that the most reproducible results were obtained using a concentration of 25 pmol for each primer and 25 cycles of PCR (data not shown).

Determination of PRLR gene expression by Q-PCR in breast samples

PRLR gene expression was studied in 25 patients with a breast tumor. For each patient, a titration was initially performed for tumor and normal tissue. In a second step, Q-PCR was performed with serial dilutions, as indicated in Fig. 3Go. In addition, PRLR gene expression was determined in cultures of normal epithelial cells obtained and prepared from 4 patients undergoing mammoplasty.



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  		Figure 3. Representative result of Q-PCR. A, Ethidium bromide staining of products separated on a 5% polyacrylamide gel. RT was carried out using 1 µg total RNA and 5 x 105 molecules of cRNA. Before PCR, 1:3 serial dilutions were prepared using one half of RT mixture. Thus, dilution curves start at 500 ng total RNA and 2.5 x 105 molecules of cRNA (lanes 1–6). Negative control reactions without total RNA in RT (lane 7) or without cDNA product (lane 8) were also amplified for 25 cycles. Position of 360-bp product from total RNA and 410-bp product from cRNA are indicated on left. M, Molecular size markers; B, bands shown in A were cut out from gel, and radioactivity counted. Radioactivity was plotted against amount of total RNA or cRNA. A linear regression of each curve was calculated, and absolute number of molecules of PRLR mRNA was estimated.

 
The results for all patients are presented in Table 2Go as a function of the histological diagnosis. A wide heterogeneity was noted among the results from tumor samples, ranging between 4500 and 4.7 x 106 molecules per microgram of total RNA, as well as in normal tissues, in which expression ranged from 1500 to 1 x 106 molecules per microgram RNA. However, expression of PRLR gene was observed in all the patients in both the tumor and the normal tissue. Finally, in primary epithelial cell cultures, expression of PRLR gene was similar to that observed in normal tissues.


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  		Table 2. Expression of PRLR gene in tumors and normal breast tissues

 
Because of the heterogeneity from one patient to another, we compared PRLR expression in tumor vs. normal tissue for each patient. As indicated in Fig. 4Go, it was always greater in the tumor than in the normal contiguous tissue with a ratio (tumor vs. normal, T/N) always greater than 1. Eleven patients had a T/N ratio between 2 and 10, and 10 patients a ratio >10.



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  		Figure 4. Representation of PRLR gene expression in 25 tumors and normal contiguous breast tissues. Results are presented from lowest to highest level of PRLR gene expression in tumors.

 
Finally, to assess the reproducibility of Q-PCR, the same sample from one patient with no strong difference between tumor and normal tissue was assayed four times. In the tumor tissue, number of molecules of the patient (LAV) was 449,000 ± 18,520 (mean ± SEM), and in normal tissue was 346,000 ± 44,510 (mean ± SEM). The ratio of T/N always remained >1.

Immunostaining of PRLR in breast tumors and normal breast tissues

Tissue sections from 25 patients with breast tumors were also studied by ICC for PRLR staining. Table 3Go reports the results of immunostaining intensity in tumor and contiguous breast tissue. In 18/25 patients, immunostaining appeared stronger in tumor (Fig. 5Go, B-D) than in normal contiguous tissue (Fig. 5AGo) and in 7 patients no difference was observed, most often in patients with low immunostaining, suggesting weak receptor expression. Never was a stronger signal seen in normal tissue compared with tumor. Staining was detected in all except two cases in at least 70% of the cells. Staining intensity was particularly intense on the luminal border of apocrine cysts. No clear correlation of PRLR gene expression assessed by Q-PCR and immunostaining was seen (data not shown).


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  		Table 3. Immunocytochemical staining in tumors and normal breast tissues

 


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  		Figure 5. Immunocytochemical localization of PRLR immunoreactivity in normal breast tissue, fibroadenoma, and breast cancer. A, Normal lobules. B, Fibroadenomas showing weak (single arrowhead) and strong (double arrowhead) immunoreactivity. C, Infiltrating carcinoma; signal is strong. Magnification, x200. D, In situ lobular carcinoma, signal is also strong. Magnification, x400.

 
Analysis of human PRL mRNA expression

Expression of hPRL mRNA was performed by RT-PCR in 16 patients. A signal of 373 bp, which corresponds the correct size for hPRL transcript, was obtained in all samples, however with high variability among the tissues studied. Expression was detected in both normal tissues and tumors, as well as in cultured mammary epithelial cells (Fig. 6Go).



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  		Figure 6. Expression of PRL mRNA in four samples of normal epithelial cells and eight patients. For each patient, results are presented in tumor (T) and normal contiguous tissue (N). A transcript of 373 bp was found with a different intensity in all tissues examined. A positive control (hPRL cDNA; C+) and a negative control (RT-PCR without RNA; C-) are also indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The requirement of PRL for growth, differentiation, and proliferation of breast tissue is well established and PRL actions are mediated through a specific membrane-bound receptor, the PRLR (21), which was only described as a long form in humans, until Clevenger et al. (11) reported an intermediate form in certain breast specimens. One of the limitations in understanding the eventual role of PRL in human breast tumorigenesis has been the difficulty to evaluate precisely the level of expression of its receptor in normal breast and tumor tissue. In fact, from 20–80% of breast tumors fail to express PRLR (5, 6, 7, 8, 9), and correlations between expression of PRLR and ER on breast cancer based on these methods have provided conflicting results (10, 22, 23, 24). Q-PCR, although not designed to measure protein levels, is a very sensitive technique (~500 molecules per microgram of RNA) (13) and is now widely used for gene expression studies (15, 16, 17). As indicated in Table 2Go and Fig. 4Go, PRLR gene expression was detected in all studied tissues, sometimes at low levels, with a great variation among patients.

An influence of previous hormonal treatments on PRLR expression is possible. Hormonal regulation of PRLR has been reported, although most studies have been performed in rat liver (25) or in human breast cancer cell lines (26). Estrogens are known to be able to increase PRLR levels (27), and it is interesting to note that all postmenopausal women (who have a low level of estradiol) had <110,000 molecules per microgram of total RNA. It can also explain why nonmenopausal women with fibroadenomas have generally higher expression of PRLR gene. In contrast, it has been suggested that progesterone can reduce PRLR (28), possibly by a direct antagonism on PRLR-PRL induction. Five patients in our study were receiving progestin therapy (10–20 days/month), but only one underwent surgery during progestin treatment. In this case, the level of PRLR gene expression was extremely low, compared with the other four patients.

In tumors as in normal tissues, there was no correlation between the level of receptor expression and the histological type of the lesion nor the ER and PR presence. However, for the three ER and PR patients with high-grade cancers, it is interesting to note that PRLR expression in the tumor was low (58,000, 115,000, and 142,000 molecules per microgram total RNA). This could be an indirect argument, suggesting that like ER and PR (29), PRLR could be a marker of differentiation. Indeed, in all except one study comparing ER and PRLR, a positive correlation has been found between the presence of lactogen and ERs (10, 22, 23, 24).

Although there is a great heterogeneity in PRLR gene expression among patients, the ratio between tumor and normal tissue was always found to be >1. This observation in 25 patients is highly suggestive of an overexpression of PRLR gene in tumors.

Immunocytochemical analysis was performed to study the expression of the PRLR at the protein level. Stronger immunostaining was found in almost all the tumors vs. normal contiguous tissues. Staining was more intense in cytoplasm, as commonly described (30), because a large proportion of the receptors are located in intracellular compartment. The lack of absolute correlation between the gene expression and immunostaining intensity for each patient might be explained by the difference of sensitivity of the methods used. Indeed, with Q-PCR, we detected PRLR gene expression in all patients, whereas by immunocytochemical analysis, PRLR could not be detected in approximately 30% of breast biopsies previously studied (5) and was absent in 16% of normal tissues we examined. In agreement with these values, ligand binding studies also appear to be less sensitive, because PRLRs were only identified in 29% of breast tumors (6). However, we cannot avoid the fact that certain tumors may express high mRNA but low protein levels.

Overexpression of PRLR in breast tumors suggests that PRL could participate in breast tumorigenesis. Using RT-PCR, we detected PRL mRNA in all the tissues we examined including epithelial cells, confirming previous reports (31, 32, 33, 34). Frequently, the signal appeared stronger in tumors vs. normal tissues; however, RT-PCR does not provide any information concerning the actual level of expression.

The exact role of PRL in breast tumorigenesis remains to be elucidated. PRL, like estradiol, is known to be important for the development and growth of experimental 7,12-dimethylbenz(a)anthracene-induced breast tumors, probably by increasing PRLR levels (1). Interestingly, PRL also has been shown to have an inhibitory influence on tumor development, depending on the time animals are exposed to elevated PRL levels (35). Finally, recent epidemiological data suggest that lactation in humans may exert a protective effect on breast cancer (36). However, little information on the role of PRL and lactation is available in vivo in undifferentiated breast tissue (37). This suggests that to better understand the role of PRL in breast tumorigenesis, new tools are necessary.

The fact that all tumors in the present study expressed more PRLRs than normal tissue coupled with the fact that mammary epithelial cells (derived from normal breast and from breast cancer) expressed the natural ligand strongly suggests that locally produced PRL may have a potential role in the process of tumor development.


   Acknowledgments
 
We thank Drs I. Mowszowicz, N. Binart, V. Goffin, and M. Edery for useful discussions. We are also grateful to Drs. E. Bourstyn, K. Clough, C. Hofmann, V. Mitz, and A. Quéré for their help in the collection of tissues and C. Coridun for excellent secretarial assistance. We gratefully appreciate Novartis Pharma SA, Rueil-Malmaison, for their support for these studies.

Received August 28, 1997.

Revised October 23, 1997.

Accepted October 30, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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