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1 Inserm Unit 808, Faculté de Médecine Descartes Paris 5 site Necker, 156 rue de Vaugirard, Paris 75015, France
2 Université Paris-Descartes, Faculté de Médecine site Necker, 75015 Paris, France
3 Department of Pathology, Saint-Louis Hospital and Institute of Hematology, 75010 Paris, France
4 Inserm, Unit 728, 75475 Paris, France
5 Department of Biomedical Sciences and Edison Biotechnology Institute, Ohio University, Athens, OH 45701, USA
(Requests for offprints should be addressed to V Goffin; Email: goffin{at}necker.fr)
(P Touraine is now at Department of Endocrinology and Reproductive Medicine, GH Pitie Salpetriere, Paris 75651, France)
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mitogenic activity of PRL has naturally led to the investigation of its role in mammary tumor growth. In rodents, the role of PRL as a promoter of mammary tumors has been described in many experimental models for almost three decades (Welsch & Nagasawa 1977). It is clearly established that hyperprolactinemia is directly related to mammary tumor development, and treatment using dopamine agonists, the physiological inhibitor of pituitary PRL production, prevents tumor growth (Wennbo et al. 1997, Vonderhaar 1998, 1999, Clevenger et al. 2003). Recently, it has been demonstrated that the development of mammary tumors induced by polyoma middle Tantigen is delayed in mice deficient in PRL (knockout (KO)), which further strengthens the promoting role of this hormone (Vomachka et al. 2000). In agreement with this, transgenic (Tg) mice that systemically express PRL (using metallothionein (Met) promoter) spontaneously develop mammary carcinomas from the age of ~1 year (Wennbo et al. 1997). These data are a few examples of the most relevant observations that confirm the major role of PRL on mammary gland tumor development in experimental models (for a review, see Clevenger et al. 2003).
In humans, PRL involvement in breast cancer remains a matter of debate, despite a large body of observations arguing for its growth-promoting actions (Clevenger et al. 2003). For example, PRL exerts mitogenic effects on human mammary tumor cell lines in vitro, irrespective of their estrogen receptor status (ER+ or ER–) (Ginsburg & Vonderhaar 1995, Llovera et al. 2000). In addition, we demonstrated some years ago that the level of PRLR expression is higher in mammary tumor biopsies than in the adjacent healthy tissue, suggesting that the former exhibits increased sensitivity to PRL than the latter (Touraine et al. 1998). At the epidemiological level, two recent publications from the large-scale Nurse Health Study (> 30 000 women) revealed that high–normal PRL levels increased the risk of developing breast cancer in post-menopausal women, establishing for the first time a convincing relationship between PRL levels and breast cancer in women (Hankinson et al. 1999, Tworoger et al. 2004). Nevertheless, the role of PRL in neoplasia still fails to be unanimously accepted at the clinical level, a situation mainly due to the fact that the treatment of breast cancer patients with dopamine agonists failed to improve their 5-year survival, suggesting that the correlation between PRL and breast cancer is probably more complex than solely related to circulating PRL levels (Clevenger et al. 2003).
For the last 10 years, many laboratories have demonstrated that, in humans, various non-pituitary tissues synthesize PRL, including the mammary and prostate glands (Clevenger et al. 1995, Ginsburg & Vonderhaar 1995, Nevalainen et al. 1997, Touraine et al. 1998, Goffin et al. 2005). Co-expression of the hormone and its receptor within the same tissue naturally led to the proposition that PRL could act via an autocrine–paracrine mechanism, and could participate in the promotion of tumor growth. The tumorigenic potential of this local PRL in human breast cancer cell cultures was first suggested from the growth-inhibitory effect of anti-PRL antibodies (Ginsburg & Vonderhaar 1995). Evidence that the expression of local PRL is increased in high-grade human prostate cancer further argued for the involvement of PRL in human tumor growth (Li et al. 2004). In contrast to humans, production of PRL in non-pituitary tissues is rather rare in the mouse. With respect to the mouse mammary gland, local PRL production seems to be restricted in terms of both the amount of hormone produced (hardly detectable) and the time window (only in gestation/lactation), although these observations remain poorly documented (Clevenger et al. 2003). Hence, various genetically modified mouse models have been used to better characterize the functional impact of this autocrine/paracrine loop of action. Transplantation of mammary epithelium from PRL-deficient mice into the mammary fat pad of immunodeficient mice revealed reduced proliferation during pregnancy compared with normal epithelium, arguing for a mitogenic role of local PRL (Naylor et al. 2003). Accordingly, permanent over-expression of a PRL transgene controlled by the mammary-specific neu-related lipocalin (NRL) promoter led to the appearance of mammary carcinoma in animals of 15–16 months (Rose-Hellekant et al. 2003), providing evidence that autocrine–paracrine PRL exhibits the same pro-oncogenic potential as endocrine PRL (Wennbo et al. 1997). Since the latter studies were focused on virgin animals, however, they emphasized the tumorigenic effect of PRL excess on undifferentiated mammary gland, while any effect on the differentiation process during gestation and/or on its ultimate biological function – lactation – were not addressed. Gourdou and colleagues (2004) generated Tg mice permanently over-expressing a constitutively active form of the PRLR in the mammary gland, the main phenotypes of which were over-development of ducts and alveoli leading to lactation failure. Although this study suggested that elevated PRLR signaling starting from the virgin state later interfered with proper mammary gland differentiation during gestation, which finally led to lactational failure, the question of whether these abnormalities developed into mammary tumors remained undetermined, since only young animals were used.
In the present study, we have used a different model to further understand the impact of locally produced PRL on the pathological development of the mammary gland once it is differentiated. We generated a Tg model expressing human PRL under the control of the promoter of whey acidic protein (WAP), a milk protein naturally expressed in the mammary gland from late pregnancy throughout lactation and until early involution. As described later, these mice displayed profound alteration in their mammary glands, including histological abnormalities and lactation defects. A striking difference with previous PRL Tg models involving virgin animals was that PRL over-expression at later stages of the differentiation process induced benign mammary lesions, but not adenocarcinomas.
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Materials and Methods |
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The WAP–hPRL fusion gene is shown in Fig. 1A
. It utilizes the murine WAP transcriptional regulatory element (–2600 to +24) to direct mammary-specific hPRL expression (Pittius et al. 1988a), and the bovine growth hormone (bGH) polyadenylation signal for proper RNA processing. The fusion gene also contains DNA sequences encoding the first intron of bGH intron A to increase expression in Tg mice. The EcoRI–BamHI DNA fragment containing this transcriptional unit was injected into fertilized mouse (B6/SJL) eggs as described previously (McGrane et al. 1988). Tg offspring were identified by DNA slot blot hybridization using a radioactive probe generated from the hPRL cDNA, and confirmed by PCR amplification of the human PRL cDNA.
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Tg lineages were initially established on the B6/SJL genetic background in the Ohio laboratory. In the French laboratory, the colony was transferred onto the Balb/c-J background by breeding F1 mice with Balb/c-J mice obtained from Charles River Laboratories (l’Arbresles, France). Routine genotyping was performed using genomic DNA isolated from tail biopsies of 12– to 14-day-old animals, using primers encompassing the initiation methionine codon (CGC GGATCC AAC ATG AAC ATC AAA GGA TCG C) and the termination codon (ATG GAT CCC GGG TTA GCA GTT GTT GTT GTG) as shown on Fig. 1A
. Animals used in this study were obtained after a minimum of six backcrosses on Balb/c-J genetic background. Wild-type littermates were used as controls when appropriate. For experiments, animals were killed by cervical dislocation. Mice were housed in the Animal Core Facility of the Necker site in France in controlled conditions (12 h light:12 h darkness cycles, feed available ad libitum), and all experimental protocols were in agreement with the procedures established by the local ethical committee.
Reproduction and pup growth studies
For reproduction studies, Tg females (or wild-type (WT) littermates) were mated with young WT males of proven fertility, and the latter were removed when pregnancy was assessed by weight gain of the females. Pups were removed from the mother upon weaning and the male was placed back with the female to ensure another pregnancy. The latency between two successive pregnancies was calculated as the time between male and female cohabitation and the first day of pregnancy (= 21 days before next parturition). For pup growth studies, litters from both WT and Tg females were normalized up to eight pups each, and the entire litter was weighed daily.
Tissue collection and homogenization
For expression studies, mammary glands were excised from WT and Tg female mice during pregnancy, lactation, involution, and post-involution. The inguinal (fourth) glands were used for analysis of mammary morphology (whole mount, histology; see below) and protein content, while the third glands were used for RNA preparation. For protein analysis, glands stored at –80 °C were homogenized by ultra-Turrax in lysis buffer (Liu et al. 1996) containing protease-inhibitor cocktails (Sigma), and the homogenates ultracentrifuged for 1 h at 50 000 g to clear out milk and lipids (top layer). The protein supernatant was used for the Bradford assay (Sigma). For RNA analysis, glands were rinsed and cut into small pieces in RNAlater (Ambion, Inc., Austin, TX, USA), homogenized using ultra-Turrax, and total RNA isolated using the RNeasy Lipid Tissue Kit (Qiagen). Pancreas, ovaries, and pituitaries were also collected for protein expression analysis. Pancreatic homogenates were obtained as described earlier for the mammary gland, except centrifugation was performed at 17 000 g. Due to the small tissue size of ovaries and pituitary, these organs were homogenized manually in a reduced volume of lysis buffer and quantified directly using the Bradford method.
Mammary gland whole mount
Inguinal glands were surgically removed and fixed overnight in Carnoy’s solution, rehydrated in ethanol baths, and stained in carmine alum solution overnight at 4 °C (0.2% carmine stain (Sigma), 0.5% alumium potassium sulfate (Sigma)). After staining, samples were dehydrated in graded ethanol baths and stored in xylene, then in histoclear II (National Diagnostics, Eurolab, Fontenay-sous-Bois, France).
Histological and immunohistochemical studies
Mammary gland tissues were fixed with 4% paraformaldehyde for 24 h at room temperature and processed for paraffin embedding according to the standard procedures. Sections (3 µm thick) were stained with hematoxylin and eosin. For immunohistochemical studies, paraffin sections were deparaffinized in xylene and ethanol, then rehydrated with PBS. Sections were then immersed in citrate buffer and micro-waved for 10 min (800 W) for antigen retrieval. After cooling to room temperature, the slides were stained overnight at 4 °C using 1:25 dilution of rabbit polyclonal anti-hPRL A0569 (DAKO, Trappes, France). For detection, the slides were incubated for 30 min at room temperature with diluted anti-rabbit Alexa-labeled secondary antibody (1:1000; Molecular Probes, Invitrogen). After three washes (5 min each) in PBS, slides were dried and mounted with Dako Antifade (DAKO) before analysis. Specific immunostaining of myoepithelial cells was performed following similar procedures, using monoclonal 
-smooth muscle actin antibody 1A4 (Sigma).
Milk composition
At day 8 of lactation, normalized litters (eight pups) from lactating females (WT or Tg) were removed 4 h before milk collection was performed as described (Prieto et al. 1995). Briefly, females were anaesthetized using isoflurane gas (Abbot Laboratories), and 15 min prior to milking, they were injected intraperitoneally with 5 units of oxytocin (Sigma) to facilitate milk extrusion through the nipple. The milk was removed from the mammary glands by massaging, drawn into a Pasteur pipette and ultracentrifuged at 50 000 g for 30 min to clear-out fat material (top layer). The bottom layer was recovered for immunoblot analysis. Milk samples were collected from seven WT and seven Tg mice. Finally, the size of lipid droplets was measured on histological slides from four WTand five Tg lactating females, using the software LUCIA Archive (VGA version 4.51, Laboratory Imaging, Prague, Czech Republic).
Reverse transcriptase (RT)-PCR
After extraction, total RNAs (2 µg each) were reverse transcribed and amplified with RT-PCR according to standard procedures. Primers and PCR conditions were as follows:
Human PRL (373 bp fragment; 30 cycles: 94 °Cx30 s, 69°Cx1 min, 72 °Cx1 min):
hPRL-forward 5'-TGCCAGGTGACCCTTCGAGACCTG-3'
hPRL-reverse 5'-GACTATCAGCTCCATGCCCTCTAG-3'
Mouse PRL (638 bp fragment; 30 cycles: 94 °Cx30 s, 56°Cx1 min, 72 °Cx1 min):
mPRL-forward: 5'-GTCAGCCCAGAAAGGGAC-3'
mPRL-reverse: 5'-CACCTCAGGACCTTGAGA-3'
Mouse PRL receptor (673 bp fragment; 30 cycles: 94 °Cx 30 s, 57 °Cx1 min, 72 °Cx1 min):
mPRLR-forward: 5'-GAGAAAAACACCTATGAATGTC-3',
mPRLR-reverse: 5'-AGCAGTTCTTCAGACTTGCC-3'
Western blot analysis
Samples (50 µg total protein homogenates, or the equivalent of 0.3 µl milk diluted in NaCl 0.9%) were mixed with 2x reducing loading buffer, boiled, electrophoresed on 12 or 15% SDS-PAGE and blotted onto pure nitrocellulose membranes of 0.45 µm (Bio-Rad). Membranes were saturated for 1 h in washing buffer containing 5% powdered skim milk and washed thrice in washing buffer. Antibodies used were: anti-hPRL A0569 at 1:500 dilution (DAKO), anti-RAM/MSP (total milk protein antiserum) at 1:5000 dilution (Nordic Labs, Tebu-Bio, Le Perray-en-Yuelines, France), anti-ß-casein and -casein S1 at 1:5000 dilution (a generous gift from Dr Itaman Barash, Institute of Animal Science, ARO, The Volcani, Centre Bet Dagan, Israel), anti-Stat5 at 1:1000 dilution, and anti-phosphorylated Stat5 at 1:10 000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation (1 h) with anti-mouse or anti-rabbit secondary antibodies coupled to horseradish peroxidase (HRP), immune complexes were detected by enhanced chemiluminescence (Amersham) according to the manufacturer’s recommendations, and autoradiographs were obtained on Konica films AX (VWR, Fontenay-sous-Bois, France).
Quantitative expression of hPRL
The expression level of hPRL (transgene product) was measured in serum (blood harvested by orbital puncture) and tissue homogenates, using the hPRL ELISA kit purchased from Diagnostic Biochemical (London, ON, Canada). This immunological assay does not cross-react with endogenous mouse PRL (Bernichtein et al. 2003b) and was performed as per the manufacturer’s recommendations. Specifically, we used 50 µl non-diluted serum, and 1.5–8 mg total tissue homogenates. Dilutions were performed occasionally to eliminate artifacts due to the hook effect.
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Results |
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Eight Tg mice were identified after egg injection (four males and four females). The Tg females were mated to non-Tg males and milk was isolated from these mothers following delivery. Two of the Tg mothers (founder nos 90 and 4) produced milk that contained elevated levels of hPRL (~2.3 and ~0.5 mg/ml respectively). Founder no. 90 displayed reproductive difficulties and died from what appeared to be an intestinal tumor approximately 3 months after giving birth. In addition, a female offspring derived from founder no. 90 also died from similar intestinal tumors following her first delivery. The only remaining lineage (no. 4) did not exhibit this phenotype and was expanded for use in this study.
Human PRL expression in lactating mammary gland
Previous reports have shown that the expression of WAP milk protein is detected from late pregnancy (day 14–16) until early involution (Pittius et al. 1988b). Accordingly, expression of the WAP–hPRL transgene was detected at the mRNA and protein levels in mammary glands of lineage no. 4 females at day 17 of gestation (G17; data not shown) and day 8 of lactation (L8) (Fig. 1B). The protein was also secreted into milk, and the expression levels averaged from several animals confirmed the value measured in the lineage no. 4 founder (0.5 mg/ml). As expected, expression of endogenous PRLR was observed in the mammary gland of all animals, with no obvious correlation with transgene expression (Fig. 1C). Due to the absence of reliable anti-mouse PRLR antibodies, any variation in the protein level could not be assessed. In contrast, although lactating mouse mammary glands have been reported to express mouse PRL (mPRL) (Clevenger et al. 2003), we were unable to detect endogenous expression of the hormone in any of the WT and Tg mice analyzed (Fig. 1C).
Immunohistochemichal studies showed that the hPRL transgene was expressed only by MECs, as shown by the absence of staining in surrounding connective tissue (Fig. 2A and B). In control experiments, no specific immunostaining was obtained for non-Tg females (Fig. 2C) or when primary antibody was omitted for Tg gland analysis (Fig. 2D). Since hPRL binds to and activates the mouse PRLR (Bernichtein et al. 2003a), these data suggest that the transgene product can act via an autocrine–paracrine loop in the mammary gland of mid-pregnant/lactating Tg animals.
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According to the human species specificity of the PRL ELISA kit used in this study, no hPRL was detected in the circulation of WT animals, irrespective of their reproductive status (data not shown). Very low levels of hPRL (2–8 ng/ml) were detected in sera of virgin Tg females (Fig. 3), indicating minimal leakage expression of the transgene regardless of pregnancy and age. Circulating levels of hPRL peaked at each pregnancy/lactation cycle, and returned to virgin-like levels in non-lactating animals (Fig. 3, right). This was especially true during the first three pregnancies, with maximal level observed at third gestation (up to 180 ng/ml). This pregnancy-related pattern of serum hPRL levels was approximately parallel to the hPRL content of mammary glands from the various stages analyzed (Fig. 3, left), suggesting that some of this locally produced hPRL was released into serum and contributed to circulating levels of the Tg protein. This correlation was much less clear after four or five pregnancies, although it should be noticed that we obtained only a very few animals achieving these stages. Finally, the quantification of Tg protein in mammary gland lysates also indicated that after the first gestation cycle, expression of autocrine hPRL was maintained to a significant and measurable level in the glands of non-pregnant (parous) females. Since the WAP promoter is PRL-responsive, it is likely that expression of the WAP–hPRL transgene remained self-induced even after pregnancy. These data indicate that once it had been turned on, the autocrine loop persisted throughout the lifespan.
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Whole mount
Figure 4 shows mammary whole mount from WT (left) and Tg (right) females at three different stages: virgin (panels A, D), day 17 of gestation (G17; panels B, E), and day 8 of lactation (L8; panels C, F, G). As expected, ducts completely reached the periphery of the mammary fat pad in WT virgin animals, and branching/end buds were prominent (Fig. 4A). The glands of Tg animals appeared to be more developed, with two major differences; ducts were much more dilated, and small alveoli were already visible (Fig. 4D). At day 14 (not shown) and day 17 of pregnancy (Fig. 4B and E), Tg and WT glands exhibited similar morphology, with extensive ductal branching and proliferation of the alveolar structures expanding into connective tissue. Again, Tg animals displayed enlarged ducts compared with WT glands. Finally, while alveoli of the lactating WT females were filled with milk and exhibited the typical structure of a functional gland (Fig. 4C), Tg glands tended to be less developed and/or milk-filled compared with WT, and the extent of this defect appeared to be variable between all the animals analyzed; panel F shows a mammary gland representative of Tg females lactating successfully, while panel G illustrates glands exhibiting lactation defects (pup death; see below). In summary, Tg mammary glands appeared to grow and differentiate prematurely (virgin), but not completely (lactating).
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shows histological sections of lactating WT (A–C) or Tg (D–L) mammary glands taken at different pregnancies (P1–P5), and it illustrates the typical mammary phenotypes observed in the majority of Tg females. WT glands displayed well-organized alveoli filled with milk (Fig. 5A), whereas Tg mammary glands displayed great heterogeneity; some areas displayed normal morphology and appeared to be functional as indicated by the presence of milk, while others seemed to be non-functional, i.e. empty or filled with dense eosinophilic secretions clearly distinct from milk (Fig. 5D–F). This heterogeneity was not correlated with the expression level of the transgene as determined by immunohistochemistry (data not shown), but was less marked in first (Fig. 5D) than subsequent (Fig. 5E and F) pregnancies. Confirming the whole-mount analysis, mammary ducts of Tg mice were more dilated and formed typical cysts in most affected glands (Fig. 5G–I). Over-expression of hPRL also strongly affected the morphology of individual cell types. In WT lactating animals, ducts and alveoli were surrounded by a wide layer of well-delineated MECs, showing intense secretory activity, with lipid globules pinched-off at the apical membrane. The basal layer of myoepithelial cells was also well identified (Figs. 5B and 6A). In contrast, alveoli (Fig. 5D–F) and ducts (Fig. 5G–I) of Tg mammary glands were surrounded by a single, sometimes discontinuous layer of flat epithelial cells with the layer dramatically atropied or even not distinguishable (Figs. 5H–I and 6B). Finally, Tg mammary glands displayed global tissue disorganization (Fig. 5J–L), increased cellularity including areas with lymphocytic infiltrates (Fig. 5J), and cyst sometimes calcified (Fig. 5L). All these phenotypes are in sharp contrast with the normal morphology of lactating WT glands (Fig. 5A–C). All the abnormalities described above were detected in more than 50% of P1 females, and accumulated were aggravated during successive pregnancies, to affect all animals in P3–P5. It is noteworthy that they were also noticed in Tg females that did not experience pup death, and to a lesser extent, in virgin animals of nearly 1 year or more.
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), as highlighted by the disappearance of the secretory epithelium (Fig. 7C). In contrast, Tg mammary glands did not undergo complete involution, as highlighted by the persistence of alveolar structures and enlarged ducts filled with dense secretions or clotted milk (Fig. 7D–F).
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). These lesions included three cases of metaplasia with keratinized epithelium and sometimes lipid crystals (Fig. 7H–J), and two cases of papilloma (Fig. 7K). These mice obviously experienced lactation problems resulting in pup loss. Such abnormalities were never detected in WT females of the same age. None of the histological analyses that we performed revealed the occurrence of malignant mammary lesions, even in the oldest animals. Pup growth and mortality
More than half of the Tg females (57%) encountered lactation problems leading to at least one pup death in one of their successive pregnancies, while the others appeared to lactate correctly (no pup death). Overall, 13% of the pups from Tg mothers died within 3 days after parturition, which is fourfold higher compared with litters of WT mothers (Fig. 8A). When pups from WT mothers were substituted for dead pups in litters from Tg mothers, they also died within days (data not shown), suggesting a lactational defect of these mothers rather than any phenotype of the pups. Also, when we performed growth curves of pups from the sub-population (43%) of Tg mothers that did not lose any pups, there was no significant difference compared with WT mothers (Fig. 8B), indicating no lactational defect in these females.
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Milk protein content was analyzed by Coomassie Blue staining (not shown) and immunoblotting of total milk proteins, ß-casein and -casein S1 (Fig. 8C). We could not detect any major difference between the protein content of milk from WT and Tg mothers, irrespective of whether they exhibited lactational defects or not. Conversely, we repeatedly observed in the course of histological analyses of mammary gland sections that the size of milk lipid droplets in Tg mammary glands was increased compared with WT animals (Fig. 8D). This parameter was quantified by measuring the area of >300 droplets by section from several animals. The average droplet size was 308 ± 8.3 and 103 ± 3.4 µm2 for Tg and WTanimals respectively (mean ± S.E.M., P<0.0001 by Student’s t-test). It is noteworthy that these experiments (milk composition, droplet size) could not be performed with mammary glands from animals showing a strong lactation defect, since their glands contained very low quantities of milk.
Non-mammary phenotypes and transgene expression in other tissues
We easily noticed that Tg females encountered fertility problems because all females (WT and Tg) originating from the same litter were often mated at the same time. The delay between first and second pregnancies was much higher for Tg animals compared with their WT counterparts (Fig. 9A). In addition to acquired infertility, many Tg females exhibited various signs of premature aging. In a test group of 14 Tg females, checked and mated regularly during 12–14 months, half remained healthy and fertile up to four gestations (achieved before the age of 12 months), while the others progressively showed fertility problems and were unable to undergo more than two or three pregnancies (they were mated up to the age of 14 months). The latter became sick (weight loss, hair loss, altered behavior) and died prematurely.
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, pancreas, ovary, and pituitary all expressed hPRL. Except for the latter, expression levels were higher in lactating than in non-lactating animals, correlating with the regulation of WAP promoter by reproduction hormones. Although absolute levels of hPRL expression were lower in these organs compared with mammary gland (Fig. 3, left), they were not negligible and were reliably measurable, indicating that these tissues are sporadically or permanently under local hyperprolactinemic conditions. |
Discussion |
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While mammary glands of young virgin Tg animals did not markedly differ from those of WT littermates, older virgin females displayed more developed mammary structures, with increased ductal branching and partial development of lobulo-alveolar structures, suggesting that the progressive increase of local PRL expression with age was sufficient to induce premature and limited mammary gland growth and differentiation. This phenotype is in good agreement with what was observed in virgin Tg mice expressing PRL or a constitutively active PRLR (Wennbo et al. 1997, Gourdou et al. 2004). From the first gestational/lactational cycle, which turned on expression of autocrine hPRL, more pronounced morphological abnormalities appeared in young WAP–hPRL animals. Despite the well-known stimulatory effects of PRL on mammopoiesis, as highlighted by PRL or PRLR KO models (Horseman et al. 1997, Ormandy et al. 1997), whole-mount analysis revealed that mammary structures tended to be less developed in many Tg animals compared with WT littermates, indicating that elevated PRL levels during mammary gland development interfered with normal differentiation. At the histological level, lactating mammary glands of our animals exhibited global disorganization mainly including: (i) enlarged and dilated ducts, surrounded by flat and discontinuous epithelium, (ii) no/less distinguishable myoepithelial cell layer, and (iii) abundance of cysts filled with clotted milk. The histological abnormalities of the mammary gland were detected in virtually all WAP–hPRL Tg, and they tended to be aggravated with successive pregnancies and age, although there was no absolute correlation between the number of pregnancies, the concentration of hPRL measured in the mammary gland, and the severity of the lesions. Since pituitary PRL production naturally increases during pregnancy up to lactation, one would have expected that even higher PRL levels would be of no consequence. In contrast, our observations indicated that amplification of PRL activity during the final stages of mammary differentiation completely prevented lobulo-alveolar formation, the phenomenon for which PRL is known to play a crucial role (Horseman et al. 1997, Ormandy et al. 1997, Kelly et al. 2002). This suggests that there is a threshold above which an excess of PRL signals is functionally detrimental. In addition, since low but detectable hPRL expression was also noticed in the mammary gland of virgin animals, one cannot exclude the possibility that premature PRL exposure of the various cell types that populate the mammary tree could also affect later development.
Although this was not the primary goal of this study, we were interested to know whether these severe histological abnormalities impacted on mammary gland functionality. Although the rate of pup mortality (13%) in litters from WAP–hPRL mothers was less dramatic than in other Tg models presenting lactation defects (Kelly et al. 2002), more than half of WAP–hPRL mothers experienced pup death, suggesting that the ability to feed correctly was altered in many animals. Neither milk production per se, nor its protein content was found to be altered. The feeding potency of milk also appeared unaffected, since pup weight gain was normal in litters undergoing no pup death. The only difference we noticed was, at the macroscopic level, the difficulty to extract milk through the nipple, and at the microscopic level, the marked increase in the size of lipid droplets observed in mammary ducts and alveoli. It is likely that the resulting increase in milk viscosity, combined with the morphological disorganization of mammary ducts and atropy of the myoepithelial layer, led to partial inability to eject milk, and ultimately to increased pup mortality. The fact that not all mothers exhibited lactational defects probably reflects the fact that the combination of these alterations needs to achieve a certain threshold to produce the phenotype. In agreement, mice displaying the most strongly altered mammary gland morphology were those exhibiting the poorest lactation.
Since previous reports investigating Tg models constitutively expressing PRL mammary gland involved virgin animals (Wennbo et al. 1997, Rose-Hellekant et al. 2003), direct comparison with our lactation phenotype was not possible. Otherwise, these reports clearly demonstrated that virgin Met-PRL and NRL–PRL Tg females developed adenocarcinomas from the age of 1 year or more (Wennbo et al. 1997, Rose-Hellekant et al. 2003). At first glance, this observation does not correlate with our model, since adenosquamous metaplasia and papillomas were the most severe phenotypes detected in the few old animals that have undergone several pregnancies. If one considers the stage of mammary differentiation at which it occurs, however, the occurrence (or not) of adenocarcinoma is probably not so surprising. The Met (Wennbo et al. 1997), NRL (Rose-Hellekant et al. 2003), and ß-lactoglobulin (Gourdou et al. 2004) promoters were shown to be constitutively active in these models, therefore, PRLR over-activation involved undifferentiated (virgin) mammary glands. At this stage, cell division appeared to be the major component of the actions induced by PRL excess, and both NRL–PRL and Met–PRL models showed that permanent and long-term (>1 year) exposure to PRLR over-signaling was necessary and sufficient to induce carcinomas. In contrast, using our model, abnormally elevated PRL levels first occurred in late gestation, i.e. when the gland is already engaged in its differentiating process, which is induced by the rise of endogenous pituitary, ovarian and placental hormones, including progesterone, estrogens, placental lactogens, and PRL (Hennighausen & Robinson 2001). The role of PRL in controlling lobulo-alveolar development and differentiation of MECs into milk-secreting cells in late gestation and pregnancy is well documented (Brisken et al. 1999). The differentiating actions of PRL have also been recently recognized as an important mechanism by which this hormone could prevent progression and metastasis of differentiated, epithelial-like breast cancer cells, but not dedifferentiated, mesenchymal-like breast cancer cells (Nouhi et al. 2006). It is thus likely that PRL over-expression in WAP–hPRL mice is not as tumorigenic as it is in the Met–PRL and NRL–PRL models, since it over-activates an already differentiated gland. Interestingly, this observation is reminiscent of the well-established fact that first full-term pregnancy protects the mammary gland against neoplastic transformation, which was shown both in animal models treated with carcinogens and in epidemiological studies (for a review, see Medina 2005). In good agreement with previous reports showing that carcinogen treatment of not completely differentiated lobulo-alveoli led to the appearance of benign tumors (adenoma, hyperplasia, fibroadenom) (Russo & Russo 1988), our model also provided evidence that local excess of PRL first acting on a differentiating mammary gland can also induce benign lesions over the long term. Interestingly, local and permanent over-expression of PRL in the prostate also led to benign tumors, with histological alterations very similar to those reported in the mammary glands of WAP–hPRL, i.e. enlarged ducts surrounded by flat epithelium and filled with abundant secretions (Kindblom et al. 2003). The occurrence of benign breast diseases (BBDs) is not rare in humans (Courtillot et al., in press), and some of them seem to predispose to breast cancer (Santen & Mansel 2005). The etiology of BBDs remains poorly established, which clearly impairs our understanding of their transformation potential and the development of preventive therapies. Based on the critical roles of PRL on mammary tissue, this hormone has been considered as a candidate potentially involved in BBDs (Courtillot et al., in press), which is in good agreement with the occurrence of benign lesions in our Tg model. In addition, the fact that the abnormalities observed in WAP–hPRL appeared to be similar to some of those reported in young NRL–PRL animals (Rose-Hellekant et al. 2003) also addressed the possibility that some benign tumors could develop into carcinoma in the mouse, provided exposure to hormonal stimuli is long enough. Since transgene expression persisted in the mammary glands of our animals once it had been turned on at the first pregnancy (Fig. 3, left), it is likely that WAP–hPRL and NRL-PRL are not so different with respect to the duration of PRL exposure. Attempts to definitively answer this question by multiplying pregnancies in order to further increase the time of exposure to very high PRL levels were hampered by the fact that WAP–hPRL females exhibited progressive infertility from the third reproduction cycle. Whatever, the major difference between WAP–hPRL and NRL–PRL or Met–PRL phenotypes –occurrence or not of carcniomas – is probably more directly correlated to the state of mammary gland differentiation at the onset of local hyperprolactinemia, rather than to PRL levels, overall PRL exposure, or any other parameters.
Various genetically modified, non-PRL-related mouse models presenting mammary phenotypes very similar to those observed in our animals have recently been reported, which include over-expression of a constitutively active form of the serine/threonine kinase Akt (Schwertfeger et al. 2001, 2003), a constitutively active form of ß-catenin (Miyoshi et al. 2002), or Rac 3, a member of the Rho family of GTPases (Leung et al. 2003). The main phenotypes of active-Akt Tg include large and distended alveoli, precocious production of milk, highly enriched in lipids, lactation defects causing significant pup death, and delayed involution, in agreement with the well-known anti-apoptotic activity of this kinase (Schwertfeger et al. 2001, Abell et al. 2005). All these phenotypes were also observed in WAP–hPRL mice. After weaning, the mammary glands of our animals did not return to a virgin-like state as expected, indicating that they underwent incomplete involution. Again, this is probably due to the fact that mammary expression of hPRL remained switched on after the first weaning (Fig. 3), leading to interference with the apoptotic process normally occurring at involution under the action of Akt, a known target of PRLR signaling (Schwertfeger et al. 2001). This mechanism, repeated at each reproductive cycle, correlates well with the aggravation of functional and morphological phenotypes as the result of the cumulative damage of involution failure. The main phenotypes of Tg mice over-expressing Rac3 are also similar, and include benign lesions, lactation defects, and delayed involution. Any functional link between Rac3 and PRL remains to be established, since only the homologous Rac1 was recently shown to be a target of PRLR signaling (Jackson et al. 2003, Miller et al. 2005). However, based on very similar phenotypes, Rac3 appears to be a good candidate to be considered. Connections between PRL, extracellular matrix proteins, and cytoskeleton play a critical role in maintaining mammary cell adhesion, morphology and function (Edwards et al. 1998, Zoubiane et al. 2004). ß-catenin is a multifunctional protein connecting cytoskeleton proteins, and it plays a critical role in Wnt signaling in the mammary gland (Muller et al. 2002). Tg mice expressing a constitutively active form of ß-catenin develop various histological abnormalities, including the accumulation of cysts and squamous (keratinized) metaplasia (Bierie et al. 2003). They also display lactation defects. ß-catenin is a target gene of PRL in pancreatic cell cultures (Collares-Buzato et al. 2001), suggesting it may also be induced in the mammary gland. The goal of further studies will be to assess whether some of these candidates are involved in the molecular pathways underlying the mammary phenotypes reported in WAP–hPRL transgenics. This could also help in understanding if and how PRL participates in BBD occurrence.
Unexpectedly, non-mammary phenotypes were also observed in WAP–hPRL females, the main of which was progressive infertility. From the second pregnancy, the interval required between the two pregnancies started to increase, finally leading to total infertility in several animals. In woman, hyperprolactinemia is known to result in infertility, mainly due to an alteration of GnRH pulsatility (Serri et al. 2003). In the mouse, hyperprolactinemia is also correlated with fertility troubles, as highlighted by the difficulty of propagating the Met–PRL lineage (J Kindblom, personal communication). The mechanism involved remains to be demonstrated, however, and may differ from that described in humans. WAP–hPRL females cannot be considered as truly hyperprolactinemic, since significantly elevated levels of circulating hPRL occur only in gestating/ lactating females. Although PRL surges might interfere with reproductive functions, we looked for alternative explanations. Leakage of the WAP-driven promoters in non-mammary tissues has been described in various studies, and we actually detected hPRL protein expression in pancreas, ovary, and pituitary (Pittius et al. 1988a). Therefore, as an alternative to systemic hyperprolactinemia, it is possible that permanent expression of PRL inside the ovary leads to local hyperprolactinemia that could disturb reproductive functions. Finally, we observed that some of the WAP–hPRL females exhibiting fertility troubles became progressively sick and died prematurely (8–10 months). Since this observation did not involve a sufficient number of animals, we cannot claim that it represents a relevant phenotype of this Tg lineage. However, we could speculate that local hyperprolactinemia due to expression of the WAP–hPRL fusion gene in various organs, such as pancreas, kidney, or liver, could lead to physiological and/or metabolic disorders leading to premature organ pathology. Interestingly, infertility and premature mortality were the most obvious phenotypes observed in the founders of the other WAP–hPRL Tg lineage (no. 90), which expressed fivefold more hPRL than the no. 4 lineage used for this study.
In summary, our data show that local over-expression of PRL in differentiating mammary glands induces dramatic functional and morphological defects, but not carcinoma. Although the features directing PRL to induce benign or malignant tumors remain to be completely elucidated, it appears that the differentiation state of the mammary gland at the onset of PRL over-expression is a key issue. The duration of hormone impregnation (continuous vs sporadic), the target-tissue involved (mammary or prostate), and the genetic background are certainly some of the other parameters that should be considered to better understand the pro-tumor effects of PRL on its target tissues.
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Acknowledgements |
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s1 and ß, and Dr Nadine Binart for her expert assistance for the mammary whole mount and for the critical reading of our manuscript. The authors are grateful to Dr C Ormandy for helpful discussions, to G Pivert for his precious help in preparing histological slides and to Dr A Bachelot for statistical analyses. This work was supported in part by Inserm and by the Comité de Paris de la Ligue Nationale contre le Cancer (Grant R05/75-15). C M was supported by student fellowships from the I.R.I.S. (France), Association pour la Recherche contre le Cancer (ARC) and La Fondation pour la Recherche Médicale (FRM). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. |
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Received in final form 13 April 2006
Accepted 18 April 2006
Made available online as an Accepted Preprint 9 May 2006
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