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[Cancer Research 59, 4276-4284, September 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 4276-4284, September 1, 1999]
© 1999 American Association for Cancer Research

Carcinogenesis

Murine Mammary Gland Carcinogenesis Is Critically Dependent on Progesterone Receptor Function1

John P. Lydon, Gouqing Ge, Francis S. Kittrell, Daniel Medina and Bert W. O’Malley2

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To define the functional relevance of progesterone-initiated intracellular signaling in mammary gland tumorigenesis, the progesterone receptor knockout (PRKO) mouse model was used in the context of an established carcinogen-induced mammary tumorigenesis system. In carcinogen-treated, 7,12-dimethylbenz(a)anthracene (DMBA), pituitary-isografted mice, there was a marked reduction in mammary tumor incidence in PRKO mice as compared with isogenic wild types (WT). Mammary tumors arose in 12 (60%) of 20 WT mice compared with 3 (15%) of 20 PRKO mice by 44 weeks after the initial DMBA treatment. In the absence of a pituitary isograft, mammary tumors developed in 4 (20%) of 20 WT mice versus 4 (20%) of 20 PRKO mice by 47 weeks. At the time of carcinogen administration, the proliferative index of the pituitary-stimulated WT gland was at least 4-fold higher than similarly treated PRKO glands, supporting the importance of PR-mediated proliferative pathways in the genesis of this tumor type. Unlike the WT gland, the PRKO gland was unable to exhibit alveologenesis in response to pituitary isograft stimulation; thus, DMBA-initiated mammary tumors observed in the PRKO were assumed to be exclusively of ductal origin. Compared with previous tested strains, by 47 weeks, a higher incidence of DMBA-induced ovarian tumors was observed in this mouse strain: (a) 4 (20%) of 20 WT mice and 9 (45%) of 20 PRKO mice with a pituitary isograft; and (b) 10 (50%) of 20 WT mice and 10 (50%) of 20 PRKO mice without a pituitary isograft. Despite the host-strain’s underlying propensity for DMBA-induced ovarian neoplasms, our studies underscore the specific importance of the PR (as distinct from the estrogen receptor) as a mandatory mediator for those intracellular signaling pathways that are essential for the initiation of the majority of murine mammary tumors induced by DMBA. Apart from providing strong support for progesterone’s role in mammary gland tumorigenesis as well as furthering our fundamental understanding of breast cancer etiology, these studies may have implications for the routine use of progestins.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clinical and epidemiological investigations have demonstrated a correlative relationship between breast cancer risk and the cyclical exposure of the mammary gland to ovarian sex steroid hormones that occurs during the premenopausal period (reviewed in Ref. 1 ). Accordingly, factors known to inhibit or reduce such steroidal exposure (for example, bilateral oophorectomy, late menarche, and early menopause) have been shown to significantly reduce this risk (2, 3, 4, 5) . The increase in breast cancer risk observed with advancing age is now believed to result from ovarian sex steroid-induced proliferation of the mammary epithelial cell that, throughout the reproductive years, facilitates the occurrence and accumulation of random genetic errors that result in neoplasia in later life (6) . With a major correlate of breast cancer risk associated with the episodic exposure of the mammary epithelial cell to ovarian sex steroids, breast cancer prevention strategies based on disabling ovarian steroidogenesis are currently being evaluated (6) .

Historically, E3 has been considered the major ovarian steroid involved in the normal proliferation of the human mammary epithelial cell as well as in the progression of this cell type to a neoplastic state (reviewed in Ref. 7 ). Conversely, based in part on its established role in endometrial differentiation, ovarian P as well as its synthetic derivatives have been assumed to exhibit antiestrogenic and, therefore, antiproliferative effects in the mammary gland and, by extension, have been judged to impart minimal effects to the promotion of mammary tumorigenesis. However, these assumptions, including recent experimental studies suggesting that P elicits insignificant mitogenic responses in the human mammary gland (8) , have not adequately explained why the proliferative index for the epithelial component of the normal mammary gland is highest during the P-dominant luteal phase of the human menstrual cycle (9) nor why progestins included in combined oral contraceptives can prevent ovarian and endometrial cancers but not breast cancers (10) . Many of the conflicting reports concerning P’s role in mammary gland biology may be partially based on the inherent experimental difficulties in using the human subject as a model system. Restrictive factors such as limited tissue availability as well as the question of whether the mammary biopsies used in human studies are completely normal [(most mammary biopsies are derived from either benign lesions (i.e., fibroadenomas) or from reduction mammoplasty procedures] coupled with the inevitable variability in the reproductive history of the test subjects have combined to limit the utility of the human model. Thus, the rodent model has been established as the experimental system of choice in studying mammary gland development and function (11) .

In the case of the mouse, we have recently generated a PRKO mouse model in which the PR was ablated through gene-targeting techniques (12 , 13) . The PRKO mouse represents a new expanding subfamily of knockout mouse models that have recently been used to examine various stages of mammary gland development in an in vivo context (reviewed in Ref. 14 ). Apart from representing a critical technological advance in facilitating our understanding of the dynamic interplay between P and E in a number of reproductive systems of the female (12 , 13 , 15 , 16) , more extensive examination of the PRKO mouse has revealed that the PR mediates essential proliferative responses in the murine mammary gland subsequent to E and P treatment (17) . In more recent studies, using the PRKO mouse in combination with established mammary gland transplantation procedures, we have unequivocally shown that the luminal epithelial compartment of the murine mammary gland is primarily responsive to the P-induced proliferative signal (18, 19, 20) ; importantly, the luminal epithelial cell has also been considered the primary site for the initial carcinogenic insult (21) . These findings have provided essential functional support for recent immunohistochemical studies that have localized PR expression predominantly to the luminal epithelial cell (22 , 23) . In addition, we have obtained strong evidence suggesting that the PR may extend its proliferative effects to neighboring mammary epithelial cells that lack PR, through as yet unidentified paracrine factors (18, 19, 20) ; these factors may represent the currently elusive molecular targets for the PR in the mammary gland.

Collectively, the above studies have substantiated and extended previous reports that have implicated a role for P in the onset of proliferative responses in the mammary gland of the virgin mouse (24, 25, 26) as well as in the development of the lobuloalveolar system in the rodent mammary gland during pregnancy (27) .

Although these recent findings represent important advances in our current understanding of P’s role in normal mammary gland development, the proposed involvement of P in mammary gland tumorigenesis has not been definitively established. Nevertheless, a number of reports have suggested a role for progestins in the active progression of certain carcinogen-induced rat and mouse mammary tumors (28, 29, 30, 31, 32, 33) . These observations have underscored the necessity to better understand P’s participation in mammary gland tumor progression not only to be able to develop a more rational basis for the current use of progestins in contraception and postmenopausal hormone replacement therapies but also to enable the design of novel diagnostic approaches and/or therapies for the future treatment and prevention of breast cancer. The present study addresses the question of P’s controversial involvement in mammary gland tumorigenesis by using the PRKO mouse in combination with the previously described chemical carcinogen-induced mouse mammary tumor model (34, 35, 36) to determine whether PR-mediated signaling pathways are essential for carcinogen-induced mammary gland tumorigenesis. Chemical carcinogen-induced mammary tumors arise from both alveolar and ductal cells (34, 35, 36) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice and Carcinogen Treatment.
For these studies, PRKO and WT female mice were originally derived by crossing chimeric male mice (F0 generation), containing the PRKO allele in a 129SvEv background, with C57BL6 mice to produce F1 mice that were 50% 129 SvEv and 50% C57BL6. F1 mice, heterozygous for the PRKO mutation, were backcrossed to C57BL6 mice to generate N2 mice that were approximately 75% C57BL6 and 25% 129SvEv. N2 mice, heterozygous for the PRKO mutation, were intercrossed to generate N3 mice that were either WT, heterozygous, or homozygous for the PRKO mutation; only WT and PRKO homozygotes, with this 129SvEvxC57BL6 mixed genetic background, were tested (12) . Mice were housed under a 12-h light and 12-h dark photocycle in a temperature-controlled environment (22 ± 2°C) and fed rodent chow meal (Purina Mills Inc., St. Louis, MO) and fresh water ad libitum. At 5 weeks of age, equal numbers (n = 24) of PRKO and WT females received, under the right kidney capsule, a pituitary isograft surgically removed from WT male siblings; pituitaries remained in situ for the duration of the experiment (34 , 35) . Three weeks after grafting the pituitary, 20 mice from both of the test groups were given, for three consecutive weeks, a 1-mg weekly dose of the chemical carcinogen DMBA (Sigma Chemical Co., St. Louis, MO) dissolved in 0.2 ml of cotton seed oil by gastric intubation (Fig. 1ACitation , treatment protocol A). As controls for treatment protocol A, coincident with the first DMBA administration, mammary glands derived from the four remaining mice from each test group were examined for the degree of ductal development resulting from implanting a pituitary for 3 weeks. For comparison, 20 PRKO and WT mice, lacking a pituitary implant, were treated with an identical weekly dose of DMBA for 6 weeks; the treatment protocol is summarized in Fig. 1BCitation . At the time of the first DMBA treatment, mammary tissue was isolated from four additional mice from the above WT and PRKO groups to serve as controls for treatment protocol B.



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  		Fig. 1. Schematic summarizing the DMBA treatment schedule for pituitary-isografted (A) and -nonisografted (B) WT and PRKO mice.

 
Mice were euthanized either when palpable tumors reached 1.0–1.5 cm in diameter or when the animal became moribund; tumor number, size, and location were scored for each mouse. Before cervical dislocation, animals were anesthetized (5 µl of anesthetic per gram of body weight) with the following triple anesthetic combination: ketamine (37.5 mg/ml), xylazine (1.9 mg/ml), and acepromazine (0.37 mg/ml). All of the animal surgical procedures and experimentation were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and were in accordance with the procedures outlined in the "Guide for Care and Use of Laboratory Animals" (NIH publication 85–23).

Whole-mount, PR, and BrdUrd Immunostaining.
Whole-mount examination of the inguinal mammary gland was performed as reported previously (12) . For immunohistochemical analysis, tissues were fixed overnight in Bouin’s fixative followed by three washes in 70% ethanol before being processed for paraffin embedding. Paraffin-embedded tissues were sectioned at 5 µm before mounting onto ProbeOn Plus microscope slides (Fisher Scientific Inc., Pittsburgh, PA) using procedures described previously (17) . Tumor classification was based on microscopic examination of H&E-stained sections representative of serial sections of a given tumor using previously published criteria (37) .

Before PR immunohistochemistry, sections were deparaffinized in Hemo-De (Fisher Scientific Inc.), rehydrated by graded concentrations of ethanol, and finally rinsed in water. After a 10-min treatment with 3% hydrogen peroxide in methanol (to inactivate endogenous peroxidase activity), sections were immersed in antigen retrieval solution (Vector Laboratories Inc., Burlingame, CA), heated for 12 min in a microwave oven (high power), and cooled to room temperature before being washed in 1x PBS. Sections were subsequently immersed in 10% normal goat serum (Vector Laboratories Inc.) for 30 min. Incubation with anti-PR antibody [a rabbit antihuman PR polyclonal antibody (A0098) that reacts with the DNA-binding domain (amino acids 533–547) of the human PR; DAKO Corporation, Carpinteria, CA; 1:100 dilution] was carried out overnight at room temperature in a humidity chamber, followed by sequential incubation with biotinylated goat antirabbit serum (Vector Laboratories Inc.; 1:200 dilution) for 1 h and streptavidin-linked horseradish peroxidase (PharMingen, San Diego, CA) for 30 min at room temperature. The PR was detected by incubation with 3,3'-diaminobenzidine tetrahydrochloride solution (Zymed Corporation, San Francisco, CA) for a time period (usually 3–5 min) sufficient to yield a dark brown color. Sections were counterstained with hematoxylin, dehydrated, cleared, and coverslipped for microscopic examination.

To measure mammary gland BrdUrd incorporation, animals received an i.p. injection of BrdUrd (30 µg of BrdUrd/g of body weight, Amersham Life Inc., Arlington Heights, IL), 2 h before being killed. After the above fixation, embedding, sectioning, deparaffinization, and blocking steps, BrdUrd immunohistochemistry was performed using the Cell Proliferation kit (Amersham Life Science Inc.) and by following the manufacturer’s protocol.

For each tissue section, cell counting consisted of scoring the number of BrdUrd-staining cells in a random field of 1000 cells. The average number of BrdUrd-staining cells in a given tissue section was obtained by taking the average obtained from counting three separate fields of 1000 cells per section. Final counts were expressed as the percentage of epithelial cells immunopositive for BrdUrd (BrdUrd-LI). Representative sections from each inguinal gland were used in these studies, and only intensely stained nuclei were scored positive.

RNA Isolation and Northern Analysis.
Immediately after the mice were killed, the inguinal gland was removed from either WT or PRKO mice and snap-frozen before isolating total RNA using the TRIzol extraction method (Life Technologies, Inc., Grand Island, N. Y.). Total RNA (20 µg) was electrophoresed through a 2.2-M formaldehyde denaturing gel containing 1.2% agarose and was subsequently transferred onto a Zetaprobe GT membrane (BioRad Laboratories, Hercules, CA). Subsequent hybridization and washing conditions were done according to the procedures of Church and Gilbert (38) . Filters were sequentially hybridized with ({alpha}-32P)dCTP-radiolabeled random primed murine {alpha}-lactalbumin (39) and ß-casein (40) cDNA probes. To control for unequal loading and transfer of RNA, filters were hybridized with a cDNA probe for murine cyclophilin (41) . Transcript induction was quantitated by exposing each filter to a phosphor screen (Molecular Dynamics Inc., Sunnyvale, CA), scanning the resultant phosphor image using a Storm 860 scanner (Molecular Dynamics, Inc.) before quantitating hybridizing bands using ImageQuant 1.1 software; for each RNA sample, hybridizing signals corresponding to each of the above transcripts were normalized to that of cyclophilin.

Statistical Analysis.
The tumor incidence data were analyzed by Fisher’s exact test.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Unlike rats (42) , mice require a pituitary isograft at the time of chemical carcinogen exposure to induce a high incidence of mammary tumors with a short latency period (34 , 35) . Free of hypothalamic control, the pituitary implant constitutively secretes prolactin (43) . In addition to its direct mammotrophic effects, prolactin exhibits strong luteotrophic effects that induce ovarian corpora lutea to synthesize and secrete P, which results in serum titers for both P and E that approximate levels observed in the pregnant mouse (44) . Collectively, this hormonal environment induces epithelial proliferation and alveolar differentiation in the mammary gland resulting in a ductal architecture that resembles the morphological phenotype attained in the mid- to-late-pregnant mouse (34 , 35) .

Whole-mount analysis was performed to evaluate the relative responses of the WT and PRKO mammary gland to pituitary isograft stimulation at the time of the first DMBA treatment (see "Materials and Methods" section; Fig. 1ACitation , treatment protocol A). In the absence of a pituitary isograft, comparative morphological analysis of the inguinal mammary glands isolated from 8-week-old WT and age-matched PRKO mice did not reveal discernible differences in gross ductal structure (Fig. 2, A and BCitation , respectively). However, after 3 consecutive weeks of pituitary isograft stimulation, the inguinal gland derived from a WT virgin mouse displayed extensive ductal arborization and lobuloalveolar development (Fig. 2C)Citation . In contrast, the PRKO gland did not exhibit the above classical morphological phenotype associated with pituitary isograft stimulation (Fig. 2D)Citation but responded with an overall ductal morphology similar to the unstimulated gland (Fig. 2B)Citation . However, closer examination revealed a marked enlargement in ductal diameter in the pituitary-stimulated PRKO gland as compared with unstimulated (compare ductal structures in Fig. 2, B and DCitation , arrows).



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  		Fig. 2. Whole-mount analysis of inguinal mammary glands isolated from pituitary-isografted WT and PRKO mice. In the absence of a pituitary isograft, WT and PRKO mice exhibit a similar ductal organization (A and B, respectively). Three weeks of pituitary-isograft stimulation results in a striking difference in branching morphogenesis and alveolar development between the WT and PRKO glands (C and D, respectively). D (arrow), marked enlargement of ductal structures in the pituitary-stimulated PRKO gland (as compared with unstimulated (B, arrow). Scale bar, 5 mm.

 
In addition to whole-mount studies, PR immunohistochemical analyses were performed to evaluate PR expression as well as to determine any changes in ductal histoarchitecture that may have arisen as a result of pituitary isograft stimulation. Fig. 3ACitation shows the PR expression pattern in the unstimulated gland of the WT virgin. The variegated expression of the PR was exclusive to the luminal epithelial cell layer as reported previously (22 , 23) ; PR expression was not detected in the myoepithelial or stromal compartments. As expected, PR expression was not detected in similarly treated glands derived from age-matched PRKO mice (Fig. 3B)Citation . However, despite the absence of PR function, the PRKO gland exhibited ductal structures equivalent to the WT virgin (Fig. 3, A and B)Citation . Fig. 3CCitation shows the response of the gland derived from the WT virgin after 3 weeks of pituitary isograft stimulation. Note the secretory acinar structures as well as a significant reduction in the percentage of epithelial cells expressing the PR as compared with the unstimulated gland (Fig. 3Citation , compare A and C). Because P has been shown to negatively regulate levels in the mammary gland during pregnancy (45) , the observed reduction in PR expression is presumably due to elevated levels of serum P in the pituitary-isografted WT mouse (43) . In contrast to WT and consistent with our whole-mount experiments described in Fig. 2DCitation , histological analysis of glands from pituitary-isografted PRKO mice did not reveal a lobuloalveolar ductal structure (Fig. 3D)Citation . Although PR expression was not detected in the pituitary-stimulated PRKO gland, unusually large ducts containing copious amounts of proteinaceous/lipid secretory material were consistently observed (Fig. 3DCitation , arrow). Northern analysis revealed that at least part of this secretory material consisted of the milk proteins: ß-casein (its gene is a target for prolactin signaling) and {alpha}-lactalbumin (Fig. 4)Citation ; however, compared with WT, the induction of both transcripts was reduced by 5- to 7-fold (Fig. 4Citation , compare Lanes 2 and 4). Because of the difference in mammary epithelial cell density between the pituitary-stimulated WT and PRKO glands (compare C and D in Figs. 2Citation and 3Citation , respectively), it could be argued that the percentage of total RNA derived from the mammary epithelial cell is greater in Lane 2 (pituitary-stimulated WT) than in Lane 4 (pituitary-stimulated PRKO), therefore, contributing to the apparent reduction in transcript levels for ß-casein and {alpha}-lactalbumin in the pituitary-stimulated PRKO gland; it is possible that the amount of message for both gene products per mammary epithelial cell is equivalent in both the pituitary-stimulated WT and the pituitary-stimulated PRKO gland. Regardless of whether the reduction in milk protein synthesis is real or apparent, this result demonstrates that, despite the absence of mammary gland alveologenesis in the pituitary-isografted PRKO mouse, signaling pathways associated with mammary gland differentiation, such as those initiated by pituitary-derived prolactin, have remained intact.



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  		Fig. 3. PR immunohistochemistry of the inguinal mammary gland isolated from pituitary-isografted WT and PRKO mice. A and B, the histoarchitecture in a representative section of a typical epithelial duct in the WT virgin and age-matched PRKO, respectively. The PR expression is limited to the luminal epithelial cell layer of the WT duct (A); as expected, the PRKO duct does not exhibit PR immunoreactivity (B). C, the typical morphological changes that occur as a result of 3 weeks of pituitary-isograft stimulation in the WT; arrow, development of lobuloalveolar structures as well as a marked reduction in the percentage of epithelial cells expressing the PR as compared with the unstimulated gland (compare C and A). Unlike WT, the PRKO gland does not develop a lobuloalveolar system in response to a pituitary isograft; however, the PRKO ducts do respond with an enlargement in ductal diameter. (D); arrow, secretory material in the ductal lumen. Scale bar, 125 µm.

 


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  		Fig. 4. ß-casein and {alpha}-lactalbumin mRNA induction in response to pituitary-isograft stimulation. Lanes 1 and 3, unstimulated WT and PRKO glands, respectively; Lanes 2 and 4, pituitary-stimulated WT and PRKO glands, respectively. The above signal intensities for ß-casein, {alpha}-lactalbumin, and cyclophilin were achieved by 2, 4, and 3 h of autoradiography, respectively. Each lane of the above Northern result, an individual mouse; this result was typical of four other Northern blots that were performed in which the RNA samples were derived from a different set of mice.

 
BrdUrd immunohistochemistry was performed on WT and PRKO control glands to determine the location and number of mammary gland cells in S phase and undergoing active cell division in response to 3 weeks of pituitary stimulation, a time point at which the carcinogen was administered. A low percentage (~2%) of epithelial cells immunoreactive for BrdUrd (the BrdUrd-LI) was observed in the untreated WT and PRKO 8-week-old gland (controls for treatment protocol B; Figs. 5, A and BCitation , and 6Citation ). However, after 3 weeks of pituitary stimulation, the 8-week-old WT gland (control for treatment protocol A) exhibited approximately a 6-fold increase in the BrdUrd-LI as compared with untreated WT (Figs. 5CCitation and 6Citation ). In the pituitary-stimulated WT gland, BrdUrd immunoreactivity was localized primarily to epithelial cells comprising the alveolar buds (Fig. 5C)Citation . In the case of the pituitary-stimulated PRKO gland (control for treatment protocol A), a nonsignificant increase in the BrdUrd-LI was observed in the large ducts as compared with PRKO untreated glands (Figs. 5DCitation and 6Citation ). The pituitary-stimulated WT gland exhibited approximately a 4-fold increase in the BrdUrd-LI as compared with the similarly treated PRKO (Figs. 5, C and DCitation , and 6Citation ). Using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay, we detected a low level of apoptosis in the above treatment groups with nonsignificant differences in apoptotic index between groups (data not shown).



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  		Fig. 5. Mammary gland BrdUrd incorporation in response to pituitary-isograft stimulation. A and B, the near absence of BrdUrd immunopositive cells in a representative section of a typical epithelial duct in the WT virgin and age-matched PRKO, respectively. C, the dramatic increase in BrdUrd labeled epithelial cells in alveolar structures (arrow) in the WT gland after 3 weeks of pituitary stimulation. D, the low level of BrdUrd labeling (arrow) in the PRKO duct after 3 weeks of pituitary stimulation. Scale bar, 100 µm.

 


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  		Fig. 6. Relative levels of BrdUrd incorporation in the untreated and pituitary-stimulated WT and PRKO mammary gland. Control, 8-week-old WT and PRKO untreated mice; Pituitary, age-matched pituitary-stimulated WT and PRKO are assigned. The counts are expressed as the mean BrdUrd-LI ± SD; n = 4.

 
To avoid possible functional impairments inherent to the PRKO pituitary, only WT pituitaries were used in the pituitary isograft test groups. To rule out the possibility of tissue rejection, all of the pituitary isografts were checked for viability after necropsy. Fig. 7ACitation shows the typical pronounced enlargement and vascularization of a WT pituitary (P) five months after transplanting into a PRKO kidney (K). Histological examination of H&E-stained serial sections of all of the pituitary isografts did not reveal evidence of rejection or necrosis (Fig. 7B)Citation .



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  		Fig. 7. WT pituitaries successfully graft into PRKO kidneys. A, gross morphological examination reveals the typical enlargement with time of the WT pituitary (P) within the renal capsule of the PRKO kidney (K); scale bar, 5 mm. B, H&E-stained section of the pituitary transplant shown in A; in B, necrosis is absent; scale bar, 1 mm.

 
Fig. 8ACitation graphically presents the number of pituitary-bearing WT and PRKO mice that developed mammary tumors in the weeks after the first administration of DMBA (treatment protocol A described in Fig. 1Citation ). The mammary tumor incidence in WT mice was significantly greater than in PRKO mice (P < 0.05). Of 20 WT mice, 12 (60%) developed mammary tumors in 15–44 weeks compared with only 3 (15%) of 20 PRKO mice within the same time period; during treatment protocol A, 6 WT and 3 PRKO mice died of DMBA-induced leukemias. The absence of a pituitary isograft from the DMBA treatment protocol (Fig. 1Citation , treatment protocol B) resulted in only 4 (20%) of 20 WT mice developing mammary tumors within 29–39 weeks (Fig. 8B)Citation ; 3 mice in this group did not develop any tumors within the time period of this study (50 weeks), and 5 mice died of DMBA-induced leukemias. In the case of the PRKO mice, only 4 (20%) of 20 developed mammary tumors within 10–27 weeks; during treatment protocol B, 7 PRKO mice died of DMBA-induced leukemias. Mammary tumors arose randomly in all of the mice, with an average of one tumor per mouse. Histological analyses of WT and PRKO mammary tumors did not reveal significant histomorphological differences nor were there obvious histological differences observed in mammary tumors generated using either of the DMBA treatment protocols, A or B (Fig. 9)Citation ; squamous adenocarcinomas and mammary adenocarcinomas type B were the most common histological type (34) .



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  		Fig. 8. Graphical representation of the number of WT and PRKO mice bearing mammary and/or ovarian tumors after the first administration of DMBA either in the presence (A) or absence (B) of a pituitary isograft.

 


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  		Fig. 9. PR immunohistochemistry of DMBA-induced mammary tumor tissue isolated from pituitary-stimulated WT and PRKO mice. A and B, typical histology of DMBA-induced mammary tumors isolated from pituitary-isografted WT and PRKO mice, respectively; arrows, the border between normal tissue (upper right corner) and the rest of the tumor section; scale bar, 100 µm. C and D are higher magnifications of A and B, respectively; arrow, a PR-expressing cell; scale bar, 100 µm.

 
Unexpectedly, ovarian tumors were observed in 4 (20%) of 20 WT mice and in 9 (45%) of 20 PRKO mice bearing pituitary isografts (Fig. 8A)Citation ; and in the absence of a pituitary isograft, 10 (50%) of 20 WT mice and 10 (50%) of 20 PRKO mice developed ovarian tumors (Fig. 8B)Citation . Ovarian tumors developed unilaterally and consisted of solid cellular masses that measured up to 1–2 cm in diameter (Fig. 10ACitation , arrow). Subsequent microscopic examination of the apparently normal contralateral ovary consistently revealed evidence of neoplastic development (data not shown). In the absence of DMBA, the ovaries of age-matched WT and PRKO mice were histologically normal with recognizable follicles and corpora lutea (Fig. 10B)Citation . In contrast, a general histological examination of DMBA-induced ovarian tumors consistently revealed a conspicuous absence of oocytes, developing follicles, and corpora lutea (Fig. 10C)Citation . For susceptible mouse strains, DMBA has been shown to induce ovarian tumors exclusively of granulosa cell origin (46, 47, 48, 49) . Extensive histological evaluation of the ovarian neoplasms generated in this study revealed a typical granulosa cell morphology (50) with a predominant cellular pattern consistent with a cylindromatous or trabecular arrangement (Fig. 10D)Citation .



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  		Fig. 10. The 129SvEvxC57BL6 mouse strain is susceptible to DMBA-induced ovarian tumorigenesis. A, the gross morphology of a typical DMBA-induced ovarian tumor in a WT mouse 30 weeks after the first DMBA treatment; scale bar, 7 mm. B, a H&E-stained representative section from an ovary of an age-matched WT mouse that did not receive DMBA; F, developing follicles; CL, corpora lutea; scale bar, 500 µm. C, a H&E-stained section of a typical DMBA-induced ovarian tumor isolated from a WT mouse; normal ovarian structures (denoted in panel B) have been replaced by neoplastic tissue. Large subcapsular hemorrhages and necroses were frequently gross features in the larger ovarian tumors (data not shown); scale bar, 500 µm. D is a higher magnification of the ovarian neoplasm depicted in C. Tumor cells display no tendency to organize into defined follicular forms but instead proliferate as a uniform mass. Granulosa cells were the predominant cell type with luteinization being a variable feature; scale bar, 100 µm.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Defining P’s controversial role in mammary tumorigenesis has proven to be one of the more challenging research areas in mammary gland biology. Carcinogen-induced rat mammary tumor models showed that although the administration of E and P at the time of carcinogen exposure potentiated the tumorigenic effect of the carcinogen, early pregnancy (high endogenous levels of E and P) or the administration of E and P before carcinogen treatment was found to effectively reduce the susceptibility of the mammary gland to chemical carcinogenesis (51, 52, 53, 54) . Furthermore, the above temporal complexity associated with P’s involvement in mammary tumorigenesis has been further exacerbated by the close temporal and spatial overlap in the functional activities of the ER and PR that has precluded a direct evaluation of P’s role in this process without also considering the complication of E’s influence. The controversy surrounding the functional relevance of progestin activity in the genesis and progression of mammary gland neoplastic development has been best exemplified by the numerous conflicting reports concerning the impact of synthetic progestins (i.e., medroxyprogesterone acetate) on mammary tumorigenesis in rodents (33 , 55 , 56) . More importantly, because of their potential influence on breast cancer risk, a considerable amount of controversy also has been linked to the use of progestins in the human (57) .

Because of the uncertainties regarding the involvement of P during mammary gland tumorigenesis coupled with the widespread clinical use of progestins, the PRKO mouse model was used to directly evaluate whether the PR, distinct from ER effects, has an important role to play in mammary tumorigenesis in vivo.

Using the carcinogen-induced murine mammary tumor model, we and others have previously provided insights into the etiopathogenesis of mammary tumor formation (34 , 35 , 58, 59, 60) . Applying this experimental approach to the PRKO mouse model revealed that the removal of PR function resulted in a significant reduction in susceptibility to DMBA-induced mammary tumorigenesis, thereby underscoring the critical importance of the PR in the genesis of this tumor type. Because alveolar development was not observed in the pituitary-stimulated PRKO mammary gland, we assume that the few mammary tumors that arose in the pituitary-stimulated PRKO gland were exclusively of ductal origin, this assumption is supported by recent studies with explant cultures of WT and PRKO glands in which DMBA-initiated hyperplastic alveolar nodules were observed in the WT but not in the PRKO gland.4

These observations demonstrate that in the absence of PR function, prolactin alone is not sufficient to facilitate the high incidence of DMBA-induced mammary tumors observed in the WT mouse. Prolactin is known to exhibit both mitogenic and differentiative effects in the mammary gland (61) , apart from its established role in normal mammary gland development, prolactin has been shown to be essential for mammary tumorigenesis in a number of rodent model systems (reviewed in Ref. 62 ). Although not fully understood, it is now believed that the response of the mammary epithelial cell to prolactin as either a mitogen, a differentiative agent, or both may depend on the hormonal environment in which the cell finds itself, and that, at the molecular level, commitment to either of the above pathways may be influenced by the type of transmembrane prolactin receptor used and/or by the particular selection of a specific prolactin-responsive kinase/phosphatase intracellular signaling cascade (reviewed in Ref. 61 ).

In light of the above, our data raises a key question concerning whether cross-talk exists within the mammary epithelial cell between P and prolactin-initiated signaling pathways that may be necessary for both the initiation and promotion of DMBA-induced mammary tumorigenesis. One interpretation that could be drawn from our observations is that P may activate key intracellular mediators of the prolactin-mitogenic response; thus, attenuation or inhibition of the prolactin-mitogenic pathway would occur in the absence of PR function; however, the prolactin differentiative pathway, if operative at the time, would remain intact, (as indicated by Northern analysis; Fig. 4Citation ). Under these conditions, a mammary epithelial cell would exhibit a low proliferative index at the time of DMBA administration and, therefore, would represent a poor candidate for neoplastic transformation (as shown in Figs. 5Citation and 6Citation ). Although a functional interaction between P and prolactin-signaling pathways has yet to be elucidated for the in vivo situation, the convergence of steroid hormone and growth factor/cytokine-signaling pathways at the level of the STATs (Signal Transducers and Activators of Transcription) may be more prevalent than previously suspected (63 , 64) .

Alternatively, or in addition to the above interpretation, the significant reduction in DMBA-induced mammary tumorigenesis observed in the PRKO may result from the absence of progenitor cells for alveologenesis that are hypothesized to exist in the WT virgin mammary epithelium but not in the PRKO (22 , 65 , 66) ; the progenitor cells for alveologenesis are thought to be the PR-expressing epithelial cells in the WT adult virgin gland that are fated to develop into alveolar cell lineages during pregnancy. Because the majority of WT mammary tumors are of alveolar cell origin, the absence of progenitors for this cell type in the PRKO gland would represent a significant reduction in the number of target cells for neoplastic transformation by DMBA.

Clearly, additional molecular and cellular approaches will be required to delineate the selective contributions of P and prolactin to DMBA-induced murine mammary tumorigenesis; however, our results unequivocally establish the PR as an essential factor in the etiopathogenesis of this mammary tumor type.

Although the WT version of this mouse strain exhibited full ductal proliferation and alveolar development in response to ectopic pituitary stimulation, DMBA-induced mammary tumorigenesis was delayed as compared with other strains (34 , 35 , 60) . In addition, and unexpectedly, this mouse strain displays an inherently high susceptibility to DMBA-induced ovarian tumorigenesis in the absence of the pituitary isograft. This surprising observation draws parallels with recent studies in which the administration of the carcinogen MNU caused leukemias in intact BALB/c mice but not in those mice that received a pituitary isograft (67) .

Because DMBA is a lipophilic polycyclic hydrocarbon requiring activation to its proximate carcinogenic form (68 , 69) , we speculate that the ovary in our 129SvEv/C57BL6 strain may contain higher levels of the requisite enzymatic activities for the conversion of DMBA to its carcinogenic form. Therefore, in the presence of a pituitary isograft and DMBA, the mammary tissue would undergo extensive proliferation and differentiation in the presence of the carcinogen resulting in tumors before ovarian tumors could be detected. Conversely, in the absence of a pituitary isograft, the mammary gland would not undergo proliferation, and, consequently, DMBA-initiated neoplasia would be retarded; in this situation, ovarian tumors would be the dominant neoplasm. An equally plausible hypothesis, though not necessarily mutually exclusive of the above, is that specific combinations of strain-specific tumor susceptibility/suppressor (tumor-modifier) genes in the 129SvEvxC57BL6 mixed genetic background may account for the significant predisposition to DMBA-induced ovarian tumors (70 , 71) . In an attempt to avoid the complication of DMBA-induced ovarian tumorigenesis, we have used the water-soluble alkylating carcinogen MNU (67) . However, despite the presence of a pituitary isograft, early observations suggest that this mouse strain is also prone to high incidences of MNU-induced leukemia (data not shown), which makes interpretations of mammary tumor incidence difficult with this carcinogen.

Despite this strain’s inherent predisposition to DMBA-induced ovarian tumorigenesis, the present studies strongly support an obligate requirement for PR activity in the carcinogen-induced murine mammary tumor model. Because mice with a 129 SvEvxC57BL6 mixed genetic background are commonly used to expeditiously introduce gene-targeted mutations into the germ line, we believe the data presented here provides important new information to aid in the design of future experiments that necessitate the use of knockout mouse models in the context of the carcinogen-induced mammary tumorigenesis system. For example, exclusively backcrossing the PRKO mutation into the FVB, C57BL6, or BALB/c mouse strains should avoid the complication of DMBA-induced ovarian tumors observed in the 129SvEvxC57BL6 strain. However, it should be noted that despite backcrossing the PRKO mutation from a 129Sv genetic background to one of the above inbred strains, the PRKO mutation will always be associated with a small region of the 129Sv genome (approximately 20 cM), after 10 backcrosses; (72) , the influence of which on phenotype has to be considered.

Finally, apart from confirming and extending those studies that previously implicated a pivotal role for P in mammary tumor development, we predict that the PRKO mouse model will be an essential research tool in the identification and isolation of novel molecular targets for the PR that may serve as useful molecular correlates not only for normal mammary gland proliferation and differentiation but also as new diagnostic, prognostic, and predicative molecular biomarkers for various stages of mammary tumor progression.


   ACKNOWLEDGMENTS
 
We thank Dr. Jeffery M. Rosen (Baylor College of Medicine, Houston, TX) for providing the murine ß-casein cDNA. The assistance of Aileen Ward in digital image capture and formatting is greatly appreciated.


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

1 Supported by NIH Grants CA-77530 (to J. P. L.), CA-11944 (to D. M.), and HD-07857 (to B. W. O.). Back

2 To whom requests for reprints should be addressed, at Department of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030. Phone: (713) 798-6205; Fax: (713) 790-1275; E-mail: berto{at}bcm.tmc.edu Back

3 The abbreviations used are: E, estrogen; P, progesterone; PR, progesterone receptor; PRKO, PR knockout; WT, wild type(s); DMBA, 7, 12-dimethylbenz(a)anthracene; BrdUrd, 5-bromo-2-deoxyuridine; BrdUrd LI, BrdUrd labeling index; MNU, N-methyl-N-nitrosourea. Back

4 Robert T. Chatterton, personal communication. Back

Received 4/14/99. Accepted 7/ 8/99.


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S.-Q. Kuang, L. Liao, S. Wang, D. Medina, B. W. O'Malley, and J. Xu
Mice Lacking the Amplified in Breast Cancer 1/Steroid Receptor Coactivator-3 Are Resistant to Chemical Carcinogen-Induced Mammary Tumorigenesis
Cancer Res., September 1, 2005; 65(17): 7993 - 8002.
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D. Medina, F. S. Kittrell, J. Hill, A. Shepard, G. Thordarson, and P. Brown
Tamoxifen Inhibition of Estrogen Receptor-{alpha}-Negative Mouse Mammary Tumorigenesis
Cancer Res., April 15, 2005; 65(8): 3493 - 3496.
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I. U. Agoulnik, X.-W. Tong, D.-C. Fischer, K. Korner, N. E. Atkinson, D. P. Edwards, D. R. Headon, N. L. Weigel, and D. G. Kieback
A Germline Variation in the Progesterone Receptor Gene Increases Transcriptional Activity and May Modify Ovarian Cancer Risk
J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6340 - 6347.
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R. R. Eason, M. C. Velarde, L. Chatman Jr, S. R. Till, Y. Geng, M. Ferguson, T. M. Badger, and R. C. M. Simmen
Dietary Exposure to Whey Proteins Alters Rat Mammary Gland Proliferation, Apoptosis, and Gene Expression during Postnatal Development
J. Nutr., December 1, 2004; 134(12): 3370 - 3377.
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H. Y. Shi, J. P. Lydon, and M. Zhang
Hormonal Defect in Maspin Heterozygous Mice Reveals a Role of Progesterone in Pubertal Ductal Development
Mol. Endocrinol., September 1, 2004; 18(9): 2196 - 2207.
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 EndocrinologyHome page
N. Gava, C. L. Clarke, K. Byth, R. L. Arnett-Mansfield, and A. deFazio
Expression of Progesterone Receptors A and B in the Mouse Ovary during the Estrous Cycle
Endocrinology, July 1, 2004; 145(7): 3487 - 3494.
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 Mol. Biol. CellHome page
Y. Yi, A. Shepard, F. Kittrell, B. Mulac-Jericevic, D. Medina, and T. K. Said
p19ARF Determines the Balance between Normal Cell Proliferation Rate and Apoptosis during Mammary Gland Development
Mol. Biol. Cell, May 1, 2004; 15(5): 2302 - 2311.
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T. A. Hopp, H. L. Weiss, S. G. Hilsenbeck, Y. Cui, D. C. Allred, K. B. Horwitz, and S. A. W. Fuqua
Breast Cancer Patients with Progesterone Receptor PR-A-Rich Tumors Have Poorer Disease-Free Survival Rates
Clin. Cancer Res., April 15, 2004; 10(8): 2751 - 2760.
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S.-Q. Kuang, L. Liao, H. Zhang, A. V. Lee, B. W. O'Malley, and J. Xu
AIB1/SRC-3 Deficiency Affects Insulin-Like Growth Factor I Signaling Pathway and Suppresses v-Ha-ras-induced Breast Cancer Initiation and Progression in Mice
Cancer Res., March 1, 2004; 64(5): 1875 - 1885.
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C. Lu, C. Speers, Y. Zhang, X. Xu, J. Hill, E. Steinbis, J. Celestino, Q. Shen, H. Kim, S. Hilsenbeck, et al.
Effect of Epidermal Growth Factor Receptor Inhibitor on Development of Estrogen Receptor-Negative Mammary Tumors
J Natl Cancer Inst, December 17, 2003; 95(24): 1825 - 1833.
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I. De Vivo, S. E. Hankinson, G. A. Colditz, and D. J. Hunter
A Functional Polymorphism in the Progesterone Receptor Gene Is Associated with an Increase in Breast Cancer Risk
Cancer Res., September 1, 2003; 63(17): 5236 - 5238.
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B. Mulac-Jericevic, J. P. Lydon, F. J. DeMayo, and O. M. Conneely
Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform
PNAS, August 19, 2003; 100(17): 9744 - 9749.
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X.-L. Zhang, D. Zhang, F. J. Michel, J. L. Blum, F. A. Simmen, and R. C. M. Simmen
Selective Interactions of Kruppel-like Factor 9/Basic Transcription Element-binding Protein with Progesterone Receptor Isoforms A and B Determine Transcriptional Activity of Progesterone-responsive Genes in Endometrial Epithelial Cells
J. Biol. Chem., June 6, 2003; 278(24): 21474 - 21482.
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D. Medina, F. S. Kittrell, A. Shepard, A. Contreras, J. M. Rosen, and J. Lydon
Hormone Dependence in Premalignant Mammary Progression
Cancer Res., March 1, 2003; 63(5): 1067 - 1072.
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 Toxicol SciHome page
J. C. Rowlands, R. Hakkak, M. J. J. Ronis, and T. M. Badger
Altered Mammary Gland Differentiation and Progesterone Receptor Expression in Rats Fed Soy and Whey Proteins
Toxicol. Sci., November 1, 2002; 70(1): 40 - 45.
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P. M. Ismail, J. Li, F. J. DeMayo, B. W. O'Malley, and J. P. Lydon
A Novel LacZ Reporter Mouse Reveals Complex Regulation of the Progesterone Receptor Promoter During Mammary Gland Development
Mol. Endocrinol., November 1, 2002; 16(11): 2475 - 2489.
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 EndocrinologyHome page
S. B. Rulli, A. Kuorelahti, O. Karaer, L. J. Pelliniemi, M. Poutanen, and I. Huhtaniemi
Reproductive Disturbances, Pituitary Lactotrope Adenomas, and Mammary Gland Tumors in Transgenic Female Mice Producing High Levels of Human Chorionic Gonadotropin
Endocrinology, October 1, 2002; 143(10): 4084 - 4095.
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T. M. Ahola, T. Manninen, N. Alkio, and T. Ylikomi
G Protein-Coupled Receptor 30 Is Critical for a Progestin-Induced Growth Inhibition in MCF-7 Breast Cancer Cells
Endocrinology, September 1, 2002; 143(9): 3376 - 3384.
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 CarcinogenesisHome page
S. Vanzulli, A. Efeyan, F. Benavides, L. A. Helguero, G. Peters, J. Shen, C. J. Conti, C. Lanari, and A. Molinolo
p21, p27 and p53 in estrogen and antiprogestin-induced tumor regression of experimental mouse mammary ductal carcinomas
Carcinogenesis, May 1, 2002; 23(5): 749 - 758.
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 Recent Prog Horm ResHome page
O. M. Conneely, B. Mulac-Jericevic, F. DeMayo, J. P. Lydon, and B. W. O'Malley
Reproductive Functions of Progesterone Receptors
Recent Prog. Horm. Res., January 1, 2002; 57(1): 339 - 355.
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 Cancer Res.Home page
K. P. L. Bhat, D. Lantvit, K. Christov, R. G. Mehta, R. C. Moon, and J. M. Pezzuto
Estrogenic and Antiestrogenic Properties of Resveratrol in Mammary Tumor Models
Cancer Res., October 1, 2001; 61(20): 7456 - 7463.
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R. G. Mehta, K. P. L. Bhat, M. E. Hawthorne, L. Kopelovich, R. R. Mehta, K. Christov, G. J. Kelloff, V. E. Steele, and J. M. Pezzuto
Induction of Atypical Ductal Hyperplasia in Mouse Mammary Gland Organ Culture
J Natl Cancer Inst, July 18, 2001; 93(14): 1103 - 1106.
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T. M. GOEPFERT, M. MCCARTHY, F. S. KITTRELL, C. STEPHENS, R. L. ULLRICH, B. R. BRINKLEY, and D. MEDINA
Progesterone facilitates chromosome instability (aneuploidy) in p53 null normal mammary epithelial cells
FASEB J, November 1, 2000; 14(14): 2221 - 2229.
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