Abstract

The vitamin D 3 receptor (VDR) is a ligand-dependent transcription factor implicated in regulation of cell cycle, differentiation and apoptosis of both normal and transformed cells derived from mammary gland. In these studies we examined whether VDR status altered mammary gland morphology or transformation in the well-characterized MMTV-neu transgenic model of breast cancer. We demonstrate that VDR protein is highly expressed in neu-positive epithelial cells of preneoplastic lesions, established tumors and lung metastases from MMTV-neu mice. Furthermore, MMTV-neu mice lacking VDR exhibit abnormal mammary ductal morphology characterized by dilated, distended ducts containing dysplastic epithelial cells. From 12 months of age on, MMTV-neu mice lacking VDR also experience body weight loss, atrophy of the mammary fat pad, estrogen deficiency and reduced survival. The limited survival of MMTV-neu mice lacking VDR precluded an accurate assessment of the impact of complete VDR ablation on tumor development. MMTV-neu mice heterozygous for VDR, however, did not exhibit body weight loss, mammary gland atrophy or compromised survival. Compared with MMTV-neu mice with two copies of the VDR gene, haploinsufficiency of VDR shortened the latency and increased the incidence of mammary tumor formation. Tumor histology and expression/subcellular localization of the neu transgene were not altered by VDR haploinsufficiency despite a significant decrease in tumor VDR expression. Collectively, these studies suggest that VDR gene dosage impacts on age-related changes in ductal morphology and oncogene-induced tumorigenesis of the mammary gland in vivo .

Introduction

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] modulates differentiation, cell cycle and apoptosis of stromal and epithelial cells derived from mammary gland and breast cancers ( 14 ). Cell cycle arrest induced by 1,25(OH) 2 D 3 is associated with inhibition of signaling by growth factors such as IGF and EGF ( 5 , 6 ) and up-regulation of the cyclin-dependent kinase inhibitors p21 ( 7 ) and p27 ( 8 ). Furthermore, 1,25(OH) 2 D 3 inhibits growth of both estrogen-dependent and estrogen-independent tumor cells ( 2 , 912 ). The effects of 1,25(OH) 2 D 3 are mediated by the vitamin D receptor (VDR), a nuclear receptor present in over 80% of human breast cancers ( 13 , 14 ). In previous studies we demonstrated presence of the VDR in normal murine mammary gland and reported accelerated mammary gland development during puberty and pregnancy in VDR knockout (VDR −/− ) mice ( 15 , 16 ). Furthermore, post-lactational involution, a process driven by apoptosis of the mammary epithelial cells, is delayed in VDR −/− mice ( 16 ). These studies have demonstrated that 1,25(OH) 2 D 3 and the VDR participate in negative growth regulation of the mammary gland.

In addition to effects of 1,25(OH) 2 D 3 and the VDR on normal mammary gland development, evidence supports a role for vitamin D 3 signaling in protection against breast cancer development. 1,25(OH) 2 D 3 inhibits carcinogen-induced preneoplastic lesions in vitro ( 17 ) and dietary vitamin D 3 negates the effects of dietary fat and chemical carcinogens on mammary tumorigenesis in vivo ( 18 , 19 ). Furthermore, treatment with synthetic VDR agonists can prevent the development of carcinogen-induced mammary tumors ( 20 , 21 ). Data from two large epidemiological studies indicate that optimal vitamin D 3 nutrition affords protection against breast cancer ( 22 , 23 ) and low serum 1,25(OH) 2 D 3 has been correlated with increased breast cancer risk and metastasis ( 24 , 25 ). Collectively, these studies suggest that vitamin D 3 signaling activates growth inhibitory pathways that may protect mammary cells from transformation.

In the studies presented here we have directly tested the hypothesis that VDR signaling modulates mammary gland morphology and/or tumorigenesis in vivo . MMTV-neu transgenic mice, an established murine model of breast cancer, were crossed with VDR −/− mice to generate MMTV-neu mice on VDR wild-type (neu/VDR +/+ ), heterozygous (neu/VDR +/− ) or knockout (neu/VDR −/− ) backgrounds. MMTV-neu mice selectively express the c- neu protooncogene in the mammary gland and predictably develop estrogen-independent mammary tumors ( 26 ). The c- neu protooncogene encodes a member of the EGF receptor family that is amplified in ∼30% of breast cancer patients and is associated with a poor prognosis ( 27 , 28 ). Since 1,25(OH) 2 D 3 and the VDR inhibit EGF receptor signaling ( 29 ), we hypothesized that disruption of VDR signaling might accelerate tumorigenesis in MMTV-neu mice. In support of this hypothesis, we demonstrate that VDR is highly expressed in mammary tumors of MMTV-neu mice and that loss of one copy of the VDR accelerates neu-driven mammary tumorigenesis. Although body weight loss and decreased survival in neu/VDR −/− mice precluded assessment of tumor development, alveolar hyperplasia and dysplastic ductal epithelial cells were consistently observed in neu/VDR −/− females. These in vivo studies implicate VDR signaling in the modulation of mammary gland function and tumorigenesis and provide a mechanistic basis for numerous epidemiological and clinical observations linking breast cancer risk to vitamin D 3 status.

Materials and methods

Animal maintenance

Wild-type (VDR +/+ ) and VDR knockout (VDR −/− ) C57BL6 mice (originally provided by Dr Marie Demay, Harvard Medical School, Boston, MA) were maintained as described by Zinser et al . ( 15 ). Male VDR −/− mice were crossed with female mice on the FVB background containing the neu protooncogene under control of the MMTV 3′-LTR promoter (MMTV-neu mice, line N202; Jackson Laboratories, Bar Harbor, ME) to produce an F 1 generation carrying the neu transgene and heterozygous (VDR +/− ) for VDR (neu/VDR +/− ). F 1 females were subsequently crossed with F 1 males to produce the F 2 generation consisting of neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− animals. This strategy was modeled after studies in which the role of the estrogen receptor α in MMTV-neu tumorigenesis was evaluated ( 30 ). Of 512 male and female mice on the three VDR backgrounds that carried the neu transgene, 111 (22%) were wild-type (neu/VDR +/+ ), 272 (53%) were heterozygous (neu/VDR +/− ) and 129 (25%) were homozygous null (neu/VDR −/− ) for VDR. Thus, the F 2 generation mice had the expected Mendelian distribution of VDR genotypes.

To prevent disturbances in calcium homeostasis and hormonal imbalances in mice lacking VDR, all mice were fed a diet containing 2% calcium, 1.25% phosphorous and 20% lactose with 2.2 IU/g vitamin D (TD96348; Teklad, Madison, WI). This diet has been shown to support normal mineral and bone homeostasis in VDR −/− mice up to 10 weeks of age, including during pregnancy ( 16 , 3133 ). All animal procedures were approved by the University of Notre Dame animal care and use committee.

Genotyping was achieved by PCR using DNA isolated from ear punches obtained at weaning. Punches were digested in lysis buffer (50 mM Tris base, pH 8, 2 mM NaCl, 10 mM EDTA and 1% SDS) containing 20 mg/ml proteinase K, diluted in ddH 2 O, boiled and allowed to cool. After purification, DNA was added to Ready-To-Go PCR beads (Amersham Pharmacia Biotech, Piscataway, NJ) along with primers designed to detect a 500 bp region of the neomyocin gene, a 752 bp region of exon 3 of the VDR or a 700 bp region of the neu transgene.

Survival and tumor development curves

Female mice (63 neu/VDR +/+ , 144 neu/VDR +/− and 72 neu/VDR −/− ) were examined weekly for mammary tumor development by palpation for up to 18.5 months. Mice that exhibited significant weight loss, morbidity or excessive tumor burden were killed and samples were collected when possible as described below. Data on survival and tumor development were subjected to Kaplan–Meier analysis using GraphPad Prism for Windows (GraphPad Software, San Diego, CA). Kaplan–Meier curves of survival data included mice that died or were killed due to tumor burden as well as mice that died from other causes.

At death, mammary glands, tumors and lungs were removed for histological examination. Both abdominal fat stores and mammary gland fat pads were assessed for signs of atrophy. Tissues and tumors were fixed in 4% neutral buffered paraformaldehyde for histology and immunohistochemistry.

Whole mounting and detection of preneoplastic lesions

For whole mount analysis, entire mammary glands were surgically removed, fixed in Carnoy's fixative, stained overnight in carmine alum, processed and mounted. For assessment of preneoplastic lesions and ductal morphology, 12 MMTV-neu mice of each VDR genotype were killed at 10.5 months of age. Two thoracic and two inguinal glands from each mouse were prepared as whole mounts and ductal architecture and preneoplastic lesions were assessed in all four glands by three investigators in a blind fashion. Preneoplastic lesions (well circumscribed, latent tumors which were not palpable) were quantitated in mammary gland whole mounts examined under an Olympus SZX12 stereoscope at 10× magnification. Data are expressed as the percentage of animals with one or more glands containing a preneoplastic lesion measuring at least 1 mm 3 in diameter.

Histology and immunohistochemistry

Formalin fixed mammary glands and tumors were embedded in paraffin, sectioned at 5 μm and stained with hematoxylin and eosin (H&E). Tissue sections were viewed under an Olympus AX70 microscope and photographed with a Spot RT Slider digital camera (Diagnostic Instruments, Sterling Heights, MI). To detect VDR, formalin fixed paraffin embedded sections were incubated in 2 N HCl at 37°C for 20 min. After rinsing in phopsphate-buffered saline for 5 min, slides were incubated overnight with a rat monoclonal antibody directed against VDR (clone 9A7; Neomarkers, Fremont, CA) at a dilution of 1:60, followed by incubation with anti-rat secondary antibody at a dilution of 1:200. To detect neu protooncoprotein, a mouse monoclonal primary antibody (clone 3B5; NeoMarkers) was prepared at a dilution of 1:70 and was incubated using the M.O.M kit according to the manufacturer's directions (Vector Laboratories, Burlingame, CA). For both VDR and neu, slides were counterstained with Harris modified hematoxylin and photographed.

Western blotting

Thoracic mammary glands or kidney (100 mg) were homogenized in Laemlli buffer containing phosphatase and protease inhibitors ( 34 ), separated by SDS–PAGE, transferred to nitrocellulose, blocked with 5% skimmed milk and immunoblotted with antibodies against VDR (clone C-20; Santa Cruz Biotechnology, Santa Cruz, CA) or c-neu (NCL-L-CBE-356; Vector Laboratories). After incubation with appropriate secondary antibody, blots were developed by enhanced chemiluminescence using products from Pierce.

Serum hormone and calcium assays

17β-Estradiol radioimmunoassay was conducted with a reagent kit from DiaSorin (Stillwater, MN) and serum calcium was determined with a colorimetric assay kit (Sigma, St Louis, MO) according to the manufacturer's directions.

Quantitative real time PCR

Total RNA was isolated from frozen mammary tumors (90–150 mg) with Trizol reagent (Gibco BRL, Rockville, MD) and used for reverse transcription reactions using TaqMan Reverse Transcription Reagents (N808-0234; Applied Biosystems, Foster City, CA). Six independent samples were analyzed for each genotype and reactions were performed in triplicate, generating three separate 1.5 µg cDNA stocks/sample. Each of the triplicate cDNA stocks were then independently analyzed for VDR expression in duplicate by real time PCR using the TaqMan PCR Core Reagent Kit (N808-0228; Applied Biosystems) and specific primer and probe sets. Gene expression was normalized against 18S RNA and reported as normalized gene expression. For each genotype, duplicate values per run were averaged and triplicate runs were then averaged to generate one value for each animal. The final data are expressed as the mean ± SE of six animals for each genotype.

Results

VDR is expressed in MMTV-neu tumors and metastatic foci

These studies were designed to test the hypothesis that VDR signaling modulates mammary gland morphology or tumorigenesis in the MMTV-neu mouse model. A direct effect of VDR on neu driven pathways in the mammary gland would be dependent on the presence of VDR in mammary lesions which develop in MMTV-neu mice. Previous studies have documented that VDR is present in normal murine mammary gland ( 1517 ), but no studies have reported VDR expression in genetically engineered murine models of mammary tumorigenesis. As demonstrated in Figure 1 , preneoplastic lesions, identified in mammary gland whole mounts of MMTV-neu mice ( Figure 1A ), express immunoreactive VDR protein ( Figure 1B ). Furthermore, epithelial tumor cells in established MMTV-neu tumors ( Figure 1C ) also express VDR ( Figure 1D ), and the percentage of cells that express VDR is higher in tumors than in normal mammary ducts ( 15 , 16 ). Expression of VDR mRNA in MMTV-neu tumors was confirmed by quantitative real time PCR, and this analysis also confirmed higher VDR expression in tumors compared with normal mammary gland (not shown). MMTV-neu tumors can metastasize to the lung ( Figure 1E ) and VDR was also detected in pulmonary metastatic foci ( Figure 1F ). The presence of VDR during all stages of neu-driven tumorigenesis suggests that the MMTV-neu mouse represents a suitable model system to test the hypothesis that VDR signaling modulates mammary tumor development or progression.

Fig. 1.

VDR expression in mammary lesions and metastatic foci from MMTV-neu mice. Mammary tumors and lungs from MMTV-neu female mice (line N202, obtained from the Jackson Laboratory) were whole mounted to visualize preneoplastic lesions ( A ) (arrow) or formalin fixed, paraffin embedded and processed for H&E staining ( C and E ) and VDR immunostaining (brown staining) ( B , D and F ). Note VDR expression in early stage lesions (B), established tumors (D) and a metastatic focus (F) within the lung. Scale bars: (A), 200 µm; (B)–(D), 50 µm; (E) and (F), 100 µm. N, normal lung; M, metastatic focus. See online supplementary material for a colour version of this figure.

Fig. 1.

VDR expression in mammary lesions and metastatic foci from MMTV-neu mice. Mammary tumors and lungs from MMTV-neu female mice (line N202, obtained from the Jackson Laboratory) were whole mounted to visualize preneoplastic lesions ( A ) (arrow) or formalin fixed, paraffin embedded and processed for H&E staining ( C and E ) and VDR immunostaining (brown staining) ( B , D and F ). Note VDR expression in early stage lesions (B), established tumors (D) and a metastatic focus (F) within the lung. Scale bars: (A), 200 µm; (B)–(D), 50 µm; (E) and (F), 100 µm. N, normal lung; M, metastatic focus. See online supplementary material for a colour version of this figure.

Effect of VDR status on mammary gland morphology in MMTV-neu mice

Female virgin MMTV-neu mice with two (neu/VDR +/+ ), one (neu/VDR +/− ) or no (neu/VDR −/− ) copies of the VDR were used for comparison of mammary gland morphology and tumor development as a function of VDR status. Since mammary tumors in MMTV-neu mice on the C57BL6 background develop slowly ( 30 , 35 , 36 ), we initially examined overall glandular morphology and preneoplastic lesions in a subset of healthy mice of each genotype at 10.5 months of age. Representative glandular morphology is presented in Figure 2 , with pathological findings summarized in Table I . The majority of glands from neu/VDR +/+ mice exhibited age-appropriate glandular development; thin ducts with secondary and tertiary branching and minimal alveolar budding ( Figure 2A and B ). Evidence of abnormal ductal thickening was present in only 2/12 (17%) neu/VDR +/+ mice. In contrast, 100% of mammary glands from neu/VDR −/− mice exhibited major changes in ductal architecture characterized by extensive lobuloalveolar budding off primary ducts and stubby, dilated ducts ( Figure 2G and H ). In glands from neu/VDR +/− mice ( Figure 2D and E ) an intermediate phenotype was observed, with predominantly normal ductal architecture, but increased incidence of ductal thickening compared with neu/VDR +/+ mice. On histological examination, the thickened and dilated ducts in glands from both neu/VDR −/− and neu/VDR +/− mice showed evidence of secretions ( Figure 2E and H ), whereas ducts of neu/VDR +/+ mice were empty ( Figure 2B ). The abnormal lobuloalveolar units branching off primary ducts in glands from neu/VDR −/− mice were acinar-like structures rather than solid dysplasias ( Figure 2H ).

Table I.

Summary of mammary gland morphology and palpable tumor development in MMTV-neu mice as a function of VDR status


 
neu /VDR +/+
 
neu /VDR +/−
 
neu /VDR −/−
 
Incidence of thick/dilated ducts at 10.5 months (%) 17  67 a  100 a 
Incidence of preneoplastic lesions at 10.5 months (%) 50 75 75 
Duration to first tumor (months) 8.5 
Tumor incidence at 12 months (%) 19.1 22.8 24.1 
Tumor incidence at 18.5 months (%) 53  74 a 59 
Tumors per mouse ( n )  1.3 1.7 1.5 
Time to 50% tumor incidence (months) 18.5  14.5 a N/A 

 
neu /VDR +/+
 
neu /VDR +/−
 
neu /VDR −/−
 
Incidence of thick/dilated ducts at 10.5 months (%) 17  67 a  100 a 
Incidence of preneoplastic lesions at 10.5 months (%) 50 75 75 
Duration to first tumor (months) 8.5 
Tumor incidence at 12 months (%) 19.1 22.8 24.1 
Tumor incidence at 18.5 months (%) 53  74 a 59 
Tumors per mouse ( n )  1.3 1.7 1.5 
Time to 50% tumor incidence (months) 18.5  14.5 a N/A 

Ductal pathology and preneoplastic lesions were evaluated in whole mounts from 12 MMTV-neu mice of each genotype (neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− ) at 10.5 months of age. Animals were considered positive for preneoplastic lesions if at least one gland (of four examined) contained a preneoplastic lesion as defined in Materials and methods. Animals were considered positive for ductal thickening if at least one gland contained two or more areas of generalized thickening and dilation of ducts. Statistical evaluation was by Fisher's exact test. Data on tumor incidence, multiplicity and latency were statistically evaluated by Kaplan–Meier analysis or Fisher's exact test.

a

Significantly ( P < 0.05) different, neu/VDR +/− or neu/VDR −/− versus neu/VDR +/+ .

Fig. 2.

Mammary gland morphology in 10.5-month-old MMTV-neu mice as a function of VDR genotype. Mammary ductal architecture ( A , D and G ), histopathology ( B , E and H ) and pre-neoplastic lesions ( C , F and I ) in 10.5-month-old MMTV-neu littermates wild-type for VDR (neu/VDR +/+ , top panels) or with germline inactivation of one (neu/VDR +/− , middle panels) or two (neu/VDR −/− , bottom panels) alleles of the VDR gene. Inguinal glands from neu/VDR +/+ mice displayed normal ductal architecture, with thin ducts (A and B, arrows) and 50% incidence of pre-neoplastic lesions (C, arrowhead). Glands from neu/VDR +/− mice showed areas of ductal thickening (D and E, arrows) and 75% incidence of pre-neoplastic lesions (F, arrowhead). Glands from neu/VDR −/− mice (G and H) had grossly distended ducts filled with secretions (arrows), stubby side branches, evidence of alveolar budding and 75% incidence of preneoplastic lesions (I, arrowheads). Scale bars: (A), (D) and (G), 1 mm; (B), (E) and (H), 100 µM; (C), (F) and (I), 300 µM. See online supplementary material for a colour version of this figure.

Fig. 2.

Mammary gland morphology in 10.5-month-old MMTV-neu mice as a function of VDR genotype. Mammary ductal architecture ( A , D and G ), histopathology ( B , E and H ) and pre-neoplastic lesions ( C , F and I ) in 10.5-month-old MMTV-neu littermates wild-type for VDR (neu/VDR +/+ , top panels) or with germline inactivation of one (neu/VDR +/− , middle panels) or two (neu/VDR −/− , bottom panels) alleles of the VDR gene. Inguinal glands from neu/VDR +/+ mice displayed normal ductal architecture, with thin ducts (A and B, arrows) and 50% incidence of pre-neoplastic lesions (C, arrowhead). Glands from neu/VDR +/− mice showed areas of ductal thickening (D and E, arrows) and 75% incidence of pre-neoplastic lesions (F, arrowhead). Glands from neu/VDR −/− mice (G and H) had grossly distended ducts filled with secretions (arrows), stubby side branches, evidence of alveolar budding and 75% incidence of preneoplastic lesions (I, arrowheads). Scale bars: (A), (D) and (G), 1 mm; (B), (E) and (H), 100 µM; (C), (F) and (I), 300 µM. See online supplementary material for a colour version of this figure.

While VDR status did not significantly alter the morphology of preneoplastic lesions ( Figure 2C, F and I ), the incidence of preneoplastic lesions was 75% in glands from neu/VDR −/− and neu/VDR +/− mice compared with 50% in neu/VDR +/+ mice ( Table I ).

VDR ablation impairs survival of MMTV-neu mice

These initial data indicated that preneoplastic lesions were present in the majority of MMTV-neu mice by 10.5 months of age and that mammary gland morphology was altered in neu/VDR +/− and neu/VDR −/− females. Based on other studies in which neu-driven mammary tumorigenesis was studied on the C57BL6 background ( 35 , 36 ), we chose to monitor the remaining neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− mice up to 18.5 months of age.

During the long-term follow-up, morbidity and mortality were unexpectedly observed in mice lacking VDR. Kaplan–Meier analysis of survival data ( Figure 3A ) revealed that neu/VDR −/− mice exhibited significantly decreased survival compared with their neu/VDR +/+ and neu/VDR +/− littermates. Mean survival time for neu/VDR −/− mice was 11 months, compared with 15.5 months for neu/VDR +/+ mice and 15 months for neu/VDR +/− mice. Unlike neu/VDR +/+ and neu/VDR +/− mice, who died or were killed due to tumor burden, the majority of premature deaths in neu/VDR −/− mice were not attributable to mammary tumor development. Morbidity in neu/VDR −/− mice was associated with significantly lower body weight compared with either neu/VDR +/+ or neu/VDR +/− mice ( Figure 3B ) and abdominal fat stores were severely depleted in MMTV-neu mice with VDR ablation (not shown). Although poor survival in neu/VDR −/− mice was clearly related to VDR deficiency, the high calcium ‘rescue’ diet was sufficient to maintain serum calcium within the normal range ( Figure 3C ), suggesting a cause other than disturbed calcium homeostasis to explain the increased morbidity in the neu/VDR −/− mice.

Fig. 3.

Effect of VDR status on health and survival of MMTV-neu mice. ( A ) Kaplan–Meier analysis of survival in MMTV-neu mice by VDR genotype. Female MMTV-neu littermates wild-type for VDR (neu/VDR +/+ ) or with germline inactivation of one (neu/VDR +/− ) or two (neu/VDR −/− ) alleles of the VDR gene were monitored for up to 18.5 months until they reached criteria for killing or died. Squares, neu/VDR +/+ mice ( n = 63); circles, neu/VDR +/− mice ( n = 144); triangles, neu/VDR −/− mice ( n = 72). Arrows indicate mean survival for each group. * Survival of neu/VDR −/− mice was significantly ( P < 0.0001) shorter than that of either neu/VDR +/+ or neu/VDR +/− littermates. ( B ) Mean body weight of MMTV-neu mice by VDR genotype. Weight of neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− mice was measured between 10 and 12.5 months of age. * Significantly ( P < 0.05, one way analysis of variance) lower body weight was observed in neu/VDR −/− mice compared with either neu/VDR +/+ or neu/VDR +/− littermates. ( C ) Serum calcium in MMTV-neu mice by VDR genotype. Total calcium was measured in serum from neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− mice at the indicated ages. Mean ± SE of 6–8 mice per group, with the exception of 18-month-old neu/VDR −/− , where n = 1.

Fig. 3.

Effect of VDR status on health and survival of MMTV-neu mice. ( A ) Kaplan–Meier analysis of survival in MMTV-neu mice by VDR genotype. Female MMTV-neu littermates wild-type for VDR (neu/VDR +/+ ) or with germline inactivation of one (neu/VDR +/− ) or two (neu/VDR −/− ) alleles of the VDR gene were monitored for up to 18.5 months until they reached criteria for killing or died. Squares, neu/VDR +/+ mice ( n = 63); circles, neu/VDR +/− mice ( n = 144); triangles, neu/VDR −/− mice ( n = 72). Arrows indicate mean survival for each group. * Survival of neu/VDR −/− mice was significantly ( P < 0.0001) shorter than that of either neu/VDR +/+ or neu/VDR +/− littermates. ( B ) Mean body weight of MMTV-neu mice by VDR genotype. Weight of neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− mice was measured between 10 and 12.5 months of age. * Significantly ( P < 0.05, one way analysis of variance) lower body weight was observed in neu/VDR −/− mice compared with either neu/VDR +/+ or neu/VDR +/− littermates. ( C ) Serum calcium in MMTV-neu mice by VDR genotype. Total calcium was measured in serum from neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− mice at the indicated ages. Mean ± SE of 6–8 mice per group, with the exception of 18-month-old neu/VDR −/− , where n = 1.

Analysis of tissues recovered from neu/VDR −/− mice older than 1 year clearly indicated that the adipose compartment of the mammary gland was severely atrophied in comparison with neu/VDR +/+ or neu/VDR +/− glands, which showed a normal morphology, with large plump adipose tissue surrounding the mammary ducts and lymph node ( Figure 4A–C ). Despite atrophy of the mammary fat pad in aging neu/VDR −/− mice, whole mounts and histological analysis showed large distended ducts containing secretions and extensive alveolar hyperplasias evident in the neu/VDR −/− glands ( Figure 4F and I ). Similar abnormal ductal morphology was observed in VDR −/− F 2 littermates that lacked neu transgene expression (compare Figure 4F and J ), indicating that this effect is related to VDR insufficiency. In the majority of glands from aging neu/VDR −/− mice one or more of the distended ducts contained dysplastic mammary epithelial cells ( Figure 4K ). Epithelial cells within these dysplastic areas were consistently positive for neu immunostaining ( Figure 4L , arrowhead), whereas epithelial cells lining the dilated ducts did not express neu ( Figure 4L , arrows). Glands from age-matched neu/VDR +/− mice (which were not atrophied) also exhibited distended ducts, hyperplasia and dysplastic cells ( Figure 4E and H ), although the phenotype was not as severe as in their neu/VDR −/− littermates. In contrast, hyperplasia and distended ducts were minimal in glands from neu/VDR +/+ mice ( Figure 4D and G ). These observations indicate that while mammary glands from neu/VDR +/+ and neu/VDR +/− mice displayed the typical epithelium:stromal content, mammary glands from neu/VDR −/− mice displayed abnormal ductal morphology, hyperplasia and an ever increasing epithelium:stromal content which became more pronounced with age.

Fig. 4.

Pathology of mammary glands in MMTV-neu mice as a function of VDR. (A–C) Post-mortem examination of inguinal mammary gland. The mammary fat pad was photographed in neu/VDR +/+ ( A ), neu/VDR +/− ( B ) and neu/VDR −/− ( C ) mice at 15 months of age. A white line is drawn around the perimeter of the fat pad and the location of the lymph node is indicated. Note atrophy of the mammary fat pad and abnormal skin in neu/VDR −/− mice compared with neu/VDR +/+ and neu/VDR +/− littermates. (D–F) Ductal architecture. Representative micrographs of inguinal whole mounts from neu/VDR +/+ ( D ), neu/VDR +/− ( E ) and neu/VDR −/− ( F ) mice. Note thin primary ducts with evidence of extensive secondary and tertiary branching in neu/ VDR+/+ mice, dilated primary ducts and some evidence of stubby secondary branching in neu/ VDR+/− mice and grossly distended primary ducts and extensive alveolar hyperplasia in neu/ VDR−/− mice. Scale bars: (D)–(F), 500 µM. (G–I) Histomorphology. Representative micrographs of H&E stained sections of mammary glands from neu/VDR +/+ ( G ), neu/VDR +/− ( H ) and neu/VDR −/− ( I ) mice. Note age-appropriate stromal:epithelial ratio in neu/ VDR+/+ mice, hyperplasia and secretions in neu/ VDR+/− mice and altered stromal:epithelial ratio secondary to adipose tissue atrophy and extensive epithelial hyperplasia in neu/ VDR−/− mice. Scale bars: 200 µM. ( J ) Ductal architecture of VDR −/− littermates lacking neu transgene. Representative micrograph of inguinal whole mount from 10-month-old virgin VDR −/− non-transgenic littermate showing dilated ducts filled with secretions similar to neu/ VDR−/− animals (F). Note lack of alveolar budding in the absence of the neu transgene. Magnification as for (D)–(F). (K and L) Intraductal epithelial dysplasia in glands from neu/VDR −/− mice. Representative micrograph of gland from neu/VDR −/− mouse stained with H&E ( K ) or processed for neu immunohistochemistry ( L ). Note that cells within the intraductal dysplastic areas are strongly positive for neu staining (arrowhead), whereas epithelial cells lining the dilated ducts are negative for neu (arrows). Scale bar: 100 µM. See online supplementary material for a colour version of this figure.

Fig. 4.

Pathology of mammary glands in MMTV-neu mice as a function of VDR. (A–C) Post-mortem examination of inguinal mammary gland. The mammary fat pad was photographed in neu/VDR +/+ ( A ), neu/VDR +/− ( B ) and neu/VDR −/− ( C ) mice at 15 months of age. A white line is drawn around the perimeter of the fat pad and the location of the lymph node is indicated. Note atrophy of the mammary fat pad and abnormal skin in neu/VDR −/− mice compared with neu/VDR +/+ and neu/VDR +/− littermates. (D–F) Ductal architecture. Representative micrographs of inguinal whole mounts from neu/VDR +/+ ( D ), neu/VDR +/− ( E ) and neu/VDR −/− ( F ) mice. Note thin primary ducts with evidence of extensive secondary and tertiary branching in neu/ VDR+/+ mice, dilated primary ducts and some evidence of stubby secondary branching in neu/ VDR+/− mice and grossly distended primary ducts and extensive alveolar hyperplasia in neu/ VDR−/− mice. Scale bars: (D)–(F), 500 µM. (G–I) Histomorphology. Representative micrographs of H&E stained sections of mammary glands from neu/VDR +/+ ( G ), neu/VDR +/− ( H ) and neu/VDR −/− ( I ) mice. Note age-appropriate stromal:epithelial ratio in neu/ VDR+/+ mice, hyperplasia and secretions in neu/ VDR+/− mice and altered stromal:epithelial ratio secondary to adipose tissue atrophy and extensive epithelial hyperplasia in neu/ VDR−/− mice. Scale bars: 200 µM. ( J ) Ductal architecture of VDR −/− littermates lacking neu transgene. Representative micrograph of inguinal whole mount from 10-month-old virgin VDR −/− non-transgenic littermate showing dilated ducts filled with secretions similar to neu/ VDR−/− animals (F). Note lack of alveolar budding in the absence of the neu transgene. Magnification as for (D)–(F). (K and L) Intraductal epithelial dysplasia in glands from neu/VDR −/− mice. Representative micrograph of gland from neu/VDR −/− mouse stained with H&E ( K ) or processed for neu immunohistochemistry ( L ). Note that cells within the intraductal dysplastic areas are strongly positive for neu staining (arrowhead), whereas epithelial cells lining the dilated ducts are negative for neu (arrows). Scale bar: 100 µM. See online supplementary material for a colour version of this figure.

Since estrogen stimulates epithelial cell proliferation and induces the MMTV-neu promoter that drives neu expression in this model and VDR ablation has been associated with impaired estrogen production ( 37 ), we measured circulating estrogen as a function of VDR status. Estrogen levels were not affected by VDR status in young mice, but decreased with age and were significantly lower in neu/VDR −/− mice than neu/VDR +/+ mice from 11.5 months on ( Figure 5 ).

Fig. 5.

Serum estrogen in MMTV-neu mice as a function of VDR genotype. 17β-Estradiol was measured by radioimmunoassay in serum from neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− mice at the indicated ages. a, P < 0.05, neu/VDR +/+ versus neu/VDR −/− ; b, P < 0.05, neu/VDR +/+ or neu/VDR +/− versus neu/VDR −/− . No differences in serum estradiol were detected between neu/VDR +/+ mice and neu/VDR +/− mice. Data are means ± SE of 6–8 mice per genotype.

Fig. 5.

Serum estrogen in MMTV-neu mice as a function of VDR genotype. 17β-Estradiol was measured by radioimmunoassay in serum from neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− mice at the indicated ages. a, P < 0.05, neu/VDR +/+ versus neu/VDR −/− ; b, P < 0.05, neu/VDR +/+ or neu/VDR +/− versus neu/VDR −/− . No differences in serum estradiol were detected between neu/VDR +/+ mice and neu/VDR +/− mice. Data are means ± SE of 6–8 mice per genotype.

VDR haploinsufficiency accelerates MMTV-neu-driven mammary tumorigenesis

Within the first year of observation palpable mammary tumors developed in a stochastic manner for all three VDR genotypes of MMTV-neu mice. Time to detection of the first tumor was 8.5 months in neu/VDR +/+ mice, 7 months in neu/VDR +/− mice and 6 months in neu/VDR −/− mice, suggesting a possible inhibitory effect of VDR. By 12 months of age tumor incidence was 19.1% in neu/VDR +/+ mice, 22.8% in neu/VDR +/− mice and 24.1% in neu/VDR −/− mice. As noted above, by 12 months of age the majority of neu/VDR −/− mice died or were killed due to morbidity and the remainder experienced significant weight loss. Despite poor overall health and a shortened lifespan, 59% of neu/VDR −/− animals developed palpable mammary tumors. However, only one of 72 neu/VDR −/− mice completed the study, making it impossible to accurately assess the effect of VDR ablation on long-term tumor development in these animals. Since haploinsufficiency of the VDR did not compromise body weight or survival, it was possible to compare long-term tumor development in neu/VDR +/+ and neu/VDR +/− mice. Kaplan–Meier analysis ( Figure 6 ) indicated that mammary tumor development was significantly enhanced in neu/VDR +/− mice compared with neu/VDR +/+ mice, and this difference became apparent during the second year of observation. The time for 50% of neu/VDR +/− mice to develop tumors was 14.5 months of age compared with 18.5 months for neu/VDR +/+ mice ( P < 0.005). Furthermore, by 18.5 months of age, mammary tumors had developed in 74% of the neu/VDR +/− mice, compared with 53% of the neu/VDR +/+ mice ( P < 0.005). The number of tumors per mouse tended to be higher in neu/VDR +/− mice (1.7) compared with neu/VDR −/− (1.5) and neu/VDR +/+ mice (1.3), however, these differences were not statistically significant. Data on tumor development for MMTV-neu mice of all three VDR genotypes are summarized in Table I .

Fig. 6.

Kinetics of mammary tumor appearance in neu/VDR +/+ and neu/VDR +/− mice. Kaplan–Meier analysis of tumor development in nulliparous female MMTV-neu littermates wild-type for VDR (neu/VDR +/+ ) or with germline inactivation of one (neu/VDR +/− ) allele of the VDR gene. Mice were monitored for mammary tumor development by palpation for up to 18.5 months. Squares, neu/VDR +/+ mice ( n = 63); circles, neu/VDR +/− mice ( n = 144). Arrows indicate mean time to 50% tumor development for each group. * The kinetics of tumor development were significantly different between neu/VDR +/− mice and their neu/VDR +/+ littermates.

Fig. 6.

Kinetics of mammary tumor appearance in neu/VDR +/+ and neu/VDR +/− mice. Kaplan–Meier analysis of tumor development in nulliparous female MMTV-neu littermates wild-type for VDR (neu/VDR +/+ ) or with germline inactivation of one (neu/VDR +/− ) allele of the VDR gene. Mice were monitored for mammary tumor development by palpation for up to 18.5 months. Squares, neu/VDR +/+ mice ( n = 63); circles, neu/VDR +/− mice ( n = 144). Arrows indicate mean time to 50% tumor development for each group. * The kinetics of tumor development were significantly different between neu/VDR +/− mice and their neu/VDR +/+ littermates.

We also assessed the percentage of lung metastases to determine if tumors with differing VDR status varied in metastatic potential. However, in these MMTV-neu animals on the C57BL6 background the incidence of lung metastases was only ∼5% and there was no effect of VDR status on the percentage of tumor-bearing animals that developed lung metastasis.

Effect of VDR status on tumor histopathology, VDR and neu expression

Representative tumors from neu/VDR +/+ and neu/VDR +/− mice at 16.5 months of age were assessed histologically ( Figure 7 ). Tumors from six neu/VDR −/− mice that survived to 16.5 months of age were available for comparison and a representative tumor is presented in Figure 7 . Regardless of VDR status, all tumors displayed the typical adenocarcinoma morphology characteristic of MMTV-neu-induced tumors ( 38 ) and were well vascularized. There were no obvious morphological differences in tumor cellularity or epithelial:stromal ratio as a function of VDR ( Figure 7A, C and E ). Expression of c-neu was detected on the plasma membrane of the epithelial cells in all tumors ( Figure 7B, D and F ) and relative c-neu expression as detected by western blotting of homogenates from these tumors was similar in all three genotypes ( Figure 7G ).

Fig. 7.

Histopathology and neu expression in primary tumors from MMTV-neu mice as a function of VDR status. (Left) Representative H&E stained sections of primary mammary tumors from neu/VDR +/+ ( A ), neu/VDR +/− ( C ) and neu/VDR −/− ( E ) littermates. (Right) Immunohistochemistry for c-neu in mammary tumors from neu/VDR +/+ ( B ), neu/VDR +/− ( D ) and neu/VDR −/− ( F ) mice. No differences in morphology or c-neu staining were detected as a function of VDR status. Scale bar: 50 µM. ( G ) Western blot for c-neu in tumors from neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− mice. Tumor homogenates were separated by SDS–PAGE, transferred to nitrocellulose and incubated with antibody against c-neu. The blot shows two independent tumor homogenates from both neu/VDR +/+ and neu/VDR +/− mice and one homogenate from a neu/VDR −/− mouse. Kidney was used as a negative control for transgene expression. See online supplementary material for a colour version of this figure.

Fig. 7.

Histopathology and neu expression in primary tumors from MMTV-neu mice as a function of VDR status. (Left) Representative H&E stained sections of primary mammary tumors from neu/VDR +/+ ( A ), neu/VDR +/− ( C ) and neu/VDR −/− ( E ) littermates. (Right) Immunohistochemistry for c-neu in mammary tumors from neu/VDR +/+ ( B ), neu/VDR +/− ( D ) and neu/VDR −/− ( F ) mice. No differences in morphology or c-neu staining were detected as a function of VDR status. Scale bar: 50 µM. ( G ) Western blot for c-neu in tumors from neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− mice. Tumor homogenates were separated by SDS–PAGE, transferred to nitrocellulose and incubated with antibody against c-neu. The blot shows two independent tumor homogenates from both neu/VDR +/+ and neu/VDR +/− mice and one homogenate from a neu/VDR −/− mouse. Kidney was used as a negative control for transgene expression. See online supplementary material for a colour version of this figure.

Finally, we assessed whether the enhanced tumor development in neu/VDR +/− mice correlated with reduced tumor VDR expression ( Figure 8 ). Mean steady-state VDR mRNA, measured by quantitative real time PCR, was lower in tumors from neu/VDR +/− mice as compared with neu/VDR +/+ mice ( Figure 8A ), although this difference did not reach statistical significance ( P = 0.17). Western blotting indicated that VDR protein expression was lower in mammary tumors, as well as kidney (a well-characterized vitamin D 3 target tissue), derived from neu/VDR +/− mice as compared with neu/VDR +/+ mice ( Figure 8B ). Immunostaining indicated diffuse VDR expression throughout tumors from both neu/VDR +/+ and neu/VDR +/− mice, suggesting that complete loss of the remaining wild-type VDR allele in neu/VDR +/− mice was unlikely. As expected, no VDR mRNA or protein was detected in homogenates of mammary tumors or kidney from neu/VDR −/− mice.

Fig. 8.

VDR expression in tumors from neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− littermates. Tumors were evaluated for VDR expression by quantitative real time PCR ( A ), western blotting ( B ) and immunohistochemistry ( C ). Data in (A) are expressed as means ± SE of 6 values per genotype. ND, not detectable. In (B) tumor and kidney homogenates were separated by SDS–PAGE and immunoblotted with a polyclonal antibody directed against VDR (clone C-20). The blot shows two independent tumor homogenates from both neu/VDR +/+ and neu/VDR +/− mice and one homogenate from a neu/VDR −/− mouse. Kidney was used as a positive control for VDR expression. Similar results were obtained in replicate experiments. In (C) formalin fixed sections derived from neu/VDR +/+ (a), neu/VDR +/− (b) and neu/VDR −/− (c) littermates were immunostained with a monoclonal antibody directed against VDR (clone 9A7). Photomicrographs from representative sections are shown. VDR expression was reduced in tumors and tissues from neu/VDR +/− mice as compared with neu/VDR +/+ mice and no VDR mRNA or protein was detected in tumors or tissues derived from neu/VDR −/− mice. Scale bar: 50 µM. See online supplementary material for a colour version of this figure.

Fig. 8.

VDR expression in tumors from neu/VDR +/+ , neu/VDR +/− and neu/VDR −/− littermates. Tumors were evaluated for VDR expression by quantitative real time PCR ( A ), western blotting ( B ) and immunohistochemistry ( C ). Data in (A) are expressed as means ± SE of 6 values per genotype. ND, not detectable. In (B) tumor and kidney homogenates were separated by SDS–PAGE and immunoblotted with a polyclonal antibody directed against VDR (clone C-20). The blot shows two independent tumor homogenates from both neu/VDR +/+ and neu/VDR +/− mice and one homogenate from a neu/VDR −/− mouse. Kidney was used as a positive control for VDR expression. Similar results were obtained in replicate experiments. In (C) formalin fixed sections derived from neu/VDR +/+ (a), neu/VDR +/− (b) and neu/VDR −/− (c) littermates were immunostained with a monoclonal antibody directed against VDR (clone 9A7). Photomicrographs from representative sections are shown. VDR expression was reduced in tumors and tissues from neu/VDR +/− mice as compared with neu/VDR +/+ mice and no VDR mRNA or protein was detected in tumors or tissues derived from neu/VDR −/− mice. Scale bar: 50 µM. See online supplementary material for a colour version of this figure.

Discussion

In this study we have examined whether disrupted VDR signaling alters murine mammary gland morphology or transformation in response to the neu protooncogene. MMTV-neu mice overexpress c-neu in mammary gland ( 26 ) and mimic human breast cancers with amplification of erbB2, a member of the EGF receptor family. MMTV-neu mice were crossed with VDR −/− mice to generate mice with enhanced neu expression and altered VDR status. Two key conclusions can be drawn from these studies in which mammary gland pathology and tumor development were monitored for up to 18.5 months. The first major conclusion is that complete loss of VDR signaling results in pathological changes, including dysplasia of the mammary ductal epithelia and atrophy of the associated fat pad, in aging female neu/VDR −/− mice. VDR ablation also significantly reduces body weight and compromises long-term survival despite dietary maintenance of normocalcemia. The second major conclusion is that haploinsufficiency of the VDR accelerates neu-driven tumorigenesis (as supported by the increased incidence and decreased latency of mammary tumors in neu/VDR +/− mice as compared with neu/VDR +/+ mice). Combined with previous findings that VDR ablation enhances chemically induced carcinogenesis in skin ( 39 ) and induces premalignant changes in colonic epithelium ( 40 ), these data implicate the vitamin D 3 signaling pathway as a general suppressor of tumorigenesis.

The observation of ductal thickening, increased lesion development and accelerated onset of neu-driven mammary tumors in mice heterozygous for the VDR provides the first evidence that VDR status affects mammary cancer development in vivo . We show that, in contrast to the estrogen and progesterone receptors (which are not expressed in neu-driven tumors) ( 41 , 42 ), VDR is highly expressed in early and late stage MMTV-neu mammary tumors as well as in pulmonary metastatic foci. Furthermore, VDR protein expression in tumors and tissues from neu/VDR +/− mice is reduced to about half that of neu/VDR +/+ littermates. Assuming that the VDR present in neu-driven tumors is functional, our data suggest that VDR signaling is ∼2-fold lower in neu/VDR +/− mice compared with neu/VDR +/+ mice. Changes in VDR expression of this magnitude correlate with parallel changes in VDR-mediated gene expression and biological effects both in vivo and in vitro ( 4345 ). 1,25(OH) 2 D 3 , the ligand for the VDR, is a negative growth regulator of both normal and transformed mammary epithelial cells ( 4648 ) and prevents the development of carcinogen-induced preneoplastic lesions in mammary organ culture ( 17 ). In addition, VDR agonists exert anti-tumor effects in animal models and these effects are dependent on expression of functional VDRs in tumor cells ( 9 , 34 ). Although our studies do not rule out the possibility that VDR haploinsufficiency might impact on tumorigenesis secondary to decreased VDR in non-mammary tissues, the most likely basis for the enhanced tumorigenesis in neu/VDR +/− mice is reduced signaling through the VDR in the mammary gland.

The best characterized action of VDR is transcriptional regulation, but the vitamin D 3 -regulated target genes in mammary gland relevant to tumorigenesis have yet to be identified. 1,25(OH) 2 D 3 inhibits signaling through the EGF family of receptors, including erbB-2, in MCF-7 breast cancer cells ( 49 ). In the current study, VDR status did not alter expression of the neu transgene, but further studies are necessary to determine if neu tyrosine kinase activity is altered by VDR status. Other potential downstream VDR targets include tumor suppressor genes such as PTEN, p21 and p27 ( 48 , 50 , 51 ). Interestingly, haploinsufficiency of PTEN and p27 increases tumorigenesis in animal models of prostate and breast cancer, respectively ( 36 , 52 , 53 ). Similar to our findings with VDR, Muraoka et al . ( 36 ) demonstrated that p27 +/− mice are more susceptible to MMTV-neu tumorigenesis than their p27 +/+ littermates. Our studies add the VDR to the list of genes that may act in a haploinsufficient manner to impact on tumorigenesis ( 53 ).

The increased incidence of mammary tumors in mice heterozygous for the VDR would predict that complete loss of the VDR would also be associated with accelerated tumorigenesis. Despite more extensive glandular hyperplasia and dysplasia in neu/VDR −/− mice compared with neu/VDR +/+ mice, the final tumor incidence in neu/VDR −/− mice (59%) was only slightly higher than that in neu/VDR +/+ mice (53%). However, tumor incidence data in neu/VDR −/− mice should be interpreted with caution, since the majority of neu/VDR −/− animals experienced poor overall health, weight loss and mortality within the first year of the study, making accurate long-term follow-up of tumor development in these animals complicated. This is particularly important since differences in tumor development between neu/VDR +/− mice and neu/VDR +/+ mice only became apparent in the second year of follow-up. The loss of body weight, atrophy of adipose stores and reduced circulating estrogen in neu/VDR −/− mice older than 12 months could theoretically have hindered the progression of early stage lesions into palpable mammary tumors and thus were confounding factors in this study. Although estrogen is not required for MMTV-neu tumorigenesis in post-pubertal animals ( 30 ), estrogen stimulates estrogen receptor-positive stromal or epithelial cells to secrete growth factors required for the outgrowth of preneoplastic lesions into palpable tumors ( 5456 ). Therefore, the reduced circulating estrogen in older neu/VDR −/− mice could have altered the incidence or timing of tumor outgrowth. In addition, final tumor development in neu/VDR −/− mice was assessed in the presence of body weight loss and mammary fat pad atrophy. The adipose and stromal compartments of the gland facilitate tumorigenesis through release of diffusible growth factors that stimulate epithelial cell receptors, including neu ( 54 , 57 , 58 ), and regulate production of extracellular matrix proteins ( 54 , 58 ). Thus, fat pad atrophy in older neu/VDR −/− mice could have prevented or delayed tumor outgrowth in comparison with neu/VDR +/+ and neu/VDR +/− littermates with abundant adipose tissue stores. A true analysis of the impact of VDR ablation on MMTV-neu tumorigenesis will require development of a mammary-specific VDR knockout animal model.

Another important outcome of this study is the observation that aging neu/VDR −/− females display altered mammary gland morphology, including alveolar outgrowths and dilated ducts filled with secretory material. While alveolar outgrowths in neu/VDR −/− mice correlated with neu expression, epithelial cells within the dilated ducts were uniformly negative for neu transgene expression. Furthermore, age-matched female VDR −/− littermates from the F 2 generation that were negative for transgene expression displayed fat pad atrophy and dilated ducts without alveolar outgrowths. This suggests that the abnormal ductal morphology in neu/VDR −/− females is secondary to VDR insufficiency rather than neu signaling. In support of this suggestion, aging female virgin mice in our VDR −/− colony on the C57BL background develop a similar ductal morphology, which is associated with inappropriate casein expression (Zinser and Welsh, unpublished results), and ongoing studies are directed at characterizing this phenotype more completely.

The mechanisms underlying the systemic wasting and shortened lifespan of neu/VDR −/− animals, reported here for the first time, remain to be elucidated. Neither phenotype was related to hypocalcemia, since the high calcium diet maintained extracellular calcium homeostasis in the neu/VDR −/− mice during the extended period of this study. Weight loss and reduced lifespan were not correlated with neu expression, since survival was also compromised (mean survival 11.6 months) in a cohort of 15 VDR −/− F 2 littermates generated in this study that lacked neu transgene expression. Furthermore, we have observed atrophy of abdominal fat stores, weight loss and reduced survival in our non-transgenic VDR knockout colony maintained on the C57BL background. Poor survival could have been related to alopecia, dermal cyst formation and thickened wrinkled skin, which is characteristic of VDR ablation and can precipitate infections ( 39 , 59 , 60 ). VDR ablation may also directly affect adipose tissue since the VDR is highly induced during adipocyte differentiation and 1,25(OH) 2 D 3 regulates expression of genes such as leptin and uncoupling protein, which participate in energy balance ( 6164 ). Therefore VDR ablation may interfere with turnover of adipose tissue, which could affect both longevity and mammary gland biology. However, since adipose tissue appears normal in young neu/VDR −/− mice, further studies are required to identify factors that might precipitate atrophy of the adipose tissue during aging of neu/VDR −/− mice.

In summary, the studies reported here demonstrate that disruption of VDR signaling induces mammary gland pathology and accelerates breast cancer development in MMTV-neu transgenic mice, a model that mimics her2/neu-positive human breast cancer. Since the ligand for VDR is derived from vitamin D 3 , an important implication of these studies, which is currently being tested in our laboratory, is that optimal vitamin D 3 status will prevent or delay neu-driven mammary tumorigenesis.

The authors are grateful to Emily Tribble for her assistance with tissue processing and histology, to Kevin McEleney for his assistance with genotyping of the colony and to Lindsay Barnett of the Freimann Life Science Center at the University of Notre Dame for care of the animal colonies. This research was supported by grants to J.W. from the National Institutes of Health (CA69700) and the DOD Breast Cancer Research Program (DAMD17-00-1-0644), and a dissertation award to G.M.Z. from the Susan G.Komen Breast Cancer Foundation (DISS 0100302).

References

1.
Lefebvre,M.F., Guillot,C., Crepin,M. and Saez,S. (
1995
) Influence of tumor derived fibroblasts and 1,25-dihydroxyvitamin D3 on growth of breast cancer cell lines.
Breast Cancer Res. Treat.
  ,
33
,
189
–197.
2.
Simboli-Campbell,M., Narvaez,C.J., Tenniswood,M. and Welsh,J. (
1996
) 1,25-Dihydroxyvitamin D3 induces morphological and biochemical markers of apoptosis in MCF-7 breast cancer cells.
J. Steroid Biochem. Mol. Biol.
  ,
58
,
367
–376.
3.
Kanazawa,T., Enami,J. and Kohmoto,K. (
1999
) Effects of 1alpha,25-dihydroxycholecalciferol and cortisol on the growth and differentiation of primary cultures of mouse mammary epithelial cells in collagen gel.
Cell Biol. Int.
  ,
23
,
481
–487.
4.
Narvaez,C.J. and Welsh,J. (
2001
) Role of mitochondria and caspases in vitamin D-mediated apoptosis of MCF-7 breast cancer cells.
J. Biol. Chem.
  ,
276
,
9101
–9107.
5.
Vink-van Wijngaarden,T., Pols,H.A., Buurman,C.J., Birkenhager,J.C. and van Leeuwen,J.P. (
1996
) Inhibition of insulin- and insulin-like growth factor-I-stimulated growth of human breast cancer cells by 1,25-dihydroxyvitamin D3 and the vitamin D3 analogue EB1089.
Eur. J. Cancer
  ,
32A
,
842
–848.
6.
Koga,M., Eisman,J.A. and Sutherland,R.L. (
1988
) Regulation of epidermal growth factor receptor levels by 1,25-dihydroxyvitamin D3 in human breast cancer cells.
Cancer Res.
  ,
48
,
2734
–2739.
7.
Liu,M., Lee,M.H., Cohen,M., Bommakanti,M. and Freedman,L.P. (
1996
) Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937.
Genes Dev.
  ,
10
,
142
–153.
8.
Wu,G., Fan,R.S., Li,W., Ko,T.C. and Brattain,M.G. (
1997
) Modulation of cell cycle control by vitamin D3 and its analogue, EB1089, in human breast cancer cells.
Oncogene
  ,
15
,
1555
–1563.
9.
Van Weelden,K., Flanagan,L., Binderup,L., Tenniswood,M. and Welsh,J. (
1998
) Apoptotic regression of MCF-7 xenografts in nude mice treated with the vitamin D3 analog, EB1089.
Endocrinology
  ,
139
,
2102
–2110.
10.
Love-Schimenti,C.D., Gibson,D.F., Ratnam,A.V. and Bikle,D.D. (
1996
) Antiestrogen potentiation of antiproliferative effects of vitamin D3 analogues in breast cancer cells.
Cancer Res.
  ,
56
,
2789
–2794.
11.
Elstner,E., Linker-Israeli,M., Umiel,T. et al . (
1996
) Combination of a potent 20-epi-vitamin D3 analogue (KH 1060) with 9-cis-retinoic acid irreversibly inhibits clonal growth, decreases bcl-2 expression and induces apoptosis in HL-60 leukemic cells.
Cancer Res.
  ,
56
,
3570
–3576.
12.
Flanagan,L., Van Weelden,K., Ammerman,C., Ethier,S.P. and Welsh,J. (
1999
) SUM-159PT cells: a novel estrogen independent human breast cancer model system.
Breast Cancer Res. Treat.
  ,
58
,
193
–204.
13.
Colston,K.W., Berger,U. and Coombes,R.C. (
1989
) Possible role for vitamin D in controlling breast cancer cell proliferation.
Lancet
  ,
1
,
188
–191.
14.
Berger,U., McClelland,R.A., Wilson,P., Greene,G.L., Haussler,M.R., Pike,J.W., Colston,K., Easton,D. and Coombes,R.C. (
1991
) Immunocytochemical determination of estrogen receptor, progesterone receptor and 1,25-dihydroxyvitamin D3 receptor in breast cancer and relationship to prognosis.
Cancer Res.
  ,
51
,
239
–244.
15.
Zinser,G., Packman,K. and Welsh,J. (
2002
) Vitamin D(3) receptor ablation alters mammary gland morphogenesis.
Development
  ,
129
,
3067
–3076.
16.
Zinser,G.M. and Welsh,J. (
2004
) Accelerated mammary gland development during pregnancy and delayed post-lactational involution in vitamin D3 receptor null mice.
Mol. Endocrinol.
  ,
18
,
2208
–2223.
17.
Mehta,R.G., Moriarty,R.M., Mehta,R.R., Penmasta,R., Lazzaro,G., Constantinou,A. and Guo,L. (
1997
) Prevention of preneoplastic mammary lesion development by a novel vitamin D analogue, 1alpha-hydroxyvitamin D5.
J. Natl Cancer Inst.
  ,
89
,
212
–218.
18.
Newmark,H.L. (
1994
) Vitamin D adequacy: a possible relationship to breast cancer.
Adv. Exp. Med. Biol.
  ,
364
,
109
–114.
19.
Jacobson,E.A., James,K.A., Newmark,H.L. and Carroll,K.K. (
1989
) Effects of dietary fat, calcium and vitamin D on growth and mammary tumorigenesis induced by 7,12-dimethylbenz(a)anthracene in female Sprague-Dawley rats.
Cancer Res.
  ,
49
,
6300
–6303.
20.
Anzano,M.A., Smith,J.M., Uskokovic,M.R. et al . (
1994
) 1Alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24–5531), a new deltanoid (vitamin D analogue) for prevention of breast cancer in the rat.
Cancer Res.
  ,
54
,
1653
–1656.
21.
Mehta,R., Hawthorne,M., Uselding,L., Albinescu,D., Moriarty,R. and Christov,K. (
2000
) Prevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis in rats by 1alpha-hydroxyvitamin D(5).
J. Natl Cancer Inst.
  ,
92
,
1836
–1840.
22.
John,E.M., Schwartz,G.G., Dreon,D.M. and Koo,J. (
1999
) Vitamin D and breast cancer risk: the NHANES I epidemiologic follow-up study, 1971–1975 to 1992. National Health and Nutrition Examination Survey.
Cancer Epidemiol. Biomarkers Prev.
  ,
8
,
399
–406.
23.
Shin,M.H., Holmes,M.D., Hankinson,S.E., Wu,K., Colditz,G.A. and Willett,W.C. (
2002
) Intake of dairy products, calcium and vitamin d and risk of breast cancer.
J. Natl Cancer Inst.
  ,
94
,
1301
–1311.
24.
Mawer,E.B., Walls,J., Howell,A., Davies,M., Ratcliffe,W.A. and Bundred,N.J. (
1997
) Serum 1,25-dihydroxyvitamin D may be related inversely to disease activity in breast cancer patients with bone metastases.
J. Clin. Endocrinol. Metab.
  ,
82
,
118
–122.
25.
Janowsky,E.C., Lester,G.E., Weinberg,C.R., Millikan,R.C., Schildkraut,J.M., Garrett,P.A. and Hulka,B.S. (
1999
) Association between low levels of 1,25-dihydroxyvitamin D and breast cancer risk.
Public Health Nutr.
  ,
2
,
283
–291.
26.
Guy,C.T., Cardiff,R.D. and Muller,W.J. (
1992
) Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.
Mol. Cell. Biol.
  ,
12
,
954
–961.
27.
Hynes,N.E. and Stern,D.F. (
1994
) The biology of erbB-2/neu/HER-2 and its role in cancer.
Biochim. Biophys. Acta
  ,
1198
,
165
–184.
28.
Slamon,D.J., Godolphin,W., Jones,L.A. et al . (
1989
) Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer.
Science
  ,
244
,
707
–712.
29.
Cordero,J.B., Cozzolino,M., Lu,Y., Vidal,M., Slatopolsky,E., Stahl,P.D., Barbieri,M.A. and Dusso,A. (
2002
) 1,25-Dihydroxyvitamin D down-regulates cell membrane growth- and nuclear growth-promoting signals by the epidermal growth factor receptor.
J. Biol. Chem.
  ,
277
,
38965
–38971.
30.
Hewitt,S.C., Bocchinfuso,W.P., Zhai,J., Harrell,C., Koonce,L., Clark,J., Myers,P. and Korach,K.S. (
2002
) Lack of ductal development in the absence of functional estrogen receptor alpha delays mammary tumor formation induced by transgenic expression of ErbB2/neu.
Cancer Res.
  ,
62
,
2798
–2805.
31.
Li,Y.C., Amling,M., Pirro,A.E., Priemel,M., Meuse,J., Baron,R., Delling,G. and Demay,M.B. (
1998
) Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets and osteomalacia, but not alopecia in vitamin D receptor-ablated mice.
Endocrinology
  ,
139
,
4391
–4396.
32.
Johnson,L.E. and DeLuca,H.F. (
2002
) Reproductive defects are corrected in vitamin d-deficient female rats fed a high calcium, phosphorus and lactose diet.
J. Nutr.
  ,
132
,
2270
–2273.
33.
Rummens,K., van Cromphaut,S.J., Carmeliet,G., van Herck,E., van Bree,R., Stockmans,I., Bouillon,R. and Verhaeghe,J. (
2003
) Pregnancy in mice lacking the vitamin D receptor: normal maternal skeletal response, but fetal hypomineralization rescued by maternal calcium supplementation.
Pediatr. Res.
  ,
54
,
466
–473.
34.
Zinser,G.M., McEleney,K. and Welsh,J. (
2003
) Characterization of mammary tumor cell lines from wild type and vitamin D3 receptor knockout mice.
Mol. Cell. Endocrinol.
  ,
200
,
67
–80.
35.
Rowse,G.J., Ritland,S.R. and Gendler,S.J. (
1998
) Genetic modulation of neu proto-oncogene-induced mammary tumorigenesis.
Cancer Res.
  ,
58
,
2675
–2679.
36.
Muraoka,R.S., Lenferink,A.E., Law,B., Hamilton,E., Brantley,D.M., Roebuck,L.R. and Arteaga,C.L. (
2002
) ErbB2/Neu-induced, cyclin D1-dependent transformation is accelerated in p27-haploinsufficient mammary epithelial cells but impaired in p27-null cells.
Mol. Cell. Biol.
  ,
22
,
2204
–2219.
37.
Kinuta,K., Tanaka,H., Moriwake,T., Aya,K., Kato,S. and Seino,Y. (
2000
) Vitamin D is an important factor in estrogen biosynthesis of both female and male gonads.
Endocrinology
  ,
141
,
1317
–1324.
38.
Rosner,A., Miyoshi,K., Landesman-Bollag,E. et al . (
2002
) Pathway pathology: histological differences between ErbB/Ras and Wnt pathway transgenic mammary tumors.
Am. J. Pathol.
  ,
161
,
1087
–1097.
39.
Zinser,G.M., Sundberg,J.P. and Welsh,J. (
2002
) Vitamin D(3) receptor ablation sensitizes skin to chemically induced tumorigenesis.
Carcinogenesis
  ,
23
,
2103
–2109.
40.
Kallay,E., Pietschmann,P., Toyokuni,S., Bajna,E., Hahn,P., Mazzucco,K., Bieglmayer,C., Kato,S. and Cross,H.S. (
2001
) Characterization of a vitamin D receptor knockout mouse as a model of colorectal hyperproliferation and DNA damage.
Carcinogenesis
  ,
22
,
1429
–1435.
41.
Tandon,A.K., Clark,G.M., Chamness,G.C., Ullrich,A. and McGuire,W.L. (
1989
) HER-2/neu oncogene protein and prognosis in breast cancer.
J. Clin. Oncol.
  ,
7
,
1120
–1128.
42.
Bacus,S.S., Chin,D., Yarden,Y., Zelnick,C.R. and Stern,D.F. (
1996
) Type 1 receptor tyrosine kinases are differentially phosphorylated in mammary carcinoma and differentially associated with steroid receptors.
Am. J. Pathol.
  ,
148
,
549
–558.
43.
Takeda,E., Yokota,I., Ito,M., Kobashi,H., Saijo,T. and Kuroda,Y. (
1990
) 25-Hydroxyvitamin D-24-hydroxylase in phytohemagglutinin-stimulated lymphocytes: intermediate bioresponse to 1,25-dihydroxyvitamin D3 of cells from parents of patients with vitamin D-dependent rickets type II.
J. Clin. Endocrinol. Metab.
  ,
70
,
1068
–1074.
44.
Schwartz,B., Smirnoff,P., Shany,S. and Liel,Y. (
2000
) Estrogen controls expression and bioresponse of 1,25-dihydroxyvitamin D receptors in the rat colon.
Mol. Cell. Biochem.
  ,
203
,
87
–93.
45.
Wietzke,J.A. and Welsh,J. (
2003
) Phytoestrogen regulation of a vitamin D3 receptor promoter and 1,25-dihydroxyvitamin D3 actions in human breast cancer cells.
J. Steroid Biochem. Mol. Biol.
  ,
84
,
149
–157.
46.
Escaleira,M.T. and Brentani,M.M. (
1999
) Vitamin D3 receptor (VDR) expression in HC-11 mammary cells: regulation by growth-modulatory agents, differentiation and Ha-ras transformation.
Breast Cancer Res. Treat.
  ,
54
,
123
–133.
47.
Welsh,J., Wietzke,J.A., Zinser,G.M., Smyczek,S., Romu,S., Tribble,E., Welsh,J.C., Byrne,B. and Narvaez,C.J. (
2002
) Impact of the vitamin D3 receptor on growth-regulatory pathways in mammary gland and breast cancer.
J. Steroid Biochem. Mol. Biol.
  ,
83
,
85
–92.
48.
Katayama,M.L., Pasini,F.S., Folgueira,M.A., Snitcovsky,I.M. and Brentani,M.M. (
2003
) Molecular targets of 1,25(OH)2D3 in HC11 normal mouse mammary cell line.
J. Steroid Biochem. Mol. Biol.
  ,
84
,
57
–69.
49.
Schneider,S.M., Offterdinger,M., Huber,H. and Grunt,T.W. (
1999
) Involvement of nuclear steroid/thyroid/retinoid receptors and of protein kinases in the regulation of growth and of c-erbB and retinoic acid receptor expression in MCF-7 breast cancer cells.
Breast Cancer Res. Treat.
  ,
58
,
171
–181.
50.
Verlinden,L., Verstuyf,A., Convents,R., Marcelis,S., Van Camp,M. and Bouillon,R. (
1998
) Action of 1,25(OH)2D3 on the cell cycle genes, cyclin D1, p21 and p27 in MCF-7 cells.
Mol. Cell. Endocrinol.
  ,
142
,
57
–65.
51.
Maenpaa,P.H., Vaisanen,S., Jaaskelainen,T., Ryhanen,S., Rouvinen,J., Duchier,C. and Mahonen,A. (
2001
) Vitamin D(3) analogs (MC 1288, KH 1060, EB 1089, GS 1558 and CB 1093): studies on their mechanism of action.
Steroids
  ,
66
,
223
–225.
52.
Kwabi-Addo,B., Giri,D., Schmidt,K., Podsypanina,K., Parsons,R., Greenberg,N. and Ittmann,M. (
2001
) Haploinsufficiency of the Pten tumor suppressor gene promotes prostate cancer progression.
Proc. Natl Acad. Sci. USA
  ,
98
,
11563
–11568.
53.
Fodde,R. and Smits,R. (
2002
) Cancer biology. A matter of dosage.
Science
  ,
298
,
761
–763.
54.
Haslam,S.Z. and Counterman,L.J. (
1991
) Mammary stroma modulates hormonal responsiveness of mammary epithelium in vivo in the mouse.
Endocrinology
  ,
129
,
2017
–2023.
55.
Elliott,B.E., Tam,S.P., Dexter,D. and Chen,Z.Q. (
1992
) Capacity of adipose tissue to promote growth and metastasis of a murine mammary carcinoma: effect of estrogen and progesterone.
Int. J. Cancer
  ,
51
,
416
–424.
56.
Elenbaas,B. and Weinberg,R.A. (
2001
) Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation.
Exp. Cell Res.
  ,
264
,
169
–184.
57.
Rahimi,N., Saulnier,R., Nakamura,T., Park,M. and Elliott,B. (
1994
) Role of hepatocyte growth factor in breast cancer: a novel mitogenic factor secreted by adipocytes.
DNA Cell Biol.
  ,
13
,
1189
–1197.
58.
Cunha,G.R., Young,P., Hom,Y.K., Cooke,P.S., Taylor,J.A. and Lubahn,D.B. (
1997
) Elucidation of a role for stromal steroid hormone receptors in mammary gland growth and development using tissue recombinants.
J. Mammary Gland Biol. Neoplasia
  ,
2
,
393
–402.
59.
Xie,Z., Komuves,L., Yu,Q.C. et al . (
2002
) Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth.
J. Invest Dermatol
  ,
118
,
11
–16.
60.
Chen,C.H., Sakai,Y. and Demay,M.B. (
2001
) Targeting expression of the human vitamin D receptor to the keratinocytes of vitamin D receptor null mice prevents alopecia.
Endocrinology
  ,
142
,
5386
–5389.
61.
Kamei,Y., Kawada,T., Kazuki,R., Ono,T., Kato,S. and Sugimoto,E. (
1993
) Vitamin D receptor gene expression is up-regulated by 1, 25-dihydroxyvitamin D3 in 3T3-L1 preadipocytes.
Biochem. Biophys. Res. Commun.
  ,
193
,
948
–955.
62.
Imagawa,M., Tsuchiya,T. and Nishihara,T. (
1999
) Identification of inducible genes at the early stage of adipocyte differentiation of 3T3-L1 cells.
Biochem. Biophys. Res. Commun.
  ,
254
,
299
–305.
63.
Menendez,C., Lage,M., Peino,R., Baldelli,R., Concheiro,P., Dieguez,C. and Casanueva,F.F. (
2001
) Retinoic acid and vitamin D(3) powerfully inhibit in vitro leptin secretion by human adipose tissue.
J. Endocrinol.
  ,
170
,
425
–431.
64.
Shi,H., Norman,A.W., Okamura,W.H., Sen,A. and Zemel,M.B. (
2002
) 1Alpha,25-dihydroxyvitamin D3 inhibits uncoupling protein 2 expression in human adipocytes.
FASEB J.
  ,
16
,
1808
–1810.