HGFL supports mammary tumorigenesis by enhancing tumor cell intrinsic survival and influencing macrophage and T-cell responses.

The Ron receptor is overexpressed in human breast cancers and is associated with heightened metastasis and poor survival. Ron overexpression in the mammary epithelium of mice is sufficient to induce aggressive mammary tumors with a high degree of metastasis. Despite the well-documented role of Ron in breast cancer, few studies have examined the necessity of the endogenous Ron ligand, hepatocyte growth factor-like protein (HGFL) in mammary tumorigenesis. Herein, mammary tumor growth and metastasis were examined in mice overexpressing Ron in the mammary epithelium with or without HGFL. HGFL ablation decreased oncogenic Ron activation and delayed mammary tumor initiation. HGFL was important for tumor cell proliferation and survival. HGFL loss resulted in increased numbers of macrophages and T-cells within the tumor. T-cell proliferation and cytotoxicity dramatically increased in HGFL deficient mice. Biochemical analysis of HGFL proficient tumors showed increased local HGFL production, with HGFL loss decreasing ß-catenin expression and NF-κB activation. Re-expression of HGFL in HGFL deficient tumor cells stimulated cell migration and invasion with coordinate activation of NF-κB and reduced apoptosis. Together, these results demonstrate critical in vivo functions for HGFL in promoting breast tumorigenesis and suggest that targeting HGFL may inhibit tumor growth and reactivate anti-tumor immune responses.

Vitamin D3-dependent VDR signaling delays ron-mediated breast tumorigenesis through suppression of ß-catenin activity.

The Ron receptor is upregulated in human breast cancers and correlates with enhanced metastasis and reduced patient survival. Ron overexpression drives mammary tumorigenesis through direct ß-catenin activation and augmented tumor cell proliferation, migration and invasion. Ron and ß-catenin are also coordinately elevated in breast cancers. The vitamin D receptor (VDR) antagonizes ß-catenin signaling. Herein, we examined mammary tumor onset and progression using a Ron-driven murine model of breast tumorigenesis crossed with VDR deficient mice. VDR ablation accelerated mammary tumor onset and led to tumors that exhibited a desmoplastic phenotype and enhanced metastases. Tumor levels of active ß-catenin were markedly increased in the absence of VDR. In vitro, VDR activation in breast cancer cells reduced ß-catenin activation and transcriptional activity leading to elevated expression of the extracellular Wnt inhibitor dickkopf-related protein 1, and a reduction in the interaction of ß-catenin with the cyclin D1 promoter. Expression of a stabilized form or ß-catenin ablated the protective effects of VDR activation.Collectively, these studies delineate a protective role for VDR signaling in Ron-induced mammary tumorigenesis through disruption of ß-catenin activation.

Loss of vitamin D receptor signaling from the mammary epithelium or adipose tissue alters pubertal glandular development.

Vitamin D3 receptor (VDR) signaling within the mammary gland regulates various postnatal stages of glandular development, including puberty, pregnancy, involution, and tumorigenesis. Previous studies have shown that vitamin D3 treatment induces cell-autonomous growth inhibition and differentiation of mammary epithelial cells in culture. Furthermore, mammary adipose tissue serves as a depot for vitamin D3 storage, and both epithelial cells and adipocytes are capable of bioactivating vitamin D3. Despite the pervasiveness of VDR in mammary tissue, individual contributions of epithelial cells and adipocytes, as well as the VDR-regulated cross-talk between these two cell types during pubertal mammary development, have yet to be investigated. To assess the cell-type specific effect of VDR signaling during pubertal mammary development, novel mouse models with mammary epithelial- or adipocyte-specific loss of VDR were generated. Interestingly, loss of VDR in either cellular compartment accelerated ductal morphogenesis with increased epithelial cell proliferation and decreased apoptosis within terminal end buds. Conversely, VDR signaling specifically in the mammary epithelium modulated hormone-induced alveolar growth, as ablation of VDR in this cell type resulted in precocious alveolar development. In examining cellular cross-talk ex vivo, we show that ligand-dependent VDR signaling in adipocytes significantly inhibits mammary epithelial cell growth in part through the vitamin D3-dependent production of the cytokine IL-6. Collectively, these studies delineate independent roles for vitamin D3-dependent VDR signaling in mammary adipocytes and epithelial cells in controlling pubertal mammary gland development.

Conditional deletion of ß-catenin in mammary epithelial cells of Ron receptor, Mst1r, overexpressing mice alters mammary tumorigenesis.

The Ron receptor tyrosine kinase (macrophage stimulating 1 receptor) is overexpressed in approximately 50% of human breast cancers. Transgenic mice overexpressing Ron in the mammary epithelium [mouse mammary tumor virus driven (MMTV)-Ron expressing mice] develop mammary tumors that exhibit up-regulation of ß-catenin and ß-catenin target genes. ß-Catenin has been shown to be a mediator of mammary tumorigenesis in various breast cancer models, including downstream of Ron. However, the in vivo impact of a conditional loss of ß-catenin downstream of Ron receptor overexpression on the onset, growth, turnover, and metastasis of mammary tumors has not been addressed. To determine the significance of ß-catenin in the context of Ron overexpression, we conditionally deleted ß-catenin in mammary epithelial cells of MMTV-Ron mice. Conditional deletion of ß-catenin in the mammary epithelium, through the use of whey acidic protein (WAP)-Cre transgenic mice, significantly delayed the onset of mammary hyperplastic nodules, the presence of palpable mammary tumors, and ultimately decreased liver metastasis. ß-Catenin loss in this model was also associated with decreased expression of cyclin D1. In total, these studies support an important role for ß-catenin downstream of Ron receptor signaling during the development of mammary tumorigenesis.

Mammary adipocytes bioactivate 25-hydroxyvitamin D3 and signal via vitamin D3 receptor, modulating mammary epithelial cell growth.

The vitamin D(3) receptor (VDR) is present in all microenvironments of the breast, yet it is hypothesized to signal through the epithelium to regulate hormone induced growth and differentiation. However, the influence or contribution of the other microenvironments within the breast that express VDR, like the breast adipose tissue, are yet to be investigated. We hypothesized that the breast adipocytes express the signaling components necessary to participate in vitamin D(3) synthesis and signaling via VDR, modulating ductal epithelial cell growth and differentiation. We utilized human primary breast adipocytes and VDR wild type (WT) and knockout (KO) mice to address whether breast adipocytes participate in vitamin D(3) -induced growth regulation of the ductal epithelium. We report in this study that breast primary adipocytes express VDR, CYP27B1 (1α-hydroxylase, 1α-OHase), the enzyme that generates the biologically active VDR ligand, 1α,25-dihydroxyvitamin D(3) (1,25D(3) ), and CYP24 (24-hydroxylase, 24-OHase), a VDR-1,25D(3) induced target gene. Furthermore, the breast adipocytes participate in bioactivating 25-hydroxyvitamin D(3) (25D(3) ) to the active ligand, 1,25D(3) , and secreting it to the surrounding microenvironment. In support of this concept, we report that purified mammary ductal epithelial fragments (organoids) from VDR KO mice, co-cultured with WT breast adipocytes, were growth inhibited upon treatment with 25D(3) or 1,25D(3) compared to vehicle alone. Collectively, these results demonstrate that breast adipocytes bioactivate 25D(3) to 1,25D(3) , signal via VDR within the adipocytes, and release an inhibitory factor that regulates ductal epithelial cell growth, suggesting that breast adipose tissue contributes to vitamin D(3) -induced growth regulation of ductal epithelium.

Age-related changes in the epithelial and stromal compartments of the mammary gland in normocalcemic mice lacking the vitamin D3 receptor.

The vitamin D(3) receptor (VDR) serves as a negative growth regulator during mammary gland development via suppression of branching morphogenesis during puberty and modulation of differentiation and apoptosis during pregnancy, lactation and involution. To assess the role of the VDR in the aging mammary gland, we utilized 12, 14, and 16 month old VDR knockout (KO) and wild type (WT) mice for assessment of integrity of the epithelial and stromal compartments, steroid hormone levels and signaling pathways. Our data indicate that VDR ablation is associated with ductal ectasia of the primary mammary ducts, loss of secondary and tertiary ductal branches and atrophy of the mammary fat pad. In association with loss of the white adipose tissue compartment, smooth muscle actin staining is increased in glands from VDR KO mice, suggesting a change in the stromal microenviroment. Activation of caspase-3 and increased Bax expression in mammary tissue of VDR KO mice suggests that enhanced apoptosis may contribute to loss of ductal branching. These morphological changes in the glands of VDR KO mice are associated with ovarian failure and reduced serum 17ß-estradiol. VDR KO mice also exhibit progressive loss of adipose tissue stores, hypoleptinemia and increased metabolic rate with age. These developmental studies indicate that, under normocalcemic conditions, loss of VDR signaling is associated with age-related estrogen deficiency, disruption of epithelial ductal branching, abnormal energy expenditure and atrophy of the mammary adipose compartment.

Chk2*1100delC Acts in synergy with the Ron receptor tyrosine kinase to accelerate mammary tumorigenesis in mice.

The CHEK2 (Chk2 in mice) polymorphic variant, CHEK2*1100delC, leads to genomic instability and is associated with an increased risk for breast cancer. The Ron receptor tyrosine kinase is overexpressed in a large fraction of human breast cancers. Here, we asked whether the low penetrance Chk2*1100delC allele alters the tumorigenic efficacy of Ron in the development of mammary tumors in a mouse model. Our data demonstrate that Ron overexpression on a Chk2*1100delC background accelerates the development of mammary tumors, and shows that pathways mediated by a tyrosine kinase receptor and a regulator of the cell cycle can act to hasten tumorigenesis in vivo.

Genomic vitamin D signaling in breast cancer: Insights from animal models and human cells.

These studies focus on identification of vitamin D regulated pathways that impact development or progression of breast cancer. In mouse experiments, we assessed genomic profiles of glandular tissue and established tumors from MMTV-neu mice fed adequate (250 IU/kg) or high (5000 IU/kg) vitamin D (cholecalciferol). Genomic profiles were also obtained in murine mammary cells that differentially express VDR that were cultured in vitro with 100 nM 1,25-dihydroxyvitamin D (1,25D). Ten candidate genes were identified that were commonly regulated in murine cells treated with 1,25D in vitro and in mammary gland of mice fed high dietary vitamin D. In complementary studies, the vitamin D pathway was evaluated in human mammary epithelial cells as a function of transformation. Genes regulated by 1,25D in human mammary epithelial cells included those involved in innate immunity (CD14), differentiation (Bmp6), extracellular matrix remodeling (Plau) and cell survival (Birc3). Transformation reduced VDR content and blunted the induction of some, but not all, target genes by 1,25D in human mammary cells. Collectively, these in vivo and in vitro data demonstrate that vitamin D signaling impacts on common pathways that drive differentiation, alter metabolism, remodel the extracellular matrix and trigger innate immunity in mammary tissue.

The Ron receptor tyrosine kinase negatively regulates mammary gland branching morphogenesis.

The Ron receptor tyrosine kinase is expressed in normal breast tissue and is overexpressed in approximately 50% of human breast cancers. Despite the recent studies on Ron in breast cancer, nothing is known about the importance of this protein during breast development. To investigate the functional significance of Ron in the normal mammary gland, we compared mammary gland development in wild-type mice to mice containing a targeted ablation of the tyrosine kinase (TK) signaling domain of Ron (TK-/-). Mammary glands from RonTK-/- mice exhibited accelerated pubertal development including significantly increased ductal extension and branching morphogenesis. While circulating levels of estrogen, progesterone, and overall rates of epithelial cell turnover were unchanged, significant increases in phosphorylated MAPK, which predominantly localized to the epithelium, were associated with increased branching morphogenesis. Additionally, purified RonTK-/- epithelial cells cultured ex vivo exhibited enhanced branching morphogenesis, which was reduced upon MAPK inhibition. Microarray analysis of pubertal RonTK-/- glands revealed 393 genes temporally impacted by Ron expression with significant changes observed in signaling networks regulating development, morphogenesis, differentiation, cell motility, and adhesion. In total, these studies represent the first evidence of a role for the Ron receptor tyrosine kinase as a critical negative regulator of mammary development.

Mammary-specific Ron receptor overexpression induces highly metastatic mammary tumors associated with beta-catenin activation.

Activated growth factor receptor tyrosine kinases (RTK) play pivotal roles in a variety of human cancers, including breast cancer. Ron, a member of the Met RTK proto-oncogene family, is overexpressed or constitutively active in 50% of human breast cancers. To define the significance of Ron overexpression and activation in vivo, we generated transgenic mice that overexpress a wild-type or constitutively active Ron receptor in the mammary epithelium. In these animals, Ron expression is significantly elevated in mammary glands and leads to a hyperplastic phenotype by 12 weeks of age. Ron overexpression is sufficient to induce mammary transformation in all transgenic animals and is associated with a high degree of metastasis, with metastatic foci detected in liver and lungs of >86% of all transgenic animals. Furthermore, we show that Ron overexpression leads to receptor phosphorylation and is associated with elevated levels of tyrosine phosphorylated beta-catenin and the up-regulation of genes, including cyclin D1 and c-myc, which are associated with poor prognosis in patients with human breast cancers. These studies suggest that Ron overexpression may be a causative factor in breast tumorigenesis and provides a model to dissect the mechanism by which the Ron induces transformation and metastasis.

Vitamin D receptor (VDR) ablation alters carcinogen-induced tumorigenesis in mammary gland, epidermis and lymphoid tissues.

The Vitamin D receptor (VDR) and its ligand, 1,25(OH)(2)D(3), regulate cell proliferation, differentiation and apoptosis in vitro, yet the physiological significance of this regulation is unclear. In these studies, we used VDR knockout (VDRKO) mice to examine the impact of VDR on chemical carcinogen-induced tumorigenesis in vivo. Wild type (WT) and VDRKO littermates were fed a high calcium diet to prevent disturbances in calcium homeostasis and were gavaged with dimethylbenzanthracence (DMBA) using a protocol designed to induce mammary tumors. Compared to WT littermates, VDRKO mice exhibited an increased incidence of mammary gland hyperplasia and a higher percentage of hormone independent tumors with squamous differentiation. VDR ablation also significantly enhanced tumor development in epidermis and lymphoid tissues, but did not affect tumor development in ovary, uterus, lung or liver. These data indicate that VDR ablation alters susceptibility to DMBA-induced carcinogenesis in a tissue specific fashion, and provide support that optimal VDR signaling may act to suppress tumorigenesis.

1,24(S)-dihydroxyvitamin D2, an endogenous vitamin D2 metabolite, inhibits growth of breast cancer cells and tumors.

BACKGROUND: 1,24-Dihydroxyvitamin D2 (1,24(OH)2D2) is a naturally occurring metabolite of vitamin D2 with low calcemic activity and potent antiproliferative activity. We evaluated the activity of 1,24(OH)2D2 in breast cancer models. MATERIALS AND METHODS: The antiproliferative activity of 1,24(OH)2D2 was quantitated against human and murine breast cancer cell lines. The antitumor activity of 1,24(OH)2D2 was quantitated using MCF-7 xenografts in nude mice. RESULTS: 1,24(OH)2D2 inhibited growth of vitamin D receptor (VDR)-positive, but not VDR-negative, breast cancer cells. 1,24(OH)2D2 (10 microg/kg or 50 microg/kg) reduced MCF-7 xenograft growth by 50% after five weeks. Tumor morphology in treated animals was consistent with replacement of epithelial cells by stromal tissue. Mice treated with 1,24(OH)2D2 showed no loss of body weight, hypercalcemia or kidney calcification. CONCLUSION: 1,24(OH)2D2 inhibits growth of breast cancer cells via VDR-dependent mechanisms; its complete lack of toxicity and significant antitumor activity supports further development for chemotherapeutic applications.

Vitamin D receptor status alters mammary gland morphology and tumorigenesis in MMTV-neu mice.

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.

Accelerated mammary gland development during pregnancy and delayed postlactational involution in vitamin D3 receptor null mice.

The vitamin D receptor (VDR) is present in mammary gland, and VDR ablation is associated with accelerated glandular development during puberty. VDR is a nuclear receptor whose ligand, 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] is generated after metabolic activation of vitamin D by specific vitamin D hydroxylases. In these studies, we demonstrate that both the VDR and the vitamin D 1-alpha hydroxylase (CYP27B1), which produces 1,25-(OH)(2)D are present in mammary gland and dynamically regulated during pregnancy, lactation, and involution. Furthermore, we show that mice lacking VDR exhibit accelerated lobuloalveolar development and premature casein expression during pregnancy and delayed postlactational involution compared with mice with functional VDR. The delay in mammary gland regression after weaning of VDR knockout mice is associated with impaired apoptosis as demonstrated by reductions in terminal deoxynucleotidyl transferase-mediated deoxyuridine nick-end labeling staining, caspase-3 activation and Bax induction. Under the conditions used in this study, VDR ablation was not associated with hypocalcemia, suggesting that altered mammary gland development in the absence of the VDR is not related to disturbances in calcium homeostasis. Furthermore, in the setting of normocalcemia, VDR ablation does not affect milk protein or calcium content. These studies suggest that the VDR contributes to mammary cell turnover during the reproductive cycle, and its effects may be mediated via both endocrine and autocrine signaling pathways. Unlike many mammary regulatory factors that exert transient, stage-specific effects, VDR signaling impacts on mammary gland biology during all phases of the reproductive cycle.

Vitamin D-3 receptor as a target for breast cancer prevention.

The vitamin D-3 receptor (VDR) is a nuclear receptor that modulates gene expression when complexed with its ligand 1-alpha,25-dihydroxycholecalciferol [1,25(OH)(2)-D(3)], which is the biologically active form of vitamin D-3. The cellular effects of VDR signaling include growth arrest, differentiation and/or induction of apoptosis, which indicate that the vitamin D pathway participates in negative-growth regulation. Although much attention has been directed in recent years toward the development of synthetic vitamin D analogs as therapeutic agents for a variety of human cancers including those derived from the mammary gland, studies on vitamin D as a chemopreventive agent for breast cancer have been quite limited. The VDR is expressed in normal mammary gland, where it functions to oppose estrogen-driven proliferation and maintain differentiation; this suggests that 1,25(OH)(2)-D(3) participates in negative-growth regulation of mammary epithelial cells. Furthermore, preclinical studies show that vitamin D compounds can reduce breast cancer development in animals, and human data indicate that both vitamin D status and genetic variations in the VDR may affect breast cancer risk. Collectively, findings from cellular, molecular and population studies suggest that the VDR is a nutritionally modulated growth-regulatory gene that may represent a molecular target for chemoprevention of breast cancer.

Characterization of mammary tumor cell lines from wild type and vitamin D3 receptor knockout mice.

1,25-Dihydroxyvitamin D(3) (1,25D(3)), the active metabolite of vitamin D(3), inhibits breast cancer cell growth in vivo and in vitro. To examine mechanisms of 1,25D(3) induced growth arrest and apoptosis, cell lines were established from DMBA induced mammary tumors derived from vitamin D(3) receptor knockout (VDRKO) and wild type (WT) mice. Two VDRKO (KO240, KO288) and two WT (WT145, WT276) cell lines were selected and characterized. All four cell lines express cytokeratins indicative of an epithelial origin, as well as vimentin, which is expressed in many transformed cell lines. The tumorigenicity of the cells was confirmed in vivo as all four cell lines form estrogen responsive tumors in nude mice. Both WT cell lines express the VDR protein and are sensitive to growth inhibition by 1,25D(3) at doses as low as 1 nM. Flow cytometric analysis indicated that 1,25D(3) induces G(0)/G(1) arrest and apoptosis in the WT cell lines. In contrast, both cell lines established from tumors that developed in VDRKO mice lack VDR mRNA and protein. Cells from WT mice exhibit 1,25D(3) inducible transcriptional activity, as measured by reporter gene assays, but cells from VDRKO mice do not. Cells from VDRKO mice are also completely resistant to 1,25D(3) mediated growth arrest and apoptosis over the range of 0.01-100 nM 1,25D(3). VDRKO cells are also resistant to the synthetic vitamin D(3) analogs EB1089 and CB1093 that are more potent growth inhibitors than 1,25D(3) in WT cells. This data conclusively demonstrate that the induction of cell cycle arrest and apoptosis in breast cancer cells by 1,25D(3), EB1089 and CB1093 is dependent on the nuclear VDR. Cells lacking VDR remain sensitive to growth arrest mediated by 9-cis retinoic acid, a ligand for the retinoid x receptor which can heterodimerize with the VDR. Sensitivity to apoptosis induced by the DNA damaging agent etoposide is not altered in VDRKO cells, indicating that VDR ablation does not impair apoptotic pathways in general. All four cell lines display equal sensitivity to tamoxifen induced growth arrest. These estrogen responsive, transformed cell lines which differentially express the VDR provide a novel model system for identification of the mechanisms by which 1,25D(3) regulates proliferation and apoptosis in breast cancer cells.

Impact of the Vitamin D3 receptor on growth-regulatory pathways in mammary gland and breast cancer.

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) interacts with the Vitamin D(3) receptor (VDR) to modulate proliferation and apoptosis in a variety of cell types, including breast cancer cells. In this review, we discuss three issues related to the role of the VDR in growth control: first, whether mammary glands lacking VDR exhibit abnormal growth; second, whether the VDR is essential for induction of apoptosis by 1,25(OH)(2)D(3); and third, whether VDR up-regulation can sensitize cells to 1,25(OH)(2)D(3). Studies from our laboratory have demonstrated that mammary glands from VDR knockout (VDR KO) mice exhibit accelerated growth and branching during puberty, pregnancy and lactation as compared to wild-type (WT) mice. In addition, involution after weaning, a process driven by epithelial cell apoptosis, proceeds at a slower rate in VDR KO mice compared to WT mice. Using cells isolated from VDR KO and WT mice, we report that both normal and transformed mammary cells derived from WT mice are growth inhibited by 1,25(OH)(2)D(3), however, cells derived from VDR KO mice are completely unresponsive to 1,25(OH)(2)D(3). In human breast cancer cells, we have identified a variety of agents, including steroid hormones, phytoestrogens and growth factors, that up-regulate VDR expression and enhance sensitivity to 1,25(OH)(2)D(3)-mediated growth inhibition. Collectively, these studies support a role for 1,25(OH)(2)D(3) and the VDR in negative growth regulation of both normal mammary gland and breast cancer cells.

Vitamin D(3) receptor ablation sensitizes skin to chemically induced tumorigenesis.

1,25-Dihydroxyvitamin D(3) (1,25D(3)) is the biologically active form of vitamin D(3) that interacts with the nuclear vitamin D(3) receptor (VDR) to modulate gene expression in a tissue-specific fashion. 1,25D(3) is a potent regulator of cell proliferation, differentiation and apoptosis in a variety of cell types, including keratinocytes. In these studies, we assessed the sensitivity of mice homozygous for a null allele of the VDR (VDR(-/-) mice) and their wild-type counterparts (VDR(+/+) mice) to oral administration of the carcinogen 7,12-dimethylbenzanthracene (DMBA). Although the protocol was optimized for the induction of mammary tumors, 85% of VDR(-/-) mice developed persistent skin tumors within 60 days of carcinogen exposure. In VDR(-/-) mice exposed to DMBA, papillomas arose on all areas of the body, with an average tumor burden of 5.3 papillomas/mouse. No papillomas or any other skin lesions were observed in age- and sex-matched VDR(+/+) mice dosed with DMBA and followed for 6 months. The majority (80%) of skin tumors that developed in VDR(-/-) mice were classified histologically as sebaceous, squamous or follicular papillomas. Other types of lesions, including basal cell carcinoma, hemangioma and melanotic foci, were occasionally observed in VDR(-/-) mice (but not in VDR(+/+) mice) exposed to DMBA. Quantification of epidermal thickness and BrdU incorporation indicated that skin from VDR(-/-) mice exhibited hyperproliferation beginning at 7 weeks of age, which was exacerbated by DMBA treatment. Untreated aging VDR(-/-) mice did not exhibit tumor formation, but did develop a progressive skin phenotype characterized by thickened wrinkled skin, dermoid cysts and long curly nails. Together with previous reports that 1,25D(3) inhibits papilloma formation induced by topical DMBA-TPA regimens, our observation of enhanced sensitivity of VDR(-/-) mice to chemically induced skin carcinogenesis offers compelling evidence that disruption of VDR signaling predisposes to neoplasia.