Susan G. Komen Big Data for Breast Cancer Initiative: How Patient Advocacy Organizations Can Facilitate Using Big Data to Improve Patient Outcomes.

Integrating different types of data, including electronic health records, imaging data, administrative and claims databases, large data repositories, the Internet of Things, genomics, and other omics data, is both a challenge and an opportunity that must be tackled head on. We explore some of the challenges and opportunities in optimizing data integration to accelerate breast cancer discovery and improve patient outcomes. Susan G. Komen convened three meetings (2015, 2017, and 2018) with various stakeholders to discuss challenges, opportunities, and next steps to enhance the use of big data in the field of breast cancer. Meeting participants agreed that big data approaches can enhance the identification of better therapies, improve outcomes, reduce disparities, and optimize precision medicine. One challenge is that databases must be shared, linked with each other, standardized, and interoperable. Patients want to be active participants in research and their own care, and to control how their data are used. Many patients have privacy concerns and do not understand how sharing their data can help to effectively drive discovery. Public education is essential, and breast cancer researchers who are skilled in using and analyzing big data are needed. Patient advocacy groups can play multiple roles to help maximize and leverage big data to better serve patients. Komen is committed to educating patients on big data issues, encouraging data sharing by all stakeholders, assisting in training the next generation of data science breast cancer researchers, and funding research projects that will use real-life data in real time to revolutionize the way breast cancer is understood and treated.

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.

Effect of the vitamin D receptor on bone geometry and strength during gestation and lactation in mice.

The vitamin D receptor (VDR) plays an important role in maintaining calcium homeostasis, acting as a mediator of transcellular calcium absorption and bone remodeling. Mice lacking a functional VDR have an abnormal skeletal phenotype, which is rescued by feeding a high-calcium diet. In this study, the role of the VDR in maintaining bone geometry and strength during gestation and lactation, when increased demands are placed on the calcium regulatory channels, was examined using a knockout mouse model. A rescue diet was used to counteract the decrease in calcium absorption in the gut that results from the absence of the VDR. Structural and compositional characteristics of the femur were compared between VDR knockout and wild-type mice following 9 and 16 days of gestation and 5 and 10 days of lactation using generalized linear models. Overall, the knockout mice had 6.5% lower cortical area, 23% lower trabecular volume fraction, and 9% lower bending stiffness than wild-type mice. However, the maximum moment of inertia of the femoral diaphyses, ultimate bending load, ash fraction, and trabecular thickness were not significantly different between knockout and wild-type mice. Only the mineral content exhibited interdependence between genotype and time point. Taken together, the results show that the VDR affects the quantity of mineralized bone tissue in the femoral diaphysis and metaphysis independently of reproductive status. However, the moments of inertia were similar between genotypes, resulting in similar bone stiffness and strength despite lower mineral content and cross-sectional area.

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.

The molecular basis of vitamin D receptor and beta-catenin crossregulation.

The signaling/oncogenic activity of beta-catenin can be repressed by activation of the vitamin D receptor (VDR). Conversely, high levels of beta-catenin can potentiate the transcriptional activity of 1,25-dihydroxyvitamin D3 (1,25D). We show here that the effects of beta-catenin on VDR activity are due to interaction between the activator function-2 (AF-2) domain of the VDR and C terminus of beta-catenin. Acetylation of the beta-catenin C terminus differentially regulates its ability to activate TCF or VDR-regulated promoters. Mutation of a specific residue in the AF-2 domain, which renders the VDR trancriptionally inactive in the context of classical coactivators, still allows interaction with beta-catenin and ligand-dependent activation of VDRE-containing promoters. VDR antagonists, which block the VDRE-directed activity of the VDR and recruitment of classical coactivators, do allow VDR to interact with beta-catenin, which suggests that these and perhaps other ligands would permit those functions of the VDR that involve beta-catenin interaction.

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.