Vitamin D and Breast Cancer: Mechanistic Update.

The presence of the vitamin D receptor (VDR) in mammary gland and breast cancer has long been recognized, and multiple preclinical studies have demonstrated that its ligand, 1,25-dihydroxyvitamin D (1,25D), modulates normal mammary gland development and inhibits growth of breast tumors in animal models. Vitamin D deficiency is common in breast cancer patients, and some evidence suggests that low vitamin D status enhances the risk for disease development or progression. Although many 1,25D-responsive targets in normal mammary cells and in breast cancers have been identified, validation of specific targets that regulate cell cycle, apoptosis, autophagy, and differentiation, particularly in vivo, has been challenging. Model systems of carcinogenesis have provided evidence that both VDR expression and 1,25D actions change with transformation, but clinical data regarding vitamin D responsiveness of established tumors is limited and inconclusive. Because breast cancer is heterogeneous, the relevant VDR targets and potential sensitivity to vitamin D repletion or supplementation will likely differ between patient populations. Detailed analysis of VDR actions in specific molecular subtypes of the disease will be necessary to clarify the conflicting data. Genomic, proteomic, and metabolomic analyses of in vitro and in vivo model systems are also warranted to comprehensively understand the network of vitamin D-regulated pathways in the context of breast cancer heterogeneity. This review provides an update on recent studies spanning the spectrum of mechanistic (cell/molecular), preclinical (animal models), and translational work on the role of vitamin D in breast cancer. © 2021 The Author. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Divergent effects of vitamins K1 and K2 on triple negative breast cancer cells.

Vitamin K serves as an essential co-factor in the γ-carboxylation of glutamate to γ-carboxyglutamate (GLA), a post-translational modification mediated by gamma-glutamyl carboxylase (GGCX) and vitamin K oxidoreductases (VKORC1 or VKORC1L1). While both phylloquinone (K1) and menaquinone (K2) support the synthesis of GLA-modified proteins, studies assessing K1 and/or K2 effects in cancer cells have reported minimal effects of K1 and anti-proliferative or pro-apoptotic effects of K2. qPCR results indicated highest expression of GGCX, VKORC1, and VKORC1L1 in triple negative breast cancer (TNBC) cell lines, Hs578T, MDA-MB-231 and SUM159PT, and in advanced stage disease. To assess differential effects of vitamin K, TNBC cells were cultured in media supplemented with K1 or K2. K1 treatment increased cell growth, and enhanced stemness and GLA-modified protein expression in TNBC lysates. Alternatively, lysates from cells exposed to vehicle, K2, or the VKOR antagonist, warfarin, did not express GLA-modified proteins. Further, K2 exposure reduced stemness and elicited anti-proliferative effects. These studies show that TNBC cells express a functional vitamin K pathway and that K1 and K2 exert distinct phenotypic effects. Clarification of the mechanisms by which K1 and K2 induce these effects may lead to relevant therapeutic strategies for manipulating this pathway in TNBC patients.

Vitamin D and testosterone co-ordinately modulate intracellular zinc levels and energy metabolism in prostate cancer cells.

Vitamin D3 and its receptor are responsible for controlling energy expenditure in adipocytes and have direct roles in the transcriptional regulation of energy metabolic pathways. This phenomenon also has a significant impact on the etiology of prostate cancer (PCa). Using several in vitro models, the roles of vitamin D3 on energy metabolism and its implication in primary, early, and late invasive PCa were investigated. BODIPY staining and qPCR analyses show that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) up-regulates de novo lipogenesis in PCa cells by orchestrating transcriptional regulation that affects cholesterol and lipid metabolic pathways. This lipogenic effect is highly dependent on the interaction of several nuclear receptors and their corresponding ligands, including androgen receptor (AR), vitamin D receptor (VDR), and retinoid X receptor (RXR). In contrast, inhibition of peroxisome proliferator-activated receptor alpha (PPARα) signaling blocks the induction of the lipogenic phenotype induced by these receptors. Furthermore, 1,25(OH)2D3, T, and 9 cis-retinoic acid (9-cis RA) together redirect cytosolic citrate metabolism toward fatty acid synthesis by restoring normal prostatic zinc homeostasis that functions to truncate TCA cycle metabolism. 1,25(OH)2D3, T, and 9-cis RA also exert additional control of TCA cycle metabolism by down-regulating SLC25A19, which limits the availability of the co-factor thiamine pyrophosphate (TPP) that is required for enzymatic catalyzation of citrate oxidation. This extensive metabolic reprogramming mediated by 1,25(OH)2D3, T, and 9-cis RA is preserved in all in vitro cell lines investigated. These data suggest that 1,25(OH)2D3 and T are important regulators of normal prostatic energy metabolism. Based on the close association between energy metabolism and cancer progression, supplementation of vitamin D3 and testosterone can restrict the energy production that is required to drive PCa progression by maintaining proper zinc homeostasis and inhibiting TCA cycle activity in PCa cells.

1,25-Dihydroxyvitamin D induces the glutamate transporter SLC1A1 and alters glutamate handling in non-transformed mammary cells.

Genomic profiling of immortalized human mammary epithelial (hTERT-HME1) cells identified several metabolic genes, including the membrane glutamate transporter, SLC1A1, as 1,25-dihydroxyvitamin D3 (1,25D) regulated. In these studies we have surveyed the effects of 1,25D on known glutamate transporters and evaluated its impact on cellular glutamate handling. We confirm that expression of SLC1A1 and all of its known transcript variants are significantly upregulated in hTERT-HME1 cells following 1,25D treatment. Expression of the full-length cognate protein, EAAT3, is correspondingly increased in 1,25D treated hTERT-HME1 cells. Under the same conditions, the expression of two other glutamate transporters--SLC1A6 (EAAT4) and SLC1A2 (EAAT2 or GLT-1)--is enhanced by 1,25D while that of SLC1A3 (EAAT1 or GLAST) and SLC7A11 (xCT) is decreased. Glutamate is not essential for growth of hTERT-HME1 cells, and supplemental glutamate (up to 0.5 mM) does not abrogate the growth inhibitory effects of 1,25D. These data suggest that extracellular glutamate is not a major contributor to cellular energy metabolism in hTERT-HME1 cells under basal conditions and that the growth inhibitory effects of 1,25D are not secondary to its effects on glutamate handling. Instead, the effects of 1,25D on glutamate transporters translated to a decrease in cellular glutamate concentration and an increase in media glutamate concentration, suggesting that one or more of these transporters functions to export glutamate in response to 1,25D exposure. The reduced cellular glutamate concentration may also reflect its incorporation into the cellular glutathione (GSH) pool, which is increased upon 1,25D treatment. In support of this concept, the expression of GCLC (which codes for the rate-limiting enzyme in GSH synthesis) and genes which generate reducing equivalents in the form of NADPH (ie, G6PD, PGD, IDH2) are elevated in 1,25D-treated cells. Taken together, these data identify 1,25D as a physiological regulator of multiple membrane glutamate transporters that impacts on overall cellular glutamate handling.

Vitamin D and cancer: integration of cellular biology, molecular mechanisms and animal models.

Epidemiologic data suggest that the incidence and severity of many types of cancer inversely correlates with indices of vitamin D status. The vitamin D receptor (VDR) is highly expressed in epithelial cells at risk for carcinogenesis including those resident in skin, breast, prostate and colon, providing a direct molecular link by which vitamin D status impacts on carcinogenesis. Consistent with this concept, activation of VDR by its ligand 1,25-dihydroxyvitamin D (1,25(OH)(2)D) triggers comprehensive genomic changes in epithelial cells that contribute to maintenance of the differentiated phenotype, resistance to cellular stresses and protection of the genome. Many epithelial cells also express the vitamin D metabolizing enzyme CYP27B1 which enables autocrine generation of 1,25(OH)(2)D from the circulating vitamin D metabolite 25-hydroxyvitamin D (25(OH)D), critically linking overall vitamin D status with cellular anti-tumor actions. Furthermore, pre-clinical studies in animal models have demonstrated that dietary supplementation with vitamin D or chronic treatment with VDR agonists decreases tumor development in skin, colon, prostate and breast. Conversely, deletion of the VDR gene in mice alters the balance between proliferation and apoptosis, increases oxidative DNA damage, and enhances susceptibility to carcinogenesis in these tissues. Because VDR expression is retained in many human tumors, vitamin D status may be an important modulator of cancer progression in persons living with cancer. Collectively, these observations reinforce the need to further define the molecular actions of the VDR and the human requirement for vitamin D in relation to cancer development and progression.

Cellular and molecular effects of vitamin D on carcinogenesis.

Epidemiologic data suggest that the incidence and severity of many types of cancer inversely correlates with indices of vitamin D status. The vitamin D receptor (VDR) is highly expressed in epithelial cells at risk for carcinogenesis including those resident in skin, breast, prostate and colon, providing a direct molecular link by which vitamin D status impacts on carcinogenesis. Consistent with this concept, activation of VDR by its ligand 1,25-dihydroxyvitamin D (1,25D) triggers comprehensive genomic changes in epithelial cells that contribute to maintenance of the differentiated phenotype, resistance to cellular stresses and protection of the genome. Many epithelial cells also express the vitamin D metabolizing enzyme CYP27B1 which enables autocrine generation of 1,25D from the circulating vitamin D metabolite 25-hydroxyvitamin D (25D), critically linking overall vitamin D status with cellular anti-tumor actions. Furthermore, pre-clinical studies in animal models has demonstrated that dietary supplementation with vitamin D or chronic treatment with VDR agonists decreases tumor development in skin, colon, prostate and breast. Conversely, deletion of the VDR gene in mice alters the balance between proliferation and apoptosis, increases oxidative DNA damage, and enhances susceptibility to carcinogenesis in these tissues. Because VDR expression is retained in many human tumors, vitamin D status may be an important modulator of cancer progression in persons living with cancer. Collectively, these observations have reinforced the need to further define the molecular actions of the VDR and the human requirement for vitamin D in relation to cancer development and progression.

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.

Tumor progression in the LPB-Tag transgenic model of prostate cancer is altered by vitamin D receptor and serum testosterone status.

Previous studies have suggested that 1,25 dihydroxyvitamin D(3) (1,25(OH)2D3) induces cell cycle arrest and/or apoptosis in prostate cancer cells in vitro, suggesting that vitamin D may be a useful adjuvant therapy for prostate cancer and a chemopreventive agent. Most epidemiological data however shows a weak link between serum 25(OH)D3 and risk of prostate cancer. To explore this dichotomy we have compared tumor progression in the LPB-Tag model of prostate in VDR knock out (VDRKO) and wild type (VDRWT) mice. On the C57BL/6 background LPB-Tag tumors progress significantly more rapidly in the VDRKO mice. VDRKO tumors show significantly higher levels of cell proliferation than VDRWT tumors. In mice supplemented with testosterone to restore the serum levels to the normal range, these differences in tumor progression, and proliferation are abrogated, suggesting that there is considerable cross-talk between the androgen receptor (AR) and the vitamin D axis which is reflected in significant changes in steady state mRNA levels of the AR, PCNA, cdk2 survivin and IGFR1 and 2 genes. These alterations may explain the differences between the in vitro data and the epidemiological studies.

Vitamin D and prevention of breast cancer.

Epidemiologic data have demonstrated that breast cancer incidence is inversely correlated with indices of vitamin D status, including ultraviolet exposure, which enhances epidermal vitamin D synthesis. The vitamin D receptor (VDR) is expressed in mammary epithelial cells, suggesting that vitamin D may directly influence sensitivity of the gland to transformation. Consistent with this concept, in vitro studies have demonstrated that the VDR ligand, 1,25-dihydroxyvitamin D (1, 25D), exerts negative growth regulatory effects on mammary epithelial cells that contribute to maintenance of the differentiated phenotype. Furthermore, deletion of the VDR gene in mice alters the balance between proliferation and apoptosis in the mammary gland, which ultimately enhances its susceptibility to carcinogenesis. In addition, dietary supplementation with vitamin D, or chronic treatment with synthetic VDR agonists, reduces the incidence of carcinogen-induced mammary tumors in rodents. Collectively, these observations have reinforced the need to further define the human requirement for vitamin D and the molecular actions of the VDR in relation to prevention of breast cancer.

Breast cancer cell regulation by high-dose Vitamin D compounds in the absence of nuclear vitamin D receptor.

1alpha,25-dihydroxyvitamin D(3) (1,25D(3)) inhibits growth and induces apoptosis in breast cancer cells in vivo and in vitro. To examine the role of the Vitamin D receptor (VDR) in mediating the actions of 1,25D(3) at nanomolar and micromolar concentrations, mammary epithelial tumor cell lines generated in wild type (WT) and VDR knockout (VDRKO) mice were utilized. WT cells express VDR and are growth inhibited by 1,25D(3) and synthetic analogs EB1089 and CB1093 at 1nM concentrations, while VDRKO cells do not express VDR and are insensitive to Vitamin D compounds at concentrations up to 100nM. In the current studies, we have confirmed and extended these previous observations. At nanomolar concentrations of 1,25D(3) and all analogs tested, including EB1089, CB1093, MC1288, and KH1230, WT cells are growth inhibited and exhibit apoptotic morphology, while VDRKO cells show no growth inhibition or apoptosis. At concentrations of 1-10microM, however, 1,25D(3) and synthetic analogs induce growth inhibition and apoptotic morphology in both WT and VDRKO cell lines. These data indicate that nanomolar concentrations of 1,25D(3) and analogs mediate growth regulatory effects via mechanisms requiring the nuclear VDR, but that micromolar concentrations of Vitamin D compounds can exert non VDR-mediated effects.

Phytoestrogen regulation of a Vitamin D3 receptor promoter and 1,25-dihydroxyvitamin D3 actions in human breast cancer cells.

1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), a steroid hormone derived from Vitamin D(3), is a negative growth regulator of breast cancer cells, and Vitamin D(3) analogs represent a novel treatment approach for human cancer. Elucidation of Vitamin D(3) receptor (VDR) regulation may reveal strategies to sensitize cancer cells to the effects of 1,25-dihydroxyvitamin D(3) and Vitamin D(3) analogs. We have previously characterized an estrogen responsive promoter region (800 bp upstream of exon 1c) in the human VDR gene, and the present studies examined regulation of this VDR promoter region by two phytoestrogens, resveratrol (present in red wine) and genistein (present in soy). We transiently transfected a VDR promoter luciferase construct into the estrogen receptor (ER) positive human breast cancer cell lines T47D and MCF-7, and treated with 0.4-4 microM resveratrol or 5-500 nM genistein. Both phytoestrogens up-regulated the transcription of the VDR promoter, as measured by reporter gene activity, approximately two-fold compared to vehicle treated cells. Co-treatment with the anti-estrogen tamoxifen (TAM) in T47D cells and transfection in an estrogen receptor negative breast cancer cell line demonstrated that the effects of phytoestrogens on the VDR promoter are dependent on estrogen receptor. Resveratrol and genistein also increased VDR protein expression as detected by Western blotting. Treatment with resveratrol had no effect on cell number or cell cycle profile, while treatment with genistein increased cell number. Because resveratrol could up-regulate VDR without increasing breast cancer cell growth, we hypothesized that resveratrol mediated increase in VDR expression would sensitize breast cancer cells to the effects of 1,25-dihydroxyvitamin D(3) and Vitamin D(3) analogs. In support of this hypothesis, both T47D and MCF-7 cells pre-treated with resveratrol exhibited increased VDR mediated transactivation of a Vitamin D(3) responsive promoter compared to cells pre-treated with vehicle. In addition, co-treatment with resveratrol enhanced the growth inhibitory effects of 1,25-dihydroxyvitamin D(3) and the Vitamin D(3) analog EB1089. These data support the concept that dietary factors, such as phytoestrogens, may impact on breast cancer cell sensitivity to Vitamin D(3) analogs through regulation of the VDR promoter.