Susan G Komen  
I've Been Diagnosed With Breast Cancer Someone I Know Was Diagnosed Share Your Story Join Us And Stay Informed Donate To End Breast Cancer
    Home > Research & Grants > Grants Program > Research Grants > Research Grants Awarded > Abstract
    Awarded Grants
    Vitamin D3 and Wnt/B-catenin Signaling in Mammary Cells

    Scientific Abstract:
    Vitamin D3 and Wnt/B-catenin signaling in mammary cells The biologically active form of vitamin D3, 1a,25 dihydroxyvitamin D3 (1,25D3), is a steroid hormone that mediates effects on calcium homeostasis through the nuclear vitamin D3 receptor (VDR). The vitamin D3 endocrine system also regulates proliferation, differentiation, and apoptosis of transformed cells of the mammary gland. In clinical studies, low serum levels of 1,25D3 correlate with increased breast cancer risk, disease progression, and metastasis. 1,25D3 inhibits growth of estrogen-dependent and estrogen-independent breast tumor cells and the VDR is present in over 80% of breast tumors, making the vitamin D3 signaling pathway an attractive target for treatment of early and late stage breast cancers. Furthermore, we have shown that the VDR is present in all of the major cell types of the normal mammary gland and that its expression is not temporally or spatially uniform. In the absence of VDR, precocious alveolar development, increased milk release and a delay in mammary gland regression is observed. Despite these consistent data supporting an important protective role for Vitamin D3 and its receptor in breast cancer, the specific signaling pathways regulated by the VDR in mammary gland have yet to be identified. Our hypothesis is that 1,25D3, through the VDR, targets the Wnt/B-catenin pathway to reduce mammary gland sensitivity to transformation. A proportion of human breast carcinomas express high levels of WNT-2, WNT-5A, and WNT-7B which control various aspects of development including cell fate determination and neoplastic transformation. Wnts are secreted factors that regulate cell growth, motility, and differentiation via stabilization and translocation of B-catenin to the nucleus. Unregulated accumulation of B-catenin in the nucleus can lead to neoplasia as a result of transcriptional activation of genes involved in the control of cell proliferation and invasiveness such as c-myc and cyclin D1. B-catenin also participates in the maintenance of cell adhesion at the cell membrane by binding E-cadherin and B-catenin, maintaining cell polarity and cell junctions, which are disrupted when B-catenin translocates to the nucleus. Animals ectopically expressing genes in the Wnt pathway develop mammary tumors with squamous differentiation. Several lines of research point to interactions between VDR and Wnt signaling. In colon cancer cells, 1,25D3 inhibits transcriptional activity of B-catenin, sequestering B-catenin at the plasma membrane with E-cadherin and may also stimulate B-catenin nuclear export. In previous Komen funded studies, we observed that mammary tumors induced by a chemical carcinogen, dimethylbenz-anthracene (DMBA), that develop in VDR knockout (VDRKO) mice exhibit squamous differentiation characteristic of activation of the Wnt pathway. In this project, we will employ both in vitro and in vivo approaches to investigate the impact of 1,25D3 on Wnt/B-catenin signaling. In Aim 1, we will utilize frozen and paraffin embedded mammary glands from wild type (WT) and VDRKO mice to examine if lack of the VDR in mammary gland alters B-catenin gene expression and/or localization. We will also compare B-catenin expression in DMBA induced mammary tumors derived from VDRKO and WT mice. In Aim 2, we will utilize DMBA induced WT and VDRKO mouse mammary tumor cell lines to assess whether B-catenin expression and localization differ between the two cell lines and to examine whether reintroduction of VDR into VDRKO mammary tumor cells will alter B-catenin localization. In Aim 3, we will characterize the effects of 1,25D3 on Wnt/B-catenin signaling in normal human mammary epithelial cells (HME-TERT) which we have already demonstrated are VDR positive and growth inhibited by 1,25D3. These studies will utilize molecular, cellular, and immunohistochemical techniques to assess the interaction of Vitamin D and the Wnt signaling pathway, offering insight into a treatment option that inhibits proliferation, induces differentiation and apoptosis, and inhibits metastatic spread of breast cancer cells. Demonstration of an interaction between Vitamin D and Wnt/B-catenin signaling cascade would suggest a novel mechanism by which VDR regulates growth within the mammary gland.

    Lay Abstract:
    Vitamin D3 and Wnt/â-catenin signaling in mammary cells Vitamin D, a steroid hormone, can be obtained from diet or produced in the skin after sun exposure. Vitamin D binds its receptor, the vitamin D receptor (VDR), and participates in the maintenance of calcium and phosphorus levels in the body. Vitamin D also participates in regulating the growth of cancer cells. Vitamin D, by binding to the VDR, induces transformed cells to stop proliferating or even undergo cell death. In clinical studies, low serum levels of Vitamin D correlate with increased breast cancer risk, disease progression, and metastasis. Vitamin D is effective at regulating the growth of estrogen-dependent as well as estrogen-independent breast cancer cells and the VDR is present in over 80% of breast tumors, making the vitamin D signaling pathway an attractive target for treatment of early and late stage breast cancers. Our lab has shown that the VDR is present in all major cell types of the developing mammary gland. Mice that lack a functional VDR have increased mammary gland development during puberty, premature mammary gland development during pregnancy, increased milk secretion during lactation, and delay in mammary gland regression after weaning. Despite these consistent data supporting an important protective role for Vitamin D and its receptor in breast cancer, the specific pathways regulated by the VDR in mammary gland have yet to be identified. Our hypothesis is that Vitamin D, by binding the VDR, targets pathways that reduce mammary gland sensitivity to mammary transformation. One particular gene that is found in human breast carcinomas is the Wnt protein which controls various aspects of development including cell fate determination and neoplastic transformation. Wnts are factors that are secreted from the cell to regulate cell growth, motility, and differentiation via stabilization and relocation of B-catenin, a protein normally found at the cell surface. In response to Wnt, B-catenin moves from the cell membrane to the nucleus and can activate growth stimulatory genes and inactivate anchoring genes. Animals that highly express these Wnt induced genes have mammary tumors with squamous differentiation. However, there is evidence that the VDR interacts with the Wnt signaling within the cell. In colon cancer cells, Vitamin D inhibits activation of B-catenin, sequesters B-catenin at the cell membrane and may also stimulate B-catenin export from the nucleus. Additionally, in previous Komen funded studies, we observed that mammary tumors that develop in mice without a functional VDR exhibit squamous differentiation characteristic of activation of the Wnt signaling pathway. In this project, we will employ both studies on cells and studies on animals to investigate the impact of Vitamin D on Wnt/B-catenin signaling. In Aim 1, we will utilize mammary glands from wild type (WT) mice containing a functional VDR, and from mice lacking a functional VDR (VDRKO) to examine if lack of the VDR in mammary gland alters B-catenin gene expression and/or where this protein is located within the cell. We will also compare B-catenin expression in mammary tumors derived from VDRKO and WT mice. In Aim 2, we will utilize WT and VDRKO mouse mammary tumor cell lines to assess whether B-catenin expression and localization differ between the two cell lines and to examine whether reintroduction of VDR into VDRKO mammary tumor cells will alter B-catenin localization. In Aim 3, we will characterize the effects of Vitamin D on Wnt/B-catenin signaling in normal human mammary epithelial cells which have a functional VDR and are growth inhibited by Vitamin D. These studies will utilize molecular, cellular, and immunohistochemical techniques to assess the interaction of Vitamin D and the Wnt signaling pathway, offering insight into a treatment option that inhibits proliferation, induces differentiation and cell death, and inhibits metastatic spread of breast cancer cells. Demonstration of an interaction between Vitamin D and Wnt/B-catenin signaling cascade would suggest a novel mechanism by which VDR regulates growth within the mammary gland.