Tumor Autonomous Effects of Vitamin D Deficiency Promote Breast Cancer Metastasis

Abstract

Patients with breast cancer (BCa) frequently have preexisting vitamin D deficiency (low serum 25-hydroxyvitamin D) when their cancer develops. A number of epidemiological studies show an inverse association between BCa risk and vitamin D status in humans, although some studies have failed to find an association. In addition, several studies have reported that BCa patients with vitamin D deficiency have a more aggressive molecular phenotype and worse prognostic indicators. However, it is unknown whether this association is mechanistically causative and, if so, whether it results from systemic or tumor autonomous effects of vitamin D signaling. We found that ablation of vitamin D receptor expression within BCa cells accelerates primary tumor growth and enables the development of metastases, demonstrating a tumor autonomous effect of vitamin D signaling to suppress BCa metastases. We show that vitamin D signaling inhibits the expression of the tumor progression gene Id1, and this pathway is abrogated in vitamin D deficiency in vivo in 2 murine models of BCa. These findings are relevant to humans, because we discovered that the mechanism of VDR regulation of Inhibitor of differentiation 1 (ID1) is conserved in human BCa cells, and there is a negative correlation between serum 25-hydroxyvitamin D levels and the level of ID1 in primary tumors from patients with BCa.

Figure 1.

Vitamin D and VDR deficiency promotes tumor aggressiveness. A, Volumes of MMTV-Wnt tumors grown in vitamin D sufficient (Con) (n = 10) and deficient (LD25) mice (n = 10). B, BLI of lungs and liver from mice with 168-FARN (n = 10) or 4T1 tumors (n = 17). C, RT-qPCR quantifying VDR mRNA expression levels in 168FARN and 4T1 cells. D, Western blottings measuring VDR levels in 168FARN cells with an empty vector control (Vec Ctrl) compared with 168FARN cells with VDR KD or VDR KD with reexpression of VDR (VDR Rescue). E, RT-qPCR quantifying the level of CYP24A1 mRNA in response to calcitriol treatment in Vec Ctrl compared with VDR KD and VDR Rescue cells. F, Transwell assays (upper) quantifying the average number of cells that migrated for each cell line (n = 3) and (lower) phase contrast images of stained cells from examples of each cell line (scale bar, 200 μm). *, P < .05; ***, P < .001. Error bars represent SEM.

Figure 2.

VDR deficiency promotes in vivo tumor autonomous growth and metastasis. A, Examples of in vivo BLI of mice with tumors generated from 168FARN cells with vector control (Vec Ctrl) or VDR KD or VDR KD with VDR reexpressed (VDR Rescue). B, Serial quantifications of BLI intensity from all mice (Vec Ctrl, n = 10; VDR KD, n = 10; VDR Rescue, n = 5) during in vivo tumor growth. Average tumor volumes (C) and tumor wet weights (D) from all mice. E, Examples and totals of BLIs of livers harvested from mice with tumors. ***, P < .001. Error bars represent SEM.

Figure 3.

Vitamin D/VDR regulates Id1 expression in BCa. A, RT-qPCR measuring Id1 expression levels in tumors isolated from mice with vitamin D deficiency (LD25) (n = 5) compared with vitamin D-sufficient mice (Con) (n = 3). B, RT-qPCR measuring Id1 expression levels in 168FARN cells with VDR KD compared with 168FARN cells with vector control (Vec Ctrl). C, RT-qPCR quantifying the level of Id1 expression in response to treating 168FARN cells with 10nM calcitriol (Cal) or vehicle control (Veh). D, Western blottings measuring ID1 and β-tubulin protein levels in Vec Ctrl and VDR KD cells in response to Cal treatment or Veh. E, Reprobe of Western blotting (Figure 1D) for ID1 protein levels in Vec Ctrl, VDR KD, and VDR Rescue cells. F, Luciferase assays quantifying the effect of 1nM Cal treatment on the transcriptional activity of the Id1 promoter and proximal elements. G, Chromatin immunoprecipitation (ChIP) of VDR in 168FARN cells showing treatment with Cal enhances occupancy of a known VDRE in the CYP24A1 gene (positive control) and there is no enrichment of VDR in the DNA region proximal to the nVDRE in ID1 (negative control). Cal increases occupancy of the nVDRE in ID1. H left, Transwell assays quantifying the number of migrating cells in Vec Ctrl (n = 3) or cells with Id1 KD (ID1 KD) (n = 3) compared with VDR KD (n = 3) and cells with both VDR and ID1 KD (VDR KD/ID1 KD) (n = 3). H right, Representative examples of phase contrast images of each of migrating cells from each condition (scale bar, 200 μm). *, P < .05; **, P < .01; ***, P < .001. Error bars represent SEM.

Figure 4.

Regulation of ID1 is conserved in human BCa. A, RT-qPCR quantifying the level of ID1 expression in response to treating MDA-MD-231 cells with 10nM calcitriol or vehicle control (Veh). B, Western blottings measuring ID1 and β-tubulin protein levels in MDA-MD-231 cells in response to a titration of calcitriol doses or Veh. C, Luciferase assays quantifying the effect of a titration of calcitriol doses or Veh on the transcriptional activity of the human ID1 promoter and proximal elements. D, Correlation between patient circulating serum 25(OH)D levels and the ID1 expression levels in their tumors. *, P < .05; ***, P < .001. Error bars represent SEM.