Gene Signatures of 1,25‐Dihydroxyvitamin D₃ Exposure in Normal and Transformed Mammary Cells

Abstract

To elucidate potential mediators of vitamin D receptor (VDR) action in breast cancer, we profiled the genomic effects of its ligand 1,25-dihydroxyvitamin D3 (1,25D) in cells derived from normal mammary tissue and breast cancer. In non-transformed hTERT-HME cells, 483 1,25D responsive entities in 42 pathways were identified, whereas in MCF7 breast cancer cells, 249 1,25D responsive entities in 31 pathways were identified. Only 21 annotated genes were commonly altered by 1,25D in both MCF7 and hTERT-HME cells. Gene set enrichment analysis highlighted eight pathways (including senescence/autophagy, TGFβ signaling, endochondral ossification, and adipogenesis) commonly altered by 1,25D in hTERT-HME and MCF7 cells. Regulation of a subset of immune (CD14, IL1RL1, MALL, CAMP, SEMA6D, TREM1, CSF1, IL33, TLR4) and metabolic (ITGB3, SLC1A1, G6PD, GLUL, HIF1A, KDR, BIRC3) genes by 1,25D was confirmed in hTERT-HME cells and similar changes were observed in another comparable non-transformed mammary cell line (HME cells). The effects of 1,25D on these genes were retained in HME cells expressing SV40 large T antigen but were selectively abrogated in HME cells expressing SV40 + RAS and in MCF7 cells. Integration of the datasets from hTERT-HME and MCF7 cells with publically available RNA-SEQ data from 1,25D treated SKBR3 breast cancer cells enabled identification of an 11-gene signature representative of 1,25D exposure in all three breast-derived cell lines. Four of these 11 genes (CYP24A1, CLMN, EFTUD1, and SERPINB1) were also identified as 1,25D responsive in human breast tumor explants, suggesting that this gene signature may prove useful as a biomarker of vitamin D exposure in breast tissue.

Constitutively active RAS signaling reduces 1,25 dihydroxyvitamin D-mediated gene transcription in intestinal epithelial cells by reducing vitamin D receptor expression

“…Although RAS transformation inhibits 1,25(OH) 2 D action by phosphorylation of the VDR heterodimeric partner RXRα in prostate epithelial cells and keratinocytes (10,34,36) , our new data suggest that the primary mechanism for RAS-mediated inhibition of 1,25(OH) 2 D action in intestinal cells is due to a 70% reduction of VDR gene expression. This is consistent with other groups that have reported that Ras-activating mutations reduce VDR mRNA and/or protein levels in NIH3T3 cells (H-Ras (37) ), non-small cell lung cancer cell lines (K-Ras (12) , and mouse mammary epithelial cells (H-Ras (11,32) ). Thus, VDR loss may be a critical contributor to Ras-mediated transformation and VDR loss may reduce the beneficial effects of high vitamin D status on cancer progression.…”

“…For example, others have found that VDR mRNA or protein levels are reduced in human colon tumors (7,28) and other cancers (29–31) . Forty percent of colon tumors have mutations in the K-RAS gene (9) and previous research has shown that RAS activating mutations suppress 1,25(OH) 2 D action in some cells (10–12,32–34) but enhance 1,25(OH) 2 D action in MG-63, HeLa, and COS-1 kidney cells (33,35) .…”

Vitamin D: Metabolism, Molecular Mechanism of Action, and Pleiotropic Effects

“…A recent whole genome profiling on cells derived from normal mammary tissue and breast cancer reveals the regulation of a set of immune genes by 1,25(OH) 2 D 3 . Among these genes, soluble CD14b, which may contribute to the protection against breast cancer, seems to be consistently induced by 1,25(OH) 2 D 3 (412). Induction of CD14 by 1,25(OH) 2 D 3 is confirmed by a microarray study on fresh tumor slices exposed to 1,25(OH) 2 D 3 (311).…”

“…Intriguingly, these results are indicative of a role of the VDR as an enhancer of cell proliferation (96). Microarray profiling of nontumorigenic mammary epithelial cells after treatment with 1,25(OH) 2 D 3 revealed the regulation of a whole set of metabolic genes such as SLC1A1 and GLUL by 1,25(OH) 2 D 3 (412). SLCA1 encodes a plasma membrane glutamate transporter that is induced by 1,25(OH) 2 D 3 , whereas GLUL, which encodes glutamine synthetase, is repressed by 1,25(OH) 2 D 3 .…”

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

“…Interestingly, the induction of SLC1A1 by 1,25D observed in hTERT-HME1, HME and DCIS.com cells was abrogated in MCF10A cells (which have MYC amplification) and in breast cancer cells MCF7 and Hs578T (10). In addition, induction of SLC1A1 gene expression by 1,25D was blunted in HME cells expressing SV-40 (HME-LT cells) and those expressing SV-40 plus oncogenic RAS (HME-PR cells) (11). These studies suggest that 1,25D enhances SLC1A1 expression in normal mammary epithelial cells but that expression of this gene and its regulation by 1,25D is often abrogated in breast cancer cells.…”