Crosstalk between the vitamin D receptor (VDR) and miR-214 in regulating SuFu, a hedgehog pathway inhibitor in breast cancer cells

doi:10.1016/j.yexcr.2016.08.012

Experimental Cell Research

Volume 349, Issue 1, 15 November 2016, Pages 15-22

https://doi.org/10.1016/j.yexcr.2016.08.012 Get rights and content

Highlights

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An interplay between VDR and miR-214 is investigated in breast cancer cells.
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VDR overexpression in breast cancer cells reduces miR-214 expression levels by 30%.
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miR-214 inhibits VDR expression and vitamin D mediated signaling in breast cancer cells.
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VDR upregulates SuFu expression whereas, miR-214 inhibits SuFu in breast cancer cells.

Abstract

The vitamin D receptor (VDR), and its ligand 1α,25-dihydroxyvitamin D₃ (1,25D₃) prevent breast cancer development and progression, yet the molecular mechanisms governing this are unclear. MicroRNAs (miRNAs) on the other hand, promote or inhibit breast cancer growth. To understand how VDR regulates miRNAs, we compared miRNA expression of wild-type (WT) and VDR knockout (VDRKO) breast cancer cells by a Mouse Breast Cancer miRNA PCR array. Compared to VDR WT cells, expressions of miR-214, miR-199a-3p and miR-199a-5p of the miR-199a/miR-214 cluster were 42, 15, and 10 fold higher in VDRKO cells respectively. Overexpression of VDR in breast cancer cells reduced the miR-199a/miR-214 cluster expression by 30%. VDR status also negatively correlated with Dnm3os expression, a non-coding RNA transcript of the dynamin-3 gene encoding the miR-199a/miR-214 cluster, suggesting that VDR represses this cluster through Dnm3os. Conversely, overexpression of miR-214 in MCF-7 and T47D cells antagonized VDR mediated signaling. Furthermore, there was a positive correlation between VDR status and the expression of Suppressor of fused gene (SuFu), a hedgehog pathway inhibitor. miR-214 on the other hand suppressed SuFu protein expression. These findings suggest a crosstalk between VDR and miR-214 in regulating hedgehog signaling in breast cancer cells, providing new therapies for breast cancer.

Introduction

The vitamin D receptor (VDR), a nuclear hormone receptor and transcription factor has been shown to play a protective role against human cancers including breast cancer [1]. Specifically, breast cancer patients with high VDR expression levels had higher survival rates compared to patients with lower VDR levels [2]. Importantly, the presence of VDR in human breast cancer tissues has been attributed to reduced metastatic potential [3].

Furthermore, epidemiological and experimental studies have shown that certain VDR genetic variants reduce breast cancer risk [4], [5]. In animal models, normal mammary glands from VDR knockout (VDRKO) mice were more proliferative in the presence of hormones such as estrogen and progesterone in contrast to VDR wild-type (WT) glands [6]. Consistent with VDR's essential role in breast cancer prevention, ductal regions in the mammary glands of VDRKO mice were undifferentiated compared with ductal regions from their WT counterparts and importantly, VDR deficiency increased mammary gland sensitivity to chemical carcinogens [7], indicating an essential role for VDR in preventing breast cancer. In addition to breast cancer, VDR has been shown to play a protective role against other cancers [8], [9]. In VDRKO mice for example, key members of the oncogenic hedgehog pathway were highly expressed in the skin, thus increasing the susceptibility of these mice to skin cancer by 85%, compared with WT mice [10].

VDR orchestrates its transcriptional activity on target genes by binding to its ligand and active vitamin D metabolite, 1α,25-dihydroxyvitamin D₃ (1,25D₃). Once bound by 1,25D₃, VDR dimerizes with the 9-cis-retinoic acid receptor (RXRα) and this heterodimer then positively regulates gene expression by acting on vitamin D response elements (VDREs) in promoters of target genes [1]. Unlike transactivation, vitamin D suppression of target genes is not clearly defined. In a VDR dependent manner, 1,25D₃ adversely affects cancer cells including breast cancer cells by promoting cell differentiation (11), inhibiting growth and inducing apoptosis [1].

1,25D₃ has recently been shown to modulate microRNA (miRNA) expression and function in various cancers [11], [12], [13]. MiRNAs are small single stranded non-coding RNA molecules that generally regulate protein expression by translational repression or mRNA degradation of target mRNAs [14]. Translational repression occurs when an miRNA's seed sequence (2–7 or 2–8 nucleotides in the 5’-untranslated region, 5’-UTR), binds to complementary sequences in the 3’UTR of its target gene [15]. MiRNAs have recently been widely implicated in breast cancer. Tumor suppressor miRNAs such as the let-7 family members for instance, inhibit breast cancer growth by reducing ERα expression [15] and oncogenic miRNAs such as miR-155 have been implicated in breast cancer progression [16]. In line with these findings a differential role for the miR-199a/miR-214 cluster has been reported in various cancer types, including breast cancer [17], [18], [19], [20], [21]. Specifically, breast tissues from women diagnosed with sporadic triple negative breast cancer expressed high levels of miR-214, correlating with a poor survival rate [22].

Although studies investigating the effect of 1,25D₃ on miRNAs are emerging [12], [13], [14], the role of unliganded VDR on miRNA expression and function in breast cancer etiology is elusive. To this end, we employed a murine breast cancer miRNA PCR array of 84 genes to compare miRNA profiles in VDRKO and WT breast cancer cells. In the present study, we show that the expression of the miR-199a/miR-214 cluster (miR-199a-5, 3p and miR-214) was inversely correlated with VDR status in both human and mouse breast cancer cells. Furthermore, ectopic expression of miR-214 reduced VDR protein and mRNA expression levels and attenuated 1,25D₃ mediated signaling in breast cancer cells, suggesting that downregulation of VDR by miR-214 which activates the oncogenic hedgehog pathway promotes breast cancer development and progression to metastatic disease. These results for the first time reveal a novel negative regulatory loop connecting miRNA and VDR which may lead to the development of prognostic tools and new therapeutic approaches for breast cancer diagnosis and treatment.

Section snippets

Cell culture and reagents

The murine VDR knockout (VDRKO- 240) and wild-type (WT-145) mammary tumor cell lines have been described previously [23]. VDRKO-240 and WT-145 designated as VDRKO and WT hereafter, were maintained in DMEM/F12 media supplemented with 5% charcoal stripped fetal bovine serum and antibiotics (Invitrogen Life Technologies, Carlsbad, CA). The MCF-7 and T47D breast cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in MEM medium (Invitrogen) as

The vitamin D receptor negatively regulates the miR-199a/miR-214 cluster expression in breast cancer cells

Consistent with a potential role for VDR in miRNA regulation, nuclear hormone receptors such as the estrogen (ERα) and androgen receptors (AR) have been shown to regulate microRNA expression and function [25]. To ascertain whether the VDR genotype shows differential expression of known breast cancer miRNAs, we used a Mouse Breast Cancer miRNA PCR Array to compare expression of 84 miRNAs in mouse mammary tumor cells derived from VDR wild-type and knockout mice. The array analysis revealed

Discussion

In this study we identified unliganded VDR as a negative regulator of the miR-199a/miR-214 cluster expression and uncovered a negative regulatory feedback loop between miR-214, of the miR-199a/miR-214 cluster and VDR in breast cancer cells for the first time. Together these findings define a novel functional link between the VDR signaling pathway and miRNAs in breast cancer cells. High miR-214 levels in circulation have been correlated with metastasis in human breast cancer patients [37]. Thus,

Acknowledgments

We thank Dr. Vittorio Sartorelli (Laboratory of Muscle Stem Cells and Gene Regulation, National Institutes of Health, Bethesda, MD) for generously providing the pcDNA-miR-214 expression plasmid. This work was supported by the UNCF/MERCK Postdoctoral Science Research Fellowship (2011) (FA) and National Institutes of Health (NIH), Grant R01-140321 (RGM).

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Research ArticleCrosstalk between the vitamin D receptor (VDR) and miR-214 in regulating SuFu, a hedgehog pathway inhibitor in breast cancer cells

Highlights

Abstract

Introduction

Section snippets

Cell culture and reagents

The vitamin D receptor negatively regulates the miR-199a/miR-214 cluster expression in breast cancer cells

Discussion

Acknowledgments

J. Investig. Dermatol.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Mol. Cell Endocrinol.

FEBS Lett.

J. Biol. Chem.

Curr. Opin. Pharm.

Trends Cell Biol.

Cell

Differentiation

J. Biol. Chem.

The role of vitamin D in reducing cancer risk and progression

Nat. Rev. Cancer

Vitamin D pathway gene polymorphisms, diet, and risk of postmenopausal breast cancer: a nested case-control study

Breast Cancer Res.

VDR status arbitrates the prometastatic effects of tumor-associated macrophages

Mol. Cancer Res.

FokI polymorphism of vitamin D receptor gene contributes to breast cancer susceptibility: a meta-analysis

Breast Cancer Res. Treat.

Functional significance of vitamin D receptor FokI polymorphism in human breast cancer cells

PLoS One

Vitamin D(3) receptor ablation alters mammary gland morphogenesis

Development

Effect of Vitamin D3 receptor ablation on murine mammary gland development and tumorigenesis

J. Steroid Biochem. Mol. Biol.

The vitamin D receptor: a tumor suppressor in skin

Disco. Med.

Using components of the vitamin D pathway to prevent and treat colon cancer

Nutr. Rev.

MicroRNA-22 is induced by vitamin D and contributes to its antiproliferative, antimigratory and gene regulatory effects in colon cancer cells

Hum. Mol. Genet.

Research Article
Crosstalk between the vitamin D receptor (VDR) and miR-214 in regulating SuFu, a hedgehog pathway inhibitor in breast cancer cells