Mammary-specific ablation of Cyp24a1 inhibits development, reduces proliferation and increases sensitivity to vitamin D

Highlights

• Vitamin D catabolism via Cyp24a1 modulates postnatal and pregnancy-associated mammary gland development.

• Cyp24a1 gene deletion in the mammary epithelium inhibits luminal epithelial proliferation but not apoptosis.

• Luminal mammary epithelial cells are more sensitive to 1,25(OH)2D with the ablation of Cyp24a1 activity.

Abstract

Active vitamin D (1,25(OH)2D) has been shown to regulate numerous cell processes in mammary cells. Degradation of 1,25(OH)2D is initiated by the mitochondrial enzyme, 25-hydroxyvitamin D 24-hydroxylase (CYP24 A1), and provides local control of 1,25(OH)2D bioactivity. Several reports of the association between elevated CYP24 A1 activity and breast cancer incidence, suggest that CYP24 A1 may be a target for therapeutic intervention. Whether CYP24 A1 activity within the mammary epithelium regulates 1,25(OH)2D levels and mammary gland development is yet to shown. We have used a conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium to demonstrate reduced terminal end bud number, ductal outgrowth and branching during puberty and alveologenesis at early pregnancy, by inhibiting proliferation but not apoptosis in both basal and luminal MECs. In vitro study showed increased sensitivity of luminal MECs to lower levels of 1,25(OH)2D with the ablation of Cyp24a1 activity. In summary, Cyp24a1 within MECs plays an important role in modulating postnatal and pregnancy-associated mammary gland development which provides support for inhibiting CYP24 A1 as a potential approach to activating the vitamin D pathway in breast cancer prevention and therapy.

Introduction

Vitamin D deficiency is well known to directly impact on numerous systems including calcium homeostasis and skeletal pathology as well as in other systems including immune function, cardiovascular health and reproductive health. The association between vitamin D deficiency and cancer incidence has also been demonstrated in numerous different settings including breast cancer [[1], [2], [3], [4]], as reviewed in [5,6]. Furthermore, several reports suggest that vitamin D may play a role in breast cancer therapy [6,7] and in predicting disease recurrence and survival [[8], [9], [10]].

The mechanisms by which vitamin D purportedly exerts breast cancer-protective actions are diverse (reviewed in [11]). These include alterations in cell cycle regulators to bring about G1 arrest, interference with estrogen receptor (ER) and Insulin-like Growth Factor-1 signaling, induction of differentiation or apoptosis [[12], [13], [14]], inhibiting tumor initiation and metastasis [15,16], regulating NF-Kappa B signaling [17], reducing cell oxidative stress and DNA damage, suppression of cell senescence inactivating oncogenes [18], activation of tumor suppressor genes [19], and the suppression of aromatase enzyme activity [20]. In human breast epithelium, vitamin D receptor (VDR) is expressed in luminal mammary epithelial cells, and was found to partially overlap with ER- and androgen receptor (AR)-expressing cells [21]. Estrogen and progesterone, signaling via their respective receptors (ER and PR), are required for mammary epithelium function, which are also modulated by VDR [1,[22], [23], [24]]. Indeed, direct interaction and genomic repression of ER activity by VDR has been reported to be a mechanism through which vitamin D exerts growth-suppressive actions in breast cancer cells [7].

In addition to VDR, the regulation of vitamin D activity at target tissue level is governed by catabolic actions of 25-hydroxyvitamin D 24-hydroxylase (CYP24A1). CYP24 A1 is expressed in most tissues throughout the body and wherever VDR is expressed [25]. Under normal circumstances, CYP24 A1 is induced in response to elevated 1,25(OH)₂D levels [26]. However, in invasive breast cancer cells and tissues, CYP24 A1 gene amplification [9,27,28], as well as higher CYP24 A1 mRNA and protein expression, have been observed when compared to normal or in situ counterparts and independent of 1,25(OH)₂D induction [29,30]. While it is uncertain whether CYP24 A1 over-activity is causal of or is a consequence of cancer progression, the therapeutic use of vitamin D or vitamin D analogues may be relatively ineffective in breast cancer where CYP24 A1 activity is shown to be aberrantly high. Even less certain is the role of CYP24 A1 activity in normal mammary epithelial function. Therefore, this study aimed to establish the effects of Cyp24a1 ablation specifically in mouse mammary epithelial cells (MECs) by assessing changes in gross morphology, proliferation and apoptosis of MECs in vivo, and the sensitivity of MECs to vitamin D in vitro.

This study demonstrates that mouse MEC-specific Cyp24a1 ablation reduces terminal end bud number, ductal outgrowth and branching during puberty and the formation of terminal branches during early pregnancy, by inhibiting proliferation in both basal and luminal MECs while not altering the rate of apoptosis. Furthermore, Cyp24a1 deletion in luminal MECs results in increased sensitivity to the growth-suppressive actions of vitamin D. Overall this study confirms that inhibiting Cyp24a1 directly targets mouse MECs to suppress growth, proliferation and development of the mammary epithelium. These findings provide support for the concept of inhibiting CYP24 A1 either as a functional alternative or an adjunct to activating the vitamin D pathway in breast cancer prevention and therapy.