Epigenetic regulation of Vitamin D hydroxylase expression and activity in normal and malignant human prostate cells

Abstract

It was previously suggested that the 25-Vitamin-D₃-1α-hydroxylase (CYP27B1) is downregulated during human prostate tumor pathogenesis while the catabolic 25-Vitamin-D₃-24-hydroxylase (CYP24) expression is increased. The latter could lead to resistance against the antimitotic, prodifferentiating activity of 1,25-dihydroxycholecalciferol. Our hypothesis was that regulation of Vitamin D hydroxylase expression during prostate tumor progression might be under epigenetic control. We demonstrate by real time RT-PCR that PNT-2 human normal prostate cells indeed possess CYP27B1, but are practically devoid of CYP24 mRNA, whereas DU-145 cancer cells have constitutive expression of CYP24, and very low levels of CYP27B1 mRNA. Treatment of PNT-2 cells with the methylation inhibitor 5-aza-2′-deoxycytidine together with the deacetylation inhibitor trichostatin A resulted in elevation of both CYP27B1 and CYP24 mRNA expression demonstrating that even in normal human prostate cells expression of Vitamin D hydroxylases may be under epigenetic control. In the DU-145 malignant cell line trichostatin A together with 5-aza-2′-deoxycytidine increased CYP27B1 mRNA expression to a smaller extent than in normal cells, however this resulted in a highly significant increase in 1α-hydroxylation capacity. This demonstrates for the first time that synthesis of 1,25-dihydroxycholecalciferol in human prostate tumors could be reinitiated by epigenetic regulators.

Introduction

1,25-Dihydroxyvitamin-D₃ (1,25-D₃), the most active metabolite of Vitamin D₃, not only regulates calcium homeostasis and bone mineralization, but also plays an important role in modulating proliferation, differentiation and function in a large number of cell types. For example, 1,25-D₃ induces cell cycle arrest, differentiation and apoptosis in a variety of malignant cell lines including prostate cancer cells, see e.g. [1]. The hormone exerts genomic actions by binding to the nuclear Vitamin D receptor (VDR), which then functions as a ligand-activated transcription factor [2], [3].

1,25-D₃ is endogenously synthesized in a multistep pathway from Vitamin D₃, which is produced under the influence of solar UV-B radiation in the skin, or is absorbed from dietary sources in the small intestine. Vitamin D₃ is hydroxylated first at position C-25 in the liver by the cytochrome P450 enzyme CYP27A1, and subsequently at C-1α by the 25-D₃-1α-hydroxylase (CYP27B1) in the kidney but also at several extra-renal sites, including the prostate gland. Hydroxylation of 1,25-D₃ at C-24 by the 25-D-24-hydroxylase, CYP24, initiates inactivation and degradation of the steroid.

Unfortunately, the antimitotic effects of 1,25-D₃ and its analogues cannot be exploited in cancer prevention or therapy due to the resultant hypercalcemia from pharmacological doses needed for patient treatment [4]. However, a new approach in cancer prevention was deemed feasible since expression of VDR and of the key enzymes in Vitamin D metabolism, i.e., CYP27B1 and CYP24, was detected also at extra-renal sites, see e.g. [5], [6], and this tissue-localized synthesis of 1,25-D₃ could be important for control of cell growth and function by 1,25-D₃ in various organs. In this respect, we demonstrated a rise in mRNA and protein expression levels of CYP27B1 and of VDR during early human colon tumor progression [7], [8], suggesting that locally produced 1,25-D₃ could be involved in an autocrine/paracrine defense mechanism for prevention of cancer progression. However, this does not appear to be the case in human prostate cancer since there is evidence that already during the transition from normalcy into the premalignant state, prostate cells loose the capability to synthesize 1,25-D₃ [9]. In addition, it has been shown that some prostate cell lines in vitro express high levels of the catabolic CYP24, which apparently degrades 1,25-D₃ and therefore renders these particular cells unresponsive to Vitamin D-mediated growth inhibition. However, when CYP24 activity was blocked with liarozole, an inhibitor of cytochrome P₄₅₀ enzymes, cells again became responsive to 1,25-D₃ [10].

It is well recognized that multiple factors contribute to the development of human prostate carcinoma and to its progression to an androgen-independent status. In addition to the expected role of androgens and their receptors in facilitating development of this malignancy, many somatic mutations, gene deletions and amplifications, as well as epigenetic DNA changes are detectable in prostate cancer cells at diagnosis. Such epigenetic modulations which do not alter the DNA base sequence are also implicated in aging and in development of many other tumors [11]. Recently, a large number of papers has appeared on regulation of gene expression by methylation during prostate cancer development. Several of them reported on global gene methylation patterns, see e.g. [12], [13], whereas others reported on methylation changes of certain genes especially during progression from normal to cancerous prostatic tissue, see e.g. [14], [15].

DNA methylation could play a role in regulating the Vitamin D system: expression of the VDR in an animal model for chemically induced colon cancer was decreased in colonic tissue compared with normal mucosa due to methylation of VDR CpG islands [16]. In human breast tumors hypermethylation of the CYP27B1 promoter was detected [17]. We have recently demonstrated in prostate tumor cells that a deacetylation inhibitor can increase transcription of CYP27B1 [18]. Thus, epigenetic mechanisms may enhance gene expression of CYP27B1 in vivo and influence the potential autocrine/paracrine mitotic control by Vitamin D in human tissue, especially if also the catabolic 24-hydroxylation would be downregulated. Since it was apparent from human prostate tissue studies that the methylation pattern of various genes was rapidly changing during progression of the disease we decided to compare the epigenetic regulation of CYP27B1 and of CYP24 expression in normal with that in a highly malignant human prostate cell line, namely PNT-2 cells and the DU-145 cell line.