Biological actions of extra-renal 25-hydroxyvitamin D -1α-hydroxylase and implications for chemoprevention and treatment

Journal of Steroid Biochemistry & Molecular Biology 97 (2005) 103–109

Biological actions of extra-renal 25-hydroxyvitamin D-1␣-hydroxylase and implications for chemoprevention and treatment Kelly Townsend a , Katie N. Evans a , Moray J. Campbell a , Kay W. Colston b , John S. Adams c , Martin Hewison a,∗ a

Division of Medical Sciences, Institute of Biomedical Research, The University of Birmingham, Birmingham B15 2TH, UK b Department of Cellular and Molecular Medicine, St. George’s Hospital Medical School, London SW17 ORE, UK c Division of Endocrinology, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA

Abstract The Vitamin D-activating enzyme 25-hydroxyvitamin D-1␣-hydroxylase (1␣-hydroxylase) is now known to be expressed in a much wider range of tissues that previously thought, suggesting a role for 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), which is more in keeping with a cytokine than a hormone. In this capacity, the function of 1␣-hydroxylase in tumors is far from clear. Studies from several groups including ours have shown altered expression of 1␣-hydroxylase in different types of neoplasm including breast, prostate and colon cancers. However, functional analysis of Vitamin D metabolism in cancer is complicated by the heterogenous composition of tumors. Immunohistochemical analysis of breast tumors has shown that 1␣-hydroxylase is expressed by both epithelial cells and by tumor-infiltrating macrophages, suggesting an immunomodulatory component to 1,25(OH)2 D3 production in some types of cancer. The demonstration of 1␣-hydroxylase activity in tumors and their equivalent normal tissues has implications for both the treatment and prevention of cancers. For example, in tumors chemotherapy options may include the use of non-1␣-hydroxylated Vitamin D analogs to increase local concentrations of active metabolites without systemic side-effects. The role of 1␣-hydroxylase in protection against cancer is likely to be more complicated and may involve anti-tumor immune responses. © 2005 Elsevier Ltd. All rights reserved. Keywords: 25-Hydroxyvitamin D-1␣-hydroxylase; Tumors; Cancer

1. Introduction The active form of Vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), is a pluripotent seco-steroid with putative applications that extend far beyond its classical role as a regulator of calcium homeostasis. In particular, the anti-proliferative and immunomodulatory properties of 1,25(OH)2 D3 have promoted its use as therapy for autoimmune disease [1–3], transplantation rejection [1,3,4] and proliferative disorders such as psoriasis [5]. However, it is the possible use of 1,25(OH)2 D3 as an anti-cancer agent that has attracted most attention [6]. Studies in vitro have shown that 1,25(OH)2 D3 is able to block cell-cycle progression and influence apoptosis in tumor cells, although translation of these effects in vivo has been compromised by ∗

Corresponding author. Tel.: +44 121 414 3776; fax: +44 121 415 8712. E-mail address: [email protected] (M. Hewison).

0960-0760/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2005.06.004

significant hypercalcemic side-effects [7]. As a consequence there has been a concerted effort to improve the specificity of 1,25(OH)2 D3 therapy through the generation of synthetic analogs or deltanoids that retain the anti-proliferative properties of the hormone while minimising calciotropic sideeffects [3]. A more rational approach to this strategy has been facilitated by recent studies which have recognized that 1,25(OH)2 D3 signaling via nuclear Vitamin D receptors (VDR) is likely to be subject to gene and tissue-specific ‘tuning’ by virtue of their interaction with accessory proteins [8]. Strategies to improve the therapeutic index of 1,25(OH)2 D3 and its analogs remain a cornerstone of Vitamin D research. However, while most of this work has focused on the treatment of cancer, there is now increasing awareness of the potential role of 1,25(OH)2 D3 in tumor prevention. This has stemmed in part from studies of the impact of Vitamin D intake and status on cancer risk and

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Table 1 Expression and activity of 1␣-hydroxylase in renal and extra-renal tissues Tissue

RNA

Protein

Activity

References

Proximal tubules Distal nephron Epidermis (statum basalis) Placenta Decidua Brain (cerebellum/purkinje cells) Pancreas Colon Breast Ovary Vasculature (endothelial cells) Peripheral blood mononuclear cells Dendritic cells Macrophages Liver Heart Adrenal cortex Adrenal medulla Parathyroid T-cells B-cells Hair follicle

** *** ** ** *** **

** *** ** ** *** **

*** ** n.d. n.d. **, 1 n.d.

[14] [14] [16] [16,18] [16,18] [16,24]

** ** * * **

** ** ** * **

* n.d. * * *, 2

[16,21] [16,23]

–

–

–, 2

[20]

*** *** – – – * * – – n.d.

*** *** – – – * * n.d. n.d. ***

***, 2 ***, 2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d.

[20]

[22] [17]

[16] [16] [16] [16] [19]

[16]

Studies carried out by our group have analysed mRNA by in situ hybridization and real-time RT-PCR, protein by Western blot and immunohistochemistry, and activity by conversion of 3 H-25OHD3 to 3 H-1,25(OH)2 D3 . ***, strong expression or activity; *, weak expression or activity; 1, first trimester decidua; 2, assays carried out using primary cultures of cells; n.d., not done.

mortality. For example, exposure to sunlight, the principal mechanism involved in generating endogenous Vitamin D, has been inversely correlated with breast and prostate cancer mortality [9–12]. As serum concentrations of 1,25(OH)2 D3 are not specifically linked to Vitamin D intake or status the question then arises as to which, if any, of the Vitamin D metabolites is associated with protection against cancer. The most direct correlate of Vitamin D intake/status is the precursor metabolite 25-hydroxyvitamin D3 (25OHD3 ), the main circulating form of Vitamin D [13]. Although biologically inactive, 25OHD3 is converted to active 1,25(OH)2 D3 by the enzyme 25-hydroxyvitamin D-1␣-hydroxylase (1␣-hydroxylase) located classically in the proximal tubules of the kidney [14]. At this renal site, 1␣-hydroxylase acts to maintain adequate circulating levels of 1,25(OH)2 D3 as part of the body’s requirements for normal calcium homeostasis and bone metabolism [15]. However, studies over the last 20 years have shown that 1␣-hydroxylase is expressed by a variety of extra-renal tissues (Table 1), indicating that there may be localized synthesis of 1,25(OH)2 D3 in a manner which is distinct from that seen in the kidney [16–25]. The presence of 1␣-hydroxylase in extra-renal tissues raises several important questions concerning the normal physiology of Vitamin D as well as its therapeutic and chemopreventative applications: (1) What are the likely physiological actions of locally synthesized 1,25(OH)2 D3 ?;

(2) What are the differences between renal and extra-renal 1␣-hydroxylase?; (3) How is the gene for 1␣-hydroxylase (CYP27B1) regulated in pathological situations such as tumors or inflammatory disease? The following review seeks to address these questions with specific emphasis on the expression and function of 1␣-hydroxylase in tumors.

2. Extra-renal functions of 1␣-hydroxylase One of the first non-kidney cell types shown to be capable of synthesising 1,25(OH)2 D3 was the macrophage. This was particularly evident in macrophages that were activated either as a consequence of inflammatory disease [26], or as a result of an immune challenge in vitro [27]. Macrophages were therefore placed at the centre of the interaction between Vitamin D and the immune system with the 1,25(OH)2 D3 produced by these cells being able to influence other leukocytes in a paracrine cytokine-like fashion. More recently this observation has been extended to include other antigen-presenting cells (APC) such as dendritic cells (DCs), which have a similar capacity for 1,25(OH)2 D3 production as macrophages [20,28]. The fact that DCs, as sentinel cells, play a pivotal role in mediating the link between innate and acquired immune responses underlines the potential importance of 1,25(OH)2 D3 production by these cells. Expression and activity of 1␣-hydroxylase is strikingly upregulated in DCs as they mature towards differentiated professional APCs so that the resulting synthesis of 1,25(OH)2 D3 may have several effects on immune responses: (1) 1,25(OH)2 D3 synthesized by DCs in lymph nodes may act in a paracrine fashion to limit T-cell activation by DCs; (2) 1,25(OH)2 D3 may act by inhibiting DC maturation. This also appears to occur in a paracrine fashion as the induction of 1␣hydroxylase expression in maturing DCs is accompanied by decreased VDR expression, similar to that seen in macrophages [20,29]. Thus, inhibition of DC maturation would also result in suppression of T-cell activation but only after the ‘licensing’ or maturation of some DCs to facilitate a normal immune response [30]; (3) 1,25(OH)2 D3 synthesized by APCs may act on other facets of the immune response such as bacterial killing [31]. Irrespective of the end effect, the ability of APCs, particularly DCs, to produce 1,25(OH)2 D3 from 25OHD3 emphasises the fact that extrarenal sources of 1␣-hydroxylase are an important conduit for Vitamin D in normal physiology. This was highlighted by the recent generation of CYP27B1 knockout mice, which presented with multiple enlarged lymph nodes consistent with exaggerated DC-mediated antigen presentation and T-cell proliferation [32]. The presence of 1␣-hydroxylase at other extra-renal sites has shown that the capacity for localized synthesis of 1,25(OH)2 D3 may be common to many tissues. For example, keratinocytes are able to synthesize 1,25(OH)2 D3 but, in vivo, this appears to be limited to defined areas of the

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epidermis suggesting a subtle role for 1␣-hydroxylase in the sequential differentiation of the cornified layer [33]. This was confirmed in elegant studies of the CYP27B1 knockout mouse by Bikle et al. who demonstrated that although these animals lacked a gross epidermal phenotype, they showed abnormal transepidermal water loss consistent with a role for locally synthesized 1,25(OH)2 D3 in maintaining barrier integrity [34]. The expression of 1␣-hydroxylase by tissues such as placenta/decidua, distal nephron, vasculature, colon, hair follicle indicates that locally synthesized 1,25(OH)2 D3 may have a more generalized role in maintaining barrier integrity [30]. This may encompass several facets of tissue function including cell proliferation [35], regulation of junction protein expression [36] and immunoregulation [37]. It therefore seems likely that in normal physiology extra-renal 1␣-hydroxylase activity will involve both immune and non-immune cells (epithelial/endothelial cells) with autocrine/paracrine consequences that reflect these different cell types. To date the link between dysregulation of these events and Vitamin D deficiency has been restricted to limit studies including the analysis of colon cell proliferation [35] and alterations in cytokine production by immune cells [38]. Thus, while it is tempting to speculate that extra-renal 1␣-hydroxylase will act as the main mediator by which Vitamin D is postulated to protect against certain tumors, further studies are required before we can fully define the precise mechanism(s) by which this occurs.

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3. Expression of 1␣-hydroxylase in tumors A seminal observation that fuelled much of the current interest in extra-renal Vitamin D metabolism and cancer was the detection of 1␣-hydroxylase in human prostate cells [39]. This raised the exciting possibility that locally synthesized 1,25(OH)2 D3 could act in an autocrine fashion to regulate prostate cell proliferation, further supporting the idea a chemopreventative role for Vitamin D in cancer [39]. Subsequently, studies using primary cultures and cell lines showed that 1␣-hydroxylase activity was much lower in prostate cancer cells compared to normal cells [40]. Thus, whereas non-neoplastic prostate cells showed a dose-dependent decrease in proliferation when treated with either 1,25(OH)2 D3 or 25OHD3 , prostate cancer cells were only responsive to 1,25(OH)2 D3 . In unpublished studies carried out in our group we have observed the same loss of 1␣-hydroxylase expression in breast cancer cell lines, suggesting that loss of endogenous 1,25(OH)2 D3 production is a common feature of tumor cells. As noted in the prostate cells, tumor loss of 1␣-hydroxylase results in decreased responsiveness to 25OHD3 although this is readily restored with a relatively modest increase in CYP27b1 expression (see Fig. 1). Collectively, the above observations support the postulate that autocrine/paracrine synthesis of 1,25(OH)2 D3 is decreased in tumor cells and cell lines. However, the same may not necessarily be true of tumors in vivo. Analysis of ex

Fig. 1. Increased expression of 1␣-hydroxylase (CYP27b1), VDR and 24-hydroxylase (CYP24) in breast tumors compared to paired normal tissue. Data are shown as the fold increase in mRNA expression for individual tumor samples compared to matched normal samples with an arbitrary expression value of 1. Results are shown in order of ascending age at diagnosis for estrogen receptor (ER)-negative (left of panel) and ER-positive breast tumors. Data are mean values for n = 3 determinations.

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Fig. 2. Over-expression of 1␣-hydroxylase (CYP27b1) induces sensitivity to 25OHD3 in MCF-7 breast cancer cells. Transient expression of CYP27b1 cDNA into 1,25(OH)2 D3 -sensitive MCF-7 cells (MCF-7 + CYP27b1): (A) increases CYP27b1 protein expression; (B) induces sensitivity to the antiproliferative effects of 25OHD3 . Parental MCF-7 cells (MCF-7) or plasmidonly transfectants (MCF-7-neo) showed anti-proliferative responses to 1,25(OH)2 D3 only. * , Statistically different from vehicle-treated cells, p < 0.001.

vivo samples of normal colon and adjacent tumors did not reveal the same pattern of decreased 1␣-hydroxylase expression seen in prostate tumor cells [41–43]. Rather it appears that the enzyme is upregulated in differentiated tumors when compared to adjacent normal tissue but then suppressed in more aggressive undifferentiated tumors [42]. In a similar fashion, we have observed increased expression of 1␣-hydroxylase mRNA in breast tumors (Fig. 2). Immunohistochemical analysis of 1␣-hydroxylase protein confirmed this observation but also indicated that expression of the enzyme was not restricted to tumor cells but was also detectable in the inflammatory infiltrate associated with breast tumors (data not shown). Indeed, we were able to correlate levels of mRNA for CYP27b1 with mRNA for the macrophage marker CD14 and the toll-like receptor 4 (TLR4), and endotoxin receptor expressed by macrophages and epithelial cells both oh which are upregulated during pathological inflammation (see Fig. 3). Thus, in vivo, the expression and function of 1␣-hydroxylase in tumors appears to be complicated by their heterogeneous composition, and in particular the presence of cells from the immune system. We have made similar observations of increased macrophage 1␣-hydroxylase activity in two other types of tumor, B-cell lymphoma [44] and dysgerminoma [22]. This was not entirely surprising as both tumors have characteristic inflammatory infiltrates. Indeed the extent of the resulting 1␣-hydroxylase activity was sufficient to cause raised circulating levels of 1,25(OH)2 D3 in a manner that was highly reminiscent of the original studies of sarcoidosis [26]. As a novel form of humoral hypercalcemia

Fig. 3. 1␣-Hydroxylase mRNA expression in breast tumors correlates with CD14 and toll-like receptor 4 (TLR4).
Ct values (representing the level of expression of target gene minus housekeeping gene) from real-time RTPCR analysis of 1␣-hydroxylase mRNA expression were compared to
Ct values for CD14 (n = 18 tumors) and toll-like receptor 4 (TLR4) (n = 41 tumors). High
Ct , low level of expression; low
Ct , high level of expression. Statistical analysis of data is shown as Pearson correlation coefficients and associated p-values.

of malignancy due to aberrant 1␣-hydroxylase activity, lymphomas and dysgerminomas probably represent the extreme end of the spectrum in terms of extra-renal 1,25(OH)2 D3 production. Nevertheless, the fact that colon and breast tumors appear to retain significant levels of 1␣-hydroxylase activity, in part due to tumor heterogeneity, poses two important questions: (1) What is the impact, if any, of 1,25(OH)2 D3 on tumor interactions with the immune system?; (2) If 1␣-hydroxylase is still functionally active in tumors why is the resulting 1,25(OH)2 D3 ineffective in counteracting the tumor? The first of these questions is difficult to answer in the absence of any specific studies. To date, studies of the interaction between Vitamin D and the immune system have focused on the role of 1,25(OH)2 D3 as an immunsuppressive agent. In this context, one would predict that extra-renal 1␣-hydroxylase would act to support immune tolerance. This would of course be counterintuitive in terms of the anti-cancer effects of Vitamin D and there may be several explanations for this paradox. The first is that locally synthesized 1,25(OH)2 D3 may act to support innate immune

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responses associated with early anti-tumor immunity. Certainly, 1,25(OH)2 D3 potently suppresses DC maturation, thereby facilitating T-cell-mediated immune tolerance, but it also actively promotes macrophage differentiation and thus supports some innate immune functions. In a similar fashion, 1,25(OH)2 D3 promotes natural killer cell activity and this may also contribute to anti-tumor immunity [45]. A second explanation is that the function of 1␣-hydroxylase in extra-renal tissues is (as outlined above) essentially to maintain barrier integrity at a pre-neoplastic level, with macrophage/DC 1␣-hydroxylase acting as a secondary response to local inflammation. If this is the case then enhanced local synthesis of 1,25(OH)2 D3 would at best be ineffective in preventing tumor progression and may in fact exacerbate the problem by promoting tissue resistance to 1,25(OH)2 D3 . Much more work is required to fully define the dynamics of 1␣-hydroxylase expression and activity in the context of tumor immunology.

calciotropic effects of Vitamin D [48]. Elevated expression of 24-hydroxylase has been described in several types of tumor and the gene for this enzyme (CYP24) has been proposed as an oncogene [49]. Studies from our group using the tumor/normal samples outlined in Fig. 1 indicated that CYP24 expression is significantly higher in breast tumors compared to paired non-neoplastic tissue (p < 0.01). Furthermore, in normal breast tissue there was close correlation of CYP24 expression with CYP27b1 (p < 0.003) and VDR levels (p < 0.008), whereas in breast tumors there was no correlation. This suggests that induction of 24-hydroxylase in non-neoplastic tissue is dependent on local synthesis of 1,25(OH)2 D3 as well as VDR levels, consistent with a regulated system of feedback control. By contrast, in tumors CYP24 appears to be dysregulated independently and may therefore attenuate Vitamin D signaling irrespective of the levels of VDR or local production of 1,25(OH)2 D3 .

4. Tumor resistance to autocrine 1,25(OH)2 D3

5. 1␣-Hydroxyalse and cancer therapy and prevention

In seeking an explanation for why locally synthesized 1,25(OH)2 D3 may be inefficient in preventing tumor progression it is important to consider two other components of the Vitamin D metabolism and signaling axis. The first of these is the Vitamin D receptor (VDR) which although ubiquitous in proliferating cells, shows considerable variation in levels of expression and is therefore likely to have a significant effect on the ability of both normal and neoplastic cells to respond to 1,25(OH)2 D3 . The function and regulation of VDR expression has been studied in many different types of cancer but the importance of receptor levels in vivo has been most clearly demonstrated by recent studies of breast tumorogenesis in the VDR knockout mouse [46]. These animals (VDR −/−) showed abnormal mammary gland development when compared to wild type (VDR +/+) mice, with haploinsufficient (VDR +/−) mice exhibiting an intermediate phenotype. The haploinsufficient mice also showed increased mouse mammary tumor virus (MMTV)-stimulated tumor formation indicating that even a partial decrease in VDR expression had a significant effect on tumorogenesis. Despite this, in wild type mice (VDR +/+) the MMTV-derived tumors themselves showed an apparent increase in VDR expression suggesting that 1,25(OH)2 D3 signaling in tumors is not only dependent on the presence of receptor protein but other epigenetic factors such as co-activator and co-repressor proteins [47]. Another important consideration in tissue-specific resistance to 1,25(OH)2 D3 is the enzyme Vitamin D-24-hydroxylase (24-hydroxylase) which acts by generating less active Vitamin D metabolites such as 25,25-dihydroxyvitamin D3 or 1,24,25-trihydroxyvitamin D3 . Expression of 24-hydroxylase is sensitively upregulated by 1,25(OH)2 D3 in almost all tissues expressing VDR as part of a feedback-control mechanism to limit the potent

The presence of 1␣-hydroxylase in a wide variety of extra-renal tissues provides a plausible mechanism for the site-specific concentration of 1,25(OH)2 D3 . However, as yet, it is difficult to conclude absolutely that this is the model linking Vitamin D status and protection against common cancers. Further studies including tissue-specific knockouts for CYP27b1 are required to fully define the role of circulating versus local levels of 1,25(OH)2 D3 but it seems likely that increased availability of substrate for 1␣-OHase (25OHD3 ) will result in increased levels of 1,25(OH)2 D3 in some peripheral tissues. For example, in macrophages and DCs the absence of any significant 24-OHase activity means that synthesis of 1,25(OH)2 D3 is directly related to the amount of 25OHD3 . This is in stark contrast to the kidney where autoregulation and induction of CYP24 limit the amount of 1,25(OH)2 D3 being produce irrespective of substrate concentrations. Although this may provide an avenue for 25OHD3 responses in the immune system it remains to be seen whether a similar relationship occurs within other tissues. Extra-renal 1␣-hydroxlase expression may also provide an alternative strategy for cancer therapy, possibly through the use of analogs or deltanoids that can be metabolised more readily in peripheral tissues. The success of such as strategy will of course not only be dependent on tissue levels of 1␣-hydroxylase and its substrate, but also on the expression of 24-hydroxylase. Indeed in terms of tumor therapy, as opposed to tumor protection, 24-hydroxylase may be the rate limiting determinant of local 1,25(OH)2 D3 levels. It is therefore encouraging to note recent studies describing enzyme-specific inhibition of 24-hydroxylase as an adjunct to treatment with Vitamin D analogs [50], and strategies similar to these may help to realise the therapeutic potential of 1␣-hydroxylase.

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Acknowledgment The authors would like to acknowledge the support for this work from The Medical Research Council (KT, MH, MJC) and Breast Cancer Campaign (KWC).

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