"Vitamin D Metabolism Implications for Treatment in Oncology"
Vitamin D Metabolism: Implications for Treatment in Oncology with increased tumour incidence. This relationship V itamin D and its analogues are poised to play an increasingly important role in the was first established nearly 30 years ago by Garland prevention and treatment of cancer. However, and Garland who reported higher colon cancer as observed in the use of other anticancer agents, mortality rates in geographical locations where tumours evolve to establish mechanisms of resistance. populations were exposed to lower amounts of In the case of vitamin D, the cytochrome P450 enzyme sunlight . Since then, numerous large scale CYP24 has emerged as a potential barrier to optimal epidemiological studies have demonstrated an therapy. Through its catabolic activity, CYP24 tightly association between low vitamin D exposure (e.g. regulates levels of vitamin D metabolites: the sunlight or diet) or low circulating 25(OH)D3 levels prohormone, 25-hydroxyvitamin D3 (25(OH)D3) and and an increased risk of cancer that include colorectal, Tina Epps, PhD, the biologically active form, 1α,25-dihydroxyvitamin breast and prostate. Conversely, higher circulating Technology Analyst at D3 (1α,25(OH)2D3). Enhanced expression of CYP24 25(OH)D3 levels has also been linked to a reduction in Cytochroma Inc, Markham, through normal and oncogenic mechanisms in cancer-related mortality and improvement in overall ON, Canada. cancerous tissue may cause decreased tumour survival in some cancers. responsiveness to 1α,25(OH)2D3. Furthermore, the Experimental studies have also provided strong combination of elevated CYP24 with increasing evidence in support of a role for vitamin D in cancer. tumour burden may also contribute to systemic In a variety of animal models, vitamin D deficiency vitamin D deficiency which itself may have has been shown to enhance tumourigenesis. Some of consequences on patient health. Selective and potent these studies have further shown that treatment with inhibition of CYP24 activity may provide a novel vitamin D metabolites or its analogues reduces therapeutic approach to maximise the antiproliferative tumour burden and metastasis. The protective action activity and effectiveness of vitamin D in the of vitamin D against carcinogenesis has been ascribed prevention and treatment of cancer. to the ability of 1α,25(OH)2D3 to induce cell cycle arrest, differentiation and apoptosis at the cellular Martin Petkovich, Vitamin D and Cancer level. Antiangiogenic activity and reduction in tumour PhD, Vitamin D deficiency, due to low dietary intake and/or invasiveness by 1α,25(OH)2D3 may further contribute Chief Scientific Officer at inadequate exposure to sunlight, has been associated to its antitumour effects in animals. Epidemiological Cytochroma Inc, Markham, ON, Canada Professor of Biochemistry, Pathology & Molecular Medicine, Figure 1: Vitamin D signalling pathway. Cancer Research Institute, Queen’s University, Kingston, ON, Canada. Correspondence: Cytochroma, Inc, 330 Cochrane Drive, Markham, ON, Canada, L3R 8E4. Tel: +1 905 479 5306 Email: martin.petkovich@ cytochroma.com 42 Volume 4 Issue 2 • May/June 2009 tissues external to the kidney, permitting localised conversion of 25(OH)D3 to 1α,25(OH)2D3 and consequent VDR activation. These observations suggested a spectrum of biological actions of 1α,25(OH)2D3 extending well beyond its classical role in regulating bone and mineral homeostasis. Indeed, activation of the vitamin D signaling cascade has been shown to directly initiate or suppress transcription of key genes involved in cell signaling pathways important in cell cycle control, differentiation and apoptosis. Downregulation of CYP27B1 and/or VDR expression has been observed in various types of human cancer, suggesting that perturbations in these mechanisms important for 1α,25(OH)2D3 synthesis and action may contribute to cancer pathogenesis and progression. The CYP24 gene encodes the enzyme 24-hydroxylase which is primarily responsible for governing the vitamin D signaling pathway through inactivation of 1α,25(OH)2D3 by 24-hydroxylation to generate 1α,24,25-tri-hydroxyvitamin D3 (1α,24,25(OH)3D3). Although 1α,25(OH)2D3 is the preferred substrate of CYP24, the circulating prohormone 25(OH)D3 can also be catabolised by CYP24 to yield 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). At present, no additional substrates of CYP24 have been identified. In vitamin D target cells, expression of CYP24 is elevated as the cellular level of 1α,25(OH)2D3 rises. From relatively low basal levels, CYP24 expression can be induced by several orders of magnitude by 1α,25(OH)2D3 via direct VDR-mediated transcriptional activation involving specific vitamin D response elements (VDREs) in the CYP24 promoter region. Thus, through its induction of CYP24 expression, 1α,25(OH)2D3 autoregulates its own cellular levels to limit potentially deleterious effects of acute or prolonged exposure. Conversely, deletion of the CYP24 gene in mice delays 1α,25(OH)2D3 clearance, leading to a prolonged elevation in 1α,25(OH)2D3 levels and signs of vitamin D toxicity that include renal calcification, hypercalcaemia and hypercalcuria . Given that CYP24 can Figure 2: CYP24 inhibitors increase sensitivity to 1α,25(OH)2D3. A) Over-expression catabolise 1α,25(OH)2D3 with very high efficiency, abnormally of CYP24 leads to decreased 1α,25(OH)2D3 levels and increased tumour resistence elevated levels of CYP24 can effectively purge vitamin D from tumour to anti-cancer effects of 1α,25(OH)2D3. B) Pure CYP24 inhibitors inhibit CYP24 cells and form a barrier, rendering cells insensitive to the anticancer activity and enhance tumour sensitivity to anti-cancer effects by increasing 1α,25(OH)2D3. action of exogenous 1α,25(OH)2D3 (Figure 2). and experimental data linking vitamin D and cancer risk provides CYP24 and Cancer compelling evidence for the potential use of vitamin D therapeutics The CYP24 gene is located in a chromosomal region frequently in cancer prevention and treatment. amplified in tumours. This region, around 20q13, also harbours a number of putative oncogenes (e.g ZNF217, NABC1) frequently Vitamin D Signaling: Role of CYP24 amplified in many types of malignancies, including breast, ovarian, Synthesis of 1α,25(OH)2D3 is a highly regulated, multistep process, pancreatic and colorectal. Amplicons constituting 20q13 gains have involving a number of enzymatic reactions (Figure 1). Vitamin D3 is prognostic relevance as such chromosomal changes have been synthesised in human skin from 7-dehydrocholesterol following associated with worsened survival and more aggressive tumour ultraviolet light exposure, and 25-hydroxylated by P450 enzymes (e.g. behaviour. Amplification of the CYP24 gene (located at 20q13.2) in CYP27A1) in the liver to form 25(OH)D3. Subsequently, 25(OH)D3 is breast cancer tissue led to the identification of CYP24 as a candidate then converted in kidney to the biologically active form, oncogene and the proposal that aberrantly elevated expression of 1α,25(OH)2D3 by the 1α-hydroxylase CYP27B1. Bound to a vitamin CYP24 in tumour cells permits their escape from the antiproliferative D binding protein, 1α,25(OH)2D3 is then circulated to target organs effects of 1α,25(OH)2D3 . The vital role of CYP24 in tightly where the biological activity of 1α,25(OH)2D3 is mediated by binding regulating the biological action of vitamin D signaling provides to its cognate high affinity nuclear vitamin D receptor (VDR) to additional support for its candidacy as a putative oncogene. initiate transcriptional activation or repression of target genes. The Overexpression of CYP24 has been observed in a wide variety of importance of vitamin D signaling in promoting the anticancer effects cancers including lung, colon, ovarian, prostate, skin and of vitamin D is strongly supported by studies demonstrating that mice oesophageal, and in some cases, linked to poor prognosis . lacking the VDR have an increased incidence of mammary and skin Although CYP24 gene amplification appears to be the mechanism tumours following treatment with carcinogens . These findings giving rise to elevated CYP24 in some cases, there appear to be indicate that optimal vitamin D-mediated VDR signaling may other mechanisms which could lead to increases in CYP24 sufficient contribute to tumour suppression and cancer prevention. to limit 1α,25(OH)2D3 activity. For example, aberrant DNA Although most abundantly expressed in kidney, CYP27B1 and methylation of the CYP24 promoter region may influence VDRs have also been shown to be widely expressed in a variety of transcriptional activation of the CYP24 gene in some cancers . In the case of vitamin D, the cytochrome P450 enzyme CYP24 has emerged as a potential barrier to optimal therapy Volume 4 Issue 2 • May/June 2009 43 Also, elevations in CYP24 gene expression can occur in response to Potential for CYP24 Inhibitors in Oncology 1α,25(OH)2D3-induced enhancement of mitogen-activated protein Recognition of the strong antineoplastic action of 1α,25(OH)2D3, as kinase (MAPK) pathways, involving extracellular-signal-regulated well as its ability to potentiate the anticancer effects of kinase (ERK) 1/2, 5 and c-Jun N-terminal kinase (JNK), and chemotherapeutic agents in vivo and in vitro, has stimulated interest phosphorylation of binding sites proximal to VDREs of the CYP24 in translating these findings into the clinic for the treatment of promoter [7,8]. Therefore, perturbations in multiple pathways various cancers. This is supported by a number of clinical trials implicated in cancer cell growth can potentially influence CYP24 completed or currently in progress investigating the potential for expression in tumour cells. vitamin D therapeutics in cancer treatment . In vitro studies Enhanced CYP24 expression has been shown to increase the indicate that supraphysiological doses of 1α,25(OH)2D3 are required growth potential of tumour cells in vitro , indicating that tumour to achieve significant antiproliferative activity in vivo. Therefore, cell sensitivity to 1α,25(OH)2D3 is, at least in part, dependent on the achieving optimal therapeutic levels of 1α,25(OH)2D3 for effective level of CYP24 activity. Tumour resistance to 1α,25(OH)2D3 can chemotherapy could be considerably hampered by the emerge as tumours progress. This is supported by observations of 1α,25(OH)2D3-mediated induction of CYP24 expression and feedback increased CYP24 expression in poorly differentiated cells relative to degradation of 1α,25(OH)2D3. Also, some tumours, as they progress, more differentiated tumours . The progressive loss of will acquire aberrations leading to vitamin D-independent elevations responsiveness to 1α,25(OH)2D3 could provide a selective growth in CYP24 expression further contributing to 1α,25(OH)2D3 resistance. advantage for tumour cells. Thus, one therapeutic approach may be The use of pure CYP24 inhibitors in these circumstances may be an to block CYP24 activity with small-molecule inhibitors to restore effective strategy to help maintain therapeutic levels of vitamin D and tumour responsiveness to the anticancer action of either endogenous increase tumour sensitivity to anticancer effects in patients. CYP24 or therapeutically administered 1α,25(OH)2D3. Thus, CYP24 inhibitors, alone or in combination with 1α,25(OH)2D3 or other inhibitors could have great therapeutic potential in the prevention vitamin D analogues, may provide a means of overcoming tumour and treatment of cancer. resistance in vitamin D sufficient patients. Pure CYP24 Inhibitors Conclusion Currently, several types of compounds capable of inhibiting CYP24 Clinical trials have shown some promising results with vitamin D enzymes belong to a class of molecules that utilise functional azole therapeutics in combination with chemoagents for the treatment of groups to target the heme moiety at the P450 enzymes catalytic core. some cancers. Blocking CYP24 activity may provide clinicians with a However, azole-based inhibitors lack specificity due to the well- new powerful approach to enable optimal penetration of target conserved heme-binding core of all P450 enzymes. This is particularly tissues and achieve therapeutic levels of 1α,25(OH)2D3 necessary for important in the clinic as non-selective binding of these compounds to tumour growth suppression. Thus, the development of selective and multiple drug-metabolising cytochrome P450 enzymes can lead to potent inhibitors of CYP24 may be a novel therapeutic class of agents drug-drug interactions, giving rise to issues of toxicity and reduced to improve the anticancer action of vitamin D therapy in the treatment effect. Non-specific P450 inhibitors have been shown to prevention and treatment of cancer. n block the main drug-metabolising enzyme CYP3A4, important for the breakdown of several chemoagents, such as taxanes. Co-administration of CYP24 azole-type inhibitors to enhance the antitumour and synergistic action of 1α,25(OH)2D3 with chemotherapy could increase the risk for chemotoxicity in these patients. Acknowledgements Selectivity is crucial when designing CYP24 inhibitors to minimise We would like to thank Dr. Christian Helvig, Dr. Dominic Cuerrier, Dr. Christopher inhibition of non-targeted enzymes, such as CYP27B1 or drug- Hosfield, Sheldon Kawarsky and Gordon Ngan for their insightful comments and helpful suggestions during the preparation of this manuscript. metabolising enzymes. Unlike other xenobiotic metabolising cytochrome P450s which have promiscuous substrate binding References pockets that can bind a wide variety of different molecular structures, 1. Garland CF, Garland FC. Do sunlight and vitamin D reduce the likelihood of the CYP24 substrate binding pocket is uniquely conformed for colon cancer? Int J Epidemiol 1980;9(3):227-31. vitamin D substrates. Developing compounds that target the CYP24 2. Bouillon R, Carmeliet G, Verlinden L et al. 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Vitamin D receptor expression, 24- 1α,25(OH)2D3 concentrations to a greater degree through hydroxylase activity, and inhibition of growth by 1α,25-dihydroxyvitamin D3 in conservation of 1α,25(OH)2D3 synthesis . This suggests that pure seven prostatic carcinoma cell lines. Clin Cancer Res 1995;1:997-1003. CYP24 inhibitors could be more advantageous in enhancing vitamin 10. Bareis P, Bises G, Bischof MG, et al. 25-hydroxy-vitamin D metabolism in D signaling and anticancer activity of 1α,25(OH)2D3 than non- human colon cancer cells during tumor progression. Biochem Biophy Res Commun 2001;285:1012-7. selective azole inhibitors currently in use. Furthermore, potential 11. Parise RA, Egorin MJ, Kanterewicz B, et al. CYP24, the enzyme that catabolizes development of dual-action vitamin D compounds that induce VDR the antiproliferative agent vitamin D, is increased in lung cancer. Int J Cancer transcriptional activity while selectively inhibiting CYP24 action 2006;119:1819-28. could serve as self-potentiating therapies that may prove to be 12. Deeb KK, Trump DL, Johnson CS. Vitamin D signaling pathways in cancer: superior than single action compounds. potential for anticancer therapeutics. Nature Rev Cancer; 7:684-700. 44 Volume 4 Issue 2 • May/June 2009