The role of vitamin D in reducing cancer risk and progression

Some observational, preclinical and clinical studies strongly suggest that vitamin D deficiency increases the risk of developing multiple malignancies. Other studies do not support this hypothesis. If adequate vitamin D concentrations do reduce risk, ensuring that people receive sufficient vitamin D would be an easily avail-able, economical and safe modality to reduce cancer incidence and mortality. Vitamin D status, which is determined by sunlight exposure, diet and supple-ments, might reduce the risk of developing cancer, and the appropriate regulation of cancer-relevant pathways by vitamin D might have a role in the treatment of can-cer. In this Review, we discuss the studies that examine this hypothesis, with an emphasis on breast, prostate and colon cancers, for which the most data have been accu-mulated. It is evident that there are strong supportive data for the vitamin D hypothesis from basic science and preclinical studies, and there are mixed findings in the epidemiological and clinical trial data. We also discuss the multiple mechanisms of the anticancer action of vitamin D and the potential reasons why the epidemi-ology data on reducing risk are inconsistent. Recent pub-lications suggesting that vitamin D can reduce cancer risk and improve prognosis have prompted physicians to screen for vitamin D deficiency and stimulated the general population to supplement their diet. Therefore, it is timely to review the data on which this hypothesis is based. Multiple recent reviews have been written about vitamin D and cancer 1–13, and a multi-authored book 14 has been written that extensively details many actions of vitamin D in cancer and other diseases.

Vitamin D is a multifunctional pro-hormone

Despite its name, vitamin D is not really a vitamin. As detailed below, it is the precursor to the potent steroid hormone calcitriol (also known as 1,25-dihydroxy-vitamin D 3 (1,25(OH)2D 3)), which mediates numerous actions in many tissues of the body. Vitamin D can be synthesized in adequate amounts in the skin using the

energy of ultraviolet (UV) radiation in sunlight (see Supplementary information S1 (figure)). Thus, it is not an essential element that is derived from the diet. Indeed, most foods have little vitamin D unless they are fortified, which means that humans are dependent on sunlight to maintain adequate vitamin D stores. However, many peo-ple do not receive adequate sunlight, owing to various life factors, including indoor occupation; avoidance of sun-light because of concerns about skin cancer risk; living far from the equator, with low levels of sunlight, especially in winter; wearing traditional attire that completely covers the body; and having dark skin that blocks the rays of the sun 15. The result is that vast numbers of individuals worldwide have been found to be vitamin D deficient 15.Vitamin D regulates calcium and phosphate metabo-lism and is essential for bone mineralization 15,16. However, over the past two decades it has become clear that vitamin D has many extraskeletal actions. Accumulating data indicate that vitamin D deficiency raises the risk of developing cancer and many other diseases, and it worsens outcomes for these diseases (BOX 1). Although many researchers and physicians embrace this idea, other investigators understand-ably do not accept these beneficial actions without

1

Department of Medicine, Division of Endocrinology, Stanford University School of Medicine, Stanford, California 94305, USA.2

Departments of Epidemiology and Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115, USA.3

Department of Pediatrics, Division of Pediatric

Endocrinology, Stanford University School of Medicine, Stanford, California 94305, USA.Correspondence to D.F . e-mail:

dfeldman@https://www.wendangku.net/doc/6614086407.html, doi:10.1038/nrc3691

Published online 4 April 2014

Randomized clinical trials

(RCT s). T rials in humans that are the gold standard for proof of efficacy of an intervention in cancer and other diseases.

Hypercalcaemia

Increased levels of blood

calcium that can lead to many symptoms, including muscle cramps, drowsiness, bone pain, kidney stones and, in severe cases, cardiac arrest and coma.

Calciotropic hormones

A complex network of hormones that regulates calcium and phosphate

metabolism to normalize bone mineralization, including

calcitriol, parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23).

confirming compelling data from human randomized clinical trials (RCTs). Indeed, the need for RCT data was highlighted in a 2010 report 17 by the Institute of Medicine (IOM) that addressed vitamin D dietary requirements. Although the IOM report endorsed the use of vitamin D to maintain bone health, without RCT data they could not support the use of vitamin D to prevent or treat cancer and other diseases (BOX 1). Their conclusions raised a controversy about what constitutes vitamin D deficiency and the doses of supplements needed to improve the vitamin D status of the popu-lace (BOX 1). A further complexity was that if vitamin D did reduce cancer and the other diseases, did this anti-cancer action require higher amounts of vitamin D than were needed to support bone health? In the absence of definitive data, the IOM set their recommendations for vitamin D requirements on the basis of bone health and not cancer, even though the vitamin D requirement for bone health is not universally agreed on either 18. Most investigators do agree that the definitive answers will require well-structured RCTs in humans.

Conversion of vitamin D to the active hormone cal-citriol. Vitamin D 3 (also known as cholecalciferol) is the precursor to the potent steroid hormone calcitriol (see Supplementary information S1 (figure)). The energy from UV radiation converts the substrate 7-dehydro-cholesterol to vitamin D 3 in the skin 15. Vitamin D 3 is sub-sequently activated to its potent hormonal form, calcitriol, by two cytochrome P450-mediated hydroxylation steps 19. The first hydroxylation step mostly occurs in the liver at C25 to yield 25-hydroxvitamin D 3 (25(OH)D 3), which is catalysed by the enzyme vitamin D-25-hydroxylase (pre-dominantly CYP2R1)20. 25(OH)D 3 is the circulating form of the hormone that is measured in the blood and clini-cally used to establish and monitor the vitamin D status of a patient. The defined cut-points for deficiency are set at 20 ng per mL (50 nmol per L) by the IOM and 30 ng

per mL (75 nmol per L) by the Endocrine Society (BOX 1). Although there is evidence in support of both positions, for reasons discussed below, we favour the Endocrine Society guidelines, and we refer the reader to the position statements for a discussion of the opposing views 18,21.Circulating 25(OH)D 3 is hydroxylated in the kidney at the C1α position (FIG. 1; see Supplementary infor-mation S1 (figure)) by the cytochrome P450 enzyme CYP27B1 (also known as 1α-hydroxylase) to yield calci-triol 19. Vitamin D 2 (ergocalciferol) is a form of vitamin D that is of plant origin, is derived from ergosterol and func-tions much like vitamin D 3 but is less active. Assays meas-uring circulating blood vitamin D metabolites, 25(OH)D 3 and 25(OH)D 2, do not distinguish the two forms or they report the total. Therefore, we will refer to the circulat-ing blood levels without any subscript as 25(OH)D or 1,25(OH)2D. Most often, the predominant active metabolite is 1,25(OH)2D 3 and then it is specified as calcitriol.Mechanism of calcitriol action. Calcitriol functions by binding to and activating the nuclear vitamin D receptor (VDR), which is a member of the steroid–thyroid–retinoid receptor superfamily of ligand-activated transcription fac-tors. As the VDR is present in most cells in the body 22 and calcitriol directly or indirectly regulates as much as 3–5% of the human genome, vitamin D activity is widespread, and it exerts actions that can alter the defences of the body 23–26 and that can seemingly limit the progression of multiple diseases, including cancer 1–16 (discussed below). Among the many genes that are induced by calcitriol, CYP24A1 (also known as 24-hydroxylase) is particularly important; it encodes the enzyme that catalyses the deg-radation of both 1,25(OH)2D 3 (calcitriol) and 25(OH)D 3 (REF . 27) (FIG. 1; see Supplementary information S1 (fig-ure)). Thus, the activity of the hormone is self-regulated, as it simultaneously induces its own inactivation. The predicted side effect of administering supraphysio l ogical concentrations of calcitriol is hypercalcaemia , which is mainly due to the actions of calcitriol in stimulating intes-tinal calcium absorption. Therefore, structural analogues of vitamin D that show reduced calcaemic effects while exerting equipotent or increased anticancer actions are being developed for therapeutic applications 28.

Vitamin D-metabolizing enzymes and cancer

Extrarenal CYP27B1 and paracrine calcitriol actions in cancer. Renal CYP27B1 and CYP24A1 are both regulated by three key calciotropic hormones — calcitriol, parathyroid hormone (PTH) and fibroblast growth fac-tor 23 (FGF23) — which are involved in mineral and skeletal homeostasis 29,30 (FIG. 1). Although the kidney is the major source of circulating calcitriol, CYP27B1 is also expressed in multiple extrarenal sites, including

| Cancer

cancer cells, where it can exert anticancer actions 31. As discussed below, this phenomenon is constantly ongo-ing in vivo in many normal human tissues, in the immune system and in cancer cells to regulate non-skeletal and non-mineral pathways 31–35. Thus, calcitriol can function both in an endocrine (systemic) manner or in an intracrine, autocrine or paracrine manner when it is locally synthesized (FIG. 1). In contrast to the renal enzyme, extrarenal CYP27B1 is not regulated by the calciotropic hormones involved in mineral homeostasis but by other factors 31–35. Local synthesis mostly depends on the cir-culating 25(OH)D 3 substrate concentration. The pres-ence of CYP27B1 in cancer cells suggests that dietary vitamin D might be used in cancer therapy, because it is easily converted to 25(OH)D 3 by the liver, and then the increased blood concentration of the substrate 25(OH)D 3 drives increased local conversion to the active hormone 1,25(OH)2D 3 (calcitriol) within the cancer tissue, where it can exert anticancer actions 36,37 (FIG. 1). This pathway has been shown to yield high local calcitriol concentra-tions in the tumor 38, with less likelihood of causing the systemic side effect of hypercalcaemia 39.

CYP24A1 and CYP27B1 expression in cancer. An aberr-antly high basal expression level of the catabolic enzyme CYP24A1 occurs in several cancer cells, thereby making them resistant to calcitriol action 40–42. A comparative gene hybridization study in patients with breast cancer identi-fied CYP24A1 as a candidate oncogene 43. Spontaneous upregulation of CYP24A1 is seen in some cancers that correlates with poor clinical outcome 40. Inhibition of CYP24A1 function amplifies the biological activity of calcitriol; indeed, the use of cytochrome P450 inhibi-tors, such as ketoconazole 44, liarazole 45 and genistein 46, increases the biological actions of calcitriol and can cause calcitriol-resistant cells to revert to sensitive cells 45,46. However, the combination with CYP24A1 inhibitors not only increases the anticancer actions of calcitriol but also augments its calcaemic effects, thereby increasing the risk of hypercalcaemia 47. Therefore, a cautious approach is necessary when using these combinations.

Data on CYP27B1 expression and activity in cancer are more varied. Depending on the organ and the tumour grade, the expression level and activity of CYP27B1 can be decreased, increased or unchanged 36,37,48–50.

| Cancer

Furthermore, CYP27B1 expression seems to be depend-ent on the degree of cellular differentiation, with well- differentiated tumours expressing higher levels than highly aggressive, poorly differentiated tumours 48. Thus, the regulation of CYP27B1 in cancer cells might depend on the tissue and the tumour stage. The reduction in

CYP27B1 expression that is seen in some cancer cells 36,37 or that is experimentally induced using RNA interference to knock down expression 51 might endow these cells with an intrinsic growth advantage because of the decrease in the local production of calcitriol, thereby decreasing an inhibitor of proliferation 36. These observations sug-gest that increases in serum 25(OH)D levels by dietary supplementation or UV exposure will contribute to the reduction of cancer development and that calcitriol signalling through the VDR could be used to regulate cancer-relevant pathways for the treatment of cancer.In all cases, the presence of VDR in the cancer cells is essential for calcitriol and vitamin D activity. High VDR expression in breast and prostate tumours is associated with a reduced risk of death from cancer and an improved prognosis 52,53. Patients with prostate cancer who had higher expression of tumour VDR had a 60%-decreased risk of dying from their cancer, even after adjusting for conventional prognostic factors 53. Greater VDR expression was associated with favourable tumour characteristics 49,54 and with better survival in breast cancer 54.

Anticancer actions of vitamin D

Mechanisms of calcitriol action. The biological actions of calcitriol are mediated by the VDR, mostly via genomic actions 23–26,55 (FIG. 2). Calcitriol binds to the VDR, thereby causing its dimerization with the retinoid X receptor (RXR), binding of the complex to vitamin D response elements (VDREs) in multiple regulatory regions located at promoters and distal sites of tar-get genes and the recruitment of co-modulators 23–26,55. Several rapid cellular actions of calcitriol through non-genomic pathways have also been described 56. One of these non-genomic pathways, which requires the VDR and the endoplasmic reticulum stress protein 57 (ERP57; also known as 1,25D 3–MARRS and GRP58)57, has been implicated in the protective effect of calcitriol against sunlight-induced DNA damage and skin cancer 58.

In 1981, Colston et al.59 showed that calcitriol inhibited the growth of malignant melanoma cells and Abe et al.60 reported that calcitriol caused the differentia-tion of HL60 leukaemia cells towards the macrophage lineage. Since then, the antineoplastic actions of calci-triol have been shown both in vitro and in vivo , in vari-ous malignancies 1–16,22–26,28,61,62. Genomic and proteomic screening approaches have identified an extensive array of VDR target genes that mediate the antineoplastic actions ascribed to calcitriol 63–68.

Calcitriol regulation of specific signalling pathways that drive colon, breast and prostate cancer growth. Calcitriol has a wide range of actions in cancer cells from many different types of cancer (BOX 2; FIG. 2). In addition, it reg-ulates specific signalling pathways in breast, colon and prostate tissues (FIG. 3), thereby mitigating the actions of key drivers of tumours arising in these tissues. Several calcitriol actions in colon cancer cells inhibit β-catenin transcriptional activity 8,69, thereby countering aberr-ant activation of WNT–β-catenin signalling, which is the most common alteration in sporadic colorectal cancer. The SNAIL transcription factor represses VDR

Figure 1 | The renal endocrine pathway and the extrarenal autocrine or paracrine pathway of calcitriol synthesis. The structure of the circulating form of vitamin D, 25?hydroxvitamin D 3 (25(OH)D 3), is shown, and the crucial carbons, at positions C1 and C24, where hydroxylations occur, are indicated. The intracellular concentration of calcitriol (also known as 1,25?dihydroxyvitamin D 3 (1,25(OH)2D 3)) and its metabolites are determined by the two key cytochrome P450 enzymes CYP27B1 (also known as 1α?hydroxylase) and CYP24A1. CYP27B1, which is the rate?limiting enzyme in the synthesis of calcitriol, is primarily expressed in the kidneys, with varied expression in other tissues. The renal CYP27B1 is tightly regulated and is the crucial determinant of the concentration of circulating calcitriol, which acts on target tissues to exert multiple effects, including anticancer actions (endocrine pathway). Several calciotropic hormones control the regulation of the renal CYP27B1 to maintain calcium and

phosphate homeostasis in the body. The principal positive regulator of renal CYP27B1 is parathyroid hormone (PTH), the level of which is inversely proportional to serum calcium concentration. Other important regulators include calcitriol itself, phosphate and the more recently recognized fibroblast growth factor 23 (FGF23). In contrast to the renal CYP27B1, the extrarenal enzyme is not regulated by the classic calciotropic hormones and mostly depends on the concentration of circulating 25(OH)D 3 substrate that

determines the extent of local synthesis. Thus, the extrarenal tissues have the capacity to locally synthesize the active hormone that can then exert anticancer actions in an autocrine, intracrine or paracrine manner, in addition to receiving signals from

kidney?synthesized circulating calcitriol. By contrast, CYP24A1 functions to protect the body from excess calcitriol. It is a mitochondrial P450 enzyme that is expressed in all cells that are responsive to calcitriol. It catalyses the hydroxylation on C24 of 25(OH) D 3 to form 24,25(OH)2D 3 and on C24 of both renally and extrarenally synthesized calcitriol to form 1,24,25(OH)3D 3, which is a molecule with reduced biological activity. Regulation of CYP24A1 has been extensively reviewed, and several factors, such as calcitriol itself, PTH and FGF23, contribute to its regulation. CYP24A1 mRNA is highly induced by calcitriol via a vitamin D receptor (VDR)?mediated genomic pathway. In normal tissue, the expression of CYP24A1 rapidly returns to basal levels once the calcitriol stimulus is removed. EGF , epidermal growth factor.

| Cancer

expression, thereby reducing the anticancer effects of calcitriol 69. Increased SNAIL expression in human colon tumours is associated with a loss of responsiveness to calcitriol, which could be used as an indicator of patients who are unlikely to respond to vitamin D therapy 69.In postmenopausal women, when ovarian oestrogen production ceases, local oestrogen that is synthesized in the breast microenvironment is the driver of oestrogen receptor-positive (ER +) breast cancer growth. By inhibit-ing both oestrogen synthesis (selective suppression of aromatase expression in breast adipose tissue 70,71) and signalling (downregulation of ERα in breast cancer cells 72–75), calcitriol might have a therapeutic benefit in the prevention or treatment of postmenopausal ER + breast cancer. However, many of the broad anticancer actions of vitamin D (BOX 2) might also benefit women with ER-negative breast cancer.

Androgens drive the growth of most prostate cancer cells through androgen receptor (AR)-mediated events. Progression of prostate cancer into castration-resistant

prostate cancer (CRPC) occurs through multiple path-ways, most of which are still mediated through AR stimulation, despite castrate levels of androgens in the circulation 76–79. Interestingly, there is crosstalk between calcitriol and androgen signalling in some prostate can-cer cells, which include: regulation of the expression of AR 80–82, as well as other androgen responsive genes, by calcitriol 64, regulation of VDR by androgens 83, induc-tion of a gene expression pattern consistent with differ-entiation 66 and growth inhibition 84 and the regulation of genes involved in androgen catabolism 64.

Vitamin D and cancer stem cells. The ‘cancer stem cell (CSC) hypothesis’ (see Supplementary information S2 (box)) posits that a unique subset of tumour cells have stem cell-like properties and are able to propagate the tumour on serial passage in vivo , thereby renewing tumour growth. Rodent studies examining prostate gland regression and regeneration during androgen deprivation and replace-ment have indicated that the prostate develops from, and

a | Both

these cells, 25(OH)D 3 is locally converted by the cytochrome P450 enzyme CYP27B1 to calcitriol. The biological actions of calcitriol, whether it is locally synthesized or derived from renal synthesis via the circulation, are mediated by the

vitamin D receptor (VDR). Calcitriol binds to the VDR, thereby causing its dimerization with the retinoid X receptor (RXR) and its translocation to the nucleus. The ligand?bound VDR–RXR complex binds to vitamin D response elements (VDREs) in multiple regulatory regions located in the promoters of target genes or at distal sites, and this causes the recruitment of co?activators or co?repressors, which leads to positive or negative transcriptional regulation of gene expression. b | These target genes are involved in diverse molecular pathways, thereby resulting in a wide range of calcitriol? mediated anticancer actions, as shown. CDK, cyclin?dependent kinase; COX2, cyclooxygenase 2; HIF1α, hypoxia? inducible factor 1α; IL?8, interleukin?8; MAPKP5, mitogen?activated protein kinase phosphatase 5; MMP9, matrix

metalloproteinase 9; NF?κB, nuclear factor?κB; PG, prostaglandin; 15?PGDH, 15?hydroxyprostaglandin dehydrogenase; PGE 2, prostaglandin E 2; POL II, polymerase II; PSA, prostate?specific antigen; TIMP1, tissue inhibitor of metalloproteinases 1; VEGF , vascular endothelial growth factor.

is maintained by, a stem cell population85. Prostate stem cells (PSCs) with multipotent, self-renewal capacity and the ability to regenerate a prostate gland were purified using the defined surface markers of LIN– SCA1+ CD133+ CD44+ CD117+(REF. 86). Although the cell of origin for prostate cancer is still not fully resolved and might vary in different subtypes of prostate cancer, purifying cells from prostate tumours using PSC markers greatly enriches for cancer cells that have stem cell-like properties87–90. Calcitriol might have potent effects on the normal PSC population, and these cells might be the origin for pros-tate cancer when mutations convert them into CSCs66. Therefore, understanding the actions of calcitriol on nor-mal PSCs might provide insight into its actions on prostate CSCs. In their paper, Maund et al.66provide evidence that the cells they model for CSCs express CYP27B1, which would facilitate local synthesis of calcitriol. Furthermore, calcitriol inhibits the proliferation of these potential PSCs, by inducing cell cycle arrest and senescence. The data of Maund et al.66 indicate that interleukin-1α could be an important VDR target gene in PSCs and CSCs, because it might regulate this senescence mechanism.

Breast cancer also contains a minority subpopula-tion of CSCs within the tumour. In these tumours too, the CSCs seem to be closely related to the normal tissue stem cells. Recent data91 implicate the number of breast stem cells present in the tissue as being a strong predictor of ultimately developing cancer, and this further supports the view that these are the cells of origin for breast cancer or are closely related to them. Few studies have tested whether the mechanism that drives the reduction in cancer risk by calcitriol occurs by an effect on the CSC population. A study using mammospheres to enrich for CSCs92 found that knocking down VDR increased mam-mosphere formation, potentially through increased proliferation. However, high-dose calcitriol had a minimal effect on the mammospheres, which suggests that calcitriol alone might not be sufficient to affect

CSC proliferation. In vitro and xenograft studies 93 indi-cated that the calcitriol analogue BXL0124 decreased tumour growth and reduced tumour CD44 levels, and this raises the possibility that the therapy was targeting the CSC population, which is thought to be CD44-positive.In summary, there is enticing preliminary evidence that vitamin D and calcitriol could be therapeutically used to target and inhibit prostate and breast CSCs. Equally important, studies such as these show that cal-citriol can be used as a powerful tool to elucidate novel pathways that are crucial for CSC activity. Once identi-fied, these pathways could be selectively targeted to yield novel therapeutics. In order to test this hypothesis, it is important that investigators use strict criteria for defin-ing CSCs. So far, there is a lack of studies that use mul-tiple markers to prospectively purify primary cells that have passed in vivo limiting-dilution assays to validate stem cell activity in the investigation of calcitriol effects on CSCs. Such investigations are most likely to reveal the most clinically relevant specific physiological and pharmacological activities of calcitriol on CSCs.

Vitamin D and microRNAs in cancer. MicroRNAs (mi R NAs) are short (~22 nucleotides) single strands of RNA that function as regulators of mRNA translation. They have integral roles in a broad array of biological pro-cesses 94. Maier et al.95 first speculated that mi R NAs might be part of the molecular mechanism by which calcitriol protects against colorectal cancer, when they identified a putative calcitriol-responsive miRNA precursor sequence within intron 3 of the cyclin D1 gene. Since then, many investigations, using various cancer cell lines and human tumour tissues, have suggested that miRNA regulation might have an important role in the anticancer actions of calcitriol 96–102 (TABLE 1). Interestingly, mi R NAs have also been implicated in the regulation of VDR and CYP24A1 expression in breast cancer cell lines and human breast tumour tissue 103,104, which suggests an impact of mi R NAs on the efficacy of vitamin D therapy (TABLE 1). Some of the diverse results that have been seen in clinical trials with calcitriol might be due to variations in intracellular calci-triol and VDR levels, owing to miRNA regulation. It will be of great interest to determine whether the expression of any mi R NAs is correlated with clinical outcomes and whether these mi R NAs can inform the ongoing clinical trials to make better predictions about which patients are likely to respond to vitamin D and calcitriol therapy.

Vitamin D in animal models of cancer

The effect of calcitriol and its analogues on the inhibi-tion of tumour growth has been extensively studied in animal models to evaluate the anticancer effects in vivo , as well as to monitor any tendency to cause hypercal-caemia. Data from animal models of various cancers

cancer cells (BOX 2; FIG. 2b), the calcitriol actions shown in this figure inhibit specific signalling pathways that drive the growth of certain cancers. These actions include the inhibition of the WNT–β-catenin signalling that drives colon cancer, inhibition of local oestrogen synthesis and signalling that drives oestrogen receptor?positive (ER +) postmenopausal breast cancer and interaction with androgen receptor (AR) signalling that drives prostate cancer. MSK, nuclear mitogen? and stress?activated protein kinase; PGE 2, prostaglandin E 2; PSA, prostate?specific antigen; VDR, vitamin D receptor.

(BOX 3; see Supplementary information S3(table)) show that dietary vitamin D, calcitriol and its analogues cause a significant reduction in tumour growth and eventual tumour burden. Vdr-null mice are more susceptible to the development of carcinogen-induced cancers105, which indicates a protective effect of calcitriol–VDR in the face of carcinogenic stressors. Combinations of calcitriol with other agents seem to exert significantly greater benefi-cial antitumour effects compared to the individual agents (see Supplementary information S3 (table)). Dietary vitamin D alone or in combination with calcium sup-plements also inhibits tumour development and tumour growth39,106,107, whereas vitamin D deficiency accelerates tumour growth, independent of calcium levels106,108,109. Higher circulating 25(OH)D levels that are achieved by vitamin D dietary supplements cause substantial tumour inhibition, whereas tumours continue to grow when ani-mals are fed the standard rodent diets39,106, which seem to be adequate to support normal mineral homeostasis and bone health. These data suggest that higher vitamin D levels than are required for bone health exert stronger anticancer actions in rodents5. The tumour-inhibitory effects of dietary vitamin D or calcitriol in multiple ani-mal models (BOX 3; see Supplementary information S3 (table)) and the calcitriol-regulated anticancer pathways mediating these actions (BOX 2;FIG. 2) provide compelling evidence and a rational framework to strongly support the hypothesis that vitamin D and calcitriol could inhibit cancer development and progression. Epidemiological studies of vitamin D and cancer Initial association studies110,111 suggested that sunlight exposure could be used as a surrogate for vitamin D con-centration and found that areas of the world that are far from the equator, with reduced sunlight, had an increased incidence of colon and prostate cancers, thereby implicat-ing vitamin D deficiency as a risk factor for cancer. The many ensuing ecological studies that mostly agreed with this concept were recently summarized112.

Among the epidemiological studies that have exam-ined the vitamin D and cancer hypothesis, greatest weight has typically been bestowed on studies that measure pre-diagnostic circulating levels of 25(OH)D. Among can-cers, associations have most consistently been observed for colorectal cancer113. Meta-analyses show a statisti-cally robust 30–40% reduction in colorectal cancer risk in those patients with high 25(OH)D levels compared with those who have low 25(OH)D levels, even after adjust-ment for other known risk factors114. For other cancers, clear associations have remained elusive. For example, a recent meta-analysis of 14 prospective studies of prostate cancer showed no inverse association with 25(OH)D lev-els115, and some evidence even suggests an increased risk with higher 25(OH)D levels116. The results for breast can-cer have been contradictory. One meta-analysis found no appreciable association between prediagnostic circulating 25(OH)D levels and breast cancer risk117. By contrast, a recent dose–response meta-analysis of plasma 25(OH)D levels suggested a stepwise inverse association beyond a threshold of 27 ng per mL, with flattening above 35 ng per mL only in postmenopausal women118. The specificity of the dose–response over a restricted range of 25(OH)D levels and the observation that the dose–response is lim-ited to menopausal women is unexplained. Recent large studies have not supported these associations119,120.

The Vitamin D Pooling Project of Rarer Cancers con-sortium121 examined prediagnostic 25(OH)D levels in various cancers in ten studies. In general, no overall asso-ciation was observed for these cancers121,122. In fact, there was an increased risk of pancreatic cancer in a smaller

Table 1 |

MicroRNAs implicated in the anticancer actions of calcitriol

subset of subjects with 25(OH)D levels of ≥40 ng per mL (39 cases), which was mostly driven by two studies123. However, another pooled analysis of five separate stud-ies reported a statistically significant 33%-decreased risk of pancreatic cancer among those with higher 25(OH)D levels and no excess risk beyond 40 ng per mL124.

In a recent systematic review of published studies, Autier et al.125 have hypothesized that inflammatory processes that are involved in disease occurrence and progression could reduce 25(OH)D levels and may explain why low 25(OH)D levels are associated with various health outcomes, including cancer. Increased inflammation that is associated with some malignancies, including colorectal cancer, could provide some basis for this hypothesis. However, substantial data (BOX 2) show that vitamin D inhibits inflammation3,34,61, which sug-gests the reverse; that is, reduced vitamin D levels might increase inflammation. Furthermore, this proposed mechanism would not account for most basic scientific findings and epidemiological evidence based on various other data. For example, multiple predictors of 25(OH)D levels, including dietary intake, supplementation, region (latitude) and sun exposure, have all been associated with cancer incidence and mortality, especially for colo-rectal cancer126. While these data might be prone to other types of confounding, we think that it is unlikely that they are susceptible to the type of confounding that have been proposed by Autier et al.125.

Some emerging findings suggest that higher 25(OH)D concentrations before or at the time of diagnosis and treatment might improve survival from various can-cers. In one study of patients with prostate cancer, men in the lowest quartile of prediagnostic 25(OH)D levels had a 60% higher risk of cancer death than those in the highest quartile127. In patients with colorectal cancer, in three large cohorts, patients with higher prediagnostic 25(OH)D levels (>30 ng per mL) had a 30–40% lower risk of cancer-specific and total mortality128. A recent meta-analysis of longitudinal epidemiological studies has found a moderate inverse association of 25(OH)D concentration with total cancer incidence and a stronger inverse association with cancer mortality129. Studies that assess post-diagnostic 25(OH)D concentrations need to be interpreted cautiously, because sicker patients could have lower 25(OH)D levels owing to behaviour (for example, less sun exposure) or systemic effects of the advanced cancer.

Other studies have examined whether common sin-gle nucleotide polymorphisms (SNPs) in the vitamin D pathway genes, particularly VDR, are associated with

cancer risk (BOX 4). Some of this research shows an association between VDR SNPs and risk for colo r ectal cancer130–132, high Gleason-grade prostate cancer133 and breast cancer131. More comprehensive studies of the multiple genes involved in vitamin D metabolism and signalling have been undertaken in these cancers134–140, which have yielded few consistently significant findings, especially after adjusting for multiple testing. A recent ‘pathway approach’, which is potentially more powerful than single SNP approaches, has suggested that multiple genes in the vitamin D pathway are jointly associated with risk of lethal prostate cancer141. As discussed in BOX 4, so far, these studies have not yielded convinc-ing data on associations between SNPs in the VDR pathway genes and cancer risk and prognosis. More definitive results might be forthcoming from consortia that allow for increased numbers of specimens for ana-lysis and larger numbers of SNPs to be studied, which will increase the power of this approach to possibly find meaningful relationships on the basis of multiple SNPs in the vitamin D pathway genes that each have a small effect.

Clinical trials

The VITamin D and OmegA-3 TriaL (VITAL), which is an ongoing RCT, is investigating whether daily diet-ary supplements of vitamin D

3

(2000 international units (IU)) or omega-3 fatty acids (1 gram of fish oil) or both reduce the risk of developing cancer, heart disease and stroke in people who do not have a prior history of these illnesses142 (https://www.wendangku.net/doc/6614086407.html, identi-fier: NCT01169259). Other RCTs in humans are also underway143. These trials may eventually shed light on the role of vitamin D in human cancer and other diseases, but the results will not be available for many

years and the findings may still engender controversy.

prostate cancer.

Vitamin D 3. In one substudy of the Women’s Health Initiative (WHI; a large group of studies of hormone replacement in postmenopausal women), modest doses of vitamin D 3 (400 IU per day) and calcium (1 g per day) showed no significant benefit in reducing the occurrence of breast or colorectal cancer 144. There was an increase in nephrolithiasis (calculi in the kidneys). As subsequently described by some WHI investigators, the limitations of this study included the ‘low’ vitamin D 3 dose provided to subjects (because 400 IU has been shown to cause only a very small or no rise in serum 25(OH)D lev-els 145), the limited adherence of the subjects to the drug, the allowance of the placebo group to also take supple-ments and the lack of serum 25(OH)D measurements 144. A reanalysis of the vitamin D 3 and calcium substudy in this RCT suggested some benefit in reducing breast and colon cancer in some subjects who were assigned to take the supplements and who were not using sup-plements at randomization, and this suggests that even low doses have a possible benefit in women who might have had low starting levels of 25(OH)D 146. However, this post-hoc and subgroup analysis was criticized and suggested to be ‘hypothesis generating’ and to be taken with caution 147.A 4-year osteoporosis and fracture RCT in 1,179 healthy postmenopausal women (https://www.wendangku.net/doc/6614086407.html, identifier: NCT00352170) had cancer incidence as the principal secondary outcome 148. The subjects received 1,400–1,500 mg supplemental calcium (Ca group), sup-plemental calcium plus 1,100 IU vitamin D 3 (Ca+D group) or placebo daily. Cancer incidence was lower in the Ca group and further decreased in the Ca+D group compared with the placebo group. Both treatments and serum 25(OH)D concentrations were significant inde-pendent predictors of cancer risk. However, the study had limitations, including being underpowered, can-cer not being the primary endpoint and the treatment including both vitamin D and calcium. Another osteo-porosis trial 149 evaluated 135 incident cases of cancer and showed no association between interventional vita-min D 3 and cancer incidence, but it did show a reduction in colorectal cancer mortality.A colon cancer trial that used dietary vitamin D 3 (800 IU per day) and calcium (2 g per day) for 6 months in 92 patients showed a reversal of biomarkers of increased colon cancer risk in biopsies of normal colo-rectal mucosa taken from patients undergoing surgery for sporadic colorectal adenoma 150. Many studies that used vitamin D 3 often combined it with calcium, and it is not clear whether the benefits were only from the vita-min D 3 or whether calcium also contributed, as seemed to be the case in some trials 148,150. A recent meta-analysis of eight major vitamin D 3 trials, which evaluated fracture incidence in more than 70,000 patients 151, concluded that vitamin D 3 plus calcium reduced the risk of death due to all causes, whereas vitamin D 3 alone did not. The fact that at least three different studies 148,150,151 suggested that the benefits of vitamin D 3 are enhanced by co- administration of calcium is certainly of interest, and further research is required to elucidate the mechanism

and confirm these findings.

Another approach used vitamin D 3 early in the course of prostate cancer in 44 patients with low-risk disease who chose active surveillance rather than pros-tatectomy 152. Vitamin D 3 supplementation (4000 IU per day) for a year was safe and caused no adverse events, but it did not change serum prostate-specific antigen (PSA) levels. However, on repeat prostate biopsy after 1 year, 55% of the subjects showed a decrease in the number of positive biopsy cores or a decrease in Gleason score of the cancer-containing biopsies, whereas 34% showed increases. Historical controls were statistically more likely to show progression. The authors concluded that patients with low-risk prostate cancer under active surveillance might benefit from vitamin D 3 supplementation.Recent meta-analyses showed an increasing risk of ischaemic heart disease, myocardial infarction and early death with decreasing plasma 25(OH)D lev-els 153. Although each of three individual intervention RCTs was negative for a vitamin D 3 benefit in can-cer 144,149,154, there was a suggestive trend to reduced cancer mortality, which was not statistically significant because each study had a relatively small number of can-cer deaths. However, when these data were pooled with other studies in the meta-analysis commissioned by the IOM and were published in the Cochrane database as a systematic review of the available vitamin D data for the prevention of mortality in adults 155, a significant trend to reduced mortality was found. Using data from multiple randomized trials, with 95,286 participants, the authors concluded that vitamin D 3 significantly decreased mor-tality in elderly people living independently or in insti-tutions (~11% decrease in mortality and ~12% decrease in cancer-related mortality), whereas vitamin D 2, alfa-calcidol (also known as 1α-hydroxyvitamin D 2) and cal-citriol had no statistically significant beneficial effects on mortality. Although the RCT evidence to date does not allow the establishment of a target level of serum 25(OH)D, it does support a causal role of vitamin D in cancer mortality.Calcitriol as a single agent or combined with other agents. Some trials have used the natural hormone calcitriol as an oral or intravenous formulation, and others have used clinically available analogues that are less calcaemic 28. Often, vitamin D-based drugs are combined with chemo-therapy or other anticancer agents 5. Trials of calcitriol as a single agent in prostate cancer report a few partial responses and some PSA responses. However, important clinical antitumour effects are infrequent. In a small pilot trial in men with early recurrent prostate cancer, calcitriol in daily oral doses of more than 2–2.5 μg led to hypercal-caemia, which limited the ability to raise the administered dose 156. At this daily dose, the rate of increase of PSA levels was stabilized in most subjects but was not significantly reduced. Failed attempts to increase dosing while avoiding hypercalcaemia led to the concept of intermittent treat-ment using extremely high doses of calcitriol, one to three

Naproxen

An anti-inflammatory drug that inhibits cyclooxygenase 2 (COX2), which is the

rate-limiting enzyme that catalyses prostaglandin synthesis.times per week12,157. These high-dose regimens required a

new, concentrated formulation of oral calcitriol (DN-101,

Novacea), and they clearly caused substantial but transient

hypercalcaemia that returned to normal before the next

dose, when given on a weekly schedule.

In the ASCENT-I trial, in men with CRPC with metas-

tases158, DN-101 (45 μg, once per week) plus docetaxel

weekly significantly improved overall survival compared

with docetaxel alone, although there was not a significant

reduction in PSA levels. This positive finding led to the

pivotal ASCENT-II trial — an open-label Phase III study

planned for 1,200 men with metastatic CRPC159. The

study had an asymmetrical design. The weekly docetaxel

dosing from ASCENT-II that was used in the DN-101

arm (45 μg DN-101 weekly, 36 mg per m2 docetaxel and

24 mg dexamethasone weekly for 3 of every 4 weeks)

was an older regimen that was shown in earlier studies

to have inferior overall survival compared with the new

and improved ‘every-3-weeks’ regimen used in the con-

trol arm (5 mg prednisone twice-daily, with 75 mg per m2

docetaxel and 24 mg dexamethasone every 3 weeks159).

In an interim analysis after 953 men were enrolled, the

DN-101 arm seemed to be associated with shorter overall

survival than the control arm, and the study was halted

early. These results were probably caused by differences

between the docetaxel regimens in the treatment and con-

trol arms. Nevertheless, these findings have discouraged

the use of calcitriol in large cancer trials.

A contemporaneous RCT used DN-101 to determine

whether calcitriol that was administered together with

naproxen, a non-steroidal anti-inflammatory agent, was

more effective than calcitriol alone in safely delaying

cancer progression (on the basis of PSA doubling time)

in men with early prostate cancer160. Weekly DN-101

and daily naproxen combination was well-tolerated

by most patients and was associated with a delay in

PSA doubling time in 75% of patients compared to the

patient’s own rate of PSA rise before the therapy. Despite

these positive findings, this trial was stopped early, when

the ASCENT-II trial was discontinued.

An RCT using alfacalcidol with docetaxel in patients

with metastatic CRPC failed to show any benefit on PSA

or overall survival161. Thus, although combined treat-

ment of calcitriol with many types of cytotoxic agents

has shown synergistic or at least additive effects in pre-

clinical studies, clinical trials testing these combinations

have been less encouraging5.

In summary, some human trials suggest the possibility

of a therapeutic benefit of vitamin D or calcitriol, especially

in early disease and on cancer mortality. However, there

have been no convincing results from RCTs. As discussed

above5, there are several unanswered questions that need

to be addressed, including questions regarding the opti-

mal form of vitamin D to use therapeutically (dietary vita-

min D

3

, calcitriol or an analogue); the target concentration

of 25(OH)D or calcitriol that needs to be achieved; the

best regimen (continuous or intermittent treatment);

the optimal drug combination; when vitamin D

3

or calci-

triol intervention should be initiated during the course of

the disease; and whether calcium supplements should be

routinely added to vitamin D therapy.

A new impediment to achieving convincing results

from RCTs is that many individuals have already started

taking vitamin D supplements, thereby making it more

difficult to recruit subjects for randomization162. A fur-

ther complication is the realization that individuals vary

in their responsiveness to vitamin D therapy — this is

partly determined by SNPs in VDR, CYP24A1 and

CYP27B1 (REF. 163)(BOX 4).

Conclusions

Vitamin D has recently received enormous attention

because of a marked rise in the number of scientific and

lay press articles suggesting that vitamin D might have a

crucial role in the prevention of cancer, as well as in a mul-

titude of non-skeletal and skeletal diseases. V arious lines of

evidence, but not all, suggest that vitamin D deficiency is

associated with an increased risk of mortality and of devel-

oping cancer and other chronic illnesses. Preclinical stud-

ies in cells and animal models, some observational studies

and smaller interventional studies support an anticancer

role for vitamin D. However, the epidemiological studies

are inconsistent, as they report both positive and nega-

tive results. Although some studies show increased cancer

risk with vitamin D deficiency, many studies using predi-

agnostic 25(OH)D levels do not support the hypothesis,

except for colorectal cancer. As approximately 150,000

colorectal cancer cases in the United States and 1,000,000

cases worldwide are annually diagnosed, even if antican-

cer effects of vitamin D were limited to colo r ectal cancer,

this would support the concept that vitamin D deficiency

should be vigorously avoided.

The vitamin D ‘pathway’ might be profoundly impor-

tant for cancer, and it needs to be functionally intact for

vitamin D intake or circulating 25(OH)D concentra-

tion to have an effect. For example, if precancerous or

malignant cells have reduced CYP27B1 activity, acquire

increased expression of CYP24A1 or lose expression of

VDR, then oral intake of vitamin D, sunlight exposure

or administration of calcitriol would be less effective or

ineffective. We therefore speculate that vitamin D intake

might be more important in early carcinogenic stages

rather than advanced cancers, with genetic instability

possibly leading to a defective vitamin D pathway.

Our conclusions are based on our assessment of the

totality of data and not on any one type of evidence.

The preclinical findings suggest how calcitriol regula-

tion of crucial molecular pathways might inhibit the

develop m ent and progression of multiple cancers.

The data suggest that higher concentrations of 25(OH)D

than are necessary for bone health would be more

effective at decreasing cancer risk and progression.

Furthermore, vitamin D metabolites or analogues might

also be helpful in the treatment of cancer, especially

when added to existing therapies. Some but not all of the

negative or inconclusive clinical trials were conducted

in patients with far-advanced disease, used a low vita-

min D or calcitriol dose or had serious design flaws.

So far, large-scale and long-term human RCTs are not

available to definitively conclude whether vitamin D

can offer preventive and therapeutic benefits in cancer.

However, meta-analyses of existing RCTs support a

1.

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2. Gocek, E. & Studzinski, G. P . Vitamin D and

differentiation in cancer. Crit. Rev. Clin. Lab Sci. 46, 190–209 (2009).

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4. Krishnan, A. V., Swami, S. & Feldman, D. Vitamin D

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This is a recent review of the evidence for and

against the role of vitamin D in the prevention and treatment of cancer.

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statistically significant benefit of vitamin D in reducing cancer mortality. We suggest that future RCTs should also aim to reduce, prevent or treat early disease and should not be implemented only in patients with far-advanced cancers in which multiple potent and current therapeutic regimens have failed.

While we await the results of RCTs, we believe that the available data are compelling enough to actively avoid vita-min D deficiency (indicated by serum 25(OH)D <30 ng per mL) to improve universal health. In our opinion, in selected patients who are not at risk of nephrolithiasis, it is probably reasonable to add safe amounts of vitamin D 3 with monitoring (that achieve serum 25(OH)D levels of

30–50 ng per mL) to existing cancer therapies, thereby enhancing their effectiveness and perhaps preventing the development of therapy resistance. Although we believe that adequate anticancer 25(OH)D levels probably exceed 30 ng per mL, future RCTs are needed to also determine the most favourable target levels of serum 25(OH)D and when supplements or drugs should optimally be intro-duced. However, the easy availability, economy and safety of this multipurpose pre-hormone indicate to us that the benefits of dietary vitamin D can be recommended, even while we await RCT data. Considerable evidence described in this Review suggests that there is a role for vitamin D in cancer risk reduction and therapy.

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Acknowledgements

D.F. acknowledges research grant support from California Breast Cancer Research Program, the American Institute for Cancer Research and the Department of Defense Breast and Prostate Cancer Research Programs. B.J.F . is supported by a US National Institutes of Health (NIH) Director’s New Innovator Award (DP2OD006740) and the California Breast Cancer Research Program.

Competing interests statement

The authors declare no competing interests.