Vitamin D signalling pathways in cancer: potential for anticancer therapeutics
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REVIEWS
Vitamin D signalling pathways
in cancer: potential for anticancer
therapeutics
Kristin K. Deeb*, Donald L. Trump‡ and Candace S. Johnson*
Abstract | Epidemiological studies indicate that vitamin D insufficiency could have an
aetiological role in various human cancers. Preclinical research indicates that the active
metabolite of vitamin D, 1α,25(OH)2D3, also known as calcitriol, or vitamin D analogues might
have potential as anticancer agents because their administration has antiproliferative
effects, can activate apoptotic pathways and inhibit angiogenesis. In addition, 1α,25(OH)2D3
potentiates the anticancer effects of many cytotoxic and antiproliferative anticancer agents.
Here, we outline the epidemiological, preclinical and clinical studies that support the
development of 1α,25(OH)2D3 and vitamin D analogues as preventative and therapeutic
anticancer agents.
The most widely accepted physiological role of vitamin The seminal finding by Garland and Garland12 of
D, which is mediated primarily by 1α,25(OH)2D3 (also higher mortality rates from colon cancer in the northeast
known as calcitriol), the most active product of vitamin and lower rates in the south, southwest and west in the
D synthesis, is in the physiological regulation of Ca2+ and United States led to the important concept that exposure
Pi transport and bone mineralization1. The importance of to ultraviolet B or sunlight, which leads to vitamin D
this role is demonstrated by studies with knockout mice synthesis, can reduce the risk of colorectal cancer. Several
deficient in key members of the vitamin D metabolic epidemiological observations have shown an associa-
pathway, such as 25-hydroxyvitamin D3‑1α-hydroxylase tion between low serum 25(OH)D3 levels (the accepted
(1α-OHase; encoded by Cyp27b1), an enzyme that gener- measure of vitamin D body stores) and increased risk for
ates 1α,25(OH)2D3, and 25-hydroxyvitamin D 24-hydrox- colorectal13, breast14 and prostate15 cancers. There are
ylase (24-OHase; encoded by Cyp24a1), the enzyme that many epidemiological studies that have sought to
degrades 1α,25(OH)2D3 (catabolism), and the vitamin D determine associations between vitamin D status and
receptor (Vdr), which binds 1α,25(OH)2D3 to affect target the risk and mortality rates of a number of cancers16–19.
gene transcription2–6 (TABLEs 1,2). Loss of these genes in Giovannucci and colleagues16 recently performed an
mice led to phenotypes with abnormal bone morphol- extensive analysis that combined major determinants
ogy. However, recent observations indicate a much of vitamin D status on cancer risk and mortality with
broader range of action for 1α,25(OH)2D3, including the 51,529 men that were enrolled in the Health Professionals
regulation of differentiation, proliferation and apoptosis. Follow-up Study (HPFS). Individuals were prospectively
Furthermore, altered expression and function of proteins followed for almost 20 years; diet, exercise and lifestyle
crucial in vitamin D synthesis and catabolism have been characteristics were analysed and health outcomes,
observed in many tumour types (TABLE 3). Interestingly, including cancer and cancer-related deaths, were
Vdr–/– mice show hyperproliferation and increased mitotic assessed. Giovannucci et al. developed a model to pre-
activity in the descending colon, suggesting a role for dict 25(OH)D3 levels based on the relationship between
Departments of
Pharmacology and 1α,25(OH)2D3-mediated signalling in tumour suppres- dietary and supplementary vitamin D, physical activity,
Therapeutics* and Medicine‡, sion7. Zinser and colleagues8–10 showed that Vdr ablation body mass index and sunlight exposure (a source of vita-
Roswell Park Cancer Institute, in the mouse increased chemical carcinogenesis in mam- min D). The model was applied to 47,800 individuals in
Buffalo, New York, USA. mary, epidermis and lymphoid tissues but not in ovary, the HPFS, and the analysis indicated a strong association
Correspondence to C.S.J.
e-mail: candace.johnson@
uterus, lung or liver (TABLE 2). However, mice deficient in between low levels of predicted 25(OH)D3 and increased
roswellpark.org key members of the vitamin D synthesis and catabolic cancer incidence and cancer-related mortality, particu-
doi:10.1038/nrc2196 pathways do not develop spontaneous cancer2,3,11. larly for cancers of the digestive system16. Furthermore,
684 | SEPTEMBER 2007 | volume 7 www.nature.com/reviews/cancer
© 2007 Nature Publishing GroupREVIEWS
At a glance resultant 25-hydroxycholecalciferol (25(OH)D3) is
1α-hydroxylated in the kidney by mitochondrial
• Epidemiological studies point to a relationship between vitamin D deficiency and 1α-hydroxylase (1α-OHase; encoded by the gene
cancer risk. CYP27B1), this yields the hormonally active secosteroid
• Alterations in vitamin D receptor expression, and in the synthesis (25-hydroxylase 1α,25(OH) 2D 3 (calcitriol) 23. 24-hydroxylation of
and 1α-hydroxylase) and catabolism (24-hydroxylase) of vitamin D metabolites are 25(OH)D 3 and 1α,25(OH) 2D 3 by the cytochrome
involved in the growth regulation of tumours; thus, compromising 1α,25(OH)2D3 P450 enzyme 25-hydroxyvitamin D 24-hydroxylase
(also known as calcitriol; the active metabolite of vitamin D signalling) sensitivity
(24-OHase; encoded by the gene CYP24A1), to the
and 1α,25(OH)2D3 signalling.
metabolites 24,25(OH) 2D 3 and 1α,24,25(OH) 2D 3,
• The antiproliferative effects of 1α,25(OH)2D3 have been demonstrated in various respectively, is the rate-limiting step for 25(OH)D 3
tumour types, as determined by preclinical trials.
and 1α,25(OH) 2 D 3 catabolism 23 . Additionally,
• The anti-tumour effects of 1α,25(OH)2D3 involve mechanisms that are associated 1α,25(OH)2D3 concentrations are feedback regulated:
with G0/G1 arrest, differentiation, induction of apoptosis and modulating
an increase in 24,25(OH)2D3 induces the synthesis of
different signalling pathways in tumour cells, as well as inhibiting tumour
angiogenesis.
1α,25(OH)2D3; whereas Ca2+, Pi and 1α,25(OH)2D3
itself suppress 1α,25(OH)2D3 synthesis23–26. CYP27B1
• Glucocorticoids potentiate the anti-tumour effects of 1α,25(OH)2D3 and decrease
(which encodes 1α-OHase) expression is induced
1α,25(OH)2D3-induced hypercalcemia. 1α,25(OH)2D3 also potentiates the anti-
tumour effects of many chemotherapeutic agents such as platinum analogues,
by parathyroid hormone (PTH) 25 and repressed
taxanes and DNA-intercalating agents. by 1α,25(OH) 2 D 3 24,27 . Furthermore, CYP24A1 is
strongly induced by 1α,25(OH)2D3 to produce the less
• Given that the major vitamin D catabolizing enzyme, CYP24A1 (24-hydroxylase), is
often amplified and overexpressed in tumour cells, agents that inhibit this enzyme active vitamin D metabolites 1α,24,25(OH)2D3 and
can potentiate 1α,25(OH)2D3 anti-tumour effects. 24,25(OH)2D323.
• Preclinical data indicate that maximal anti-tumour effects are seen with
There are instances of tissue-specific regulation
pharmacological doses of 1α,25(OH)2D3, and can be safely achieved in animals of the vitamin D synthetic enzymes. 1α,25(OH)2D3
using a high-dose, intermittent schedule of administration. Some clinical trial data functions in an autocrine and paracrine manner to
indicates that 1α,25(OH)2D3 is well-tolerated in cancer patients within a proper modulate vitamin D function and signalling. 1α-
dosing schedule. OHase is expressed at extra-renal sites such as nor-
• Data support the hypothesis that vitamin D compounds may have an important mal colon, brain, placenta, pancreas, lymph nodes
role in cancer therapy and prevention, and merit further investigation. and skin 28, allowing local conversion of 25(OH)D 3
to 1α,25(OH)2D3. Importantly, increased CYP27B1
expression is observed in breast29 and prostate30 can-
Giovannucci et al. reported that an increase of 25 nmol cers and during early colon tumour progression in
per L in predicted 25(OH)D3 level is associated with a well-to-moderately differentiated states, but decreased
29% reduction in cancer-related mortality and a 17% in poorly differentiated colon carcinomas 31–33 .
reduction in cancer incidence, suggesting that high Increased expression of CYP27B1 in cancer tis-
25(OH)D3 levels might be associated with a decreased risk sues could provide local conversion of 25(OH)D 3
of some cancers16. A meta-analysis of case–control and to 1α,25(OH)2D3, and may support the notion that
cohort studies found that individuals with ≥ 33 ng per ml 25(OH)D3 and 1α,25(OH)2D3 might have a role in the
(82 nmol per L) 25(OH)D3 had a 50% lower incidence of chemoprevention of these cancers. However, CYP24A1
colorectal cancer20. Additionally, patients with early stage (encoding 24-OHase) mRNA expression is upregu-
non-small-cell lung cancer with high 25(OH)D3 levels lated in tumours, and may counteract 1α,25(OH)2D3
and high vitamin D intake at the time of diagnosis and antiproliferative activity, presumably by decreasing
initiation of treatment had improved overall and recur- 1α,25(OH)2D3 levels34,35 (TABLE 3). Cross et al.35 have
rence-free survival21. Therefore, these data suggest that demonstrated that the upregulation of CYP24A1 and
low levels of 25(OH)D3 are an important risk factor for downregulation of CYP27B1 can occur in high-grade
cancer incidence. colon carcinomas. The chromosomal region 20q13.2,
containing the CYP24A1 gene, is amplified in human
Synthesis and catabolism of vitamin D breast tumours36, and CYP24A1 mRNA expression
1α,25(OH) 2D 3 is synthesized from vitamin D in a is upregulated in samples from human lung, colon
Secosteroid hormones highly regulated multistep process (FIG. 1). The first and ovarian tumours, suggesting that 1α,25(OH)2D3
Molecules that are very similar
in structure to steroids but with
step in vitamin D synthesis is the formation of vita- levels would be reduced in these cases35,37,38 (TABLE 3).
a ‘broken’ ring; two of the min D3 in the skin through the action of ultraviolet This suggests that inhibition of CYP24A1 expression
B‑ring carbon atoms (C-9 and irradiation; vitamin D3 can also be taken in the diet and activity is essential for prevention to be effective.
10) of the four steroid rings are but in North America and Europe dietary vitamin D3 Small-molecule inhibitors with varying specificity for
not joined.
intake is a minor component of vitamin D3 acquisi- 24-OHase39–42 render tumour cells more sensitive to the
Autocrine tion because dairy products, eggs, fish and fortified action of 1α,25(OH)2D3 and its analogues. Consistent
A substance secreted by a cell foods contain only small quantities of vitamin D22. with the epidemiological studies discussed above, these
that acts on the surface Decreased sun exposure further limits vitamin D findings indicate that 1α,25(OH)2D3 catabolism could
receptors of the same cell. synthesis. modulate tumour growth in some tissues, indicat-
Paracrine
Vitamin D 3 (cholecalciferol) is hydroxylated by ing the potential for the development of 24-OHase
A substance secreted by a cell liver mitochondrial and microsomal 25-hydroxylases inhibitors as cancer preventative and/or anticancer
that acts on adjacent cells. (25-OHase) 23, encoded by the gene CYP27A1. The therapeutic agents.
nature reviews | cancer volume 7 | SEPTEMBER 2007 | 685
© 2007 Nature Publishing GroupREVIEWS
Table 1 | Mouse models of vitamin D metabolic enzymes and receptor signalling
Genetic modification Mouse phenotype Cancer phenotype Refs
Cyp27b1 (which
–/–
Pseudo-vitamin D deficiency rickets (PDDR); decreased serum Ca and 2+
None 2,3
encodes 1α-OHase) Pi; secondary hyperthyroidism; undetectable 1α,25(OH)2D3 (calcitriol)
levels; disorganized growth plate structure and osteomalacia. In addition:
infertile females; uterine hypoplasia; decreased ovarian size; compromised
folliculogenesis; reduction in CD4+ and CD8+ peripheral T lymphocyte
Cyp24a1–/– (24-OHase) Lethal hypercalcemia; impaired intramembranous bone mineralization None 11
Vdr (VDR signalling)
–/–
Vitamin D deficiency rickets type II (VDDR II) and osteomalacia; alopecia, Hyperproliferation of descending 4,5,6,7
hypocalcemia, hyperparathyroidism; impaired bone formation, growth colon (increased PCNA positivity
retardation; female infertility, uterine hypoplasia, impaired folliculogenesis and cyclin D1 expression)
PCNA, proliferating cell nuclear antigen.
1α,25(OH)2D3-mediated transcription of target genes. in a transcriptionally repressed state48. Conformational
1α,25(OH)2D3 exerts transcriptional activation and change also repositions the VDR activation function
repression of target genes by binding to the VDR (BOX 1). 2 (AF2) domains to bind to stimulatory coactivators,
The VDR is a member of the steroid hormone receptor consisting of the steroid receptor coactivators (SRCs),
superfamily and regulates gene expression in a ligand- nuclear coactivator 62 kDa–SKI-interacting protein
dependent manner43 (FIG. 2). Interestingly, N‑terminal VDR (NCoA62–SKIP) and the chromatin modifiers, CREB
variants show tissue-specific expression44,45 that might also binding protein (CBP)–p300 and PBAF (polybromo- and
contribute to the differential specificity of 1α,25(OH)2D3- SWI‑2-related gene 1 associated factor), which acetylates
mediated regulation. 1α,25(OH)2D3–VDR-dependent histones in the nucleosomes to unravel DNA for transcrip-
transcriptional activity is modulated through synergistic tion49. Once the chromatin is de-repressed, the vitamin
ligand-binding and dimerization with retinoic X receptor D receptor-interacting proteins (DRIPs) form a complex
(RXR). The activated 1α,25(OH)2D3–VDR–RXR com- that binds to the AF2 domain of VDR and interacts with
plex specifically binds to vitamin D response elements the transcription machinery, such as TF2B (transcription
(VDREs), composed of two hexanucleotide repeats inter- factor 2B) and RNA polymerase II, and initiates transcrip-
spaced by varying numbers of nucleotides (for example, tion (for a review see REF. 23). Recently, it has been shown
GGTCCA-NNN-GGTCCA, where N is any nucleotide; that epigenetic regulation of VDR through increased
this is denoted DR3), in the promoter regions of target expression of NCoR1 and SMRT repress VDR-mediated
genes46. For transcriptional activation, VDR occupies the 3′ signalling in prostate50 and breast cancer51 cell lines, and
half-site whereas RXR binds the 5′ half-site of VDRE47. may have a role in mediating the antiproliferative effects
Co-factor proteins also have the ability to modulate of 1α,25(OH)2D3 in these tissues.
VDR-mediated gene expression; these proteins possess The mechanism by which 1α,25(OH)2D3 represses
intrinsic chromatin-modifying enzymatic activities, act gene expression through the binding of VDR to negative
as a platform for the recruitment of chromatin-modifying VDREs (DR3-type), placing VDR on the 5′ half-site of
proteins and recruit basal transcription factors to the pro- the VDRE52,53, such as is the case with human PTH, may
moters23. 1α,25(OH)2D3 binding induces phosphoryla- involve interference with transcriptional machinery but
tion and conformational changes in VDR, which causes is less understood. Recently, transcriptional repression
the release of co-repressors (such as nuclear receptor by 1α,25(OH)2D3 has been further elucidated for the
co-repressors (NCoRs) and the silencing mediator for human CYP27B1 (REFS 27,54,55) and PTH56 genes. The
retinoid and thyroid hormone receptors (SMRT)–histone VDR–RXR heterodimer represses gene transcription in
deacetylase (HDAC) complex) that maintain chromatin a 1α,25(OH)2D3-dependent manner through E‑box-type
Table 2 | Vdr knockout mice and carcinogenesis
Oncogene/ Tissue Cancer phenotype Ref
carcinogen
MPA plus DMBA Skin 40% sebaceous, 25% squamous and 15% follicular papillomas 9
carcinogens compared with WT littermates; other infrequent lesions include
basal cell carcinoma and haemangioma
Mammary Higher incidence of alveolar and ductal hyperplasias in 8
Vdr–/– mice compared with WT mice; development of palpable
mammary tumours was not altered by Vdr ablation
Lymph nodes and/ Lymphoblastic and thymic lymphoma higher in Vdr–/– (27%) 8
or thymus compared with WT mice (11%)
Vdr–/– ;neu oncogene Mammary Decreased survival of Vdr–/–; neu mice compared with their 10
Vdr+/+; neu and Vdr+/–; neu littermates; increased development of
mammary tumours driven by the neu oncogene
DMBA, 7,12-dimethylbenzanthracene; MPA, medroxyprogesterone acetate; WT, wild-type.
686 | SEPTEMBER 2007 | volume 7 www.nature.com/reviews/cancer
© 2007 Nature Publishing GroupREVIEWS
Table 3 | Expression of molecules that function in vitamin D metabolism and signalling in human cancers.
Protein Altered expression observed Prognostic or histological Types of cancer
(gene) in human cancer tissues observations
Vitamin D metabolic enzymes
25-OHase Increased mRNA NA Breast34, cervical34 and ovarian cancer34, HCC172
(CYP27A1)
1α-OHase Increased mRNA NA Basal cell carcinoma173, breast34,174, cervical34
(CYP27B1) and ovarian cancer34
Increased mRNA Moderately differentiated Colon cancer31,35,175
Decreased mRNA Poorly differentiated Colon cancer35
Splice variants (Hyd‑V5, ‑V6, ‑V7 NA Glioblastoma multiforme176, melanoma177,
and ‑V8) cervical cancer177
Immunoreactivity NA Pancreatic132, breast132 and colon cancer 33,
renal cell carcinoma132
Increased immunoreactivity Moderately differentiated Colon cancer33,35
Decreased immunoreactivity Poorly differentiated Colon cancer33,35
24-OHase Amplified at 20q13.2 locus NA Gastric adenocarcinoma37, breast cancer36
(CYP24A1)
Increased mRNA NA Basal cell carcinoma173, SCC (cutaneous)178,
lung38,42, breast34,36, colon35,38, cervical34 and
ovarian cancer34,38
Increased mRNA Poor prognosis Oesophageal cancer179
Decreased mRNA NA Breast cancer38
Increased mRNA and activity Poorly differentiated Colon cancer32
Increased protein NA Lung cancer (NSCLC)42
Vitamin D receptor
VDR Increased mRNA NA Basal cell carcinoma173, SCC (cutaneous)178,
(VDR) colon cancer31
Decreased mRNA Poorly differentiated Colon cancer31
Increased immunoreactivity NA Breast34, cervical34 and ovarian cancer34
Increased, predominantly Well differentiated Colon cancer166
cytoplasmic
Decreased immunoreactivity Moderately and poorly Colon cancer166
differentiated
Poorly differentiated Colon cancer35
HCC, hepatocellular carcinoma; NA, non applicable; NSCLC, non-small cell lung carcinoma; SCC, squamous cell carcinoma.
negative VDREs, comprised of a CANNTG-like motif control of 1α,25(OH)2D3 biosynthesis55, as well as negative
in the promoter regions of the CYP27B1 (REFS 27,54,55) regulation of other genes with nVDREs in their promot-
and PTH56 genes, which are distinct from the DR3- ers. Furthermore, Kim et al.57 demonstrated that not only
type response elements. VDR-interacting repressor is histone deacetylation crucial for chromatin structure
(VDIR), when bound to E‑box-type elements, induces remodelling in suppression of the CYP27B1 gene, but
the transcriptional activation of CYP27B154. However, that transrepression by VDR requires DNA methylation
the binding of 1α,25(OH)2D3 to VDR causes VDR to of the CYP27B1 gene promoter, suggesting complicated
interact with VDIR. 1α,25(OH)2D3-induced association epigenetic modifications for transcriptional regulation
between VDR and VDIR induces dissociation of the of the CYP27B1 gene. Epigenetic regulation of CYP27B1
histone acetyltransferase (HAT) co-activator and recruit- and CYP24A1 has been previously reported for the PNT‑2
ment of HDAC co-repressor for 1α,25(OH)2D3-induced human normal prostate cells and DU‑145 prostate cancer
transrepression of CYP27B1 gene expression54. In addi- cell line58. Histone methylation and demethylation are cru-
tion, Williams syndrome transcription factor (WSTF) cial events that impose ligand- and signal-dependent gene
potentiates 1α,25(OH)2D3-induced transrepression by activation by nuclear receptors and prevent the recruit-
VDR of the CYP27B1 gene promoter by facilitating the ment of unliganded nuclear receptors and transcription
association between WINAC, a multifunctional, ATP- factors from binding to their target promoters and causing
dependent chromatin-remodelling complex, and chro- constitutive gene activation59.
matin55. This transrepression mechanism is an important Examples of genes with DR3-type response elements
biological function of VDR to allow negative-feedback that are transcriptionally activated by 1α,25(OH)2D3
nature reviews | cancer volume 7 | SEPTEMBER 2007 | 687
© 2007 Nature Publishing GroupREVIEWS
UV-B
D3
DBP
7-dehydrocholesterol D3 D3
Circulation
Pre-D3
D3
Skin
Intestine
Liver
Pi, Ca2+ and 25-OHase
other factors D3
24-OHase
25(OH)D3
+/–
24,25(OH)2D3
Parathyroid PTH Kidney + Excretion
glands
1α,24,25(OH)2D3
1α-OHase
Dietary sources
24-OHase of vitamin D
1α,25(OH)2D3
Intestine Tumour
Increases microenvironment
absorption • Inhibits proliferation
of Ca2+ Bone Immune cells • Induces differentiation
and Pi Increases bone Induces • Inhibits angiogenesis
mineralization differentiation
Figure 1 | Vitamin D metabolism. Photochemical synthesis of vitamin D3 (cholecalciferol, D3) occurs cutaneously
where pro-vitamin D3 (7-dehydrocholesterol) is converted to pre-vitamin D3 (pre‑D3) in response to ultraviolet B
Nature
(sunlight) exposure. Vitamin D3, obtained from the isomerization of pre-vitamin D3 in the epidermal Reviews
basal layers| Cancer
or
intestinal absorption of natural and fortified foods and supplements, binds to vitamin D‑binding protein (DBP) in the
bloodstream, and is transported to the liver. D3 is hydroxylated by liver 25-hydroxylases (25-OHase). The resultant
25‑hydroxycholecalciferol (25(OH)D3) is 1α-hydroxylated in the kidney by 25-hydroxyvitamin D3‑1α-hydroxylase
(1α‑OHase). This yields the active secosteroid 1α,25(OH)2D3 (calcitriol), which has different effects on various target
tissues23. The synthesis of 1α,25(OH)2D3 from 25(OH)D3 is stimulated by parathyroid hormone (PTH) and suppressed by
Ca2+, Pi and 1α,25(OH)2D3 itself. The rate-limiting step in catabolism is the degradation of 25(OH)D3 and 1α,25(OH)2D3
to 24,25(OH)D3 and 1α,24,25(OH)2D3, respectively, which occurs through 24-hydroxylation by 25-hydroxyvitamin D 24-
hydroxylase (24-OHase), encoded by the CYP24A1 gene. 24,25(OH)D3 and 1α,24,25(OH)2D3 are consequently excreted.
The main effects of 1α,25(OH)2D3 on various target tissues are highlighted above.
consist of CYP24A1 (REF. 60) (encoding 24-OHase), Nongenomic action of 1α,25(OH)2D3. Nongenomic
BGLAP61 (osteocalcin; expressed in bone osteoblasts), actions mediated by 1α,25(OH)2D3 are rapid and not
and CDKN1A62 (which encodes the cyclin depend- dependent on transcription. However, nongenomic
ent kinase (CDK) inhibitor p21). Those repressed signalling may indirectly affect transcription through
by 1α,25(OH)2D3 include PTH53. Although VDREs cross-talk with other signalling pathways68,69. Although
are traditionally thought to occur in the promoter there is no agreement on how the nongenomic actions
regions of the target genes, a DR3-type VDRE was are initiated, data suggest that these effects begin at the
recently identified in exon 4 of the growth arrest plasma membrane and involve a non-classical membrane
and DNA-damage-inducible (GADD45) gene 63 . receptor (memVDR; FIG. 2) described in intestinal caveo-
1α,25(OH)2D3-mediated repression or activation of lae70, and a 1α,25(OH)2D3-membrane-associated rapid-
many proto-oncogenes or tumour-suppressor genes response steroid binding protein (1α,25D3-MARRS)
is described in normal and tumour tissues 62,64–67; isolated from chick intestinal basal-lateral membrane71.
however, only a few such genes contain VDREs in the The most well-described nongenomic effect of
promoter regions and are under the direct transcrip- 1α,25(OH)2D3 is the rapid intestinal absorption of Ca2+
tional control of 1α,25(OH)2D3, such as CDKN1A62 (REF. 72). Binding of 1α,25(OH)2D3 to the proposed mem-
and CCNC (which encodes cyclin C, containing a brane receptor can result in the activation of numerous
DR4-type VDRE)65. This suggests that 1α,25(OH)2D3 signalling cascades68,69 (FIG. 2). Activation of these signalling
exerts many of its effects indirectly by modulating cascades, such as protein kinase C (PKC), can result in the
signalling cascades or by unknown nongenomic rapid opening of voltage-gated Ca2+ channels and an increase
mechanisms (FIGS 2,3). in intracellular Ca2+ (REF. 73), which may subsequently
688 | SEPTEMBER 2007 | volume 7 www.nature.com/reviews/cancer
© 2007 Nature Publishing GroupREVIEWS
Box 1 | The vitamin D receptor
The human VDR gene (which encodes the vitamin D receptor), located on chromosome 12q, is composed of promoter and
regulatory regions (1a–1f) and exons 2–9, which encode 6 domains (A – F) of the full length VDR protein (see figure)23. VDR
nuclear localization signals (blue) direct the receptor into the nucleus155,156 along microtubule tracts to the nuclear pores157.
Upon 1α,25(OH)2D3 binding to the hormone ligand-binding domain (red), VDR is stabilized by the phosphorylation of
serine 51 in the DNA-binding domain (green) by protein kinase C76, and serine 208 in the hinge region by casein kinase II158.
VDR associates with the retinoic acid receptor (RXR) through the dimerization domains (yellow). The 1α,25(OH)2D3–VDR–
RXR complex binds to the vitamin D response elements (VDREs) through the DNA-binding domain in the promoters of
target genes. Conformational change in the VDR results in the dissociation of the co-repressor, silencing mediator for
retinoid and thyroid hormone receptors (SMRT), and allows interaction of the VDR activation function 2 (AF2)
transactivation domain (light grey) with stimulatory coactivators, such as steroid receptor coactivators (SRCs), vitamin D
receptor-interacting proteins complex and nuclear coactivator‑62 kDa–Ski-interacting protein (NCoA62–SKIP)23 that
mediate transcriptional activation.
Non-synonymous (FokI) and synonymous (BsmI, ApaI, TaqI and Tru9I) single-nucleotide polymorphisms (SNPs) have
been identified in VDR (defined by restriction enzymes, polymorphisms are indicated in parentheses). FokI
polymorphism at translation initiation codon results in a smaller VDR that interacts with transcription factor 2B (TF2B)
more efficiently and has greater transcriptional activity than the full length VDR159. Although the functional effects of
these SNPs remain unknown, they have been reported to be associated with increased susceptibility to primary and
metastatic breast cancer17, squamous cell carcinoma160, colorectal cancer161,162, and prostate cancer163,164, but may be
protective against head and neck cancer165.
The expression of VDR is an important determinant of the tumour cell response to 1α,25(OH)2D3. The VDR is
overexpressed or repressed in several histological types of cancer (TABLE 3), demonstrating tissue-type variations in
1α,25(OH)2D3 signalling (supplemental information S1 (table)). VDR expression increases in hyperplastic polyps and in
the early stages of tumorigenesis, but declines in late-stage poorly differentiated tumours and is absent in associated
metastases. Tumours of the colon with the highest expression of VDR were most responsive to 1α,25(OH)2D3
treatment85,166. However, downregulation of the VDR in colon cancer cells through the transcription factor SNA1L167
reduces the anticancer effect of the vitamin D analogue EB1089.
VDR gene
Chromosome 12
1f 1e 1a 1d 1b 1c 2 3 45 6 7 8 9
q13–14 ~75 kb
Bsml (A60890G)
Fokl (C27823T) Tru9l (G61050A)
Apal (G61888T) Taql (T61938C)
DNA binding
(aa 24–90, 91–115) VDR protein
Nuclear localization S51 S208
(aa 49–55, 79–105) P P
AF-2
Hormone ligand binding N Hinge region 48 kDa
(aa 227–244, 268–316, 396–422)
1 24 49 91 115 227244268 317 396 422 427 aa
Dimerization
(aa 37, 91–92, 244–263, 317–395) A/B C D E/F
Transactivation
(aa 246, 416–422)
Nature Reviews | Cancer
activate the Raf–mitogen-activated protein kinase extracel- Anti-tumour effects of 1α,25(OH)2D3 signalling
lular signal-regulated kinase kinase (MEK)–mitogen-acti- 1α,25(OH)2D3 has been examined preclinically for its
vated protein kinase (MAPK)–extracellular signal-regulated therapeutic efficacy in chemopreventive and anticancer
kinase (ERK) cascade in skeletal muscle cells74. Activation activity. A chemoprevention study used Nkx3‑1;Pten
of the Raf–MEK–MAPK–ERK cascade, which mediates mutant mice to recapitulate prostate carcinogenesis,
proliferative cellular effects, may be a response to increased and showed that 1α,25(OH)2D3 administration delayed
Ca2+ in normal colon73 and skeletal muscle cells74, and may the onset of prostate intraepithelial neoplasias (PIN) and
not have a direct role in the antiproliferative activities of had better anti-tumour activity when administered to
1α,25(OH)2D3 in tumour cells (discussed below). In addi- mice with early-stage (PIN) rather than advanced-stage
tion, ERK can also increase the transcriptional activity of prostate disease77. Furthermore, studies using model
the VDR75, and nongenomic activation of PKC may stabi- systems of squamous cell carcinoma (SCC)78, prostate
lize VDR (through phosphorylation)23,76, thereby affecting adenocarcinoma79, cancers of the ovary80, breast81 and
the transcriptional activity of the receptor. Therefore, the lung82 showed that the administration of 1α,25(OH)2D3
nongenomic activation of these pathways may cooperate or vitamin D analogues had significant anticancer effects.
with the classical genomic pathway to transactivate VDR The effects of 1α,25(OH)2D3 and its derivatives have
and elicit the antiproliferative effects of 1α,25(OH)2D3, but been shown to function through the VDR to regulate
this remains to be elucidated. proliferation, apoptosis and angiogenesis62,83–87.
nature reviews | cancer volume 7 | SEPTEMBER 2007 | 689
© 2007 Nature Publishing GroupREVIEWS
SOC 1α,25(OH)2D3 GPCR
1α,25(OH)2D3 channels
P
VDR
1α,25(OH)2D3 PLCγ
d AC
Caveolae Ca2+ PI3K
mem ? [cAMP]
PKC Ras
VDR
9cRA PKA
RXR P
VDR Raf isoforms
MEK1/2
PBAF
a CBP/ SWI/SNF
ERK–MAPK1/2
P300 Chromatin
NCoA62– remodeling
1α,25(OH)2D3 SKIP
9cRA SRC-1 (histone
RXR VDR P acetylation) Nucleus
Transcriptional 9cRA
P Cross-talk
activation RXR VDR
Transcriptional
repression
5′ 3′
VDREs NCoA62– b Gene
SKIP 05 transcription
IP2
9cRA DR DRIPs RNA CDKN1A
c TF2B Pol II
WINAC P CYP24A1
HDAC Gene RXR VDR SPP1
complexes NCoR–
SMRT WSTF repression
9cRA CYP27B1 3′
Chromatin PTH 5′
remodelling RXR VDR P VDREs
(histone VDIR
deacetylation)
nVDREs
Nature Reviews | Cancer
Figure 2 | 1α,25(OH)2D3-mediated transcriptional regulation. Classical action of 1α,25(OH) D is mediated by
2 3
binding of the vitamin D receptor (VDR)−9-cis-retinoic acid receptor (RXR) complex at the vitamin‑D response
elements (VDREs). a | Transcriptional activation involves the co-activators, steroid receptor coactivators (SRCs),
nuclear coactivator‑62 kDa–Ski-interacting protein (NCoA62–SKIP) and histone acetyltransferases (HATs), CREB
binding protein (CBP)–p300 and polybromo- and SWI‑2-related gene 1 associated factor (PBAF–SNF) to acetylate
histones to derepress chromatin. b | Binding of the vitamin D receptor-interacting protein 205 (DRIP205) to the
activation function 2 (AF2) of VDR (and RXR) attracts a mediator complex containing other vitamin D receptor-
interacting proteins (DRIPs) that bridge the VDR–RXR–NCoA62–SKIP–DRIP205 complex with transcription factor
2B (TF2B) and RNA polymerase II (RNA Pol II) for transcription initiation. The presence of the multiprotein complex
facilitates increased transcription of genes, such as CDKN1A (which encodes the cyclin-dependent kinase
inhibitor p21), CYP24A1 (which encodes 24-OHase) and SPP1 (which encodes osteopontin)23.
c | 1α,25(OH)2D3-mediated transcriptional repression involves VDR–RXR heterodimer association with VDR-
interacting repressor (VDIR) bound to E‑box-type negative VDREs (nVDREs), dissociation of the HAT co-activator
and recruitment of histone deacetylase (HDAC) co-repressor54. Williams syndrome transcription factor (WSTF)
potentiates transrepression by interacting with a multifunctional, ATP-dependent chromatin-remodelling
complex (WINAC) and chromatin55. This leads to the repression of genes, such as CYP27B1 (which encodes 1α-
OHase) and PTH (which encodes parathyroid hormone). d | Non-genomic, rapid actions of 1α,25(OH)2D3 are
hypothesized to involve 1α,25(OH)2D3 binding to cytosolic (VDR) and membrane VDR (memVDR), also found in
caveolae, and speculated to activate the mitogen-activated protein kinase (MAPK)–extracellular signal-regulated
kinase (ERK) 1 and 2 cascade68 through the phosphorylation (P) and activation of Raf by protein kinase C (PKC) by
Ca2+ influx through store-operated Ca2+ (SOC) channels. 1α,25(OH)2D3 stimulates SOC Ca2+ influx (in muscle cells)
by trafficking of the classic VDR to the plasma membrane, where the VDR interacts with the SOC channel. Ca2+
influx activates Ca2+ messenger systems, such as PKC. Activated PKC can phosphorylate VDR. 1α,25(OH)2D3
binding to G‑protein coupled receptors (GPCRs) activates phospholipase Cγ (PLCγ), Ras, phosphatidylinositol
3‑kinase (PI3K) and protein kinase A (PKA) pathways, and induces MAPK–ERK1 and 2 signalling. Activated Raf–
MAPK–ERK may engage in cross-talk with the classical VDR pathway to modulate gene expression. AC, adenylate
cyclase; cAMP, cyclic adenosine monophosphate.
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© 2007 Nature Publishing GroupREVIEWS
a b c d e f g
1α,25(OH)2D3
IGF1
TGFβ ↑E-cadherin Wnt
TGFβR2
TGFβR1
TGF1R
EGFR
β-catenin Frizzled
Ras P13K Cytosol
BCL2 BAX SMADs β-catenin
Raf isoforms BCL-XL BCL-XS APC
Akt β-catenin VDR
MEK1/2 Degradation
Telomerase Effector
caspases
ERK–MAPK1/2
p15 p21 p27 SKP2 β-catenin
Apoptosis MYC MYC
TCF4
TCF1
Differentiation CD44
Apoptosis CDK4/6 CDK2 Degradation PARG
Growth inhibition
Cyclin D1,2,3 Cyclin E
p107/p130 Nucleus
PPP E2F4/5
pRB pRB +
DP1 Cell cycle
E2F1,2,3
Growth arrest
Figure 3 | Key cancer-related signalling pathways targeted by 1α,25(OH)2D3. 1α,25(OH)2D3 inhibits mitogen-activated
Nature Reviews | Cancer
protein kinase (MAPK)–extracellular signal-regulated kinase (ERK) 1 and 2 signalling through suppression of epidermal
growth factor (EGFR; a) and insulin-like growth factor 1 (IGF1; b), which both target Ras. 1α,25(OH)2D3 induces apoptosis
through the IGFR1−phosphatidylinositol 3‑kinase (PI3K)−Akt-dependent signalling pathway (b), inhibiting telomerase (c),
downregulating BCL2, inducing BAX and activating caspase cleavage (d). Cell-cycle progression is perturbed by
1α,25(OH)2D3 through S‑phase kinase-associated protein ubiquitin ligase (SKP2; targeting p27 for degradation; e), and
MYC, which results in pRB dephosphorylation; and transforming growth factor-β (TGFβ; f) cross-talk. Cell-cycle
perturbation by 1α,25(OH)2D3 ultimately affects the association of retinoblastoma pocket proteins (pRB and p107/p130)
and the E2F family of transcription factors and DP polypepitide (DP1) heterodimers that mediate the transcription of cell-
cycle genes. Association of E2F1, 2 and 3 with pRB in its hypophosphorylated state and interaction of the E2F4 and 5
transcriptional repressors and DP1 with p107/p130 prevent transcription of cell-cycle genes and restrain cell-cycle
progression. Activation of VDR by 1α,25(OH)2D3 induces the expression of E‑cadherin (g), thereby promoting the
translocation of β‑catenin from the nucleus to the plasma membrane and competing with T-cell transcription factor 4
(TCF4) for β‑catenin binding; thus inhibiting the Wnt–β-catenin–TCF4 signalling pathway, which leads to the induction of
MYC, TCF1 (transcription factor 1), CD44 and PPARG (peroxisome proliferator-activated receptor-γ). APC, adenomatosis
polyposis coli; CDK, cyclin-dependent kinase; pRB, phosphorylated retinoblastoma; Wnt, wingless-related MMTV
integration site.
Antiproliferative effects of 1α,25(OH)2D3. Cell-cycle contain VDREs, and their transcriptional activation
perturbation is central to 1α,25(OH) 2D3-mediated or repression may not be directly mediated by VDR.
antiproliferative activity in tumour cells (supplemen- 1α,25(OH) 2D 3–VDR transcriptional activation of
tal information S1 (table)). Progression through the CDKN1A induces cell-cycle exit (differentiation) and
cell cycle is regulated by cyclins, and their association cell-cycle arrest in human U937 myelomonocytic cells62.
with CDKs and CDK inhibitors (CKIs). Expression Treatment of human breast cancer MCF7 cells with
of the CKIs p21 and p27 inhibits proliferation, in part 1α,25(OH)2D3 also increases the expression of CDKN1A
by inducing G1 cell-cycle arrest and withdrawal from and CDKN1B, (which encodes p27) and represses
the cell cycle (G0). CDKN1A and GADD45A contain CCND1 (encoding cyclin D1), CCND3 (encoding cyc-
a functional VDRE and are direct transcriptional tar- lin D3), CCNA1 (which encodes cyclin A1) and CCNE1
gets of 1α,25(OH)2D3–VDR. However, many genes are (which encodes cyclin E1), and hence leads to the inhibi-
transcriptionally affected by 1α,25(OH)2D3 but do not tion of CDK activity and pRb hypophosphorylation88,89.
nature reviews | cancer volume 7 | SEPTEMBER 2007 | 691
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Similarly, the treatment of SCC cells with 1α,25(OH)2D3 of 1α,25(OH)2D3102. Recent findings reported by Palmer
induces G0/G1 cell-cycle arrest owing to the tran- et al.103 indicate that 1α,25(OH)2D3 promotes differentia-
scriptional activation of CDKN1B and consequent tion through the induction of CDH1 (which encodes E
pRb hypophosphorylation90. However, in this context cadherin) in adenomatosis polyposis coli (APC)-mutated
CDKN1A expression was repressed, indicating that the human colorectal cancer SW480 cells. CDH1 activation
cell-cycle arrest is an indirect effect of 1α,25(OH)2D3 consequently restrained cell growth by facilitating the
treatment or that cell-type specificity might determine translocation of β‑catenin from the nucleus to the plasma
the ability of activated 1α,25(OH)2D3–VDR to induce membrane, thus inhibiting β‑catenin-mediated tran-
CDKN1A expression90. Other genes have been shown scription and allowing activated VDR to compete with
to be transcriptionally affected by 1α,25(OH)2D3 in β‑catenin for transcription factor binding. Again, there
colon cancer, ovarian carcinoma and leukaemia cells, appears to be no specific mechanism regarding the ability
such as activation of GADD45 (REF 63), which is involved of 1α,25(OH)2D3 to induce differentiation in tumour cells
in DNA damage responses, repression of TYMS (supplemental information S1 (table)).
(which encodes thymidylate synthetase)91 and TK1
(which encodes thymidine kinase)91, which are involved Apoptosis. In addition to the antiproliferative effects
in DNA replication, and activation of the INK4 fam- of 1α,25(OH) 2D 3, there is increasing evidence that
ily92 of cyclin D‑dependent kinase inhibitors, which 1α,25(OH)2D3 exerts anti-tumour effects by regulat-
mediate G1 cell-cycle arrest; whereas cyclin E–CDK2 ing key mediators of apoptosis, such as repressing the
and the SKP2 (S-phase kinase-associated protein 2) expression of the anti-apoptotic, pro-survival proteins
ubiquitin ligase, which targets CKIs to the proteasome, BCL2 and BCL-XL, or inducing the expression of pro-
are downregulated93 by 1α,25(OH)2D3. 1α,25(OH)2D3 apoptotic proteins (such as BAX, BAK and BAD). It
treatment also results in the repression of the proto- has been reported that 1α,25(OH)2D3 downregulates
oncogene MYC89,94, which significantly contributes to BCL2 expression in MCF‑7 breast tumour and HL‑60
the antiproliferative effects of 1α,25(OH)2D3. leukaemia cells and upregulates BAX and BAK expres-
1α,25(OH)2D3 can have many indirect effects on sion in prostate cancer, colorectal adenoma and carci-
cell-cycle regulation owing to cross-talk with other noma cells84. In addition to regulating the expression
pathways; for example, 1α,25(OH)2D3 treatment can of the BCL2 family, 1α,25(OH)2D3 might also directly
result in the upregulation of IGFBP3 (which encodes activate caspase effector molecules, although it is
insulin growth factor binding protein 3) and trans- unclear whether 1α,25(OH)2D3-induced apoptosis is
forming growth factor‑β (TGFβ)–SMAD3 signalling caspase-dependent84. In support of this idea, the treat-
cascades and by downregulating the epidermal growth ment of mouse SCC tumour cells with 1α,25(OH)2D3
factor receptor (EGFR) signalling pathway67,95,96 (FIG. 3). increased VDR expression and concomitantly inhib-
Although there appears to be an overall inhibition of ited the phosphorylation of ERK104. Upstream of ERK,
cell-cycle progression in tumour cells treated with the growth-promoting and pro-survival signalling
1α,25(OH)2D3, the precise molecular basis for such molecule MEK is cleaved and inactivated in a caspase-
an effect differs from one tumour cell type to another dependent manner in cells that undergo apoptosis
such that a unifying hypothesis with regard to the after treatment with 1α,25(OH)2D3. Recently, a novel
exact mechanism of 1α,25(OH) 2D 3-mediated cell- mechanism of 1α,25(OH)2D3-mediated apoptosis in
cycle perturbation has not been possible (supplemental epithelial ovarian cancer cells was proposed by Jiang
information S1 (table)). et al. 105, wherein they showed that 1α,25(OH) 2D 3
Activation of the VDR by 1α,25(OH)2D3 can also destabilizes telomerase reverse transcriptase (TERT)
inhibit tumour cell proliferation by inducing differentia- mRNA, therefore inducing apoptosis through tel-
tion in various myeloid leukaemia cell lines and freshly omere attrition resulting from the down-regulation
isolated leukaemia cells62,83, which is dependent on the of telomerase activity. The diverse effects observed
formation of activated VDR and phosphatidylinositol 3- for 1α,25(OH) 2D 3-mediated apoptosis suggest that
kinase (PI3K) complexes97. However, in haematopoeitic although anti-proliferative effects directed against
progenitor cells, 1α,25(OH)2D3 inhibits differentiation the tumour are clear in vitro and in vivo (supple-
through VDR-independent suppression of interleukin mental information S1 (table)), dissecting the exact
12 (IL12) protein secretion and down-regulation of other mechanism(s) central to these activities remains a
co-stimulatory molecules (CD40, CD80 and CD86)98. In challenge.
cell lines of head and neck, colon and prostate tumours,
administration of 1α,25(OH)2D3 or vitamin D analogues Angiogenesis. 1α,25(OH)2D3 inhibits the proliferation
induces the expression of genes that are associated with the of endothelial cells in vitro and reduces angiogenesis
differentiated cell of origin91,99,100. In various colon cancer in vivo106–108. Vascular endothelial growth factor (VEGF)-
cells, treatment with 1α,25(OH)2D3 induces differentia- induced endothelial cell tube formation and tumour
tion either by increasing PKC- and JNK-dependent JUN growth are inhibited in vivo by 1α,25(OH)2D3 admin-
activation101 or by differentially regulating the expression istration to mice with VEGF-overexpressing MCF‑7
of inhibitor of DNA binding 1 and 2 (ID1 and ID2), which xenografts86. 1α,25(OH)2D3 can increase VEGF mRNA
encode proteins that are transcriptional regulators of epi- levels in vascular smooth muscle cells109 and upregu-
thelial cell proliferation (ID2) and differentiation (ID1); late mRNA levels of the potent anti-angiogenic factor
the repression of ID2 mediated the antiproliferative effects thrombospondin 1 (THBS1) in SW480-ADH human
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© 2007 Nature Publishing GroupREVIEWS
colon tumour cells 102. In SCC cells, 1α,25(OH) 2D 3 prostaglandin activity, the induction of their degradation
induces the angiogenic factor interleukin 8 (IL8)110, but through the upregulation of 15-hydroxyprostaglandin
in prostate cancer cells 1α,25(OH)2D3 interrupts IL8 dehydrogenase, and reduction of prostaglandin recep-
signalling leading to the inhibition of endothelial cell tors120. These findings support the rationale for clinical
migration and tube formation111. A significant inhibition evaluation of a combination of 1α,25(OH)2D3 and non-
of metastasis is observed in prostate and lung murine steroidal anti-inflammatory drugs (NSAIDs) for prostate
models treated with 1α,25(OH)2D3, and these effects cancer therapy120. Increased anti-tumour effects with
may be based, at least in part, on the anti-angiogenic 1α,25(OH)2D3 combination therapy offers the opportu-
effects described79,82. Interestingly, in tumour-derived nity for the clinical use of 1α,25(OH)2D3 across several
Platinum analogues endothelial cells (TDECs), 1α,25(OH)2D3 induces apop- tumour types where modest effects are observed with
Platinum-based tosis and cell-cycle arrest; however, these effects are not chemotherapy alone.
chemotherapeutics that seen in endothelial cells isolated from normal tissues
crosslink DNA and therefore or from Matrigel plugs (Matrigel-derived endothelial Clinical trials of 1α,25(OH)2D3
impair the progression of DNA
replication machinery.
cells)106. Recently, Chung et al.112 demonstrated that With the recognition of the preclinical antiproliferative
TDECs may be more sensitive to 1α,25(OH)2D3 owing and pro-differentiating effects of vitamin D in the 1970s
Taxanes to the epigenetic silencing of CYP24A1. Therefore, and 1980s, a number of attempts were made to trans-
Drugs that inhibit microtubule direct effects of 1α,25(OH)2D3 on endothelial cells may late these findings into the clinic. Several investigators
dynamics by stabilizing GDP-
have a primary role in the 1α,25(OH)2D3-mediated attempted to administer 1α,25(OH)2D3 as a differenti-
bound tubulin. Microtubules
form the mitotic spindle and so anti-tumour activity that is observed in animal models ating agent in myelodysplasia and acute leukaemia121–123.
taxanes prevent the of cancer. Although some patients seemed to respond to the ther-
completional of mitosis. apy, these improvements were not enough to encour-
Preclinical combination studies age further trials, as 20–30% of patients who received
Myelodysplasia
Any of a group of bone marrow
In vitro and in vivo analyses indicate that 1α,25(OH)2D3 a daily dose of 1α,25(OH)2D3 developed hypercalcemia.
disorders that have markedly acts synergistically with chemotherapeutic agents. Such findings have reinforced the conviction that less
abnormal reduction in one or 1α,25(OH)2D3 potentiates the anticancer activity of hypercalcemic analogues of vitamin D, with modified
more types of circulating blood agents such as platinum analogues113–115, taxanes116,117 and chemical structures to make them less prone to degra-
cells owing to defective growth
DNA-intercalating agents117,118. Optimal potentiation dation by 24-OHase (FIG. 4a), must be developed if the
and maturation of blood-
forming cells in the bone is seen when 1α,25(OH)2D3 is administered before or therapeutic advantages of vitamin D biological effects are
marrow. simultaneously with chemotherapy treatment; admin- to be realized124,125. It is important to note that the early
istration of 1α,25(OH)2D3 after the cytotoxic agent anticancer studies of 1α,25(OH)2D3 were conducted
Hypercalcemia does not provide potentiation114,116. The combination of using dosing schedules optimized for the treatment
Excess of Ca2+ in the blood.
Chronic elevated serum levels
1α,25(OH)2D3 and cisplatin in SCC cells in vitro induced of renal osteodystrophy and osteoporosis, and the doses
of Ca2+ (12.0 mg dL) can result tumour cell apoptosis characteristic of 1α,25(OH)2D3 important for anticancer effects were not investigated
in urinary calculi (renal or alone. The pro-apoptotic signalling molecule MEKK1 separately. Had the administration of 1α,25(OH)2D3
bladder stones) and abnormal (mitogen-activated protein kinase kinase kinase 1), is been developed from an anticancer standpoint, the
heart rhythms. Severe
up-regulated in both apoptotic and pre-apoptotic SCC following considerations would have been determined:
hypercalcemia (above 15–16
mg dL) can result in coma and cells treated with 1α,25(OH)2D3 (REF 104). This up-regu- first, optimal biologically-effective dose and maximum
cardiac arrest. lation of MEKK1 was potentiated in combination with tolerated dose (MTD) across several cancers; second,
cisplatin treatment, suggesting that 1α,25(OH)2D3 pre- the most effective dosing schedules to achieve antican-
Osteodystrophy treatment commits cells to undergo apoptosis through cer activity; third, 1α,25(OH)2D3-dependent signalling
Defective bone ossification that
occurs when the kidney fails to
specific molecular pathways (probably the MEK signal- targets and molecular end-points; fourth, 1α,25(OH)2D3
maintain proper levels of Pi and ling pathway), and that this effect is increased when cells interactions with other cytotoxic or other anticancer
Ca2+. This results in slowed are treated with an additional genotoxic stimulus 113. drugs that may be therapeutically advantageous; and
bone growth and causes bone Similar effects are seen in MCF‑7 cells treated with the finally, design of clinical trials that mirror, as much as
deformities in children. In
vitamin D analogue ILX 23‑7553 in combination with possible, the exposures active in preclinical models to
adults, renal osteodystrophy
results in thin and weak bones, doxorubicin or ionizing radiation118. In these studies, ILX determine whether biological effects can be achieved in
bone and joint pain and 23‑7553 increased doxorubicin cytotoxicity and blocked human tumours in clinical therapy (FIG. 4b).
vulnerability to osteoporosis. the induction of p53 expression. Increased anti-tumour Several studies have attempted to define a safe
activity with 1α,25(OH)2D3 and the taxane paclitaxel is and effective clinical treatment regimen126–129. These
Osteoporosis
A condition that is
associated with a significant decrease in p21 expression, investigations were based on the recognition that most
characterized by a decrease in which sensitizes cells to both DNA-damaging agents positive preclinical studies used high-dose, intermit-
bone mass with decreased (such as cisplatin and doxorubicin) and microtubule- tent 1α,25(OH)2D3. Although it is clear that 20–30%
density and enlargement of disrupting agents (such as paclitaxel and docetaxel)116. of patients receiving 1α,25(OH)2D3 at a dose of 1.5–2.0
bone spaces producing
In SCC and PC‑3 (prostate cancer) xenografts, pre- µg a day develop hypercalcemia130, there have been few
porosity and brittleness of the
bone. treatment with 1α,25(OH)2D3 resulted in an increased studies that have compared continuous and intermittent
anti-tumour effect in combination with paclitaxel116. dosing regimens in cancer patients. Muindi and col-
Pharmacokinetics Similar results have also been observed in vivo with leagues131 have determined the pharmacokinetic profile of
The characteristic interactions MCF‑7 xenografts in which mice were treated with a 1α,25(OH)2D3 regimen that is active in a preclinical
of a drug and the body in
terms of its absorption,
vitamin D analogues and paclitaxel119. 1α,25(OH)2D3- animal model. High-dose 1α,25(OH)2D3 (daily for 3
distribution, metabolism and mediated downregulation of cyclooxygenase 2 (COX2) days 0.125 µg per mouse ~6.25 µg per kg (body weight))
excretion. expression in prostate cancer cells leads to decreased resulted in growth inhibition of the syngeneic mouse SCC
nature reviews | cancer volume 7 | SEPTEMBER 2007 | 693
© 2007 Nature Publishing GroupREVIEWS
a 21 Side chain
18 20 22
24
12
17 23
11 13 25 OH
C 14D
16 OH
9
8 15
7
6
Seco-B-ring
5 19
4 10 Vitamin D3 25(OH)D3 1α,25(OH)2D3
A HO
1 Cholecalciferol 25-Hydroxycholecalciferol Calcitriol
HO 3 HO OH
2
Vitamin D analogues Vitamin D receptor modulators
OH
OH S
HO O
S
O
LY2108491 O
Paricalcitol EB1089
HO OH S
HO OH HO
OH
O O
LY2109866
O
OH
OH
O O
HO OH ILX23-7553 O LG190119 O
HO OH OCT
b Epidemiology/risk factors 1 ,25(OH)2D3 or new analogues
Prediction of and drug combinations
• Nutritional composition
1α,25(OH)2D3 response
• UV exposure
• VDR polymorphisms In vitro systems
• Genetic background • Tumour cells Mechanisms of action
• Stromal cells • Genomic (VDR)
Vitamin D levels • Progenitor cancer stem cells • Non-genomic (memVDR)
(serum and cancer tissue) 1α,25(OH)2D3 1α,25(OH)2D3 • Apoptosis
or new associated toxicities: • Cell cycle
• Effectors of 1α,25(OH)2D3 • Angiogenesis
metabolism: expression analogues Improve drug dosing
and drug Pharmacokinetics In vivo systems • Cell signaling cross-talk
and activity of 25-OHase, • Preclinical animal models: • Cell–cell interaction
1α-OHase, 24-OHase, VDR combinations Pharmacodynamics
syngeneic, xenografts and
genetically-modified (VDR–/–)
Clinical assessment
Cancer • Therapeutic impact, Clinical trials
patient response and biomarkers • Prevention Clinical dosing
evaluation • Anti-tumour therapy schedules
Figure 4 | Development of 1α,25(OH)2D3 and vitamin D analogues as anticancer drugs. a | Cholecalciferol (vitamin
Nature Reviews | Cancer
D3) is 25-hydroxylated at C‑25 (denoted by carbon atom number on the structure of cholecalciferol) to form 25-hydroxyc-
holecalciferol (25(OH)D3). This is 1α-hydroxylated at C‑1 by 1α-OHase to yield 1α,25(OH)2D3 (calcitriol). 1α,25(OH)2D3 is a
secosteroid that is similar in structure to steroids but with a ‘broken’ B‑ring (denoted seco‑B-ring) where two of the carbon
atoms (C‑9 and C‑10) of the four steroid rings are not joined. Many vitamin D analogues (left) retain the secosteroid
structure with modified side chain structures around the C‑24 position, which makes them less hypercalcemic and less
prone to degradation by 24-OHase170,171. Several structures of vitamin D analogues referred to in the text are shown:
paricalcitol (19-nor‑1α(OH)2D2), ILX23‑7553 (16-ene‑23-yne‑1α,25(OH)2D3), OCT (Maxacalcitol, 22-oxa‑1α,25(OH)2D3)
and EB1089 (Seocalcitol, 1α-dihydroxy‑22,24-diene‑24,26,27-trihomo-vitamin D3). Vitamin D receptor modulators
(VDRMs, right) are non-secosteroidal in structure. Some of the representative compounds described are LY2108491,
LY2109866 and LG190119 (REFs 146,147). b | Paradigm for development and clinical translation of 1α,25(OH)2D3 as an
anticancer agent. Establishment of in vitro and in vivo experimental systems is crucial to developing 1α,25(OH)2D3 or
vitamin D analogues that target vitamin D metabolism and signalling. These systems allow the mechanisms of action of
1α,25(OH)2D3 to be studied along with novel analogues (also in combination with cytotoxic drugs) in multiple transformed
cell types and their biological effects (tumour and normal tissues) in animals. Importantly, studies on the pharmacokinetics
and pharmacodynamics of drug action will enable the development of better designed clinical dosing schedules for
clinical trials that will mirror the exposures active in preclinical models where optimal biological effects of 1α,25(OH)2D3
are demonstrated and are achievable in human tumours in clinical therapy.
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