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.
Departments of
Pharmacology and
Therapeutics* and Medicine‡,
Roswell Park Cancer Institute,
Buffalo, New York, USA.
Correspondence to C.S.J.
e-mail: candace.johnson@
roswellpark.org
doi:10.1038/nrc2196
The most widely accepted physiological role of vitamin
D, which is mediated primarily by 1α,25(OH)2D3 (also
known as calcitriol), the most active product of vitamin
D synthesis, is in the physiological regulation of Ca2+ and
Pi transport and bone mineralization1. The importance of
this role is demonstrated by studies with knockout mice
deficient in key members of the vitamin D metabolic
pathway, such as 25-hydroxyvitamin D3‑1α-hydroxylase
(1α-OHase; encoded by Cyp27b1), an enzyme that generates 1α,25(OH)2D3, and 25-hydroxyvitamin D 24-hydroxylase (24-OHase; encoded by Cyp24a1), the enzyme that
degrades 1α,25(OH)2D3 (catabolism), and the vitamin D
receptor (Vdr), which binds 1α,25(OH)2D3 to affect target
gene transcription2–6 (TABLEs 1,2). Loss of these genes in
mice led to phenotypes with abnormal bone morphology. However, recent observations indicate a much
broader range of action for 1α,25(OH)2D3, including the
regulation of differentiation, proliferation and apoptosis.
Furthermore, altered expression and function of proteins
crucial in vitamin D synthesis and catabolism have been
observed in many tumour types (TABLE 3). Interestingly,
Vdr–/– mice show hyperproliferation and increased mitotic
activity in the descending colon, suggesting a role for
1α,25(OH)2D3-mediated signalling in tumour suppression7. Zinser and colleagues8–10 showed that Vdr ablation
in the mouse increased chemical carcinogenesis in mammary, epidermis and lymphoid tissues but not in ovary,
uterus, lung or liver (TABLE 2). However, mice deficient in
key members of the vitamin D synthesis and catabolic
pathways do not develop spontaneous cancer2,3,11.
684 | SEPTEMBER 2007 | volume 7
The seminal finding by Garland and Garland12 of
higher mortality rates from colon cancer in the northeast
and lower rates in the south, southwest and west in the
United States led to the important concept that exposure
to ultraviolet B or sunlight, which leads to vitamin D
synthesis, can reduce the risk of colorectal cancer. Several
epidemiological observations have shown an association between low serum 25(OH)D3 levels (the accepted
measure of vitamin D body stores) and increased risk for
colorectal13, breast14 and prostate15 cancers. There are
many epidemiological studies that have sought to
determine associations between vitamin D status and
the risk and mortality rates of a number of cancers16–19.
Giovannucci and colleagues16 recently performed an
extensive analysis that combined major determinants
of vitamin D status on cancer risk and mortality with
51,529 men that were enrolled in the Health Professionals
Follow-up Study (HPFS). Individuals were prospectively
followed for almost 20 years; diet, exercise and lifestyle
characteristics were analysed and health outcomes,
including cancer and cancer-related deaths, were
assessed. Giovannucci et al. developed a model to predict 25(OH)D3 levels based on the relationship between
dietary and supplementary vitamin D, physical activity,
body mass index and sunlight exposure (a source of vitamin D). The model was applied to 47,800 individuals in
the HPFS, and the analysis indicated a strong association
between low levels of predicted 25(OH)D3 and increased
cancer incidence and cancer-related mortality, particularly for cancers of the digestive system16. Furthermore,
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REVIEWS
At a glance
•Epidemiological studies point to a relationship between vitamin D deficiency and
cancer risk.
•Alterations in vitamin D receptor expression, and in the synthesis (25-hydroxylase
and 1α-hydroxylase) and catabolism (24-hydroxylase) of vitamin D metabolites are
involved in the growth regulation of tumours; thus, compromising 1α,25(OH)2D3
(also known as calcitriol; the active metabolite of vitamin D signalling) sensitivity
and 1α,25(OH)2D3 signalling.
•The antiproliferative effects of 1α,25(OH)2D3 have been demonstrated in various
tumour types, as determined by preclinical trials.
•The anti-tumour effects of 1α,25(OH)2D3 involve mechanisms that are associated
with G0/G1 arrest, differentiation, induction of apoptosis and modulating
different signalling pathways in tumour cells, as well as inhibiting tumour
angiogenesis.
•Glucocorticoids potentiate the anti-tumour effects of 1α,25(OH)2D3 and decrease
1α,25(OH)2D3-induced hypercalcemia. 1α,25(OH)2D3 also potentiates the antitumour effects of many chemotherapeutic agents such as platinum analogues,
taxanes and DNA-intercalating agents.
•Given that the major vitamin D catabolizing enzyme, CYP24A1 (24-hydroxylase), is
often amplified and overexpressed in tumour cells, agents that inhibit this enzyme
can potentiate 1α,25(OH)2D3 anti-tumour effects.
•Preclinical data indicate that maximal anti-tumour effects are seen with
pharmacological doses of 1α,25(OH)2D3, and can be safely achieved in animals
using a high-dose, intermittent schedule of administration. Some clinical trial data
indicates that 1α,25(OH)2D3 is well-tolerated in cancer patients within a proper
dosing schedule.
•Data support the hypothesis that vitamin D compounds may have an important
role in cancer therapy and prevention, and merit further investigation.
Giovannucci et al. reported that an increase of 25 nmol
per L in predicted 25(OH)D3 level is associated with a
29% reduction in cancer-related mortality and a 17%
reduction in cancer incidence, suggesting that high
25(OH)D3 levels might be associated with a decreased risk
of some cancers16. A meta-analysis of case–control and
cohort studies found that individuals with ≥ 33 ng per ml
(82 nmol per L) 25(OH)D3 had a 50% lower incidence of
colorectal cancer20. Additionally, patients with early stage
non-small-cell lung cancer with high 25(OH)D3 levels
and high vitamin D intake at the time of diagnosis and
initiation of treatment had improved overall and recurrence-free survival21. Therefore, these data suggest that
low levels of 25(OH)D3 are an important risk factor for
cancer incidence.
Secosteroid hormones
Molecules that are very similar
in structure to steroids but with
a ‘broken’ ring; two of the
B‑ring carbon atoms (C-9 and
10) of the four steroid rings are
not joined.
Autocrine
A substance secreted by a cell
that acts on the surface
receptors of the same cell.
Paracrine
A substance secreted by a cell
that acts on adjacent cells.
Synthesis and catabolism of vitamin D
1α,25(OH) 2D 3 is synthesized from vitamin D in a
highly regulated multistep process (FIG. 1). The first
step in vitamin D synthesis is the formation of vitamin D3 in the skin through the action of ultraviolet
irradiation; vitamin D3 can also be taken in the diet
but in North America and Europe dietary vitamin D3
intake is a minor component of vitamin D3 acquisition because dairy products, eggs, fish and fortified
foods contain only small quantities of vitamin D22.
Decreased sun exposure further limits vitamin D
synthesis.
Vitamin D 3 (cholecalciferol) is hydroxylated by
liver mitochondrial and microsomal 25-hydroxylases
(25-OHase) 23, encoded by the gene CYP27A1. The
nature reviews | cancer
resultant 25-hydroxycholecalciferol (25(OH)D3) is
1α-hydroxylated in the kidney by mitochondrial
1α-hydroxylase (1α-OHase; encoded by the gene
CYP27B1), this yields the hormonally active secosteroid
1α,25(OH) 2D 3 (calcitriol) 23. 24-hydroxylation of
25(OH)D 3 and 1α,25(OH) 2D 3 by the cytochrome
P450 enzyme 25-hydroxyvitamin D 24-hydroxylase
(24-OHase; encoded by the gene CYP24A1), to the
metabolites 24,25(OH) 2D 3 and 1α,24,25(OH) 2D 3,
respectively, is the rate-limiting step for 25(OH)D 3
and 1α,25(OH) 2 D 3 catabolism 23 . Additionally,
1α,25(OH)2D3 concentrations are feedback regulated:
an increase in 24,25(OH)2D3 induces the synthesis of
1α,25(OH)2D3; whereas Ca2+, Pi and 1α,25(OH)2D3
itself suppress 1α,25(OH)2D3 synthesis23–26. CYP27B1
(which encodes 1α-OHase) expression is induced
by parathyroid hormone (PTH) 25 and repressed
by 1α,25(OH) 2 D 3 24,27 . Furthermore, CYP24A1 is
strongly induced by 1α,25(OH)2D3 to produce the less
active vitamin D metabolites 1α,24,25(OH)2D3 and
24,25(OH)2D323.
There are instances of tissue-specific regulation
of the vitamin D synthetic enzymes. 1α,25(OH)2D3
functions in an autocrine and paracrine manner to
modulate vitamin D function and signalling. 1αOHase is expressed at extra-renal sites such as normal colon, brain, placenta, pancreas, lymph nodes
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 cancers and during early colon tumour progression in
well-to-moderately differentiated states, but decreased
in poorly differentiated colon carcinomas 31–33 .
Increased expression of CYP27B1 in cancer tissues could provide local conversion of 25(OH)D 3
to 1α,25(OH)2D3, and may support the notion that
25(OH)D3 and 1α,25(OH)2D3 might have a role in the
chemoprevention of these cancers. However, CYP24A1
(encoding 24-OHase) mRNA expression is upregulated in tumours, and may counteract 1α,25(OH)2D3
antiproliferative activity, presumably by decreasing
1α,25(OH)2D3 levels34,35 (TABLE 3). Cross et al.35 have
demonstrated that the upregulation of CYP24A1 and
downregulation of CYP27B1 can occur in high-grade
colon carcinomas. The chromosomal region 20q13.2,
containing the CYP24A1 gene, is amplified in human
breast tumours36, and CYP24A1 mRNA expression
is upregulated in samples from human lung, colon
and ovarian tumours, suggesting that 1α,25(OH)2D3
levels would be reduced in these cases35,37,38 (TABLE 3).
This suggests that inhibition of CYP24A1 expression
and activity is essential for prevention to be effective.
Small-molecule inhibitors with varying specificity for
24-OHase39–42 render tumour cells more sensitive to the
action of 1α,25(OH)2D3 and its analogues. Consistent
with the epidemiological studies discussed above, these
findings indicate that 1α,25(OH)2D3 catabolism could
modulate tumour growth in some tissues, indicating the potential for the development of 24-OHase
inhibitors as cancer preventative and/or anticancer
therapeutic agents.
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REVIEWS
Table 1 | Mouse models of vitamin D metabolic enzymes and receptor signalling
Genetic modification
Mouse phenotype
Cyp27b1 (which
encodes 1α-OHase)
Pseudo-vitamin D deficiency rickets (PDDR); decreased serum Ca and
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
None
2,3
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,
hypocalcemia, hyperparathyroidism; impaired bone formation, growth
retardation; female infertility, uterine hypoplasia, impaired folliculogenesis
Hyperproliferation of descending
colon (increased PCNA positivity
and cyclin D1 expression)
–/–
–/–
Cancer phenotype
2+
Refs
4,5,6,7
PCNA, proliferating cell nuclear antigen.
1α,25(OH)2D3-mediated transcription of target genes.
1α,25(OH)2D3 exerts transcriptional activation and
repression of target genes by binding to the VDR (BOX 1).
The VDR is a member of the steroid hormone receptor
superfamily and regulates gene expression in a liganddependent manner43 (FIG. 2). Interestingly, N‑terminal VDR
variants show tissue-specific expression44,45 that might also
contribute to the differential specificity of 1α,25(OH)2D3mediated regulation. 1α,25(OH)2D3–VDR-dependent
transcriptional activity is modulated through synergistic
ligand-binding and dimerization with retinoic X receptor
(RXR). The activated 1α,25(OH)2D3–VDR–RXR complex specifically binds to vitamin D response elements
(VDREs), composed of two hexanucleotide repeats interspaced by varying numbers of nucleotides (for example,
GGTCCA-NNN-GGTCCA, where N is any nucleotide;
this is denoted DR3), in the promoter regions of target
genes46. For transcriptional activation, VDR occupies the 3′
half-site whereas RXR binds the 5′ half-site of VDRE47.
Co-factor proteins also have the ability to modulate
VDR-mediated gene expression; these proteins possess
intrinsic chromatin-modifying enzymatic activities, act
as a platform for the recruitment of chromatin-modifying
proteins and recruit basal transcription factors to the promoters23. 1α,25(OH)2D3 binding induces phosphorylation and conformational changes in VDR, which causes
the release of co-repressors (such as nuclear receptor
co-repressors (NCoRs) and the silencing mediator for
retinoid and thyroid hormone receptors (SMRT)–histone
deacetylase (HDAC) complex) that maintain chromatin
in a transcriptionally repressed state48. Conformational
change also repositions the VDR activation function
2 (AF2) domains to bind to stimulatory coactivators,
consisting of the steroid receptor coactivators (SRCs),
nuclear coactivator 62 kDa–SKI-interacting protein
(NCoA62–SKIP) and the chromatin modifiers, CREB
binding protein (CBP)–p300 and PBAF (polybromo- and
SWI‑2-related gene 1 associated factor), which acetylates
histones in the nucleosomes to unravel DNA for transcription49. Once the chromatin is de-repressed, the vitamin
D receptor-interacting proteins (DRIPs) form a complex
that binds to the AF2 domain of VDR and interacts with
the transcription machinery, such as TF2B (transcription
factor 2B) and RNA polymerase II, and initiates transcription (for a review see REF. 23). Recently, it has been shown
that epigenetic regulation of VDR through increased
expression of NCoR1 and SMRT repress VDR-mediated
signalling in prostate50 and breast cancer51 cell lines, and
may have a role in mediating the antiproliferative effects
of 1α,25(OH)2D3 in these tissues.
The mechanism by which 1α,25(OH)2D3 represses
gene expression through the binding of VDR to negative
VDREs (DR3-type), placing VDR on the 5′ half-site of
the VDRE52,53, such as is the case with human PTH, may
involve interference with transcriptional machinery but
is less understood. Recently, transcriptional repression
by 1α,25(OH)2D3 has been further elucidated for the
human CYP27B1 (REFS 27,54,55) and PTH56 genes. The
VDR–RXR heterodimer represses gene transcription in
a 1α,25(OH)2D3-dependent manner through E‑box-type
Table 2 | Vdr knockout mice and carcinogenesis
Oncogene/
carcinogen
Tissue
Cancer phenotype
MPA plus DMBA
carcinogens
Skin
40% sebaceous, 25% squamous and 15% follicular papillomas
compared with WT littermates; other infrequent lesions include
basal cell carcinoma and haemangioma
9
Mammary
Higher incidence of alveolar and ductal hyperplasias in
Vdr–/– mice compared with WT mice; development of palpable
mammary tumours was not altered by Vdr ablation
8
Lymph nodes and/
or thymus
Lymphoblastic and thymic lymphoma higher in Vdr–/– (27%)
compared with WT mice (11%)
8
Mammary
Decreased survival of Vdr–/–; neu mice compared with their
Vdr+/+; neu and Vdr+/–; neu littermates; increased development of
mammary tumours driven by the neu oncogene
Vdr–/– ;neu oncogene
Ref
10
DMBA, 7,12-dimethylbenzanthracene; MPA, medroxyprogesterone acetate; WT, wild-type.
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REVIEWS
Table 3 | Expression of molecules that function in vitamin D metabolism and signalling in human cancers.
Protein
(gene)
Altered expression observed
in human cancer tissues
Prognostic or histological
observations
Types of cancer
Vitamin D metabolic enzymes
25-OHase
(CYP27A1)
Increased mRNA
NA
Breast34, cervical34 and ovarian cancer34, HCC172
1α-OHase
(CYP27B1)
Increased mRNA
NA
Basal cell carcinoma173, breast34,174, cervical34
and ovarian cancer34
Increased mRNA
Moderately differentiated
Colon cancer31,35,175
Decreased mRNA
Poorly differentiated
Colon cancer35
Splice variants (Hyd‑V5, ‑V6, ‑V7
and ‑V8)
NA
Glioblastoma multiforme176, melanoma177,
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
Amplified at 20q13.2 locus
NA
Gastric adenocarcinoma37, breast cancer36
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
Increased mRNA
NA
Basal cell carcinoma173, SCC (cutaneous)178,
colon cancer31
Decreased mRNA
Poorly differentiated
Colon cancer31
Increased immunoreactivity
NA
Breast34, cervical34 and ovarian cancer34
Increased, predominantly
cytoplasmic
Well differentiated
Colon cancer166
Decreased immunoreactivity
Moderately and poorly
differentiated
Colon cancer166
Poorly differentiated
Colon cancer35
24-OHase
(CYP24A1)
Vitamin D receptor
VDR
(VDR)
HCC, hepatocellular carcinoma; NA, non applicable; NSCLC, non-small cell lung carcinoma; SCC, squamous cell carcinoma.
negative VDREs, comprised of a CANNTG-like motif
in the promoter regions of the CYP27B1 (REFS 27,54,55)
and PTH56 genes, which are distinct from the DR3type response elements. VDR-interacting repressor
(VDIR), when bound to E‑box-type elements, induces
the transcriptional activation of CYP27B154. However,
the binding of 1α,25(OH)2D3 to VDR causes VDR to
interact with VDIR. 1α,25(OH)2D3-induced association
between VDR and VDIR induces dissociation of the
histone acetyltransferase (HAT) co-activator and recruitment of HDAC co-repressor for 1α,25(OH)2D3-induced
transrepression of CYP27B1 gene expression54. In addition, Williams syndrome transcription factor (WSTF)
potentiates 1α,25(OH)2D3-induced transrepression by
VDR of the CYP27B1 gene promoter by facilitating the
association between WINAC, a multifunctional, ATPdependent chromatin-remodelling complex, and chromatin55. This transrepression mechanism is an important
biological function of VDR to allow negative-feedback
nature reviews | cancer
control of 1α,25(OH)2D3 biosynthesis55, as well as negative
regulation of other genes with nVDREs in their promoters. Furthermore, Kim et al.57 demonstrated that not only
is histone deacetylation crucial for chromatin structure
remodelling in suppression of the CYP27B1 gene, but
that transrepression by VDR requires DNA methylation
of the CYP27B1 gene promoter, suggesting complicated
epigenetic modifications for transcriptional regulation
of the CYP27B1 gene. Epigenetic regulation of CYP27B1
and CYP24A1 has been previously reported for the PNT‑2
human normal prostate cells and DU‑145 prostate cancer
cell line58. Histone methylation and demethylation are crucial events that impose ligand- and signal-dependent gene
activation by nuclear receptors and prevent the recruitment of unliganded nuclear receptors and transcription
factors from binding to their target promoters and causing
constitutive gene activation59.
Examples of genes with DR3-type response elements
that are transcriptionally activated by 1α,25(OH)2D3
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UV-B
D3
7-dehydrocholesterol
DBP
D3
D3
Circulation
Pre-D3
D3
Skin
Intestine
Liver
Pi, Ca2+ and
other factors
25-OHase
25(OH)D3
+/–
Parathyroid
glands
PTH
D3
24-OHase
24,25(OH)2D3
Kidney
Excretion
+
1α,24,25(OH)2D3
1α-OHase
Intestine
Increases
absorption
of Ca2+
and Pi
1α,25(OH)2D3
Bone
Increases bone
mineralization
Dietary sources
of vitamin D
24-OHase
Immune cells
Induces
differentiation
Tumour
microenvironment
• Inhibits proliferation
• Induces differentiation
• Inhibits angiogenesis
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
Reviews
(sunlight) exposure. Vitamin D3, obtained from the isomerization of pre-vitamin D3 in the epidermal
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 24hydroxylase (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),
BGLAP61 (osteocalcin; expressed in bone osteoblasts),
and CDKN1A62 (which encodes the cyclin dependent kinase (CDK) inhibitor p21). Those repressed
by 1α,25(OH)2D3 include PTH53. Although VDREs
are traditionally thought to occur in the promoter
regions of the target genes, a DR3-type VDRE was
recently identified in exon 4 of the growth arrest
and DNA-damage-inducible (GADD45) gene 63 .
1α,25(OH)2D3-mediated repression or activation of
many proto-oncogenes or tumour-suppressor genes
is described in normal and tumour tissues 62,64–67;
however, only a few such genes contain VDREs in the
promoter regions and are under the direct transcriptional control of 1α,25(OH)2D3, such as CDKN1A62
and CCNC (which encodes cyclin C, containing a
DR4-type VDRE)65. This suggests that 1α,25(OH)2D3
exerts many of its effects indirectly by modulating
signalling cascades or by unknown nongenomic
mechanisms (FIGS 2,3).
688 | SEPTEMBER 2007 | volume 7
Nongenomic action of 1α,25(OH)2D3. Nongenomic
actions mediated by 1α,25(OH)2D3 are rapid and not
dependent on transcription. However, nongenomic
signalling may indirectly affect transcription through
cross-talk with other signalling pathways68,69. Although
there is no agreement on how the nongenomic actions
are initiated, data suggest that these effects begin at the
plasma membrane and involve a non-classical membrane
receptor (memVDR; FIG. 2) described in intestinal caveolae70, and a 1α,25(OH)2D3-membrane-associated rapidresponse steroid binding protein (1α,25D3-MARRS)
isolated from chick intestinal basal-lateral membrane71.
The most well-described nongenomic effect of
1α,25(OH)2D3 is the rapid intestinal absorption of Ca2+
(REF. 72). Binding of 1α,25(OH)2D3 to the proposed membrane receptor can result in the activation of numerous
signalling cascades68,69 (FIG. 2). Activation of these signalling
cascades, such as protein kinase C (PKC), can result in the
rapid opening of voltage-gated Ca2+ channels and an increase
in intracellular Ca2+ (REF. 73), which may subsequently
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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)
Tru9l (G61050A)
Apal (G61888T)
Fokl (C27823T)
DNA binding
(aa 24–90, 91–115)
Taql (T61938C)
VDR protein
Nuclear localization
(aa 49–55, 79–105)
Hormone ligand binding
(aa 227–244, 268–316, 396–422)
Dimerization
(aa 37, 91–92, 244–263, 317–395)
Transactivation
(aa 246, 416–422)
S51
S208
P
P
AF-2
48 kDa
Hinge region
N
1
24
A/B
49 91 115
C
activate the Raf–mitogen-activated protein kinase extracellular signal-regulated kinase kinase (MEK)–mitogen-activated protein kinase (MAPK)–extracellular signal-regulated
kinase (ERK) cascade in skeletal muscle cells74. Activation
of the Raf–MEK–MAPK–ERK cascade, which mediates
proliferative cellular effects, may be a response to increased
Ca2+ in normal colon73 and skeletal muscle cells74, and may
not have a direct role in the antiproliferative activities of
1α,25(OH)2D3 in tumour cells (discussed below). In addition, ERK can also increase the transcriptional activity of
the VDR75, and nongenomic activation of PKC may stabilize VDR (through phosphorylation)23,76, thereby affecting
the transcriptional activity of the receptor. Therefore, the
nongenomic activation of these pathways may cooperate
with the classical genomic pathway to transactivate VDR
and elicit the antiproliferative effects of 1α,25(OH)2D3, but
this remains to be elucidated.
nature reviews | cancer
D
227244268 317
E/F
396 422 427 aa
Nature Reviews | Cancer
Anti-tumour effects of 1α,25(OH)2D3 signalling
1α,25(OH)2D3 has been examined preclinically for its
therapeutic efficacy in chemopreventive and anticancer
activity. A chemoprevention study used Nkx3‑1;Pten
mutant mice to recapitulate prostate carcinogenesis,
and showed that 1α,25(OH)2D3 administration delayed
the onset of prostate intraepithelial neoplasias (PIN) and
had better anti-tumour activity when administered to
mice with early-stage (PIN) rather than advanced-stage
prostate disease77. Furthermore, studies using model
systems of squamous cell carcinoma (SCC)78, prostate
adenocarcinoma79, cancers of the ovary80, breast81 and
lung82 showed that the administration of 1α,25(OH)2D3
or vitamin D analogues had significant anticancer effects.
The effects of 1α,25(OH)2D3 and its derivatives have
been shown to function through the VDR to regulate
proliferation, apoptosis and angiogenesis62,83–87.
volume 7 | SEPTEMBER 2007 | 689
© 2007 Nature Publishing Group
REVIEWS
SOC
channels
1α,25(OH)2D3
P
1α,25(OH)2D3
d
Caveolae
mem
VDR
9cRA
RXR
VDR
1α,25(OH)2D3
VDR
GPCR
PLCγ
Ca2+
?
PI3K
PKC
Ras
AC
[cAMP]
PKA
P
Raf isoforms
MEK1/2
a
9cRA
1α,25(OH)2D3
RXR VDR P
Transcriptional
activation
Transcriptional
repression
NCoA62–
SKIP
CBP/
P300
SRC-1
9cRA
RXR VDR
PBAF
SWI/SNF
Chromatin
remodeling
(histone
acetylation)
Nucleus
P
5′
3′
VDREs
c
WINAC
HDAC
NCoR–
complexes
WSTF
SMRT
9cRA
Chromatin
RXR VDR P
remodelling
(histone
VDIR
deacetylation)
nVDREs
ERK–MAPK1/2
Gene
repression
CYP27B1
PTH
Cross-talk
NCoA62–
SKIP
b
Gene
transcription
RNA
DRIPs
9cRA
CDKN1A
TF2B Pol II
P
CYP24A1
RXR VDR
SPP1
05
IP2
DR
3′
5′
VDREs
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 receptorinteracting 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 VDRinteracting 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 1αOHase) 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 Group
REVIEWS
a
b
c
d
e
g
TGFβ
↑E-cadherin
1α,25(OH)2D3
Wnt
TGF1R
TGFβR1
TGFβR2
IGF1
EGFR
f
Frizzled
β-catenin
P13K
Ras
Raf isoforms
Cytosol
Akt
β-catenin
SMADs
BCL2
BAX
BCL-XL
BCL-XS
APC
β-catenin VDR
Degradation
MEK1/2
Telomerase
ERK–MAPK1/2
Apoptosis
Differentiation
Apoptosis
Growth inhibition
Effector
caspases
MYC
p15
CDK4/6
p21
p27
CDK2
β-catenin
SKP2
Degradation
TCF4
MYC
TCF1
CD44
PARG
Cyclin D1,2,3 Cyclin E
Nucleus
p107/p130
PPP
pRB
pRB
+
E2F1,2,3
E2F4/5
DP1
Cell cycle
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 cellcycle 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
perturbation is central to 1α,25(OH) 2D3-mediated
antiproliferative activity in tumour cells (supplemental information S1 (table)). Progression through the
cell cycle is regulated by cyclins, and their association
with CDKs and CDK inhibitors (CKIs). Expression
of the CKIs p21 and p27 inhibits proliferation, in part
by inducing G1 cell-cycle arrest and withdrawal from
the cell cycle (G0). CDKN1A and GADD45A contain
a functional VDRE and are direct transcriptional targets of 1α,25(OH)2D3–VDR. However, many genes are
transcriptionally affected by 1α,25(OH)2D3 but do not
nature reviews | cancer
contain VDREs, and their transcriptional activation
or repression may not be directly mediated by VDR.
1α,25(OH) 2D 3–VDR transcriptional activation of
CDKN1A induces cell-cycle exit (differentiation) and
cell-cycle arrest in human U937 myelomonocytic cells62.
Treatment of human breast cancer MCF7 cells with
1α,25(OH)2D3 also increases the expression of CDKN1A
and CDKN1B, (which encodes p27) and represses
CCND1 (encoding cyclin D1), CCND3 (encoding cyclin D3), CCNA1 (which encodes cyclin A1) and CCNE1
(which encodes cyclin E1), and hence leads to the inhibition of CDK activity and pRb hypophosphorylation88,89.
volume 7 | SEPTEMBER 2007 | 691
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REVIEWS
Similarly, the treatment of SCC cells with 1α,25(OH)2D3
induces G0/G1 cell-cycle arrest owing to the transcriptional activation of CDKN1B and consequent
pRb hypophosphorylation90. However, in this context
CDKN1A expression was repressed, indicating that the
cell-cycle arrest is an indirect effect of 1α,25(OH)2D3
treatment or that cell-type specificity might determine
the ability of activated 1α,25(OH)2D3–VDR to induce
CDKN1A expression90. Other genes have been shown
to be transcriptionally affected by 1α,25(OH)2D3 in
colon cancer, ovarian carcinoma and leukaemia cells,
such as activation of GADD45 (REF 63), which is involved
in DNA damage responses, repression of TYMS
(which encodes thymidylate synthetase)91 and TK1
(which encodes thymidine kinase)91, which are involved
in DNA replication, and activation of the INK4 family92 of cyclin D‑dependent kinase inhibitors, which
mediate G1 cell-cycle arrest; whereas cyclin E–CDK2
and the SKP2 (S-phase kinase-associated protein 2)
ubiquitin ligase, which targets CKIs to the proteasome,
are downregulated93 by 1α,25(OH)2D3. 1α,25(OH)2D3
treatment also results in the repression of the protooncogene MYC89,94, which significantly contributes to
the antiproliferative effects of 1α,25(OH)2D3.
1α,25(OH)2D3 can have many indirect effects on
cell-cycle regulation owing to cross-talk with other
pathways; for example, 1α,25(OH)2D3 treatment can
result in the upregulation of IGFBP3 (which encodes
insulin growth factor binding protein 3) and transforming growth factor‑β (TGFβ)–SMAD3 signalling
cascades and by downregulating the epidermal growth
factor receptor (EGFR) signalling pathway67,95,96 (FIG. 3).
Although there appears to be an overall inhibition of
cell-cycle progression in tumour cells treated with
1α,25(OH)2D3, the precise molecular basis for such
an effect differs from one tumour cell type to another
such that a unifying hypothesis with regard to the
exact mechanism of 1α,25(OH) 2D 3-mediated cellcycle perturbation has not been possible (supplemental
information S1 (table)).
Activation of the VDR by 1α,25(OH)2D3 can also
inhibit tumour cell proliferation by inducing differentiation in various myeloid leukaemia cell lines and freshly
isolated leukaemia cells62,83, which is dependent on the
formation of activated VDR and phosphatidylinositol 3kinase (PI3K) complexes97. However, in haematopoeitic
progenitor cells, 1α,25(OH)2D3 inhibits differentiation
through VDR-independent suppression of interleukin
12 (IL12) protein secretion and down-regulation of other
co-stimulatory molecules (CD40, CD80 and CD86)98. In
cell lines of head and neck, colon and prostate tumours,
administration of 1α,25(OH)2D3 or vitamin D analogues
induces the expression of genes that are associated with the
differentiated cell of origin91,99,100. In various colon cancer
cells, treatment with 1α,25(OH)2D3 induces differentiation either by increasing PKC- and JNK-dependent JUN
activation101 or by differentially regulating the expression
of inhibitor of DNA binding 1 and 2 (ID1 and ID2), which
encode proteins that are transcriptional regulators of epithelial cell proliferation (ID2) and differentiation (ID1);
the repression of ID2 mediated the antiproliferative effects
692 | SEPTEMBER 2007 | volume 7
of 1α,25(OH)2D3102. Recent findings reported by Palmer
et al.103 indicate that 1α,25(OH)2D3 promotes differentiation through the induction of CDH1 (which encodes E
cadherin) in adenomatosis polyposis coli (APC)-mutated
human colorectal cancer SW480 cells. CDH1 activation
consequently restrained cell growth by facilitating the
translocation of β‑catenin from the nucleus to the plasma
membrane, thus inhibiting β‑catenin-mediated transcription and allowing activated VDR to compete with
β‑catenin for transcription factor binding. Again, there
appears to be no specific mechanism regarding the ability
of 1α,25(OH)2D3 to induce differentiation in tumour cells
(supplemental information S1 (table)).
Apoptosis. In addition to the antiproliferative effects
of 1α,25(OH) 2D 3, there is increasing evidence that
1α,25(OH)2D3 exerts anti-tumour effects by regulating key mediators of apoptosis, such as repressing the
expression of the anti-apoptotic, pro-survival proteins
BCL2 and BCL-XL, or inducing the expression of proapoptotic proteins (such as BAX, BAK and BAD). It
has been reported that 1α,25(OH)2D3 downregulates
BCL2 expression in MCF‑7 breast tumour and HL‑60
leukaemia cells and upregulates BAX and BAK expression in prostate cancer, colorectal adenoma and carcinoma cells84. In addition to regulating the expression
of the BCL2 family, 1α,25(OH)2D3 might also directly
activate caspase effector molecules, although it is
unclear whether 1α,25(OH)2D3-induced apoptosis is
caspase-dependent84. In support of this idea, the treatment of mouse SCC tumour cells with 1α,25(OH)2D3
increased VDR expression and concomitantly inhibited the phosphorylation of ERK104. Upstream of ERK,
the growth-promoting and pro-survival signalling
molecule MEK is cleaved and inactivated in a caspasedependent manner in cells that undergo apoptosis
after treatment with 1α,25(OH)2D3. Recently, a novel
mechanism of 1α,25(OH)2D3-mediated apoptosis in
epithelial ovarian cancer cells was proposed by Jiang
et al. 105, wherein they showed that 1α,25(OH) 2D 3
destabilizes telomerase reverse transcriptase (TERT)
mRNA, therefore inducing apoptosis through telomere attrition resulting from the down-regulation
of telomerase activity. The diverse effects observed
for 1α,25(OH) 2D 3-mediated apoptosis suggest that
although anti-proliferative effects directed against
the tumour are clear in vitro and in vivo (supplemental information S1 (table)), dissecting the exact
mechanism(s) central to these activities remains a
challenge.
Angiogenesis. 1α,25(OH)2D3 inhibits the proliferation
of endothelial cells in vitro and reduces angiogenesis
in vivo106–108. Vascular endothelial growth factor (VEGF)induced endothelial cell tube formation and tumour
growth are inhibited in vivo by 1α,25(OH)2D3 administration to mice with VEGF-overexpressing MCF‑7
xenografts86. 1α,25(OH)2D3 can increase VEGF mRNA
levels in vascular smooth muscle cells109 and upregulate mRNA levels of the potent anti-angiogenic factor
thrombospondin 1 (THBS1) in SW480-ADH human
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© 2007 Nature Publishing Group
REVIEWS
Platinum analogues
Platinum-based
chemotherapeutics that
crosslink DNA and therefore
impair the progression of DNA
replication machinery.
Taxanes
Drugs that inhibit microtubule
dynamics by stabilizing GDPbound tubulin. Microtubules
form the mitotic spindle and so
taxanes prevent the
completional of mitosis.
Myelodysplasia
Any of a group of bone marrow
disorders that have markedly
abnormal reduction in one or
more types of circulating blood
cells owing to defective growth
and maturation of bloodforming cells in the bone
marrow.
Hypercalcemia
Excess of Ca2+ in the blood.
Chronic elevated serum levels
of Ca2+ (12.0 mg dL) can result
in urinary calculi (renal or
bladder stones) and abnormal
heart rhythms. Severe
hypercalcemia (above 15–16
mg dL) can result in coma and
cardiac arrest.
Osteodystrophy
Defective bone ossification that
occurs when the kidney fails to
maintain proper levels of Pi and
Ca2+. This results in slowed
bone growth and causes bone
deformities in children. In
adults, renal osteodystrophy
results in thin and weak bones,
bone and joint pain and
vulnerability to osteoporosis.
Osteoporosis
A condition that is
characterized by a decrease in
bone mass with decreased
density and enlargement of
bone spaces producing
porosity and brittleness of the
bone.
Pharmacokinetics
The characteristic interactions
of a drug and the body in
terms of its absorption,
distribution, metabolism and
excretion.
colon tumour cells 102. In SCC cells, 1α,25(OH) 2D 3
induces the angiogenic factor interleukin 8 (IL8)110, but
in prostate cancer cells 1α,25(OH)2D3 interrupts IL8
signalling leading to the inhibition of endothelial cell
migration and tube formation111. A significant inhibition
of metastasis is observed in prostate and lung murine
models treated with 1α,25(OH)2D3, and these effects
may be based, at least in part, on the anti-angiogenic
effects described79,82. Interestingly, in tumour-derived
endothelial cells (TDECs), 1α,25(OH)2D3 induces apoptosis and cell-cycle arrest; however, these effects are not
seen in endothelial cells isolated from normal tissues
or from Matrigel plugs (Matrigel-derived endothelial
cells)106. Recently, Chung et al.112 demonstrated that
TDECs may be more sensitive to 1α,25(OH)2D3 owing
to the epigenetic silencing of CYP24A1. Therefore,
direct effects of 1α,25(OH)2D3 on endothelial cells may
have a primary role in the 1α,25(OH)2D3-mediated
anti-tumour activity that is observed in animal models
of cancer.
Preclinical combination studies
In vitro and in vivo analyses indicate that 1α,25(OH)2D3
acts synergistically with chemotherapeutic agents.
1α,25(OH)2D3 potentiates the anticancer activity of
agents such as platinum analogues113–115, taxanes116,117 and
DNA-intercalating agents117,118. Optimal potentiation
is seen when 1α,25(OH)2D3 is administered before or
simultaneously with chemotherapy treatment; administration of 1α,25(OH)2D3 after the cytotoxic agent
does not provide potentiation114,116. The combination of
1α,25(OH)2D3 and cisplatin in SCC cells in vitro induced
tumour cell apoptosis characteristic of 1α,25(OH)2D3
alone. The pro-apoptotic signalling molecule MEKK1
(mitogen-activated protein kinase kinase kinase 1), is
up-regulated in both apoptotic and pre-apoptotic SCC
cells treated with 1α,25(OH)2D3 (REF 104). This up-regulation of MEKK1 was potentiated in combination with
cisplatin treatment, suggesting that 1α,25(OH)2D3 pretreatment commits cells to undergo apoptosis through
specific molecular pathways (probably the MEK signalling pathway), and that this effect is increased when cells
are treated with an additional genotoxic stimulus 113.
Similar effects are seen in MCF‑7 cells treated with the
vitamin D analogue ILX 23‑7553 in combination with
doxorubicin or ionizing radiation118. In these studies, ILX
23‑7553 increased doxorubicin cytotoxicity and blocked
the induction of p53 expression. Increased anti-tumour
activity with 1α,25(OH)2D3 and the taxane paclitaxel is
associated with a significant decrease in p21 expression,
which sensitizes cells to both DNA-damaging agents
(such as cisplatin and doxorubicin) and microtubuledisrupting agents (such as paclitaxel and docetaxel)116.
In SCC and PC‑3 (prostate cancer) xenografts, pretreatment with 1α,25(OH)2D3 resulted in an increased
anti-tumour effect in combination with paclitaxel116.
Similar results have also been observed in vivo with
MCF‑7 xenografts in which mice were treated with
vitamin D analogues and paclitaxel119. 1α,25(OH)2D3mediated downregulation of cyclooxygenase 2 (COX2)
expression in prostate cancer cells leads to decreased
nature reviews | cancer
prostaglandin activity, the induction of their degradation
through the upregulation of 15-hydroxyprostaglandin
dehydrogenase, and reduction of prostaglandin receptors120. These findings support the rationale for clinical
evaluation of a combination of 1α,25(OH)2D3 and nonsteroidal anti-inflammatory drugs (NSAIDs) for prostate
cancer therapy120. Increased anti-tumour effects with
1α,25(OH)2D3 combination therapy offers the opportunity for the clinical use of 1α,25(OH)2D3 across several
tumour types where modest effects are observed with
chemotherapy alone.
Clinical trials of 1α,25(OH)2D3
With the recognition of the preclinical antiproliferative
and pro-differentiating effects of vitamin D in the 1970s
and 1980s, a number of attempts were made to translate these findings into the clinic. Several investigators
attempted to administer 1α,25(OH)2D3 as a differentiating agent in myelodysplasia and acute leukaemia121–123.
Although some patients seemed to respond to the therapy, these improvements were not enough to encourage further trials, as 20–30% of patients who received
a daily dose of 1α,25(OH)2D3 developed hypercalcemia.
Such findings have reinforced the conviction that less
hypercalcemic analogues of vitamin D, with modified
chemical structures to make them less prone to degradation by 24-OHase (FIG. 4a), must be developed if the
therapeutic advantages of vitamin D biological effects are
to be realized124,125. It is important to note that the early
anticancer studies of 1α,25(OH)2D3 were conducted
using dosing schedules optimized for the treatment
of renal osteodystrophy and osteoporosis, and the doses
important for anticancer effects were not investigated
separately. Had the administration of 1α,25(OH)2D3
been developed from an anticancer standpoint, the
following considerations would have been determined:
first, optimal biologically-effective dose and maximum
tolerated dose (MTD) across several cancers; second,
the most effective dosing schedules to achieve anticancer activity; third, 1α,25(OH)2D3-dependent signalling
targets and molecular end-points; fourth, 1α,25(OH)2D3
interactions with other cytotoxic or other anticancer
drugs that may be therapeutically advantageous; and
finally, design of clinical trials that mirror, as much as
possible, the exposures active in preclinical models to
determine whether biological effects can be achieved in
human tumours in clinical therapy (FIG. 4b).
Several studies have attempted to define a safe
and effective clinical treatment regimen126–129. These
investigations were based on the recognition that most
positive preclinical studies used high-dose, intermittent 1α,25(OH)2D3. Although it is clear that 20–30%
of patients receiving 1α,25(OH)2D3 at a dose of 1.5–2.0
µg a day develop hypercalcemia130, there have been few
studies that have compared continuous and intermittent
dosing regimens in cancer patients. Muindi and colleagues131 have determined the pharmacokinetic profile of
a 1α,25(OH)2D3 regimen that is active in a preclinical
animal model. High-dose 1α,25(OH)2D3 (daily for 3
days 0.125 µg per mouse ~6.25 µg per kg (body weight))
resulted in growth inhibition of the syngeneic mouse SCC
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REVIEWS
a
21
18
12
11
9
C
13
16
OH
25
OH
15
8
5
HO 3
23
17
14D
Side chain
24
7
6
4
20 22
19
10
A
Seco-B-ring
Vitamin D3
Cholecalciferol
1
25(OH)D3
25-Hydroxycholecalciferol
HO
2
HO
1α,25(OH)2D3
Calcitriol
OH
Vitamin D analogues
Vitamin D receptor modulators
OH
OH
S
HO
O
O
LY2108491
EB1089
Paricalcitol
HO
OH
HO
S
HO
OH
O
LY2109866
O
b
OH ILX23-7553
Epidemiology/risk factors
• Nutritional composition
• UV exposure
• VDR polymorphisms
• Genetic background
Cancer
patient
HO
OH OCT
Prediction of
1α,25(OH)2D3 response
Vitamin D levels
(serum and cancer tissue)
• Effectors of 1α,25(OH)2D3
metabolism: expression
and activity of 25-OHase,
1α-OHase, 24-OHase, VDR
Clinical assessment
• Therapeutic impact,
response and biomarkers
evaluation
OH
O
O
OH
OH
HO
S
O
1α,25(OH)2D3
or new
analogues
and drug
combinations
1α,25(OH)2D3
associated toxicities:
Improve drug dosing
Pharmacokinetics
Pharmacodynamics
Clinical trials
• Prevention
• Anti-tumour therapy
LG190119
O
O
O
1 ,25(OH)2D3 or new analogues
and drug combinations
In vitro systems
• Tumour cells
• Stromal cells
• Progenitor cancer stem cells
In vivo systems
• Preclinical animal models:
syngeneic, xenografts and
genetically-modified (VDR–/–)
Mechanisms of action
• Genomic (VDR)
• Non-genomic (memVDR)
• Apoptosis
• Cell cycle
• Angiogenesis
• Cell signaling cross-talk
• Cell–cell interaction
Clinical dosing
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-hydroxycholecalciferol (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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REVIEWS
Box 2 | The pharmacology of 1α,25(OH)2D3
In developing an agent for human use, it is crucial to understand the
pharmacokinetics and, when possible, the pharmacodynamic parameters
associated with drug administration. Although the pharmacodynamics of
1α,25(OH)2D3 have not been well studied, the pharmacokinetics of 1α,25(OH)2D3
have been extensively investigated using a standard, commercial formulation of
1α,25(OH)2D3 (Rocaltrol, Hoffman–LaRoche). Importantly, these pharmacokinetic
studies revealed that dose escalation did not result in the escalation of systemic
exposure. Both groups found that the desirable linear relationship between dose
administered and systemic exposure (area under the curve (AUC) and Cmax) was
lost at doses >16 µg127,129,131. In addition, there was marked variation (5–10×) in
AUC and Cmax among patients receiving the same dose of 1α,25(OH)2D3. Two
approaches indicate that these pharmacokinetic findings are a product of the
pharmaceutical characteristics of Rocaltrol when administered at high dose
rather than reduced absorption or increased catabolism of 1α,25(OH)2D3 within
the patients studied. Novocea, Inc.168 has developed a new formulation of
1α,25(OH)2D3 (DN‑101, Ascentar) for high-dose applications (15 µg and 45 µg
caplets) and pharmacokinetic studies indicate a linear relationship between dose
and exposure at oral doses up to 168 µg, indicating that there is no ‘barrier’ to
gastrointestinal absorption of 1α,25(OH)2D3 at the doses studied168.
High-dose intravenous 1α,25(OH)2D3 (Calcijex, Abbott Pharmaceuticals) has been
investigated in a phase I clinical trial136 (supplemental information S2 (table)). A linear
relationship between dose and exposure was observed across a wide dose range
(10–125 µg), indicating that 1α,25(OH)2D3 administration is not associated with rapid
induction of 1α,25(OH)2D3 catabolism. The same patients monitored on multiple
occasions had no convincing evidence that the pharmacokinetics of 1α,25(OH)2D3 on
day 1 of a once a day for 3 days a week schedule are different from the
pharmacokinetics on day 28; neither does the administration of either paclitaxel or
dexamethasone modify 1α,25(OH)2D3 pharmacokinetics. Although formal
bioavailability studies of 1α,25(OH)2D3 have not been done, inspection of
pharmacokinetic curves in our studies of intravenous 1α,25(OH)2D3 and those of the
DN‑101 study suggest that oral absorption of a suitable formulation is very efficient
(80–90%) even at a high dose136,169.
Area under the curve
(AUC). In pharmacokinetics, the
area under the curve is a plot
of concentration of drug in
serum over time that
represents the measure of an
individual’s exposure to the
drug.
Bioavailability
Measurement of an
administered dose of a
therapeutically active drug that
reaches the systemic
circulation and depends on the
mode of administration.
Cmax
Maximum or ‘peak’
concentration of a drug
observed after its
administration.
Glucocorticoids
Corticosteroids are involved in
carbohydrate, protein and fat
metabolism to regulate liver
glycogen and blood sugar by
increasing gluconeogenesis;
clinically used for antiinflammatory and
immunosuppressive effects.
cells. At these doses, the area under the curve (AUC) 0–24 h
(37 ± 2.5 ng•hr ml) and Cmax (22 nM) of 1α,25(OH)2D3
were within the concentration range that does not
cause toxicity in patients131. Although SCC is a sensitive
model, concentrations and exposure to 1α,25(OH)2D3
that inhibit SCC tumour growth are also active in many
human tumour xenograft models41,78,80–82,132,133.
In developing an anticancer agent, more aggressive
management of toxicity and use of supportive care
approaches often allow one to overcome mild to moderate side effects. Beer and colleagues129 conducted a
standard phase I dose-escalation trial of 1α,25(OH)2D3
administered orally once a week, and found that 2.8 µg
per kg (body weight) can be safely administered without any side effects. Dose escalation was not continued
because at doses higher than 2.4 µg per kg (body weight)
oral absorption was found to be incomplete and unreliable (BOX 2). Several phase I trials have been conducted in
which an MTD of 1α,25(OH)2D3 has been sought127,134,135.
Dose-limiting toxicity of oral 1α,25(OH)2D3 has not
been observed in these studies.
As discussed above, combinations of 1α,25(OH)2D3
with other anticancer agents demonstrate synergistic
interactions. Phase I studies of 1α,25(OH)2D3 plus
paclitaxel127 and 1α,25(OH)2D3 plus gefitinib136 for the
treatment of advanced malignancies, and phase II studies of 1α,25(OH)2D3 plus carboplatin and 1α,25(OH)2D3
plus docetaxel for the treatment of prostate cancer137,138
nature reviews | cancer
have been completed (supplemental information S2
(table)). Most of these studies are based on persuasive
preclinical data, but confront a problem not usually
encountered in single-agent or combination phase I
studies in cancer: dose, toxicity and pharmacokinetic
data for 1α,25(OH)2D3 as a single agent are limited.
Although investigators working with the Novacea
formulation of 1α,25(OH)2D3 (DN‑101) have defined
suitable pharmacokinetics and have shown the feasibility of very high doses of 1α,25(OH)2D3, an aggressive
MTD in cancer patients has not been determined.
Trump and colleagues139 completed a 43-patient study
of 1α,25(OH)2D3 in escalating oral doses to a maximum
of 12 µg 1α,25(OH)2D3 three times a week together
with dexamethasone. Minimal hypercalcemia was
observed and high-dose intermittent 1α,25(OH)2D3
plus dexamethasone was safe and feasible139. At present,
dexamethasone in combination with weekly intravenous 1α,25(OH)2D3 plus gefitinib is being explored
in a phase I clinical trial to determine whether an aggressive MTD can be achieved (supplemental information
S2 (table)). Glucocorticoids are used clinically to ameliorate hypercalcemia in a number of situations including
1α,25(OH)2D3 intoxication140. Dexamethasone also significantly improves 1α,25(OH)2D3 anti-tumour efficacy,
in vitro and in vivo, through direct effects on the VDR141.
In studies of tumour-bearing animals, dexamethasone
increases VDR receptor number without changing the
ligand affinity (Kd) in SCC tumour tissue xenograft
and the kidney, but not in gastrointestinal mucosa141.
The ability of dexamethasone to increase anti-tumour
activity in certain tissues and decrease toxicity is mediated through the modulation of VDR expression. The
combined use of a glucocorticoid and 1α,25(OH)2D3 is
a viable approach to reducing side-effects experienced
by patients treated with 1α,25(OH)2D3.
Another approach to reducing potential toxicity and
increasing anti-tumour activity is the development of
vitamin D analogues and vitamin D receptor modulators (VDRMs) (FIG. 4a) that are less prone to cause
hypercalcemia. However, considerable data indicate
that when 1α,25(OH)2D3 is given at an intermittent
schedule, clinical use is not limited by hypercalcemia
or hypercalciuria126–129,137,138,142. Vitamin D analogues have
been synthesized and their properties examined143.
Although many appear to be less hypercalcemogenic
than 1α,25(OH)2D3, the complexities of in vitro–in vivo
data and dose extrapolation limit the conclusions that
analogues which cause less hypercalcemia are equipotent in terms of anticancer effects. For example, most
analogues that cause less hypercalcemia bind less tightly
to the VDR, a property that probably reduces their
anti-tumour effects143. As non-steroidal tissue-selective
oestrogen-receptor (ER) modulators (SERMs) such
as tamoxifen have proven clinically successful for the
prevention of breast cancer in high-risk women, and
for the treatment of ER‑positive breast cancer144, recent
development of non-secosteroidal VDRMs have shown
potential in anticancer therapy145. The novel non-secosteroidal VDRMs LY2108491 and LY2109866 (FIG. 4a)
were identified as potent tissue-selective agonists in
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REVIEWS
1α,25(OH)2D3 intoxication
The symptoms of
hypervitaminosis D (excessive
doses of vitamin D) are a result
of hypercalcemia caused by
increased intestinal Ca2+
absorption. Gastrointestinal
symptoms include anorexia,
nausea and vomiting.
Hypercalciuria
Excessive urinary Ca2+
excretion. The morbidity
associated with hypercalciuria
is related to kidney stone
disease and bone
demineralization leading to
osteopaenia (decrease in bone
density) and osteoporosis.
Thromboembolic
complications
Associated with blockage of a
blood vessel by a particle that
has dislodged from a blood
clot at its primary formation
site.
keratinocytes, human peripheral blood mononuclear
cells and osteoblasts, but have weak potency in intestinal
cells146. Furthermore, the non-secosteroidal, tissue-selective VDRMs were less calcemic in vivo compared with
1α,25(OH)2D3, and show efficacy in an animal model of
psoriasis146; however, their potential in anticancer therapy
has not been determined. Polek and collegues reported
that a novel VDRM, LG190119 (FIG. 4a), inhibited LNCaP
xenograft tumour growth without hypercalcemia147. Nonsecosteroidal VDRMs represent promising therapeutic
agents for the treatment of cancers with tissue-selectivity
and potential evasion of hypercalcemia.
Clinical studies of vitamin D analogues have
focused primarily on continuous daily administration
of EB1089 (FIG. 4a; seocalcitol, 1α-dihydroxy‑22,24diene‑24,26,27-trihomovitamin D3) to patients with
breast cancer, colorectal cancer or hepatocellular
carcinoma148–150. EB1089 failed to show evidence of
anti-tumour activity in these studies, and potentially
problematic hypercalcemia was seen but was not dose
limiting. Paricalcitol (FIG. 4a; 19-nor‑1α,25-(OH)2D2,
Zemplar), an analogue developed by Abbott, appears
to be more effective than 1α,25(OH)2D3 in the management of renal osteodystrophy and chronic renal
disease151, and preclinical data indicate that paricalcitol
has anti-tumour effects in prostate, pancreas, lung and
breast cancers, as well as multiple myeloma152. Current
phase I clinical trials have been initiated for paricalcitol
plus gemcitabine and paricalcitol plus zoledronic acid
(a bisphosphonate) in patients with advanced solid
tumours and multiple myeloma, respectively, to establish whether very high doses of these analogues can be
safely administered intravenously when an intermittent schedule is used (supplemental information S2
(table)).
In patients with prostate cancer that progresses
despite castration (so-called androgen-independent
prostate cancer or AIPC), 1α,25(OH)2D3 has been studied in ‘standard’ dose and schedule and at a high dose.
The most striking indication of 1α,25(OH)2D3 antitumour effects in AIPC is the randomized trial reported
by Beer et al.138 that used docetaxel (36 µg once a week)
and 1α,25(OH)2D3 (DN‑101, 45 µg one day before
docetaxel). The survival in the DN‑101 plus docetaxeltreated patients was improved, but further confirmation
is required because survival was not the primary end
point of this phase II study. Novacea is currently conducting a 1,000 patient phase III trial to further evaluate
this survival difference. It is also striking that severe or
life-threatening side effects, including thromboembolic
complications, were reduced in the DN‑101 arm153. The
results of this trial point to potentially clinically relevant
anti-tumour effects of 1α,25(OH)2D3 in combination
with docetaxel.
Conclusions and future perspectives
The data described above support the continued
exploration of vitamin D supplementation and
1α,25(OH) 2D 3 as approaches to cancer prevention
and treatment, respectively. The epidemiological data
indicate that vitamin D deficiency is relatively com-
696 | SEPTEMBER 2007 | volume 7
mon, at least in some parts of the US and Europe, and
that inadequate levels of 25(OH)D3 are associated with
an increased risk and poor prognosis of several types
of cancer16. In view of the numerous other potential
consequences of vitamin D deficiency to human health,
such as rickets and osteomalacia, one could easily recommend more aggressive monitoring of 25(OH)D3
levels as part of a health maintenance programme.
Meta-analysis and cancer-prevention trials indicate
that vitamin D3 supplementation to achieve a level of
>82 nmol per L 25(OH)D3 can lower the incidence of
colorectal cancer by 50%20. To achieve serum levels in
this range, individuals require a daily 4,000 IU (international unit) supplement of vitamin D3 (REF. 20), which
is achievable with the current formulations that range
from 200–2,000 IU and a liquid formulation of 2,000
IU per drop. Formal randomized studies to optimize
replacement strategies and to evaluate vitamin D3 as a
cancer-preventative approach should be considered.
Changes in the expression of proteins important
in vitamin D synthesis and catabolism (25-OHase,
1α-OHase, 24-OHase) and those crucial for mediating the biological effects of 1α,25(OH)2D3 (VDR) have
been shown to be associated with poor differentiation
status and prognosis of several types of cancer, such as
colon cancer31–33,35. The overexpression of vitamin D
catabolic enzymes in cancer suggests that low cellular
1α,25(OH)2D3 is also associated with poor prognosis,
but this has not yet been addressed convincingly. In
addition, the steady-state level of cellular 1α,25(OH)2D3
in tumour tissue is difficult to measure. Assessment
of the catabolic enzyme that degrades 1α,25(OH)2D3,
such as 24-OHase (encoded by CYP24A1), may have
merit in the development of prognostic models.
Indeed, CYP24A1 is overexpressed in many cancers
(TABLE 3). Of interest, epigenetic silencing of CYP24A1
in tumour-derived endothelial cells renders the tumour
sensitive to the anti-angiogenic effects of 1α,25(OH)2D3
(REF. 112) . Various molecules can inhibit 24-OHase
(such as azoles and vitamin D analogues). These merit
exploration and further development as specific smallmolecule 24-OHase inhibitors, especially in combination with high-dose intermittent 1α,25(OH) 2D 3
or other vitamin D analogues. These may maximize
intracellular 1α,25(OH)2D3 content and exert optimal
antiproliferative effects.
The growth restraining, differentiation and apoptosis-inducing effects of 1α,25(OH)2D3 in different
tumour cell types is well documented (supplemental
information S1 (table)). Across various tumour cell
lines, different molecular markers of cell cycle, differentiation and apoptosis can be observed with no clear
pattern of modulation by 1α,25(OH)2D3; perhaps these
studies demonstrate the importance of heterogeneity
to the 1α,25(OH)2D3 response, even among similar
tumour cell types. The antiproliferative actions of
1α,25(OH)2D3 may depend on the differentiation status
of the tumour cells and VDR expression level, as well
as genomic or post-translational modifications of coactivator proteins that are essential for the assembly of
the transcriptionally active VDR complex154.
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REVIEWS
1α,25(OH) 2D 3 has prominent antiproliferative,
anti-angiogenic and pro-differentiative effects in a
broad range of cancers (supplemental information S1
(table)). These effects are mediated through perturbation of several important signalling pathways mediated through genomic and non-genomic mechanisms.
1α,25(OH)2D3 potentiates the anti-tumour effects of
many anticancer therapeutic compounds, and several clinical trials indicate that the administration of
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Acknowledgements
D.L.T. and C.S.J. are supported by grants from the US
National Cancer Institute, Department of Defense, American
Cancer Society and The Roswell Park Alliance Foundation.
C.S.J. is also supported by the Robert, Lew and Ann Wallace
Endowment Fund.
700 | SEPTEMBER 2007 | volume 7
Competing interests statement
The authors declare competing financial interests, see web
version for details.
DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
BCL2 | BGLAP | CBP | CCNA1 | CCNC | CCND1 | CCND3 |
CCNE1 | CDH1 | CDKN1A | CDKN1B | COX2 | CYP24A1 |
CYP27A1 | CYP27B1 | EGFR | GADD45 | HDAC | ID1 | ID2 |
IGFBP3 | MEKK1 | MYC | PI3K | PTH | SKIP | SKP2 | SMAD3 |
SMRT | TGFβ | THBS1 | TF2B | TK1 | TYMS | VDR | VEGF | WSTF
National Cancer Institute: http://www.cancer.gov
breast cancer | colorectal cancer | prostate cancer
FURTHER INFORMATION
Authors’ homepage: http://roswellpark.org
SUPPLEMENTARY INFORMATION
See online article: S1 (table) | S2 (table)
All links are active in the online pdf
www.nature.com/reviews/cancer
© 2007 Nature Publishing Group