Abstract. Colorectal cancer is a major health problem
worldwide. Aberrant activation of the Wingless-type mouse
mammary tumour virus integration site family (Wnt)/β-catenin
signalling pathway due to mutation of adenomatous polyposis
coli (APC), β-catenin (CTNNB1) or AXIN genes is the most
common and initial alteration in sporadic colorectal tumours.
Numerous epidemiological and experimental studies have
indicated a protective action of vitamin D against colorectal
cancer. Previous work has demonstrated that the most active
vitamin D metabolite, 1α,25-dihydroxyvitamin D3(1,25(OH)2D3)
inhibits β-catenin transcriptional activity by promoting vitamin
D receptor (VDR) binding to β-catenin and the induction of E-
cadherin expression. Recently, 1,25(OH)2D3has been shown
to distinctly regulate two genes encoding the extracellular Wnt
inhibitors DICKKOPF-1 and DICKKOPF-4 (DKK-1, DKK-4).
By an indirect transcriptional mechanism, 1,25(OH)2D3
increases the expression of DKK-1 RNA and protein, which
acts as a tumour suppressor in human colon cancer cells
harbouring endogenous mutations in the Wnt/β-catenin
transcription by inducing direct VDR binding to its promoter.
Unexpectedly, DKK-4 is a target of the Wnt/β-catenin pathway
and is up-regulated in colorectal tumours, and it has been
shown to increase cell migration and invasion and to promote
a proangiogenic phenotype. Together, these results show that
1,25(OH)2D3exerts a complex set of regulatory actions
leading to the inhibition of the Wnt/β-catenin pathway in colon
cancer cells that is in line with its protective effect against this
Vitamin D and Colon Cancer – Brief Overview
Colorectal cancer is the second most frequent malignancy
and the second leading cause of death from cancer in
Europe, with 412,900 cases diagnosed and 207,400 deaths in
2006. By sex, it constitutes the second most frequent tumour
in women after breast cancer and the third in men after lung
and prostate tumours (1).
There is strong evidence supporting the hypothesis that
vitamin D may reduce the risk of colorectal cancer (2). It is
now becoming clear that adult vitamin D deficiency is
endemic and epidemiological data suggest a link between
UV-B exposure or vitamin D deficiency and cancer (3).
Several studies have recently revealed an inverse relationship
between 25-hydroxyvitamin D3(calcidiol, 25(OH)D3) levels
and colorectal cancer mortality (4-7), showing that the
improvement of vitamin D status may reduce the risk and the
incidence of cancer (8-10). The involvement of calcium in
this effect is unclear.
The majority of the pleiotropic actions of 1α,25-
dihydroxyvitamin D3(calcitriol, 1,25(OH)2D3), the most
active vitamin D metabolite, are mediated by its nuclear
receptor (VDR), a ligand-regulated transcription factor and a
member of the nuclear receptors superfamily, that binds to
specific sequences (vitamin D response elements, VDRE) in
its target genes and modulates their expression (11, 12).
VDR is expressed in nearly all human tissues and although
initially considered to be exclusively nuclear, is now believed
to shuttle constantly between the nucleus and the cytoplasm
upon ligand activation.
Abbreviations:1,25(OH)2D3, 1α,25-Dihydroxyvitamin D3; 25(OH)D3,
25-hydroxyvitamin D3; APC, adenomatous polyposis coli; DKK,
DICKKOPF; FAP, familial adenomatous polyposis; LEF/TCF,
lymphoid enhancer factor/T-cell factor; LRP, low density lipoprotein
receptor-related protein;VDR, vitamin D receptor.
Correspondence to: Alberto Muñoz, Instituto de Investigaciones
Biomédicas “Alberto Sols”, Arturo Duperier 4, E-28029 Madrid,
Spain. Tel: +34 915854451, Fax: +34 915854401, e-mail:
Key Words: Vitamin D, Wnt, β-catenin, Dickkopf, colon cancer,
ANTICANCER RESEARCH 28: 2613-2624 (2008)
Vitamin D and Wnt/β-catenin Pathway in Colon Cancer:
Role and Regulation of DICKKOPF Genes
NATALIA PENDÁS-FRANCO1, ÓSCAR AGUILERA1, FABIO PEREIRA1,2,
JOSÉ MANUEL GONZÁLEZ-SANCHO1and ALBERTO MUÑOZ1
1Instituto de Investigaciones Biomédicas “Alberto Sols”, Consejo Superior de Investigaciones Científicas,
Universidad Autónoma de Madrid, E-28029 Madrid, Spain;
2Faculty of Nutrition and Food Sciences of the University of Porto, Porto, Portugal
In addition to its classical actions on the normal
development and mineralization of a healthy skeleton,
1,25(OH)2D3suppresses tumour progression by restraining
cell proliferation and inducing cell differentiation and
apoptosis in a large variety of tumour cells, including cells of
1,25(OH)2D3, be it pro-differentiative, anti-proliferative or
pro-apoptotic, depends largely on the differentiation status,
the VDR expression level and the cancer cell type (17).
Briefly, cell-cycle arrest may result from the induction of
cyclin-dependent kinase inhibitors such as p21WAF1/CIP1and
p27KIP1and the repression of cyclin D1, or direct induction of
CLL/lymphoma 2 (BCL2) and the activation of BCL2-
associated X (BAX) and BCL2-antagonist/killer (BAK)
contribute to the apoptosis sensitization (reviewed in (12,
High VDR expression has been reported to be associated
with a favourable prognosis in colorectal cancer (18, 19).
dedifferentiation, which correlates with the up-regulation of
SNAIL1, a transcriptional repressor of VDR (20, 21). This
may help to explain the loss of responsiveness to the
antitumour effects of 1,25(OH)2D3and its analogues in vitro
and in vivo, and therefore be used as an indicator of patients
who are unlikely to respond to this therapy (reviewed in (22)).
Many genes are regulated by 1,25(OH)2D3either directly,
through VDR binding to their regulatory regions, or indirectly,
via intermediate genes, or by affecting other pathways such as
Wnt/β-catenin (see below). The blockade of β-catenin
transcriptional activity and the induction of E-cadherin, a
major contributor to intercellular adhesion that is lost in the
adenoma to carcinoma transition, must be important for the
phenotypic change of tumour cells towards a normal epithelial
phenotype induced by 1,25(OH)2D3(13, 23).
The Wnt/β-catenin Signalling Pathway
intestine (13-16). The predominanteffect of
VDRexpression is lost duringtumour
The Wnts comprise a large family of highly conserved growth
factors that are responsible for important developmental and
homeostatic processes throughout the animal kingdom (24).
Secreted Wnt proteins may bind to a plethora of potential
Wnt membrane receptors which include Frizzleds, low
density lipoprotein receptor-related proteins (LRPs), RYK
receptor-like tyrosine kinase (RYK)/Derailed, retinoid-related
orphan receptor (Ror)-2, and FRL1/Cripto, and elicit different
types of intracellular responses. In the best understood Wnt/β-
catenin or Wnt canonical signalling pathway, Wnt binding to
Frizzled and LRP5/6 co-receptors induces β-catenin protein
stabilization and entry into the nucleus where it modulates the
transcription of target genes (Figure 1). In the absence of Wnt
ANTICANCER RESEARCH 28: 2613-2624 (2008)
Figure 1. Extracellular inhibitors of the Wnt/β-catenin pathway. Wnt
signalling leads to stabilization of cytosolic β-catenin through the
inactivation of a multiprotein complex which phosphorylates
β-catenin and targets it for degradation by the proteasome. Stabilised
β-catenin enters the cell nucleus and associates with LEF/TCF
transcription factors, modulating the transcription of Wnt-target
genes. There are two types of Wnt/β-catenin pathway extracellular
inhibitors: on one hand, secreted Frizzled-related proteins (SFRPs),
Wnt inhibitory factor-1 (WIF-1), and Xenopus Cerberus that bind
directly to Wnt factors and block their interaction with Frizzled
proteins; and on the other hand, DKK -1 and -4, and in some cases
DKK-2, and Wise that bind to LRP5/6 and block Wnt signal
transduction by preventing Wnt-Frizzled-LRP interaction and/or
inducing LRP endocytosis in the presence of the DKK co-receptors
ligands, free β-catenin is phosphorylated by casein kinase 1
(CK1) and glycogen synthase kinase 3β (GSK3β) in a
destruction complex that contains the scaffolding proteins
axin and APC (Figure 1). Phosphorylated β-catenin is
recognized by the E3 ubiquitin ligase β-transducin repeat
containing protein (β-TrCP) and targeted for proteasomal
degradation. Wnt binding to Frizzled/LRP induces the co-
clustering of receptors in LRP-signalosomes, which leads to
the phosphorylation of LRP by GSK3β and CK1γ (25, 26).
Axin docking to the phosphorylated residues in LRP
promotes the inactivation of the destruction complex and the
accumulation of β-catenin. Then a population of β-catenin
molecules translocates into the cell nucleus where it partners
with members of the lymphoid enhancer factor/T-cell factor
(LEF/TCF) family of transcription factors to activate the
transcription of target genes (Figure 1). Numerous TCF or
Wnt target genes have been identified in diverse biological
systems. For a comprehensive, updated overview of TCF
target genes, the reader is referred to the Wnt homepage
Wnt reception is modulated by secreted extracellular Wnt
antagonists which can be divided into two functional classes:
those that bind directly to Wnts (secreted Frizzled-related
proteins (SFRPs), Wnt inhibitory factor-1 (WIF-1), and
Xenopus Cerberus), thereby altering their ability to bind to
the Wnt receptors; and those that inhibit Wnt signalling by
binding to LRP5/6 (Dickkopf (Dkk) proteins, and Wise) (27)
(Figure 1). The Dickkopf family encodes secreted proteins of
255-350 aminoacids and consists of four main members in
vertebrates (Dkk-1 to -4) (28). Dkk-1, the most widely
studied member of this family, and Dkk-4 proteins act as pure
inhibitors of Wnt/β-catenin signalling. In contrast, Dkk-2 and
Dkk-3 can activate or inhibit the pathway depending on the
cellular context (28-30). The inhibitory effect of Dkks may
be brought about by two mechanisms. First, Dkk binding to
LRP5/6 can directly block the LRP-Wnt interaction (31). And
second, Dkks can form a ternary complex with LRP5/6 and
another class of high affinity Dkk receptors named Kremen
(Krm1/2), which induces rapid endocytosis and removal of
LRP56 from the plasma membrane, thereby presumably
blocking Wnt/β-catenin signalling (32, 33).
Abnormal Wnt/β-catenin signalling is associated with
many human diseases, including cancer, osteoporosis,
degenerative disorders and with aging (34, 35). Mutations
that strongly and constitutively activate the Wnt/β-catenin
pathway are involved in the initiation and progression of
several types of cancer. The best-known example of a disease
involving a Wnt pathway mutation that produces tumours is
familial adenomatous polyposis (FAP), an autosomal,
dominantly inherited disease in which patients inherit one
defective APC allele (36, 37) and as a consequence develop
large numbers of colon adenomas, or polyps, in early
adulthood. Polyps are benign, clonal outgrowth of epithelial
cells in which often the second APC allele is later inactivated
adenocarcinoma. Loss of both APC alleles occurs in the large
majority of sporadic colorectal carcinomas (38) leading to
inappropriate stabilization of β-catenin. In rare cases where
APC is not mutated, AXIN2 is mutant (39) or activating
mutations in CTNNB1 (β-catenin) are found (40).
some of themto progress into malignant
Functional Interplay between 1,25(OH)2D3and
the Wnt/β-catenin Pathway
Results from our group have demonstrated that 1,25(OH)2D3
and several analogues can antagonize canonical Wnt signalling
in human colorectal cancer cells (13). In SW480-ADH cells,
1,25(OH)2D3inhibits the transcriptional activity of β-catenin
by two mechanisms. Firstly, it rapidly increases the amount of
VDR bound to β-catenin, thus reducing the interaction
between β-catenin and TCF4. Therefore, 1,25(OH)2D3
modulates LEF/TCF target genes in the opposite way to
β-catenin. This effect is independent of E-cadherin, as it takes
places in LS-174T cells that lack E-cadherin expression (13).
Secondly, the reduction of β-catenin transcriptional activity
caused by 1,25(OH)2D3is accompanied by the nuclear export
of β-catenin and its relocalization to the plasma membrane, an
effect that has recently been shown to be abolished in vitro
and in vivo by SNAIL1 (41). The nuclear export of β-catenin
is concomitant to E-cadherin protein induction. These results
indicate that 1,25(OH)2D3down-regulates the Wnt/β-catenin
signalling pathway, which may control the phenotype of colon
epithelial cells. Upon β-catenin stabilization in colon cancer
cells, due to its own mutation or that of APC or AXIN, binding
to VDR may buffer its stimulatory action on TCF4 target
genes, a protective effect which can be lost along with VDR
expression during malignant progression. Additionally, we
found that nuclear β-catenin transiently potentiates VDR
transcriptional activity before β-catenin moves out of the
nucleus and/or VDR is extinguished (13).
Shah and colleagues have confirmed our results and showed
that the effects of β-catenin on VDR activity were due to
interaction between the activator function-2 (AF-2) domain of
theVDR and the C-terminal region of β-catenin (42). Moreover,
acetylation of the β-catenin C-terminal region differentially
regulates its ability to activate LEF/TCF or VDR-regulated
promoters and the mutation of a specific residue in the AF-2
domain, which renders a VDR that can bind hormone, but is
transcriptionally inactive in the context of classical co-activators,
still allows interaction with β-catenin and ligand-dependent
activation of VDRE-containing promoters. Interestingly, VDR
antagonists, which block theVDRE-directed activity of theVDR
and recruitment of classical co-activators, do allow VDR to
interact with β-catenin, which suggests that these and perhaps
other ligands would permit those functions of the VDR that
involve β-catenin interaction (42).
Pendás-Franco et al: Vitamin D Antagonizes Wnt Signalling (Review)
In the skin, the canonical Wnt pathway controls both
epidermal stem cell renewal and lineage selection (43-45).
Likewise, VDR is essential for adult epidermal homeostasis
(46) and mutations in the VDR gene in humans result in
familial 1,25(OH)2D3-resistant rickets, which can be
associated with alopecia (47). In vivo, the expression of a
mutant VDR that can bind β-catenin, but not 1,25(OH)2D3
rescues alopecia in Vdr null mice, demonstrating ligand-
independent functions of VDR in the skin (48). Recently,
two independent groups have shown that the absence of VDR
impairs canonical Wnt signalling in keratinocytes and leads
to the development of alopecia (49, 50). Cianferotti and
colleagues found a gradual decrease in the size of the stem
cell compartment in Vdr–/–epidermis and this correlated with
a failure of β-catenin to induce proliferation (49). In
contrast, Palmer et al. saw no evidence that VDR loss
impaired the proliferative response to β-catenin (50, 51).
Alternatively, they have demonstrated that β-catenin is a co-
activator of VDR in epidermal keratinocytes and that a
number of Wnt target genes in the skin are likely to be
regulated through VDREs. For these researchers, the primary
role of the VDR/β-catenin interaction in the skin is to
differentiation of the hair follicle lineages. Constitutive
activation of the Wnt pathway leads to ectopic hair follicle
formation and, subsequently, to a type of benign tumour
differentiation of ectopic hair follicles is stimulated and
trichofolliculloma development is blocked. Conversely, in the
absence of VDR, differentiation of ectopic follicles is
inhibited and the tumours that develop in response to β-
catenin are undifferentiated basal cell carcinomas (50). Thus,
vitamin D analogues may be beneficial in the treatment of
tumours in which the canonical Wnt pathway is activated
inappropriately. An interesting corollary to this work is that
β-catenin can no longer be considered as chiefly an activator
of LEF/TCF target genes. The interaction of β-catenin with
other transcription factors, such as VDR, is likely to
contribute to the pleiotropic effects of the Wnt pathway,
which has different target genes in different cell types.
The skeleton is also a direct target of vitamin D action,
which modulates the proliferation of osteoblast precursors,
their differentiation into mature osteoblasts and their
1,25(OH)2D3are reminiscent of those orchestrated by the
Wnt signalling pathway (53). Indeed, the Wnt co-receptor
LRP5 is now known to play a particularly important role in
bone formation such that loss of this component results in a
reduction in osteoblast number, a delay in mineralization and
a reduction in peak bone mineral density (54, 55).
Interestingly, it has recently been reported that the LRP5
gene is a direct transcriptional target of 1,25(OH)2D3(56)
the transcriptionof genes associatedwith
In the presence
activity(52). Some ofthese effectsof
highlighting the existence of a functional interaction between
the Wnt and 1,25(OH)2D3pathways in this tissue.
1,25(OH)2D3Induces the Expression of
Dickkopf (Dkk)-1, the founding member of the Dkk gene
family, was originally identified as an embryonic head
inducer and Wnt antagonist in Xenopus (57). Since then,
Dkks have been identified in other vertebrates including
humans as well as in invertebrates such as Dictyostelium,
cnidarians, urochordates and ascidians (58-61), but not in
Drosophila or Caenorhabditis elegans. Thus, Dkks are an
evolutionarily ancient gene family that was already present
in the last common ancestor of cnidarians and bilaterians and
which was probably secondarily lost during evolution in
protostomes. A distant Dkk family member, soggy (sgy; also
called Dickkopf-like protein 1, Dkkl1), has been described in
vertebrates (61) and shows unique homology to Dkk3.
Human DKK-1 seems to have wide and complex effects
on cell proliferation and differentiation, it induces the
proliferation of human adult bone marrow stem cells (62)
and inhibits osteoblastic differentiation (63) which is in line
with the finding that high circulating levels of DKK-1 in
patients with multiple myeloma are associated with
osteolytic lesions (64). Moreover, glucocorticoids, which are
associated with bone loss and osteoporosis (65), enhance
DKK-1 expression in osteoblasts (66). In contrast, DKK-1
expression promotes preadipocyte differentiation (67), and
in the mouse small intestine and colon, forced Dkk-1
expression inhibits the proliferation of the crypt progenitor
cells that is induced by the transcriptional activity of β-
catenin/TCF (68, 69). Additionally, human DKK-1 was
reportedly induced by p53 (70), although in another study
DKK-1 was induced by DNA damage and sensitized to
apoptosis in a p53-independent manner (71).
Thus, DKK-1 seems to have distinct effects depending on
the cell type, which agrees with the different effects of
Wnt/β-catenin signalling. While Wnt/β-catenin promotes
proliferation and blocks differentiation in colon epithelial
osteoblastogenesis and represses differentiation to alternative
cell types, such as adipocytes (72, 73). Also, the role of Wnt
signalling in melanoma cells is controversial as it probably
depends upon a regulated, temporal expression pattern of
Wnts. Expression at a particular time will lead a cell to
tumourigenesis and invasion, while expression at other times
may have the opposite effect, resulting instead in cellular
In breast cancer, the role of DKK-1 is controversial. It has
been reported that DKK-1 is expressed in hormone-resistant
breast tumours and thus it has been proposed as a new
prognosis marker (75). On the other hand, Mikheev et al.
inmesenchymal precursors itstimulates
ANTICANCER RESEARCH 28: 2613-2624 (2008)
described tumour suppression in breast carcinoma cells
mediated by DKK-1. Ectopic expression of DKK-1 in these
cells was associated with increased phosphorylation and
degradation of β-catenin and inhibition of cyclin D1 (CCND1)
and c-MYC oncogenes (76). Likewise, the overexpression of
DKK-1 in hepatocellular carcinoma cell lines down-regulates
CCND1 and c-MYC, so inhibiting cell growth and migration
during the metastatic process (77). Moreover, DKK-1 is down-
regulated by the neural (n)-MYC (MYCN) oncogene and
inhibits neuroblastoma cell proliferation (78). The DKK-1-
inducible neuroblastoma IMR32-DKK-1 cell line showed
impaired proliferation upon DKK-1 expression. Surprisingly,
DKK-1 expression did not inhibit the canonical Wnt/β-catenin
pathway, suggesting a role of DKK-1 in an alternative route
(78). Also in lung and oesophageal carcinomas, DKK-1 has
been proposed as prognostic and a serological marker. Gene
expression profiling of both carcinomas has revealed that
DKK-1 was highly transactivated in the majority of lung
squamous cell carcinomas and serum DKK-1 levels were
higher in lung and oesophageal cancer patients than in healthy
controls (79). In conclusion, the antitumoural activity of
DKK-1 is strictly dependent on the tissue and cancer type.
Curiously, over-expression of DKK-1 has also been
associated with neuronal degeneration in the brain of
Alzheimer’s patients. In this case, the cascade was triggered
by the β-amyloid peptide which up-regulates TP53.
Subsequently, DKK-1 expression was enhanced and the
Wnt/β-catenin pathway inhibited (80).
We and others have observed that the transcription of the
DKK-1 gene is enhanced by β-catenin/TCF acting on several
sites in the promoter region (81-83). Our group reported also
that DKK-1 is down-regulated in colon cancer (82),
indicating the loss of a negative feedback control of the
Wnt/β-catenin pathway in this neoplasia. We also showed
that DKK-1 down-regulation occurs, at least in part, due to
promoter methylation, which is specifically found in 25% of
advanced, less differentiated tumours (Dukes’ stages C and
D) (84). Interestingly, DKK-1 seems to have antitumoural
effects independently of the antagonism of β-catenin/TCF
transcriptional activity in H28 and MS-1 mesothelioma,
HeLa cervical, and JAR and JEG3 human placental
choriocarcinoma cancer cells (23, 85, 86). Activation of the
Jun N-terminal kinase (JNK) pathway is involved in some
of these tumour suppressor effects (23, 85). Also in DLD-1
colon cancer cells, which bear a truncated APC gene and so
transfection of DKK-1 decreases cell growth in vitro and
tumour formation in immunodeficient mice (84). These data
indicate that DKK-1 can inhibit tumourigenesis by different
mechanisms. Nevertheless, further studies will be necessary
to reveal whether DKK-1 may be acting in ways other than
inhibiting the canonical Wnt signalling pathway or holding
back the pathway in an unknown manner.
a constitutively active Wnt/β-cateninpathway,
The gene expression profile associated with exposure of
human SW480-ADH colon cancer cells to 1,25(OH)2D3has
shown that numerous genes are modulated by this hormone,
including many involved in transcription, cell adhesion, DNA
synthesis, apoptosis, redox status, and intracellular signalling
(23). Among them, DKK-1 seemed to be up-regulated by
1,25(OH)2D3. We have validated that 1,25(OH)2D3increases
the level of DKK-1 RNA and protein in SW480-ADH cells.
This effect is slow and depends on the presence of a
transcription-competent VDR (87). The regulation of DKK-
1 expression by 1,25(OH)2D3is transcriptional, but indirect.
The slow kinetics of DKK-1 RNA accumulation and the lack
of VDR binding to the promoter region that is activated by
the hormone, together with the absence of effect on the half-
life of DKK-1 RNA and the requirement of VDR
transcriptional activity strongly suggest that 1,25(OH)2D3
up-regulates the transcription of DKK-1 via intermediate
proteins encoded by early response genes that remain
uncharacterized. The induction of DKK-1 by 1,25(OH)2D3
constitutes a third mechanism by which this hormone
antagonizes the Wnt/β-catenin pathway. The existence of
several mechanisms of Wnt/β-catenin signalling antagonism
by 1,25(OH)2D3reinforces the importance of this pathway
and of its regulation for the biology of the colonic
Another interesting finding is that DKK-1 is up-regulated
by ectopic E-cadherin in SW480-ADH cells and that a
blocking antibody against E-cadherin inhibits 1,25(OH)2D3-
mediated DKK-1 induction. These data strongly indicate that
the regulatory effect of 1,25(OH)2D3is an indirect
consequence of the induction of the epithelial adhesive
The finding that DKK-1 expression is silenced by promoter
methylation in a subset of advanced, typically dedifferentiated
colorectal tumours and the association of DKK-1 with the
differentiated phenotype suggest the interesting hypothesis
that DKK-1 silencing is not only concomitant with, but also
plays a role in the dedifferentiation process. This may thus
explain the correlation between DKK-1 and VDR expression
in human tumours:VDR expression has been reported to be a
marker of differentiation in colon carcinoma cells (18, 88)
and is lost through colon cancer progression together with
that of E-cadherin in parallel to the up-regulation of SNAIL1
(20, 21, 41).
DICKKOPF-4 Induces a Malignant
Phenotype in Colon Cancer Cells and
is Repressed by 1,25(OH)2D3
characterized member of the DKK family. This gene was first
described by Krupnik and collegues in 1999 (61) but
information is limited in the scientific literature. Probably,
Pendás-Franco et al: Vitamin D Antagonizes Wnt Signalling (Review)
one of the main reasons is that during adult life DKK-4 is
not expressed or its levels are very low. In fact, its pattern of
expression is unclear. Although Northern blot analysis of
several adult and fetal human tissues did not detect DKK-4
RNA, a survey of a human cDNA library panel by PCR with
specific primers generated products from libraries prepared
from cerebellum, activated human T-lymphocytes, lung and
oesophagus (61). Dkk-4 mRNA was also detected in the pre-
placodes in a murine model, being expressed at sites of
presumptive epithelial-mesenchymal interactions during
appendage morphogenesis including the dental lamina,
mammary gland, eccrine gland, and primary and secondary
hair follicles (89).
Interestingly, the expression of DKK-4 has also recently
been detected in some pathological processes such as
inflammation, cancer and squizophrenia. Aung et al. have
reported that the RNA level of DKK-4 was increased in 11
out of 44 (25%) gastric cancer biopsies, even though they
only detected DKK-4 protein in 2 out of 151 (1.3%) by
immunohistochemistry (90). In another study, significantly
higher expression of DKK-1 and DKK-4 RNA and protein
was detected in the distal squamous mucosa of the
oesophagus in oesophagitis patients compared to healthy
controls and patients with Barrett’s oesophagus. In this case,
the authors suggested that those genes might play a role in
the development of different injuries in response to
pathological gastro-oesophageal acid reflux (91). Also,
DKK-4 was the molecular marker that showed the highest
quantitative RT-PCR (138-fold increase) analyses in the
endometrial cancer (92). Moreover, DKK-4 RNA levels
were increased in patients with ulcerative colitis (93) and
also with colon cancer (94, 95). Finally, Proitsi et al.
identified single nucleotide polymorphisms (SNPs) in the
DKK-4 gene, which is located in genome regions previously
linked to schizophrenia, suggesting that DKK-4 might play
a role in this disease (96).
Regarding its biological activity, DKK-4 protein has been
described as an antagonist of Wnt/β-catenin signalling (33,
61) and has been shown to be transcriptionally induced by
this pathway (89, 95) as is DKK-1 (81-83) (Figure 2).
DKK-4 is a weaker Wnt inhibitor than DKK-1, although its
effect is increased if Kremen 2 is overexpressed ((33) and
our unpublished data). In apparent contradiction, DKK-4
inhibits the Wnt/β-catenin pathway and is overexpressed in
several pathological diseases including some types of cancer
(90-92, 94, 95). As stated above, we and others have found
DKK-4 RNA expressed in human colorectal tumours while
it was undetectable in normal adjacent tissue (94, 95). This
result contrasts with the common silencing of the DKK-4
gene in colon cancer cell lines that we and others (97) have
found and that may be related to cell culture conditions.
in microarray (46.9-foldincrease) and
ofHong KongChinese womenwith
To investigate whether DKK-4 up-regulation in human
colon cancer could have functional implications for tumour
progression, we expressed DKK-4 ectopically in two human
colon cell lines, SW480-ADH, which expresses low levels
of the endogenous gene, and DLD-1, with undetectable
expression. Exogenous DKK-4 enhanced the migratory and
invasive potential in vitro of both cell lines. These effects
were partially inhibited by the transfection of DKK-4
siRNA oligonucleotides. Moreover, the migration and
morphogenetic capacity of primary human microvascular
endothelial cells (HMVEC) were robustly increased in the
presence of conditioned medium from DKK-4-expressing
cells or recombinant DKK-4 protein (95). The ability to
induce and sustain angiogenesis is essential for incipient
neoplasias to grow, and the capability for invasion enables
cancer cells to metastasise. Thus, although DKK-4 can act
as a Wnt inhibitor, these findings support new roles for this
protein in human colon cancer, probably inducing β-
catenin-independent actions during the progression of this
Wnt antagonists other than DKK-4 are also up-regulated
and may contribute to tumourigenesis in different systems.
For example, SFRP4 is expressed in the stromal cells
surrounding endometrial and breast carcinomas, but is barely
detectable in the stroma of secretory or menstrual
endometrium (98). Moreover, the expression of SFRP1 and
SFRP2 is up-regulated in glioma-derived cell lines, and
SFRP2 promotes tumour growth in nude mice (99).
chorioallantoic membranes and increases migration and
organization of endothelial cells into capillary-like structures
(100). Strong DKK-3 expression has been detected in tumour
endothelial cells of glioma, high-grade non-Hodgkin’s
lymphomas, melanoma and colorectal carcinoma (101, 102),
and the authors proposed that DKK-3 might be a marker for
endothelial cell activation during tumour angiogenesis. In
contrast, DKK-3 is frequently inactivated in lung cancer by
regulation of DKK-4 and several Wnt inhibitors in some
cancer cell types imply their involvement in roles other than
the control of this signalling pathway.
Unlike DKK-1, the available data indicate that DKK-4 is a
target gene induced by Wnt/β-catenin that remains up-
regulated in colon tumours. The pro-tumourigenic actions of
DKK-4 in cultured cells suggest that they overcome its weak
inhibitory effect on the Wnt/β-catenin pathway (Figure 2).
The discrepancy between the regulation of DKK-4 in colon
and breast tumours, in which variable levels but no up-
regulation was found in a first study (95), reveal tissue-
specific actions and promotes interest in extending the study
of DKK-4 effects to other types of carcinomas. Notably,
1,25(OH)2D3inhibits DKK-4 expression in colorectal cancer
cells and diminishes transcription from the DKK-4 promoter
inducesangiogenesis in chick
hypermethylation (29).In conclusion,up-
ANTICANCER RESEARCH 28: 2613-2624 (2008)
in SW480-ADH and in three human breast cancer cell lines
repression appears to be direct as, again in contrast to DKK-
1, 1,25(OH)2D3promotes the binding of VDR and also of the
silencing mediator of retinoic acid and thyroid hormone
receptor (SMRT) co-repressor to a consensus sequence
adjacent to the transcription initiation site and the abrogation
of histone H4 acetylation. Interestingly, the inverse
correlation found between VDR and DKK-4 RNA levels in
human colorectal tumours suggests that the regulation of
DKK-4 observed in cell lines also occurs in patients.
The regulation and expression of DKK-1 and DKK-4
occur in opposite directions in human colorectal cancer.
While the induction of DKK-1 by 1,25(OH)2D3is slow and
requires intermediate proteins (87), 1,25(OH)2D3represses
DKK-4 rapidly promoting direct VDR binding to the gene
promoter region (95). Both, DKK-1 and DKK-4 proteins
have β-catenin-independent activitiesthat,
however, must differ markedly. While DKK-1 has anti-
tumoural effects (84), the effects of DKK-4 on the
phenotype of colon cancer cells and its up-regulation in
colon cancer indicate tumour-promoting actions (95). The
induction of DKK-1 and the repression of DKK-4 by
1,25(OH)2D3agrees with and may contribute to its
protective effects against this neoplasia. The elucidation of
the roles of DKK-1 and DKK-4 proteins in colon cancer
cells may be important for understanding the biology of
colon cancer and 1,25(OH)2D3action.
1 Ferlay J, Autier P, Boniol M, Heanue M, Colombet M and
Boyle P: Estimates of the cancer incidence and mortality in
Europe in 2006. Ann Oncol 18: 581-592, 2007.
Grant WB and Garland CF: A critical review of studies on
vitamin D in relation to colorectal cancer. Nutr Cancer 48: 115-
Pendás-Franco et al: Vitamin D Antagonizes Wnt Signalling (Review)
Figure 2. Multifaceted effects of 1,25(OH)2D3on the Wnt/β-catenin pathway in colorectal cancer through the induction of direct β-catenin–VDR
interaction and the opposite modulation of DKK-1 and DKK-4 genes. Both DKK genes are transcriptionally induced by Wnt/β-catenin signalling,
and the corresponding encoded proteins are secreted and inhibit this pathway. However, DKK-1 is down-regulated in colon cancer while DKK-4 is
overexpressed. Additionally, DKK-1 has other anticancer effects still to be characterized. In contrast, DKK-4 potentiates the migratory, invasive
and pro-angiogenic phenotype of tumour cells.
3Cannell JJ, Hollis BW, Zasloff M and Heaney RP: Diagnosis
Pharmacother 9: 107-118, 2008.
Peters U, Hayes RB, Chatterjee N, Shao W, Schoen RE, Pinsky P,
Hollis BW and McGlynn KA: Circulating vitamin D metabolites,
polymorphism in vitamin D receptor, and colorectal adenoma
risk. Cancer Epidemiol Biomarkers Prev 13: 546-552, 2004.
Giovannucci E, Liu Y, Rimm EB, Hollis BW, Fuchs CS,
Stampfer MJ and Willett WC: Prospective study of predictors
of vitamin D status and cancer incidence and mortality in men.
J Natl Cancer Inst 98: 451-459, 2006.
Freedman DM, Looker AC, Chang SC and Graubard BI:
Prospective study of serum vitamin D and cancer mortality in
the United States. J Natl Cancer Inst 99: 1594-1602, 2007.
Jacobs ET, Alberts DS, Benuzillo J, Hollis BW, Thompson PA
and Martinez ME: Serum 25(OH)D levels, dietary intake of
vitamin D, and colorectal adenoma recurrence. J Steroid
Biochem Mol Biol 103: 752-756, 2007.
Autier P and Gandini S: Vitamin D supplementation and total
mortality: a meta-analysis of randomized controlled trials. Arch
Intern Med 167: 1730-1737, 2007.
Lappe JM, Travers-Gustafson D, Davies KM, Recker RR and
Heaney RP: Vitamin D and calcium supplementation reduces
cancer risk: results of a randomized trial. Am J Clin Nutr 85:
10 Grau MV, Baron JA, Sandler RS, Haile RW, Beach ML, Church
TR and Heber D: Vitamin D, calcium supplementation, and
colorectal adenomas: results of a randomized trial. J Natl
Cancer Inst 95: 1765-1771, 2003.
11 Carlberg C and Dunlop TW: An integrated biological approach
to nuclear receptor signaling in physiological control and
disease. Crit Rev Eukaryot Gene Expr 16: 1-22, 2006.
12 Campbell MJ and Adorini L: The vitamin D receptor as a
therapeutic target. Expert Opin Ther Targets 10: 735-748, 2006.
13 Palmer HG, Gonzalez-Sancho JM, Espada J, Berciano MT, Puig
I, Baulida J, Quintanilla M, Cano A, de Herreros AG, Lafarga M
and MunozA:Vitamin D(3) promotes the differentiation of colon
carcinoma cells by the induction of E-cadherin and the inhibition
of beta-catenin signaling. J Cell Biol 154: 369-387, 2001.
14 Lamprecht SA and Lipkin M: Chemoprevention of colon cancer
by calcium, vitamin D and folate: molecular mechanisms. Nat
Rev Cancer 3: 601-614, 2003.
15 Ordonez-Moran P, Larriba MJ, Pendas-Franco N, Aguilera O,
Gonzalez-Sancho JM and Munoz A: Vitamin D and cancer: an
update of in vitro and in vivo data. Front Biosci 10: 2723-
16 Gonzalez-Sancho JM, Larriba MJ, Ordonez-Moran P, Palmer
HG and Munoz A: Effects of 1alpha,25-dihydroxyvitamin D3in
human colon cancer cells. Anticancer Res 26: 2669-2681, 2006.
17 Deeb KK, Trump DL and Johnson CS: Vitamin D signalling
pathways in cancer: potential for anticancer therapeutics. Nat
Rev Cancer 7: 684-700, 2007.
18 Cross HS, Bajna E, Bises G, Genser D, Kallay E, Potzi R,
Wenzl E, Wrba F, Roka R and Peterlik M: Vitamin D receptor
and cytokeratin expression may be progression indicators in
human colon cancer. Anticancer Res 16: 2333-2337, 1996.
19 Evans SR, Nolla J, Hanfelt J, Shabahang M, Nauta RJ and
Shchepotin IB: Vitamin D receptor expression as a predictive
marker of biological behavior in human colorectal cancer. Clin
Cancer Res 4: 1591-1595, 1998.
treatment ofvitaminD deficiency.ExpertOpin
20 Palmer HG, Larriba MJ, Garcia JM, Ordonez-Moran P, Pena C,
Peiro S, Puig I, Rodriguez R, de la Fuente R, Bernad A, Pollan
M, Bonilla F, Gamallo C, de Herreros AG and Munoz A: The
transcription factor SNAIL represses vitamin D receptor
expression and responsiveness in human colon cancer. Nat Med
10: 917-919, 2004.
21 Pena C, Garcia JM, Silva J, Garcia V, Rodriguez R, Alonso I,
Millan I, Salas C, de Herreros AG, Munoz A and Bonilla F:
E-cadherin and vitamin D receptor regulation by SNAIL and
ZEB1 in colon cancer: clinicopathological correlations. Hum
Mol Genet 14: 3361-3370, 2005.
22 Larriba MJ and Munoz A: SNAIL vs. vitamin D receptor
expression in colon cancer: therapeutics implications. Br J
Cancer 92: 985-989, 2005.
23 Palmer HG, Sanchez-Carbayo M, Ordonez-Moran P, Larriba
MJ, Cordon-Cardo C and Munoz A: Genetic signatures of
differentiation induced by 1alpha,25-dihydroxyvitamin D3in
human colon cancer cells. Cancer Res 63: 7799-7806, 2003.
24 Logan CY and Nusse R: The Wnt signaling pathway in
development and disease. Annu Rev Cell Dev Biol 20: 781-810,
25 Bilic J, Huang YL, Davidson G, Zimmermann T, Cruciat CM,
Bienz M and Niehrs C: Wnt induces LRP6 signalosomes and
Science 316: 1619-1622, 2007.
26 Zeng X, Huang H, Tamai K, Zhang X, Harada Y, Yokota C,
Almeida K, Wang J, Doble B, Woodgett J, Wynshaw-Boris A,
Hsieh JC and He X: Initiation of Wnt signaling: control of Wnt
dishevelled and axin functions. Development 135: 367-375,
27 Kawano Y and Kypta R: Secreted antagonists of the Wnt
signalling pathway. J Cell Sci 116: 2627-2634, 2003.
28 Niehrs C: Function and biological roles of the Dickkopf family
of Wnt modulators. Oncogene 25: 7469-7481, 2006.
29 Yue W, Sun Q, Dacic S, Landreneau RJ, Siegfried JM,Yu J and
Zhang L: Downregulation of Dkk3 activates beta-catenin/TCF-4
signaling in lung cancer. Carcinogenesis 29: 84-92, 2008.
30 Wang XY,YinY,Yuan H, Sakamaki T, Okano H and Glazer RI:
Musashi1 modulates mammary progenitor cell expansion
through proliferin-mediated activation of the Wnt and Notch
pathways. Mol Cell Biol 28: 3589-99, 2008.
31 Semenov MV, Tamai K, Brott BK, Kuhl M, Sokol S and He X:
Head inducer Dickkopf-1 is a ligand for Wnt coreceptor LRP6.
Curr Biol 11: 951-961, 2001.
32 Mao B, Wu W, Davidson G, Marhold J, Li M, Mechler BM,
Delius H, Hoppe D, Stannek P, Walter C, Glinka A and Niehrs
C: Kremen proteins are Dickkopf receptors that regulate
Wnt/beta-catenin signalling. Nature 417: 664-667, 2002.
33 Mao B and Niehrs C: Kremen2 modulates Dickkopf2 activity
during Wnt/LRP6 signaling. Gene 302: 179-183, 2003.
34 Clevers H: Wnt/beta-catenin signaling in development and
disease. Cell 127: 469-480, 2006.
35 Moon RT, Kohn AD, De Ferrari GV and Kaykas A: WNT and
beta-catenin signalling: diseases and therapies. Nat Rev Genet
5: 691-701, 2004.
36 Kinzler KW, Nilbert MC, Su LK, Vogelstein B, Bryan TM,
Levy DB, Smith KJ, Preisinger AC, Hedge P, McKechnie D et
al: Identification of FAP locus genes from chromosome 5q21.
Science 253: 661-665, 1991.
ANTICANCER RESEARCH 28: 2613-2624 (2008)
37 Nishisho I, Nakamura Y, Miyoshi Y, Miki Y, Ando H, Horii A,
Koyama K, Utsunomiya J, Baba S and Hedge P: Mutations of
chromosome 5q21 genes in FAP and colorectal cancer patients.
Science 253: 665-669, 1991.
38 Kinzler KW and Vogelstein B: Lessons from hereditary
colorectal cancer. Cell 87: 159-170, 1996.
39 Liu W, Dong X, Mai M, Seelan RS, Taniguchi K, Krishnadath
KK, Halling KC, Cunningham JM, Boardman LA, Qian C,
Christensen E, Schmidt SS, Roche PC, Smith DI and Thibodeau
SN: Mutations in AXIN2 cause colorectal cancer with defective
mismatch repair by activating beta-catenin/TCF signalling. Nat
Genet 26: 146-147, 2000.
40 Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H,
Vogelstein B and Kinzler KW: Activation of beta-catenin-Tcf
signaling in colon cancer by mutations in beta-catenin or APC.
Science 275: 1787-1790, 1997.
41 Larriba MJ, Valle N, Palmer HG, Ordonez-Moran P, Alvarez-
Diaz S, Becker KF, Gamallo C, de Herreros AG, Gonzalez-
Sancho JM and Munoz A: The inhibition of Wnt/beta-catenin
signalling by 1alpha,25-dihydroxyvitamin D3is abrogated by
Snail1 in human colon cancer cells. Endocr Relat Cancer 14:
42 Shah S, Islam MN, Dakshanamurthy S, Rizvi I, Rao M, Herrell
R, Zinser G, Valrance M, Aranda A, Moras D, Norman A, Welsh
J and Byers SW: The molecular basis of vitamin D receptor and
beta-catenin crossregulation. Mol Cell 21: 799-809, 2006.
43 Andl T, Reddy ST, Gaddapara T and Millar SE: WNT signals
are required for the initiation of hair follicle development. Dev
Cell 2: 643-653, 2002.
44 Lowry WE, Blanpain C, Nowak JA, Guasch G, Lewis L and
Fuchs E: Defining the impact of beta-catenin/Tcf transactivation
on epithelial stem cells. Genes Dev 19: 1596-1611, 2005.
45 Silva-Vargas V, Lo Celso C, Giangreco A, Ofstad T, Prowse
DM, Braun KM and Watt FM: Beta-catenin and Hedgehog
signal strength can specify number and location of hair follicles
in adult epidermis without recruitment of bulge stem cells. Dev
Cell 9: 121-131, 2005.
46 Bikle DD: Vitamin D and skin cancer. J Nutr 134: 3472S-
47 Hughes MR, Malloy PJ, O’Malley BW, Pike JW and Feldman
D: Genetic defects of the 1,25-dihydroxyvitamin D3receptor. J
Recept Res 11: 699-716, 1991.
48 Skorija K, Cox M, Sisk JM, Dowd DR, MacDonald PN,
Thompson CC and Demay MB: Ligand-independent actions of
the vitamin D receptor maintain hair follicle homeostasis. Mol
Endocrinol 19: 855-862, 2005.
49 Cianferotti L, Cox M, Skorija K and Demay MB: Vitamin D
receptor is essential for normal keratinocyte stem cell function.
Proc Natl Acad Sci USA 104: 9428-9433, 2007.
50 Palmer HG, Anjos-Afonso F, Carmeliet G, Takeda H and Watt
FM: The vitamin D receptor is a Wnt effector that controls hair
follicle differentiation and specifies tumor type in adult
epidermis. PLoS ONE 3: e1483, 2008.
51 Palmer HG, Martinez D, Carmeliet G and Watt FM: The
vitamin D receptor is required for mouse hair cycle progression
but not for maintenance of the epidermal stem cell
compartment. J Invest Dermatol, doi: 10.1038/jid.2008.43.
52 Aubin JE and Heersche JNM: Vitamin D and osteoclast. In:
Vitamin D. Feldman D, Pike, JW, Glorieux and FH (eds.).
Burlington, Elsevier Academic Press, pp. 649-663, 2005.
53 Hartmann C: A Wnt canon orchestrating osteoblastogenesis.
Trends Cell Biol 16: 151-158, 2006.
54 Kato M, Patel MS, Levasseur R, Lobov I, Chang BH, Glass DA
II, Hartmann C, Li L, Hwang TH, Brayton CF, Lang RA,
Karsenty G and Chan L: Cbfa1-independent decrease in
osteoblast proliferation, osteopenia, and persistent embryonic
eye vascularization in mice deficient in Lrp5, a Wnt coreceptor.
J Cell Biol 157: 303-314, 2002.
55 Holmen SL, Giambernardi TA, Zylstra CR, Buckner-Berghuis
BD, Resau JH, Hess JF, Glatt V, Bouxsein ML, Ai M, Warman
ML and Williams BO: Decreased BMD and limb deformities in
mice carrying mutations in both Lrp5 and Lrp6. J Bone Miner
Res 19: 2033-2040, 2004.
56 Fretz JA, Zella LA, Kim S, Shevde NK and Pike JW:
1,25-Dihydroxyvitamin D3regulates the expression of low-
downstream of the start site of transcription. Mol Endocrinol
20: 2215-2230, 2006.
57 Glinka A, Wu W, Delius H, Monaghan AP, Blumenstock C and
Niehrs C: Dickkopf-1 is a member of a new family of secreted
proteins and functions in head induction. Nature 391: 357-362,
58 Hino K, Satou Y, Yagi K and Satoh N: A genomewide survey
of developmentally relevant genes in Ciona intestinalis. VI.
Genes for Wnt, TGFbeta, Hedgehog and JAK/STAT signaling
pathways. Dev Genes Evol 213: 264-272, 2003.
59 Fedders H, Augustin R and Bosch TC: A Dickkopf-3-related
gene is expressed in differentiating nematocytes in the basal
metazoan Hydra. Dev Genes Evol 214: 72-80, 2004.
60 Guder C, Pinho S, Nacak TG, Schmidt HA, Hobmayer B,
Niehrs C and Holstein TW: An ancient Wnt-Dickkopf
antagonism in Hydra. Development 133: 901-911, 2006.
61 Krupnik VE, Sharp JD, Jiang C, Robison K, Chickering TW,
Amaravadi L, Brown DE, Guyot D, Mays G, Leiby K, Chang
B, Duong T, Goodearl AD, Gearing DP, Sokol SY and
McCarthy SA: Functional and structural diversity of the human
Dickkopf gene family. Gene 238: 301-313, 1999.
62 Gregory CA, Perry AS, Reyes E, Conley A, Gunn WG and
Prockop DJ: Dkk-1-derived synthetic peptides and lithium
chloride for the control and recovery of adult stem cells from
bone marrow. J Biol Chem 280: 2309-2323, 2005.
63 Morvan F, Boulukos K, Clement-Lacroix P, Roman Roman S,
Suc-Royer I, Vayssiere B, Ammann P, Martin P, Pinho S,
Pognonec P, Mollat P, Niehrs C, Baron R and Rawadi G:
Deletion of a single allele of the Dkk1 gene leads to an increase
in bone formation and bone mass. J Bone Miner Res 21: 934-
64 Tian E, Zhan F, Walker R, Rasmussen E, MaY, Barlogie B and
Shaughnessy JD Jr: The role of the Wnt-signaling antagonist
DKK1 in the development of osteolytic lesions in multiple
myeloma. N Engl J Med 349: 2483-2494, 2003.
65 Mazziotti G, Angeli A, Bilezikian JP, Canalis E and Giustina A:
Endocrinol Metab 17: 144-149, 2006.
66 Ohnaka K, Taniguchi H, Kawate H, Nawata H and Takayanagi
R: Glucocorticoid enhances the expression of dickkopf-1 in
human osteoblasts: novel mechanism of glucocorticoid-induced
osteoporosis. Biochem Biophys Res Commun 318: 259-264,
osteoporosis:an update. Trends
Pendás-Franco et al: Vitamin D Antagonizes Wnt Signalling (Review)
67 Christodoulides C, Laudes M, Cawthorn WP, Schinner S, Soos
M, O’Rahilly S, Sethi JK and Vidal-Puig A: The Wnt
antagonist Dickkopf-1 and its receptors are coordinately
regulated during early human adipogenesis. J Cell Sci 119:
68 Pinto D, Gregorieff A, Begthel H and Clevers H: Canonical Wnt
signals are essential for homeostasis of the intestinal epithelium.
Genes Dev 17: 1709-1713, 2003.
69 Kuhnert F, Davis CR, Wang HT, Chu P, Lee M, Yuan J, Nusse
R and Kuo CJ: Essential requirement for Wnt signaling in
proliferation of adult small intestine and colon revealed by
adenoviral expression of Dickkopf-1. Proc Natl Acad Sci USA
101: 266-271, 2004.
70 Wang J, Shou J and Chen X: Dickkopf-1, an inhibitor of the
Wnt signaling pathway, is induced by p53. Oncogene 19: 1843-
71 Shou J, Ali-Osman F, Multani AS, Pathak S, Fedi P and
Srivenugopal KS: Human Dkk-1, a gene encoding a Wnt
antagonist, responds to DNA damage and its overexpression
sensitizes brain tumor cells to apoptosis following alkylation
damage of DNA. Oncogene 21: 878-889, 2002.
72 Kennell JA and MacDougald OA: Wnt signaling inhibits
adipogenesis through beta-catenin-dependent and -independent
mechanisms. J Biol Chem 280: 24004-24010, 2005.
73 Kang S, Bennett CN, Gerin I, Rapp LA, Hankenson KD and
blastogenesis of mesenchymal precursors by suppressing
CCAAT/enhancer-binding protein alpha and peroxisome
proliferator-activated receptor gamma. J Biol Chem 282:
74 Weeraratna AT: A Wnt-er wonderland–the complexity of Wnt
signaling in melanoma. Cancer Metastasis Rev 24: 237-250, 2005.
75 Forget MA, Turcotte S, Beauseigle D, Godin-Ethier J, Pelletier
S, Martin J, Tanguay S and Lapointe R: The Wnt pathway
regulator DKK1 is preferentially expressed in hormone-resistant
breast tumours and in some common cancer types. Br J Cancer
96: 646-653, 2007.
76 Mikheev AM, Mikheeva SA, Maxwell JP, Rivo JV, Rostomily
R, Swisshelm K and Zarbl H: Dickkopf-1 mediated tumor
suppression in human breast carcinoma cells. Breast Cancer Res
Treat, doi: 10.1007/s10549-007-9867-2.
77 Qin X, Zhang H, Zhou X, Wang C, Zhang X and Ye L:
Proliferation and migration mediated by Dkk-1/Wnt/beta-
catenin cascade in a model of hepatocellular carcinoma cells.
Transl Res 150: 281-294, 2007.
78 Koppen A, Ait-Aissa R, Hopman S, Koster J, Haneveld F,
Versteeg R and Valentijn LJ: Dickkopf-1 is down-regulated by
MYCN and inhibits neuroblastoma cell proliferation. Cancer
Lett 256: 218-228, 2007.
79 Yamabuki T, Takano A, Hayama S, Ishikawa N, Kato T,
Miyamoto M, Ito T, Ito H, Miyagi Y, Nakayama H, Fujita M,
Hosokawa M, Tsuchiya E, Kohno N, Kondo S, NakamuraY and
Daigo Y: Dikkopf-1 as a novel serologic and prognostic
biomarker for lung and esophageal carcinomas. Cancer Res 67:
80 Caricasole A, Copani A, Caraci F, Aronica E, Rozemuller AJ,
Caruso A, Storto M, Gaviraghi G, Terstappen GC and Nicoletti
F: Induction of Dickkopf-1, a negative modulator of the Wnt
Alzheimer’s brain. J Neurosci 24: 6021-6027, 2004.
OA: Wnt signaling stimulatesosteo-
is associated withneuronaldegenerationin
81 Niida A, Hiroko T, Kasai M, Furukawa Y, Nakamura Y, Suzuki
Y, Sugano S and Akiyama T: DKK1, a negative regulator of
Wnt signaling, is a target of the beta-catenin/TCF pathway.
Oncogene 23: 8520-8526, 2004.
82 Gonzalez-Sancho JM, Aguilera O, Garcia JM, Pendas-Franco
N, Pena C, Cal S, Garcia de Herreros A, Bonilla F and Munoz
A: The Wnt antagonist DICKKOPF-1 gene is a downstream
target of beta-catenin/TCF and is downregulated in human
colon cancer. Oncogene 24: 1098-1103, 2005.
83 Chamorro MN, Schwartz DR, Vonica A, Brivanlou AH, Cho
KR and Varmus HE: FGF-20 and DKK1 are transcriptional
targets of beta-catenin and FGF-20 is implicated in cancer and
development. EMBO J 24: 73-84, 2005.
84 Aguilera O, Fraga MF, Ballestar E, Paz MF, Herranz M, Espada
J, Garcia JM, Munoz A, Esteller M and Gonzalez-Sancho JM:
Epigenetic inactivation of the Wnt antagonist DICKKOPF-1
(DKK-1) gene in human colorectal cancer. Oncogene 25: 4116-
85 Lee AY, He B, You L, Xu Z, Mazieres J, Reguart N, Mikami I,
Batra S and Jablons DM: Dickkopf-1 antagonizes Wnt signaling
independent of beta-catenin in human mesothelioma. Biochem
Biophys Res Commun 323: 1246-1250, 2004.
86 Mikheev AM, Mikheeva SA, Liu B, Cohen P and Zarbl H: A
functional genomics approach for the identification of putative
tumor suppressor genes: Dickkopf-1 as suppressor of HeLa cell
transformation. Carcinogenesis 25: 47-59, 2004.
87 Aguilera O, Pena C, Garcia JM, Larriba MJ, Ordonez-Moran P,
Navarro D, Barbachano A, Lopez de Silanes I, Ballestar E,
Fraga MF, Esteller M, Gamallo C, Bonilla F, Gonzalez-Sancho
JM and Munoz A: The Wnt antagonist DICKKOPF-1 gene is
induced by 1alpha,25-dihydroxyvitamin D3associated with the
differentiation of human colon cancer cells. Carcinogenesis 28:
88 Shabahang M, Buras RR, Davoodi F, Schumaker LM, Nauta RJ
and Evans SR: 1,25-Dihydroxyvitamin D3receptor as a marker
of human colon carcinoma cell line differentiation and growth
inhibition. Cancer Res 53: 3712-3718, 1993.
89 Bazzi H, Fantauzzo KA, Richardson GD, Jahoda CA and
Christiano AM: The Wnt inhibitor, Dickkopf 4, is induced by
morphogenesis. Dev Biol 305: 498-507, 2007.
90 Aung PP, Oue N, MitaniY, Nakayama H,Yoshida K, Noguchi T,
BosserhoffAK andYasui W: Systematic search for gastric cancer-
specific genes based on SAGE data: melanoma inhibitory activity
and matrix metalloproteinase-10 are novel prognostic factors in
patients with gastric cancer. Oncogene 25: 2546-2557, 2006.
91 Ali I, Rafiee P, Hogan WJ, Jacob HJ, Komorowski RA, Haasler
GB and Shaker R: Dickkopf homologs in squamous mucosa of
esophagitis patients are overexpressed compared with Barrett’s
patients and healthy controls. Am J Gastroenterol 101: 1437-
92 Wong YF, Cheung TH, Lo KW, Yim SF, Siu NS, Chan SC, Ho
TW, Wong KW,Yu MY, Wang VW, Li C, Gardner GJ, Bonome
T, Johnson WB, Smith DI, Chung TK and Birrer MJ:
Identification of molecular markers and signaling pathway in
endometrial cancer in Hong Kong Chinese women by genome-
wide gene expression profiling. Oncogene 26: 1971-1982, 2007.
93 You J, Nguyen AV, Albers CG, Lin F and Holcombe RF: Wnt
pathway-related gene expression in inflammatory bowel
disease. Dig Dis Sci 53: 1013-1019, 2008.
Wnt signalingduring ectodermalappendage
ANTICANCER RESEARCH 28: 2613-2624 (2008)
94. Xi Y, Formentini A, Nakajima G, Kornmann M and Ju J: Download full-text
Validation of biomarkers associated with 5-fluorouracil and
thymidylate synthase in colorectal cancer. Oncol Rep 19: 257-
95 Pendas-Franco N, Garcia JM, Pena C, Valle N, Palmer HG,
Heinaniemi M, Carlberg C, Jimenez B, Bonilla F, Munoz A and
Gonzalez-Sancho JM: DICKKOPF-4 is induced by TCF/beta-
catenin and upregulated in human colon cancer, promotes
tumour cell invasion and angiogenesis and is repressed by
1alpha,25-dihydroxyvitamin D3. Oncogene, doi: 10.1038/
96 Proitsi P, Li T, Hamilton G, Di Forti M, Collier D, Killick R,
Chen R, Sham P, Murray R, Powell J and Lovestone S: Positional
pathway screen of wnt signaling genes in schizophrenia:
association with DKK4. Biol Psychiatry 63: 13-16, 2008.
97 Sato H, Suzuki H, Toyota M, Nojima M, Maruyama R, Sasaki
S, Takagi H, Sogabe Y, Sasaki Y, Idogawa M, Sonoda T, Mori
M, Imai K, Tokino T and Shinomura Y: Frequent epigenetic
gastrointestinal tumors. Carcinogenesis 28: 2459-2466, 2007.
98 Abu-Jawdeh G, Comella N, Tomita Y, Brown LF, Tognazzi K,
Sokol SY and Kocher O: Differential expression of frpHE: a
novel human stromal protein of the secreted frizzled gene
family, during the endometrial cycle and malignancy. Lab Invest
79: 439-447, 1999.
ofDICKKOPFfamily genesin human
99 Roth W, Wild-Bode C, Platten M, Grimmel C, Melkonyan HS,
Dichgans J and Weller M: Secreted Frizzled-related proteins
inhibit motility and promote growth of human malignant glioma
cells. Oncogene 19: 4210-4220, 2000.
100 Dufourcq P, Couffinhal T, Ezan J, Barandon L, Moreau C, Daret
D and Duplaa C: FrzA, a secreted frizzled related protein,
induced angiogenic response. Circulation 106: 3097-3103, 2002.
101 Untergasser G, Steurer M, Zimmermann M, Hermann M, Kern
J, Amberger A, Gastl G and Gunsilius E: The Dickkopf-
homolog 3 is expressed in tumor endothelial cells and supports
capillary formation. Int J Cancer 122: 1539-1547, 2008.
102 Zitt M, Untergasser G, Amberger A, Moser P, Stadlmann S,
Muller HM, Muhlmann G, Perathoner A, Margreiter R,
Gunsilius E and Ofner D: Dickkopf-3 as a new potential marker
for neoangiogenesis in colorectal cancer: expression in cancer
tissue and adjacent non-cancerous tissue. Dis Markers 24: 101-
Received May 16, 2008
Revised July 15, 2008
Accepted August 11, 2008
Pendás-Franco et al: Vitamin D Antagonizes Wnt Signalling (Review)