Clinical & Experimental Metastasis

https://doi.org/10.1007/s10585-018-9937-3

RESEARCH PAPER

Dickkopf-1 (Dkk1) protein expression in breast cancer with special

reference to bone metastases
Mariz Kasoha1   · Rainer M. Bohle2 · Anita Seibold2 · Christoph Gerlinger1 · Ingolf Juhasz‑Böss1 ·
Erich‑Franz Solomayer1

Received: 11 May 2018 / Accepted: 18 September 2018

© Springer Nature B.V. 2018

Abstract
Dysregulation of the Wnt inhibitor dickkopf-1 protein (Dkk1) has been reported in a variety of cancers. In addition, it has
been linked to the progression of malignant bone disease by impairing osteoblast activity. This study investigated serum-
and tissue levels of Dkk1 in breast cancer patients with- or without bone metastases. Serum Dkk1 levels were measured
by ELISA in 89 breast cancer patients and 86 healthy women. Tissue levels of Dkk1 and β-catenin, a major downstream
component of Wnt transduction pathway, were tested with immunohistochemical staining in 143 different tissues, including
adjacent non-tumoral breast tissues, primary breast tumours, lymph nodes metastases, and bone metastases. Serum levels
of Dkk1 were significantly increased in breast cancer patients without metastases compared with healthy controls and even
more increased in patients with bone metastases. Tissue expression of Dkk1 was positive in 70% of tested primary breast
cancer tissues and demonstrated significant correlation with histological type and PR status. Less frequent expression of
Dkk1 was found in lymph nodes metastases and bone metastases compared with adjacent non-tumoral breast tissues and
primary breast tumours. Tissue expression of β-catenin was positive in the vast majority of all tested tissue types indicating
activated Wnt/β-catenin signalling. Our results suggested that Wnt/β-catenin signalling in breast tumours and their second-
ary lymph nodes- and bone metastases is dysregulated and this could be related to aberrant Dkk1 expression levels. Hence,
Dkk1 protein might provide insights into the continued development of novel comprehensive and therapeutic strategies for
breast cancer and its bone metastases.

Keywords  Breast cancer · Dickkopf-1 (Dkk1) · Bone metastases · β-Catenin · Target therapy

Introduction
Wnt signalling is a fundamental transduction pathway that
mediates a wide variety of processes during embryonic
development and adult tissue homeostasis [1]. Components
of Wnt signalling have been classified according to their
activity in cell lines or in vivo assays in two functional cat-
egories, noncanonical and canonical. Noncanonical Wnts
activate Wnt signalling during different development pro-
cesses including Wnt/Ca2+ and Wnt/planar cell polarity
* Mariz Kasoha
mariz.kasoha@uks.eu
1
Department of Gynecology, Obstetrics and Reproductive
Medicine, University Medical School of Saarland,
D‑66421 Homburg, Germany
2
Institute of General and Special Pathology, University
Medical School of Saarland, D‑66421 Homburg, Germany
pathway (PCP) pathways [2, 3]. On the other hand, canoni-
cal Wnts stabilize β-catenin by inhibiting its phosphoryla-
tion and its consequent proteasomal degradation. Stabilized
β-catenin accumulates in the cytoplasm and thereby trans-
located into the nucleus resulting in activation of transcrip-
tion of T cell factor/lymphoid enhancing factor (TCF/LEF)
and enhancing expression of their target genes [4]. Wnt/β-
catenin pathway is regulated by two different classes of
secreted proteins including secreted frizzled related protein
(sFRP) family and Wnt inhibitory protein (WIF) family that
are known to antagonize both canonical and noncanonical
pathways by directly binding Wnts proteins [5] and dickkopf
(Dkk) family and the sclerostin (SOST) family that bind to
the LRP5/LRP6 component of the Wnt receptor complex,
inhibiting only canonical pathway [6]. Wnt/β-catenin signal-
ling is illustrated in Fig. 1 [7].
Breast cancer (BC) is a multifactorial disease in which
diverse carcinogenetic process at genetic and epigenetic

13
Vol.:(0123456789)

LRP5/LRP6
FZD
Wnt
Kremen
Dvl
Axin
GSK3β CK1
APC
β-Cat β-Cat
β-Cat
TCF Genes
β-Cat
LEF transcription
Nucleus
On state
Fig. 1  Canonical Wnt/β-catenin signalling pathway. In the absence of
Wnts, glycogen synthase kinase (GSK3β), axin, adenomatous polypo-
sis coli (APC), and casein kinase I (CKI) form the β-catenin destruc-
tion complex, which phosphorylates β-catenin, enabling it to be
degraded by proteasomes. In the ʻoff stateʼ, extracellular Wnt ligands
can interact with various secreted antagonists and cells maintain low
cytoplasmic and nuclear levels of β-catenin. When Wnt concentra-
tions exceed the buffering capacity of Wnt inhibitors, Wnt signalling
is initiated by binding of the Wnt protein to one of the 10 members of
levels are involved. Wnt/β-catenin pathway has been
reported to play an important regulatory role during the
development of human breast carcinomas and tumour metas-
tasis via different mechanisms. Wnt/β-catenin signalling was
found to regulate the self-renewal and migration of cancer
stem cells (CSCs) in BC, thereby promoting tumour growth
and metastasis [8]. The phosphoinositide 3-kinase (PI3K)
pathway is one of the most commonly activated signalling
pathways in human cancer. Upregulation of Wnt/β-catenin
has been shown to be a mechanism of resistance to PI3K
inhibitors, and the use of β-catenin inhibitors may sensitize
PIK3CA mutant BC patients to PI3K inhibition [9]. In addi-
tion, a significant association between increased β-catenin
expression and poor overall survival was documented in
triple negative BC patients [10]. Other studies have sug-
gested that in BC, dysregulation of this signalling pathway
may be caused by disruption of its negative regulators. Con-
sistent with this notion, expression of the sFRP1 is found
to be significantly down-regulated in many breast cancers
and is associated with poor survival and a poor therapeutic
response [11]. Moreover, dickkopf-1 protein (Dkk1), a major
13
Clinical & Experimental Metastasis
SFRP
Wnt WIF
LRP5/LRP6 Wnt
FZD
Dkk1
Kremen
Dvl
Axin
GSK3β CK1
APC
P
β-Cat Proteosome
P
TCF Gene
LEF transcription
Nucleus
Off state
the frizzled (FZD) receptor family; Wnt-FZD then binds low-density
lipoprotein-related protein-5 (LRP5) or LRP6. The resultant com-
plex activates dishevelled (Dvl), a protein that draws axin away from
the destruction complex and antagonizes its ability to phosphoryl-
ate β-catenin, thereby preventing β-catenin destruction. Thus the ʻon
stateʼ involves increasing the post-translational stability of β-catenin.
As β-catenin levels rise, the protein accumulates and translocates to
the nucleus, where it interacts with TCF/LEF transcription factors
and enhances expression of their target genes
member of the Dkk family proteins, has been linked by dif-
ferent correlative and functional studies to the pathology
of BC [12, 13]. Thus, understanding the interplay of Wnt
agonists and antagonists is critical to derive new approaches
for BC management.
BC continues to be the most frequently diagnosed form
of cancer and the leading cause of cancer deaths among
females globally [14]. Metastatic disease, either at the time
of first diagnosis in patients with advanced disease or at
disease recurring after months or even years, is responsible
for the vast majority of cancer patient deaths [15]. Bone is
a frequent site of metastatic recurrence, arising in > 70% of
patients with stage IV disease [16] and metastatic sequelae
account for approximately two-thirds of the costs associ-
ated with disease treatment [17]. Molecular and cellular
mechanisms involved in skeletal metastases secondary to
BC have been well explained and attributed to different
mechanical factors and pathophysiologic process between
host and tumour cells. Aberrant activation of Wnt/β-catenin
signalling has been showed to be involved in the meta-
static process of BC to the bone [18, 19]. Subsequently, the
Clinical & Experimental Metastasis

plausible enrolment of the Wnt/β-catenin signalling negative
regulators, mainly Dkk1, in the pathogenesis of BC-induced
bone metastasis was investigated and established by some
studies [7]. Dkk1 has been particularly described to play an
important role in bone cancer progression and metastasis
development in multiple myeloma (MM) [20, 21]. Results
of these studies clearly provided the beneficial effect of anti-
Dkk1 treatment for MM and supported its use in clinical
trials [22]. Targeted therapy is a new approach to facilitate
the treatment of cancer and has yielded promising results
in clinical practice such as using Trastuzumab in the treat-
ment of patients with HER2-positive BC [23]. Therefore, we
sought in our study to further investigate Dkk1 involvement
in BC with special reference to bone metastases.
Materials and methods
Patients and specimens
Our study included a cohort of 189 women consisting of
86 healthy women and 103 women with histologically
confirmed BC. All BC cases were diagnosed according
to the WHO classification in effect at the time of initial
diagnosis. For each patient, demographic, clinical, patho-
logical and follow-up data were retrieved from patient’s
records and pathology records. Details on the case series
are given in Fig. 2.
Serum samples were available from 89 BC cases of
which 69 samples were taken from patients with primary
BC at the time of diagnosis, prior to surgery and phar-
macological treatment of the disease. The other 20 sam-
ples were taken from patients who had bone metastases at
different time from the first diagnosis. These included 3
samples that were obtained from patients who had bone
metastases at the time of the first diagnosis, 8 samples
were obtained from patients that were treated with sur-
gery followed with chemotherapy and/or radiation in addi-
tion to endocrine therapy at the time of bone metastases
involvement, and 9 samples were taken from patients who
had already bone metastases and were treated as the later
in addition to antiresorptive therapy (denosumab and/or
bisphosphonate) (Fig. 2). Serum samples were also col-
lected from age matched 86 healthy women [mean age
(range) 58 (34–88) years vs 59 (35–83) years of controls
and patients respectively]. These healthy control cohorts
were recruited from those who had undergone medical
screening at our department and who had no history of

All study cases: 189 subjects

Serum (175 samples) Paraffin tissue (143 samples)

86 Healthy women IHC staining of Dkk1 was applied on:

77 primary breast tumours
89 BC patients 41 normal breast tissues
• Serum concentrations of Dkk1
were tested in all samples. 14 malignant lymph nodes tissues
• Serum concentrations of 25- 11 malignant bone tissues
OH-D and ALP were tested in
27 samples.

IHC staining of β-catenin was applied on:

69 patients with primary BC (M0) 20 BC patients with bone 66 primary breast tumours
(Blood sampling was at the time of metastases 25 normal breast tissues
the first diagnosis) 8 malignant lymph nodes tissues
• Serum concentrations of Dkk1, 25-OH-D, 10 malignant bone tissues
• Serum concentrations of Dkk1 were tested and ALP were tested in all samples.
in all samples.
• Serum concentrations of 25-OH-D and
ALP were tested in 68 samples.

3 patients with primary
metastasized BC (M1).
(Blood sampling was
at the time of the first
diagnosis).
11 BC patients who were treated with
surgery followed with chemotherapy
and/or endocrine therapy and had later
bone metastases.
(Blood sampling was at the time of
skeletal metastases involvement).
9 BC patients who were treated with
surgery followed with chemotherapy
and/or endocrine therapy and had later
bone metastases.
(Blood sampling was after application of
antiresorptive therapy).

Fig. 2  Details on the study case series

13

cancer or bone disease. All serum samples were frozen in
aliquots at − 80 °C until analysed.
143 Representative formalin-fixed paraffin-embedded
blocks were retrieved from the archives of the institute
of pathology (Saarland University Medical Center) and
consisted of 41 normal breast tissues, 77 primary breast
tumours, 14 lymph nodes metastases, and 11 bone metasta-
ses. 3 Consecutive sections of 4-µm thickness were prepared
from the blocks for hematoxylin-eosin (HE) staining and for
Dkk1- and β-catenin immunohistochemistry (IHC) analysis.
All HE stained sections were tested by a pathologist to con-
firm the histology and to verify tumour content.
Serum markers analysis
Serum concentrations of Dkk1 protein were measured by
enzyme-linked immunosorbent assay (ELISA) using com-
mercially available kit, Human Dkk-1 Quantikine ELISA Kit
(DKK100B) produced at R&D Systems®, according to man-
ufacturer’s instructions. The optical density was measured at
450 nm and referenced to 570 nm on a 96-well microplate
reader (Sunrise-Tecan, Life Science). Dkk-1 concentrations
were obtained with a four-parameter logistic curve fitted
against a standard curve and multiplied by the dilution factor
using Magellan 7.2 Ink Data Analysis Software (Life Sci-
ence-Tecan). All measurements were performed in duplicate.
Serum concentrations of 25-hydroxyvitamin D (25-OH-
D) were measured by chemiluminescent immunoassay
(CLIA) using LIAISON® 25 OH Vitamin D TOTAL Assay
Kit and LIAISON® Analyzer and alkaline phosphatase
(ALP) concentrations were measured by colorimetric assay
using alkaline phosphatase acc. to IFCC Gen.2 Kit and
Roche/Hitachi cobas c System. These two tests were per-
formed in the Central Laboratory of our hospital. Concen-
trations of calcium, tumour marker CA 15–3, and creatinine
were obtained from patient’s records in our department.
Immunohistochemistry (IHC) staining of Dkk1‑
and β‑catenin expression in different tissue types
IHC-staining of β-catenin was performed on an automated
staining instrument (BenchMark XT, Ventana Medical
System, Inc., Tucson, AZ, USA). First of all, sections were
deparaffinized using EZ prep solution (Ventana Medical
Systems, Tucson, AZ) followed by rehydration. Then, heat-
induced antigen retrieval was achieved using CC1 solution
(Ventana Medical Systems, Tucson, AZ). Afterwards, incu-
bation with β-catenin rabbit polyclonal antibody (H-102)
(Santa Cruz Biotechnology), dilution 1:50, was done for
32 min at 36 °C and developed with a detection system with
ultraView™ Universal Alkaline Phosphatase Red Detection
Kit (Code 760-501-Ventana) according to the manufacturer’s
instructions. Finally, section were manually dehydrated in
13
Clinical & Experimental Metastasis
ascending concentrations of ethanol, cleared in xylene, and
then counterstained with hematoxylin.
IHC-staining of Dkk1 was manually performed using
Dako REAL™ Detection System, Alkaline Phosphatase/
RED, Rabbit/Mouse Detection Kit (Code K5005-DAKO)
and Dkk1 rabbit polyclonal antibody (H-120) from Santa
Cruz Biotechnology at a dilution of 1:400. In brief, sec-
tions were deparaffinized by passing the specimens through
xylene for 3 times each for 15 min and rehydrated through
serial dilutions of ethanol (100%, 96% and 70%) each for
5 min. Antigen retrieval that was performed using a retrieval
solution (S1699-DAKO), at a dilution of 1:10, for 20 min at
95 °C. Then, sections were blocked with 3% bovine serum
albumin (BSA fraction V, Sigma-Aldrich) for 30 min at room
temperature. Incubation with the primary antibody was done
for 1 h at 37 °C. Secondary antibody detection and visualiza-
tion were done according to the manufacturer’s instructions
of the used kit. After washing in PBS buffer, slides were
counterstained with hematoxylin for 6 min, raised in water,
dehydrated in ascending concentrations of ethanol followed
by clearance with xylene and cover slipped.
Staining specificity was checked using negative controls
which underwent the entire staining procedure except that
the primary antibody was replaced by phosphate-buffered
saline (PBS). Placenta specimens served as positive control
for Dkk1 staining. Breast cancer tissues with known positive
expression of β-catenin used as positive control for β-catenin
staining.
Assessment of IHC staining
All procedures included in this step of work were performed
by a pathologist unaware of the clinical and pathological
data. All tissue sections were analyzed under Zeiss micro-
scope (Axioskop 40, Carl Zeiss, Germany) and selected pic-
tures were captured with attached digital camera (AxioCam
MRC, Carl Zeiss, Germany) using Axiovision Documenta-
tion Rel.4.8 program.
Using HE stained slides; the percentage of tumour cells
was estimated in each tested slide. This was performed
by marking manually the tumour area within the section.
Then, 5 areas (hot-spots) that enclose the highest number
of tumour cells were identified. The number of tumour- and
normal cells was counted in these selected spots and the
average of the percentage of tumour cells was calculated.
Expression of Dkk1 and β-catenin was quantified accord-
ing to Remmele and Stegner [24]. Staining intensity was
scored semi quantitatively as negative (0), weak (1), moder-
ate (2) or strong (3). Extent of staining was scored as per-
centage of cells stained (0, 0% of cells; 1, < 10% of cells;
2, 10–50% of cells; 3, 51–80% of cells; 4, > 80% of cells).
The final immune reactive score (IRS) was determined by
multiplying the scores of intensity and extent of staining,
Clinical & Experimental Metastasis

ranging from 0 to 12. IRS < 2 was considered as negative Results

staining, 3–4 as weak staining, 6–8 as moderate staining and
9–12 as strong staining.
Statistical analysis
Continuous variables were analysed descriptively using
mean, standard deviation, median and range of the obser-
vations. Categorical variables were analysed descriptively
using absolute and relative frequencies. Times to event vari-
ables were described using Kaplan–Meier curves.
The null hypothesis that there are no differences between
the controls and/or the two patient groups was tested against
its alternative using the nonparametric Kruskal–Wallis-test
for continuous variables, Fisher’s exact test for categorical
variables, and the log-rank test for time to event variables.
Correlations between two continuous variables were evalu-
ated using the non-parametric spearman correlation coef-
ficient and the corresponding test of the null hypothesis that
the correlation coefficient is equal to zero. All statistical tests
were performed at a two-sided comparison-wise significance
level of 5%. As appropriate for exploratory studies no adjust-
ments for multiple testing was performed. Data were ana-
lyzed using SAS version 9.4 (SAS Inc., Durham, NC, USA).
Serum concentrations of Dkk1 are increased in BC
patients
Serum concentrations of Dkk1 were measured in all
obtained serum samples (Fig. 2). Values of 25-OH-D and
ALP were tested in 27 and 88 cases of healthy women and
BC patients respectively. Serum values of tumour marker
CA 15–3, calcium, and creatinine-GFR were available only
for some study cases in patient’s records. All obtained data
and results are shown in Table 1.
Serum concentrations of Dkk1 in primary BC patients
[2845 pg/mL (1780–8198)] were significantly higher than
those in controls [1899 pg/mL (786–5161)] (p < 0.001).
Moreover, patients with bone metastases had signifi-
cantly higher serum concentrations of Dkk1 [3502 pg/mL
(1288–5464)] when compared with healthy controls and
with primary BC patients (p < 0.001and p = 0.001 respec-
tively) (Fig. 3). Using Spearman’s rank correlation test,
serum Dkk1 concentrations in primary BC patients and in
BC patients with bone metastases showed no significant
correlation with age or with any of other serum markers,
including 25-OH-D, ALP, calcium, and creatinine-GFR
(all p ≥ 0.05). Serum levels of creatinine-GFR, as an indica-
tor of kidney function, and calcium, as indicator of bone
turnover, showed no significant differences between patients
with primary BC and patients with BC and bone metas-
tases. In addition, serum levels of 25-OH-D and ALP, as
further indicators of bone turnover, showed no significant

Table 1  Serum markers of all study groups

Serum marker Study group
Control cases BC patients with primary BC patients with bone
tumour metastases

Dkk1 (pg/mL) (n = 86) (n = 69) (n = 20) p < 0.0001*

[1899 (786–5161)] [2845 (1780–8198)] [3502 (1288–5464)]
25-OH-D (ng/mL) (n = 27) (n = 68) (n = 20) NS**
[18.8 (4.0–32.9)] [15.4 (4.0-39.8)] [13.6 (8.3–35.0)]
ALP (U/L) (n = 27) (n = 68) (n = 20) NS**
[65 (36–122)] [66 (36–172)] [75 (35–382)]
Tumour marker CA 15–3 (U/mL) No data (n = 65) (n = 18) p = 0.001*
[17.8 (5.8–76.4)] [64.5 (11.0–615.2)]
Calcium (mmol/L) No data (n = 42) (n = 14) p = 0.048*
[2.4 (2.1–2.7)] [2.5 (2.2–2.8)]
Creatinine-GFR (mL/min) No data (n = 56) (n = 11) NS*
[79.4 (28.9–117.4)] [80.3 (53.9–102.2)]

Results are showed as (number of cases) [median (range)]

NS not significant, n number of tested cases
*p value is tested with Mann–Whitney U-test
**p value is tested with Kruskal–Wallis-H

13

Fig. 3  Serum Dkk1 concentra-
tions in healthy women, in BC 10000
patients with primary tumor,
and in BC patients with bone
metastases
8000
Serum Dkk1 (pg/ml)
6000
4000
2000
Control group
differences between tested cases of healthy controls, patients
with primary BC, and patients with BC and bone metasta-
ses. Moreover, BC patients with bone metastases had sig-
nificantly higher concentrations of tumour marker CA 15–3
compared with primary BC patients. However, there was
no significant association between serum levels of Dkk1
and of tumour marker CA 15–3 within each patients group.
Together, these data indicate that differences in serum Dkk1
levels between different study groups might be related to the
presence of breast tumour with- or without bone metastases.
Within the patients group with bone metastases (Fig. 2),
serum Dkk1 concentrations showed no significant differ-
ence between 10 patients who had metastases only in the
bone [3477 ± 1046 pg/mL] and 10 patients who had metas-
tases in the bone and in other organs [3566 ± 1003 pg/mL]
(p ≥ 0.05). In addition, there was no significant difference
in serum Dkk1 concentrations between patients who had
no antiresorptive therapy [3559 ± 977 pg/mL, N = 11] and
patients who had denosumab or bisphosphonate therapy
[3475 ± 1082 pg/mL; N = 9] (p ≥ 0.05).
The relationships between serum Dkk1 levels and clin-
icopathologic characteristics were analysed in 72 patients
by whom blood sampling was at the time of first diagno-
sis (Fig. 2). Patients’ characteristics are shown in Table 2.
Results showed no significant correlation between serum
Dkk1 levels and different characteristics including age,
menstrual status, histological type, pathologic tumour (pT)
stage, pathologic node (pN) stage, metastatic (pM) stage,
ER status, PR status, HER2 status and Ki-67expression (all
p ≥ 0.05). Next, serum concentrations of Dkk1in patients
13
Clinical & Experimental Metastasis
p<0.0001
p<0.0001 P=0.001
BC patients BC patients
with primary tumor with bone metastases
with M0 status were classified as high or low in relation
to the median value and Kaplan–Meier method for OS and
MFS was applied. Patients were followed for a mean of 66
months (range 5–87 months). Results showed that there was
no significant difference in OS or in MFS between patients
with high concentrations of Dkk1 and patients with low
concentrations [Log Rank (Mantel Cox): p = 0.382 and
p = 0.345 respectively].
IHC staining of Dkk1 protein in tumoral breast
tissues, in adjacent non‑tumoral tissues, in lymph
nodes metastases, and in bone metastases
IHC staining of Dkk1 protein was assessed in 143 tissue
samples (Fig. 2), including 77 primary BC tissues, 41 adja-
cent non-tumoral breast tissues, 14 lymph nodes metastases,
and 11 bone metastases. An example of Dkk1 staining in the
different types of tissues is shown in Fig. 4. In tumoral breast
tissues, Dkk1 showed to be stained in 70% (54/77) of tested
cases and ranged between weak and strong (Table 3). Stain-
ing scores of Dkk1 in breast tumours were near-borderline
significance correlated to its staining scors in the adjacent
non-tumoral tissues in 41 matched cases (Spearman test:
p = 0.050, correlation coefficient = 0.308). In addition, Dkk1
appeared to be stained in lymph nodes metastases as well as
in bone metastases (28% and 50% respectively) and ranged
between weak and moderate (Table 3). A significant correla-
tion between Dkk1 staining scores in 14 primary tumours
and their matched lymph nodes metastases was observed
(Spearman test: p = 0.014, correlation coefficient = 0.627).
Clinical & Experimental Metastasis

Table 2  Characteristics of patients in whom Dkk1 levels were tested (all p ≥ 0.05). Patients’ characteristics are shown in Table 4.
in serum Dkk1 was found to be more stained in 64 patients with
Characteristics Number Percentage (%) invasive ductal BC compared to 12 patients with invasive
lobular BC [IRS median (range): 2, 4 (0–12) vs 2 (0–12)
Case; [age at diagnosis: median (range) years]
respectively; p = 0.046]. In addition, patients with positive
 Control subjects; [58 (34–88) years] 86
PR status (n = 52) showed stronger Dkk1 staining compared
 BC patients; [59 (35–83) years] 72
to patients with negative PR status (n = 25) [IRS median
Menopause
(range): 3, 4 (0–12) vs 3 (0–8) respectively; p = 0.028].
 Control subjects
Then, Dkk1staining levels were classified as negative or pos-
  Premenopausal 34 40
itive according to the IRS value (IRS = 0–2 and IRS = 3–12
  Postmenopausal 52 60
respectively) and Kaplan–Meier method for OS and MFS
 BC patients
was applied. Patients were followed for a mean of 65 months
  Premenopausal 22 31
(range 5–312 months) for OS and for a mean of 60 months
  Postmenopausal 50 69
(range 5–292) for MFS. Results showed that there was no
Histology
significant difference in OS or MFS between patients with
 Invasive ductal 63 88
negative Dkk1 staining and patients with positive Dkk1stain-
 Invasive lobular 9 12
ing [Log Rank (Mantel Cox): p = 0.436 and p = 0.389
T stage: (unknown: 1 case)
respectively]. Moreover, in 55 patients with primary BC,
 T1 44 62
Dkk1 staining level in the primary breast tumours was not
 T2 23 32
correlated with its concentrations in the serum (Spearman
 T3 4 6
test: p = 0.752, correlation coefficient = 0.044).
N stage: (unknown: 1 case)
 N0 47 66
IHC staining of β‑catenin protein in tumoral breast
 N1 19 27
tissues, in adjacent non‑tumoral tissues, in lymph
 N2 5 7
nodes metastases, and in bone metastases
Grading
 G1 6 8
IHC staining of of β-catenin was assessed in 109 tissue sam-
 G2 53 74
ples (Fig. 2), including 66 primary BC tissues, 25 adjacent
 G3 13 18
non-tumoral breast tissues, 8 lymph nodes metastases, and
Receptor status
10 bone metastases. An example of β-catenin staining in
 ER− 13 18
the different types of tissues is shown in Fig. 5. β-Catenin
 ER+ 59 82
appeared to be stained in the vast majority of tested tumoral
 PR− 27 38
tissues including breast tumours (60/66), lymph nodes
 PR+ 45 62
metastases (7/8), and bone metastases (9/10) and ranged
 Her2Neu− 58 81
between weak and strong (Table 5). β-catenin staining in
 Her2Neu+ 14 19
tumoral breast tissues was neither significantly correlated
 Triple negative 10 14
with its staining in the adjacent non-tumoral breast tissues
Ki-67 Index (%): (unknown: 4 cases)
(25 matched pairs) nor with its staining in the other metasta-
 = 5%–10% 39 53
sised tumours including lymph nodes metastases (8 matched
 = 15%–25% 16 24
pairs) and bone metastases (7 matched pairs) (Spearman test:
 ≥ 30% 13 23
both p ≥ 0.05).
M status
IHC staining of β-catenin protein in the primary breast
 M0 69 96
tumours was significantly increased in invasive ductal
 M1 3 4
tumours compared with invasive lobular tumours [IRS
median (range): 3, 8 (0–12) vs 3 (0–12) respectively;
p = 0.006] and in HER2 negative tumours compared with
The relationships between Dkk1-IRS in the primary HER2 positive tumours [IRS median (range): 4, 8 (0–12) vs
BC tissues and clinicopathologic variables were tested and 4 (0–12) respectively; p = 0.002 (Mann–Whitney U-test)].
demonstrated significant correlation only with histological There was no significant relation between β-catenin stain-
type and with PR status but not with other characteristics ing scores in the primary breast tumours and other clinico-
including age, menstrual status, pathologic tumour (pT) pathologic features. Likewise, β-catenin staining levels were
stage, pathologic node (pN) stage, pathologic metastatic classified as negative or positive according to the IRS value
(pM) stage, ER status, HER2 status and Ki-67expression (IRS = 0–2 and IRS = 3–12 respectively) and Kaplan–Meier

13
 Clinical & Experimental Metastasis

Normal breast tissue Breast tumour tissue Lymph node metastases Bone metastases

Fig. 4  IHC staining of Dkk1 in normal breast tissue, in breast tumour tissue, in lymph node metastases, and in bone metastases. A negative
staining (IRS = 0–2). B positive staining (IRS = 4–12)

Table 3  Dkk1 protein Negative Weak Moderate Strong All

expression in the tissues IRS: 0–2 IRS: 3–4 IRS: 6–8 IRS: 9–12

Dkk1 in adjacent non-tumoral tissues 12 (29%) 20 (49%) 9 (22%) 0 (0%) 41 (100%)

Dkk1 in tumoral breast tissues 23 (30%) 37 (48%) 15 (19%) 2 (3%) 77 (100%)
Dkk1 in malignant lymph nodes tissues 10 (72%) 2 (14%) 2 (14%) 0 (0%) 14 (100%)
Dkk1 in malignant bone tissues 5 (46%) 3 (27%) 3 (27%) 0 (0%) 11 (100%)

Data showed as number of cases and percentage

method for OS and MFS was applied, results showed that
there was no significant difference in OS or MFS between
patients with negative β-catenin staining and patients
with positive β-catenin staining [Log Rank (Mantel Cox):
p = 0.308 and p = 0.509 respectively].
The relation between Dkk1‑ and β‑catenin protein
staining levels in different tissue types
Staining levels of Dkk1 and β-Catenin proteins were com-
pared in 66, 25, 8, and 10 matched cases of breast tumours,
adjacent non-tumoral breast tissues, lymph nodes metasta-
ses, and bone metastases respectively. We found that stain-
ing levels of β-catenin in all tissue types were significantly
stronger compared with staining levels of Dkk1 (Table 6).
Then, staining levels of Dkk1 and β-catenin were dichoto-
mized as negative and positive on behalf to the IRS values
(IRS = 0–2 and IRS = 3–12 respectively) and Fisher’s exact
test was applied to test the association between proteins
staining in all tissue groups. Results showed that staining
levels of Dkk1 and β-catenin were associated in the primary
breast tumours (p = 0.043) but not in other types of tested
tissues.
Discussion
Dkk1 functions in cancers have been well revised by sev-
eral authors and showed to represent an attractive target for
oncology across diverse fields including disease diagno-
sis, prognosis and treatment [25–28]. The role and expres-
sion pattern of Dkk1 vary according to the location of the
cancer. While, Dkk1 found to function as a tumour sup-
pressor gene in colon cancer [29], its overexpression has
been reported to be significantly associated with tumour
progression and decreased survival in lung cancer, hepa-
tocellular carcinoma, and glioma [30–32]. In this study
we reported that serum concentrations of Dkk1 were sig-
nificantly increased in BC patients compared with healthy
controls. This is in agreement with the results from other
studies [13, 33]. We observed no relationship between
serum concentrations of Dkk1 and different clinicopatho-
logic features. In addition, serum Dkk1 concentrations
had no prognostic significance in our study collective.
These findings are in contradiction with those reported
by Zhou et al. [13] who showed that high serum levels
of Dkk1 were significantly correlated with TNM stage,

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Clinical & Experimental Metastasis

Table 4  Characteristics of patients in whom Dkk1 levels were tested primary tumours from patients with axillary lymph node
in tumoral breast tissues invasion. Sato et al. [38] demonstrated elevated levels of
Characteristics Number Percentage (%) Dkk1 in four out of six human breast tumours and in 6 of
14 breast cancer cell lines and found that 65.1% of BC
Age at diagnosis 77 100
patients involved in their study had positive Dkk1 serum.
Median (range) years: 57 (35–86) years
Moreover, increased concentrations of Dkk1 were con-
Premenopausal 21 27
firmed in hormone-resistant BC cell lines, and were asso-
Postmenopausal 56 73
ciated with poor outcome in patients with triple negative
Histology: (unknown: 1 case)
cancers [11]. These paradoxical findings could be due to
 Invasive ductal 64 84
different tumour properties such as tissue origin (epithelial
 Invasive lobular 12 16
or mesenchymal), tumour stage, and tumour histotype.
T Stage: (unknown: 2 cases)
Metastatic bone is a long-term complication of BC
 T1 37 49
and skeletal metastases are a major problem in the man-
 T2 29 39
agement of advanced disease. The influence of Wnts and
 T3 9 12
Wnt inhibitors on breast tumour biology has been extended
N stage: (unknown: 3 cases)
well beyond the bone microenvironment. Osteoblasts are
 N0 41 55
the supreme cellular targets of the canonical Wnt signal-
 N1 20 27
ling and osteoblast-selective deficiency of β-catenin affects
 N2 13 18
bone resorption as well as bone formation [39, 40]. Dkk1,
Grading: (unknown: 1 case)
as an inhibitor of Wnt/β-catenin pathway, has been found
 G1 4 6
to act as a stimulator of osteoclast activity and inhibitor of
 G2 52 68
osteoblast formation and differentiation, thereby counteract-
 G3 20 26
ing Wnt-mediated effects on the bone [41, 42]. In addition,
Receptor status
Dkk1-mediated inhibition of Wnt signalling was shown to
 ER− 12 16
shift OPG:RANKL ratio in favour of bone resorption via
 ER+ 65 84
limiting OPG expression [43]. Thus, we further tested serum
 PR− 25 33
levels of Dkk1 in BC patients with bone metastases. Our
 PR+ 52 67
results showed that concentrations of serum Dkk1 were sig-
 Her2Neu− 67 87
nificantly increased in patients who had bone metastases
 Her2Neu+ 10 13
secondary to BC compared with healthy controls and with
 Triple negative 10 13
patients without metastases. Our data are in agreement with
Ki-67 Index (%): (unknown: 6 cases)
the observations of Voorzanger-Rousselot et al. [44, 45]
 =5%-10% 37 52
who demonstrated that median Dkk1serum levels in patients
 =15%–25% 18 25
with BC and bone metastases were significantly higher than
 ≥30% 16 23
its levels in patients in complete remission and in healthy
M status
women. While 19 patients with metastatic BC who were
 M0 67 87
on stable antineoplastic therapy and did not receive previ-
 M1 10 13
ous treatment with bisphosphonates were included in the
later study, our group of BC patients with bone metasta-
ses consisted of 20 patients of which 9 cases had previous
tumour grade, lymph node metastasis, HER2 expression, denosumab or bisphosphonate therapy. We observed no sig-
and shorter OS and RFS. This discrepancy between find- nificant difference in serum Dkk1 concentrations between
ings could be related to the differences between patient’s patients treated with antiresorptive drugs and patients who
characteristics including number of tested cases and treat- had no treatment. In addition, each subgroup of patients with
ment with radiation and/or chemotherapy. The function metastatic BC (treated or not treated) showed separately
of Dkk1 within the context of BC pathogenesis remains significant increment in serum Dkk1 concentrations when
inconclusive. It has been shown that Dkk1 acts as a puta- compared with those in patients without metastases and in
tive tumour suppressor in BC cells either via Wnt signal- healthy controls (data are not shown). There is little evidence
ling suppression [34, 35] or via mechanisms independent available about how serum Dkk1 levels can be affected by
of β-catenin-dependent transcription [36]. On the other antiresorptive drugs used in BC therapy. Rachner and co-
hand, Forget et al. [37] showed that in breast tumours from workers [46] showed that Dkk1 serum levels were decreased
women with a family history of BC, Dkk1 was preferen- by 60% comparing with baseline values in ER-negative and
tially expressed in ER and PR-negative tumours and in non-metastatic BC patients who received zoledronic acid

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 Clinical & Experimental Metastasis

Normal breast tissue Breast tumour tissue Lymph node metastases Bone metastases

Fig. 5  IHC staining of β-catenin in normal breast tissue, in breast tumour tissue, in lymph node metastases, and in bone metastases. A negative
staining (IRS = 0–2). B positive staining (IRS = 3–12)

Table 5  β-catenin protein Negative Weak Moderate Strong All

expression in the tissues IRS: 0–2 IRS: 3–4 IRS: 6–8 IRS: 9–12

β-Catenin in adjacent non-tumoral tissues 1 (4%) 7 (28%) 13 (52%) 4 (16%) 25 (100%)

β-Catenin in tumoral breast tissues 6 (9%) 9 (14%) 30 (45%) 21 (32%) 66 (100%)
β-Catenin in malignant lymph nodes tissues 1 (13%) 1 (13%) 5 (62%) 1 (12%) 8 (100%)
β-Catenin in malignant bone tissues 1 (10%) 1 (10%) 2 (20%) 6 (60%) 10 (100%)

A negative staining (IRS = 0–2), b positive staining (IRS = 4–12)

Data showed as number of cases and percentage

Table 6  Dkk1- and β-catenin Tissue type Number of Dkk1-IRS β-catenin-IRS p

protein expression in different matched cases Median (range) Median (range)
tissue types
Adjacent non-tumoral tissues 25 4 (0–8) 8 (2–12) 0.001
Tumoral breast tissues 66 4 (0–8) 8 (0–12) 0.001
Malignant lymph nodes tissues 8 2 (0–8) 8 (1–12) 0.032
Malignant bone tissues 10 2.5 (0–8) 10.5 (0–12) 0.004

IRS data showed as median and range

for 12 months whereas placebo patients showed an increase
of 44%. A more recent study from this research group dem-
onstrated that Dkk1 levels in women with primary ER-posi-
tive BC were reduced by 35% after tamoxifen treatment and
remained unaltered after aromatase inhibitors treatment [47].
One limitation of our study is the absence of baseline Dkk1
values for patients group of metastasised BC.
The role of Dkk1 in promoting osteolytic metastases has
been mainly demonstrated by studies of multiple myloma
(MM)-associated bone disease [48, 49]. In BC studies, Dkk1
was found to be expressed by osteolytic BC cell lines but
not by osteoblastic lines and mice that were inoculated with
MDA-231-BO cells, a subpopulation of BC-MDA-MB-231
cells that metastasize exclusively in bone, had developed
skeletal lesions and had six-fold higher bone marrow levels
of Dkk1 compared with control non-inoculated mice [45].
In addition, Dkk1 expression and activated Wnt/β-catenin
signalling were increased in MDA-231-BO cells compared
with MDA-MB-231 cells and these variances were asso-
ciated with decreased osteoblast differentiation and OPG
expression which were reversed upon treatment with a spe-
cific anti-Dkk1 antibody [50]. All in all, these statements
support the active role of Dkk1 in the pathogenesis of BC
skeletal metastases.

13
Clinical & Experimental Metastasis
A further observation from our study demonstrated no
significant differences in serum concentrations of Dkk1
between patients who had metastases only in bone and
patients who had metastases in bone and in other organs.
Unfortunately, there is no previous data concerning this issue
to be compared with ours. However, Voorzanger-Rousselot
et al. [45] showed that Dkk1 levels in serum of BC patients
with bone metastases were significantly higher compared
to them in BC patients with metastases at non-bone sites.
Moreover, a very recent study of Zhuang et al. [51] reported
that Dkk1 was significantly downregulated in cells that tend
to metastasized to the lung but significantly upregulated in
those favouring bone metastases. In addition, compared to
non-metastatic BC patients, serum levels of Dkk1 increased
significantly in patients with bone metastases and decreased
in patients with lung metastases suggesting a dichotomous
role of Dkk1 in lung and bone metastases.
In order to further analyse the plausible related molecular
mechanisms of Dkk1 in BC, we have examined IHC staining
of Dkk1and β-catenin proteins. Results showed that Dkk1
was detected in 70% of tested tumoral breast tissues, una-
like serum where it was detectable in all tested samples.
In matched serum-tissue cases, levels of Dkk1showed no
significant correlation suggesting that serum levels could
not be used to predict tissue levels. In furtherance of inves-
tigating whether Dkk1 levels are dysregulated in tumoral
breast tissues, we analysed its staining pattern in adjacent
non-tumoral tissues and found that it was stained with the
similar frequency and strength as in tumoral breast tissues.
Dkk1 staining levels in matched normal-tumoral tissues
were significantly and positively correlated. These findings
suggest that Dkk1 levels might be dysregulated in non-
affected cells as well as in tumoral cells in the cancerous
breast. Another limitation of our study is the absence of
normal breast tissues from completely healthy women to
better scrutinize the differences in Dkk1 levels among dif-
ferent groups. We observed that levels of Dkk1 in tumoral
tissues increased significantly in PR-positive patients and in
invasive ductal tumours compared with PR-negative patients
and invasive lobular tumours respectively. This is in agree-
ment with Tulac et al. [52] observations showing that Dkk1
is regulated by progesterone in normal endometrial stroma
cells. Similar to serum levels of Dkk1, its tissue levels had
no prognostic significance in our study cohort. Regarding
the analysis of β-catenin staining, we found that this pro-
tein was strongly stained in the vast majority of adjacent
non-tumoral breast tissues as well as tumoral breast tissues
(96% and 91% respectively). Increased staining scores were
notified in invasive ductal tumours and in HER2-negative
tumours compared with invasive lobular tumours and HER2-
positive tumours respectively. No significant correlation was
found with other pathologic parameters or with OS or MFS.
Interestingly, in tumoral breast tissues, staining scores of
β-catenin showed to be positively correlated with staining
scores of Dkk1. As Dkk1 is a Wnt target gene, this eleva-
tion may actually indicate the activation of pathway activity.
Activated β-catenin-dependent Wnt signalling can result in
the up-regulation of Dkk1, potentially as a negative feedback
mechanism under physiological conditions [53]. In tumours,
which stain for Dkk1 and β-catenin, the negative feedback
may have been disrupted by, for example, stabilizing muta-
tions in β-catenin that would render the inhibitory activity of
Dkk1 inoperative. Protein levels of both β-catenin and Dkk1
in human breast tissues has been detected in limited number
of studies. Our observations are in part in accordance with
other studies. Acar et al. [54] found that the immunohisto-
chemical investigation of Dkk1 showed positive staining in
31%, 17%, and 33% of primary breast tumours, adjacent
non-tumoral tissues and normal breast tissues respectively.
Dkk1 protein levels were not associated with clinicopatho-
logical factors. Furthermore, 61% and 65% of 85 triple nega-
tive breast tumours showed positive immunohistochemical
staining of Dkk1 and β-catenin respectively. Dkk1 levels
correlated significantly with β-catenin levels and elevated
levels of both proteins indicated poor outcome of patients
[11]. In addition, nuclear staining of β-catenin and overex-
pression of its downstream target cyclin D1 has been associ-
ated with a worse prognosis of BC patients with metastases
[55].
In regard to more exploring the role of Dkk1in meta-
static BC, we have investigated protein levels of Dkk1 and
β-catenin in lymph nodes- and bone metastases. β-catenin
staining in both tissue types appeared to be also positive
in the vast majority of tested cases. However, the strongest
staining was observed in the malignant bone tissues. On the
other hand, staining of Dkk1 showed to be less frequent in
lymph node metastases (28%) and bone metastases (54%)
compared to its staining in tumoral breast tissues (65%) and
in adjacent non-tumoral tissues (71%). In addition, staining
scores of Dkk1 in 14 lymph node metastases were also sig-
nificantly correlated to its staining scores in the correspond-
ing primary breast tumours. Further shortcoming in the
present study is lacking immunohistochemical analysis of
Dkk1 and β-catenin in all tissue types from the same patient
in many cases resulting in a small representative dataset.
Taken together, our results highlight the dysregulated lev-
els of Dkk1which in turn might be related to the activated
Wnt/β-catenin signalling in breast tumours and their second-
ary lymph nodes- and bone metastases.
In spite of the paradoxical behaviour of Dkk1 in varied
cancer, it has been viewed by some as a promising target for
cancer therapy. Currently, two different anti-Dkk1 antibod-
ies are being tested clinically for potential use in cancers.
BHQ88 has completed phase 1B in MM and DKN-01which
is being evaluated in phase 1b trials for advanced cholan-
giocarcinoma and relapsed gastric cancer [22, 56]. Primary
13

data from these studies have demonstrated an advantageous
and favourable safety profile. In sum, Dkk1 is considered as
an attractive diagnostic and therapeutic target for oncology
given its potential for broad clinical applicability. However,
we must not over-simplified the complexity of Wnt signal-
ling which involves a large number of ligands and offers
multiple steps that may be considered for clinical interven-
tion. Serum Dkk1 is considered as an advantageous bio-
marker because of its non-invasive, inexpensive, and simple
detection protocol, but the great variability and non-normal
distribution of its levels affected the chances of identifying
a clear cut-off value or significant differences related to the
clinical features of the groups. Furthermore, as a secretory
glycoprotein, a difference in secondary modifications on
it or change in relative abundance of its ligands and their
secondary modifications could impact its activity. There-
fore, particularly in BC, a number of questions concerning
the discrepancy in Dkk1 function as tumour suppressor or
metastasis promotor still exist, further studies to address
these clinical and research claims are warranted.
Compliance with ethical standards  16. Manders K, van de Poll-Franse LV, Creemers GJ et al (2006)
Conflict of interest  The authors declare that they have no conflict of
interest.
Ethical approval  This study was approved by the local Ethic Commit-
tee of the Medical Association of the Saarland (Reference Number:
09/14). All procedures performed in studies involving human partici-
pants were in accordance with the ethical standards of the institutional
and/or national research committee and with the 1964 Helsinki declara-
tion and its later amendments or comparable ethical standards.
Informed consent  Informed consent was obtained from all individual
participants included in the study.
References
1. Clevers H, Loh KM, Nusse R (2014) Stem cell signaling. An
integral program for tissue renewal and regeneration: Wnt signal-
ing and stem cell control. Science. https​://doi.org/10.1126/scien​
ce.12480​12
2. Gao C, Chen YG (2010) Dishevelled: the hub of Wnt signaling.
Cell Signal 22:717–727
3. De A (2011) Wnt/Ca2+ signalling pathway: a brief overview. Acta
Biochim Biophys Sin 43:745–756
4. Clevers H (2006) Wnt/β-catenin signaling in development and
disease. Cell 127:469–480
5. Bovolenta P, Esteve P, Ruiz JM, Cisneros E, Lopez-Rios J (2008)
Beyond Wnt inhibition: new functions of secreted Frizzled-related
proteins in development and disease. J Cell Sci 121:737–746
6. He X, Semenov M, Tamai K et al (2004) LDL receptor-related
proteins 5 and 6 in Wnt/β-catenin signaling: arrows point the way.
Development 131:1663–1677
7. Mariz K, Ingolf JB, Daniel H, Teresa NJ, Erich-Franz S (2015)
The Wnt inhibitor dickkopf-1: a link between breast cancer and
bone metastases. Clin Exp Metastasis 32:857–866
13
Clinical & Experimental Metastasis
8. Gyu-Beom J, Ji-Young K, Sung-Dae C, Ki-Soo P, Ji-Youn J,
Hwa-Yong L, In-Sun H, Jeong-Seok N (2015) Blockade of Wnt/β-
catenin signaling suppresses BC metastasis by inhibiting CSC-like
phenotype. Sci Rep. https​://doi.org/10.1038/srep1​2465
9. Merino VF, Cho S, Liang X, Park S, Jin K, Chen Q, Pan D,
Zahnow CA, Rein AR, Sukumar S (2017) Inhibitors of STAT3,
beta-catenin, and IGF-1R sensitize mouse PIK3CA mutant breast
cancer to PI3K inhibitors. Mol Oncol 11:552–566
10. Shen T, Zhang K, Siegal GP, Wei S (2016) Prognostic value of
E-cadherin and beta-catenin in triple-negative breast cancer. Am
J Clin Pathol 146:603–610
11. Veeck J, Niederacher D, An H, Klopocki E et al (2006) Aber-
rant methylation of the Wnt antagonist SFRP1 in breast cancer is
associated with unfavourable prognosis. Oncogene 25:3479–3488
12. Xu WH, Liu ZB, Yang C et  al (2012) Expression of dick-
kopf-1 and beta-catenin related to the prognosis of breast cancer
patients with triple negative phenotype. PLoS ONE. https​://doi.
org/10.1371/journ​al.pone.00376​24
13. Zhou SJ, Zhou SR, Yang XQ et al (2014) Serum Dickkopf-1
expression level positively correlates with a poor prognosis
in breast cancer. Diagn Pathol. https​://doi.org/10.1186/s1300​
0-014-0161-4
14. Jemal A, Bray F, Center MM et al (2011) Global cancer statistics.
CA Cancer J Clin 61:69–90
15. Weigelt B, Peterse JL, van‘t Veer LJ (2005) Breast cancer metas-
tasis: markers and models. Nat Rev Cancer 5:591–602. 2005
Clinical management of women with metastatic breast cancer: a
descriptive study according to age group. BMC Cancer 6:179
17. Lipton A (2005) Management of bone metastases in breast cancer.
Cur Treat Options Oncol 6:161–171
18. Johnson RW, Merkel AR, Page JM, Ruppender NS, Guelcher SA,
Sterling JA (2014) Wnt signaling induces gene expression of fac-
tors associated with bone destruction in lung and breast cancer.
Clin Exp Metastasis 31:945–959
19. Johnson RW, Mai PN, Susan SP et al (2010) TGF-β promotion of
Gli2-induced expression of parathyroid hormone-related protein,
an important osteolytic factor in bone metastasis, is independent
of canonical hedgehog signaling. Cancer Res 71:822–831
20. Gunn WG, Conley A, Deininger L et  al (2006) A crosstalk
between myeloma cells and marrow stromal cells stimulates pro-
duction of DKK1 and interleukin-6: a potential role in the devel-
opment of lytic bone disease and tumor progression in multiple
myeloma. Stem Cells 24:986–991
21. Heath DJ, Chantry AD, Buckle CH, Coulton L, Shaughnessy JD
Jr, Evans HR, Snowden JA, Stover DR, Vanderkerken K, Croucher
PI (2009) Inhibiting Dickkopf-1 (Dkk1) removes suppression of
bone formation and prevents the development of osteolytic bone
disease in multiple myeloma. J Bone Miner Res 24:425–436
22. Iyer SP, Beck JT, Stewart A et al (2014) A Phase IB multicentre
dose-determination study of BHQ880 in combination with anti-
myeloma therapy and zoledronic acid in patients with relapsed or
refractory multiple myeloma and prior skeletal-related events. Br
J Haematol 167:366–375
23. Durkee BY, Qian Y, Pollom EL, King MT, Dudley SA, Shaffer
JL, Chang DT, Gibbs IC, Goldhaber-Fiebert JD, Horst KC (2016)
Cost-effectiveness of pertuzumab in human epidermal growth
factor receptor 2-positive metastatic breast cancer. J Clin Oncol
34:902–909
24. Remmele W, Stegner HE (1987) Recommendation for uniform
definition of an immunoreactive score (IRS) for immunohisto-
chemical estrogen receptor detection (ER-ICA) in breast cancer
tissue. Pathologe 8:138–140
25. Shao YC, Wei Y, Liu JF, Xu XY (2017) The role of Dickkopf
family in cancers: from Bench to Bedside. Am J Cancer Res
7:1754–1768
Clinical & Experimental Metastasis
26. Kagey MH, He X (2017) Rationale for targeting the Wnt signal-
ling modulator Dickkopf-1 for oncology. Br J Pharmacol. https​://
doi.org/10.1111/bph.13894​
27. Mazon M, Masi D, Carreau M, Modulating (2016) Dickkopf-1: a
strategy to monitor or treat cancer? Cancers (Basel). https​://doi.
org/10.3390/cance​rs807​0062
28. Liu Y, Tang W, Xie L et al (2014) Prognostic significance of dick-
kopf-1 overexpression in solid tumors: a meta-analysis. Tumour
Biol 35:3145–3154
29. González-Sancho JM, Aguilera O, García JM, Pendás-Franco N,
Peña C, Cal S, García de Herreros A, Bonilla F, Muñoz A (2005)
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
30. Sheng SL, Huang G, Yu B, Qin WX (2009) Clinical significance
and prognostic value of serum Dickkopf-1 concentrations in
patients with lung cancer. Clin Chem 55:1656–1664
31. Fouad YM, Mohamed HI, Kamal EM, Rasek MA (2016) Clini-
cal significance and diagnostic value of serum dickkopf-1 in
patients with hepatocellular carcinoma. Scand J Gastroenterol
51:1133–1137
32. Zhou Y, Liu F, Xu Q, Wang X (2010) Analysis of the expression
profile of Dickkopf-1 gene in human glioma and the association
with tumor malignancy. J Exp Clin Cancer Res 28:129–138
33. Liu JT, Guo WB, Sun JY (2017) Serum Dickkopf-1 acts as a new
biomarker in human breast cancer. Minerva Med 108:334–340
34. Zhou XL, Qin XR, Zhang XD et al (2010) Downregulation of
Dickkopf-1 is responsible for high proliferation of breast cancer
cells via losing control of Wnt/beta-catenin signaling. Acta Phar-
macol Sin 31:202–210
35. Qiao L, Xu ZL, Zhao TJ, Ye LH, Zhang XD (2008) Dkk-1 secreted
by mesenchymal stem cells inhibits growth of breast cancer cells
via depression of Wnt signalling. Cancer Lett 269:67–77
36. Mikheey AM, Mikheeva SA, Maxwell JP et al (2008) Dickkopf-1
mediated tumor suppression in human breast carcinoma cells.
Breast Cancer Res Treat 112:263–273
37. Forget MA, Turcotte S, Beauseigle D et al (2007) The Wnt path-
way regulator DKK1 is preferentially expressed in hormone-
resistant breast tumours and in some common cancer types. Br J
Cancer 96:646–653
38. Sato N, Yamabuki T, Takano A et al (2010) Wnt inhibitor Dick-
kopf-1 as a target for passive cancer immunotherapy. Cancer Res
70:5326–5336
39. Kato M, Patel MS, Levasseur R et al (2002) 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
40. Glass DA 2nd, Bialek P, Ahn JD et al (2005) Canonical Wnt sign-
aling in differentiated osteoblasts controls osteoclast differentia-
tion. Dev Cell 8:751–764
41. Christodoulides C, Scarda A, Granzotto M et al (2006) WNT10B
mutations in human obesity. Diabetologia 49:678–684
42. Cheng SL, Shao JS, Cai J, Sierra OL, Towler DA (2008) Msx2
exerts bone anabolism via canonical Wnt signaling. J Biol Chem
283:20505–20522
43. Diarra D, Stolina M, Polzer K et al (2007) Dickkopf-1 is a master
regulator of joint remodeling. Nat Med 13:156–163
44. Voorzanger-Rousselot N, Journe F, Doriath V et al (2009) Assess-
ment of circulating Dickkopf-1 with a new two-site immunoas-
say in healthy subjects and women with breast cancer and bone
metastases. Calcif Tissue Int 84:348–354
45. Voorzanger-Rousselot N, Goehrig D, Journe F et  al (2007)
Increased Dickkopf-1 expression in breast cancer bone metasta-
ses. Br J Cancer 97:964–970
46. Rachner TD, Göbel A, Thiele S (2014) Dickkopf-1 is regulated
by the mevalonate pathway in breast cancer. Breast Cancer Res.
https​://doi.org/10.1186/bcr36​16
47. Göbel A, Kuhlmann JD, Link T, Wimberger P, Browne AJ, Rauner
M, Hofbauer LC, Rachner TD (2017) Adjuvant tamoxifen but not
aromatase inhibitor therapy decreases serum levels of the Wnt
inhibitor dickkopf-1 while not affecting sclerostin in breast cancer
patients. Breast Cancer Res Treat 164:737–743
48. Hideshima T, Mitsiades C, Tonon G et al (2007) Understanding
multiple myloma pathogenesis in the bone marrow to identify new
therapeutics targets. Nat Rev Cancer 7:585–595
49. Qiang YW, Chen Y, Stephens O et al (2008) Myeloma derived
Dickkopf-1 disrupts Wnt-regulated osteoprotegrin and RANKL
production by osteoblasts: a potential mechanism underlying
osteolytic bone lesions in multiple myeloma. Blood 112:196–207
50. Bu G, Lu W, Liu CC, Selander K, Yoneda T, Hall C, Keller ET, Li
Y (2008) Breast cancer-derived Dickkopf1 inhibits osteoblast dif-
ferentiation and osteoprotegerin expression: implication for breast
cancer osteolytic bone metastases. Int J Cancer 123:1034–1042
51. Zhuang X, Zhang H, Li X, Li X, Cong M, Peng F, Yu J, Zhang
X, Yang Q, Hu G (2017) Differential effects on lung and bone
metastasis of breast cancer by Wnt signalling inhibitor DKK1.
Nat Cell Biol 19:1274–1285
52. Tulac S, Overgaard MT, Hamilton AE, Jumbe NL, Suchanek E,
Giudice LC (2006) Dickkopf-1, an inhibitor of Wnt signaling, is
regulated by progesterone in human endometrial stromal cells. J
Clin Endocrinol Metab 91:1453–1461
53. Chen W, Zahng UW, Li Y, Zahng JW, Zahng T, Fu BS, Zahng Q,
Jiang N (2016) Constitutive expression of Wnt/β-catenin target
genes promotes proliferation and invasion of liver cancer stem
cells. Mol Med Rep 13:3466–3474
54. Acar M, Çora T, Toy H, Acar H (2012) Analysis of promoter
methylation of Dickkopf1 (DKK1) gene in breast cancer. Turk J
Med Sci 42:1379–1387
55. Dolled-Filhart M, McCabe A, Giltnane J, Cregger M, Camp RL,
Rimm DL (2006) Quantitative in situ analysis of beta-catenin
expression in breast cancer shows decreased expression is asso-
ciated with poor outcome. Cancer Res 66:5487–5494
56. Eads J, El-Khoueiry A, Manji G et al (2016) Phase I study of
DKN-01 (D), an anti-DKK1 monoclonal antibody, in combina-
tion with gemcitabine (G) and cisplatin (C) in patients (pts) with
advanced biliary cancer (ABC). Ann Oncol 27(suppl_6):698
13