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NEOPLASIA
Increasing Wnt signaling in the bone marrow microenvironment inhibits the
development of myeloma bone disease and reduces tumor burden in bone in vivo
Claire M. Edwards,1,2 James R. Edwards,2,3 Seint T. Lwin,3 Javier Esparza,2 Babatunde O. Oyajobi,2 Brandon McCluskey,2
Steven Munoz,3 Barry Grubbs,2 and Gregory R. Mundy2,3
1Vanderbilt Center for Bone Biology, Department of Cancer Biology, Vanderbilt University, Nashville, TN; 2Department of Cellular and Structural Biology,
University of Texas Health Science Center at San Antonio; and 3Vanderbilt Center for Bone Biology, Department of Medicine/Clinical Pharmacology, Vanderbilt
University, Nashville, TN
There is increasing evidence to suggest
that the Wnt signaling pathway plays a
critical role in the pathogenesis of myeloma bone disease. In the present study,
we determined whether increasing Wnt
signaling within the bone marrow microenvironment in myeloma counteracts development of osteolytic bone disease.
C57BL/KaLwRij mice were inoculated intravenously with murine 5TGM1 myeloma
cells, resulting in tumor growth in bone
and development of myeloma bone disease. Lithium chloride (LiCl) treatment
activated Wnt signaling in osteoblasts,
inhibited myeloma bone disease, and decreased tumor burden in bone, but increased tumor growth when 5TGM1 cells
were inoculated subcutaneously. Abrogation of ␤-catenin activity and disruption
of Wnt signaling in 5TGM1 cells by stable
overexpression of a dominant-negative
TCF4 prevented the LiCl-induced increase
in subcutaneous growth but had no effect
on LiCl-induced reduction in tumor burden within bone or on osteolysis in
myeloma-bearing mice. Together, these
data highlight the importance of the local
microenvironment in the effect of Wnt
signaling on the development of myeloma bone disease and demonstrate that,
despite a direct effect to increase tumor
growth at extraosseous sites, increasing
Wnt signaling in the bone marrow microenvironment can prevent the development of myeloma bone disease and inhibit myeloma growth within bone in vivo.
(Blood. 2008;111:2833-2842)
© 2008 by The American Society of Hematology
Introduction
There have been many advances in our understanding of the
biology of multiple myeloma and the associated bone disease, yet a
number of critical questions remain unanswered and myeloma
remains an incurable malignancy. One such question, with important therapeutic implications, is the exact nature of myeloma bone
disease—specifically the dysregulation of both osteoclastic bone
resorption and osteoblastic bone formation. Histomorphometric
studies have demonstrated that bone resorption is increased in
patients with multiple myeloma, and for many years, the osteoclast
was considered to be the primary mechanism involved in the
development of myeloma bone disease.1-3 Although early stages of
multiple myeloma have been associated with an increase in
osteoblast recruitment, a very marked impairment of bone formation due to reduced osteoblast number and activity is a common
feature in later stages of the osteolytic bone disease.3-5 This has
been confirmed in recent studies that demonstrate that markers of
bone formation are decreased in patients with multiple myeloma.6,7
Although the cellular and molecular mechanisms involved in this
reduction of osteoblast activity are poorly understood, it is clear
that the regulation of bone formation plays a critical role in the
pathogenesis of myeloma bone disease and represents an important
therapeutic target for the treatment of this destructive bone disease.
The Wnt signaling pathway plays a key role in the regulation of
bone mass, and there is increasing data to suggest a role for this
pathway in the development of multiple myeloma.8 Human genetic
bone diseases and in vivo mouse models provide strong evidence
for the function of the Wnt signaling pathway in bone biology.
Inactivating mutations in the gene for LRP5 result in osteoporosispseudoglioma syndrome in humans, whereas “gain of function”
mutations in LRP5 are associated with a syndrome of hereditary
high bone density.9-11 Overexpression of ␤-catenin in osteoblasts
has been demonstrated to induce a high bone mass phenotype.12
Transgenic mice overexpressing the soluble antagonist of Wnt,
Dickkopf 1 (Dkk1), in osteoblasts develop severe osteopenia,
whereas deletion of a single allele of Dkk1 caused an increase in
bone mass.13,14 In multiple myeloma, patients have increased serum
levels of Dkk1, which correlate with the presence of bone lesions.15
Serum taken from these patients was also demonstrated to inhibit
osteoblast differentiation in vitro, and this inhibitory effect was
found to be mediated by Dkk1. Furthermore, a recent study has
demonstrated that inhibition of Dkk1 in a severe combined
immunodeficient 11-rabbit (SCID-rab) model of myeloma reduced
both osteolytic bone resorption and tumor burden.16 Myeloma cells
have also been found to release sFRP2, which can inhibit osteoblast
differentiation in vitro.17 Taken together, these studies provide
strong evidence to suggest that soluble antagonists of the Wnt
signaling pathway, Dkk1 and sFRP2, may play a role in the
development of myeloma bone disease.
Submitted March 1, 2007; accepted November 14, 2007. Prepublished online
as Blood First Edition paper, December 18, 2007; DOI 10.1182/blood-2007-03077685.
of Hematology, Atlanta, GA, December 11, 2007.
Parts of this work were presented in abstract form at the 28th annual meeting of
the American Society of Bone and Mineral Research, Philadelphia, PA,
September 15, 2006, and at the 49th Annual Meeting of the American Society
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2008 by The American Society of Hematology
2833
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2834
EDWARDS et al
The aim of the present study was to determine whether
increasing Wnt signaling within the bone microenvironment in
myeloma can prevent the development of myeloma bone disease,
using the 5TGM1 murine model of myeloma. By specific inhibition
of ␤-catenin activity in myeloma cells combined with systemic
stimulation of the Wnt signaling pathway, our results suggest that
increasing Wnt signaling in myeloma has dual effects; first, to
directly increase myeloma growth at nonosseous sites, and second
to enhance bone formation and thus indirectly reduce tumor burden
in bone, highlighting the importance of the bone marrow microenvironment in regulating myeloma growth and survival.
Methods
Reagents
Recombinant Wnt-3A was from R & D Systems (Minneapolis, MN).
Dominant negative TCF4 (⌬NTCF4), in which amino acids 2 to 53
(␤-catenin–binding domain) had been deleted, was kindly provided by Dr
Osamu Tetsu, University of California at San Francisco.18 Unless stated
otherwise, all other chemical and tissue culture reagents were from Sigma
Chemical (St Louis, MO).
Cell culture
The 5TGM1-GFP myeloma cell line was cultured as described previously.19,20 Cells were plated at 5 ⫻ 105/mL and treated with 10 mM lithium
chloride (LiCl) or 50 ng/mL Wnt-3A for 24 hours. Proliferation was
assessed using a colorimetric MTT assay (Promega, Madison, WI)
following the manufacturer’s instructions.
Stable cell lines
5TGM1-GFP cells were transfected with 2 ␮g pcDNA3 or ⌬NTCF4 by
electroporation using nucleofector technology (solution V, program H023;
Amaxa, Gaithersburg, MD). Cells were cultured in RPMI 1640 media as
referred to in “Cell culture,” supplemented with 1.6 mg/mL geneticin
(Invitrogen, Carlsbad, CA). Dead cells were removed by magnetic bead
separation using a Dead Cell Removal Kit (Miltenyi Biotech, Auburn, CA),
according to the manufacturer’s instructions. Overexpression of ⌬NTCF4
was confirmed by Western blotting for myc expression, and cells were
continuously cultured in geneticin to maintain overexpression.
Real-time PCR
RNA was extracted using a RNeasy kit (Qiagen, Valencia, CA). Total RNA
(1 ␮g) was used for first-strand cDNA synthesis using Superscript II reverse
transcriptase with random hexamers (Invitrogen). Real-time polymerase
chain reaction (PCR) was performed using primers and probes for axin2
(Applied Biosystems, Foster City, CA) on an Applied Biosystems 7300
Real-Time PCR System according to the manufacturer’s instructions.
Western blotting
Total cell lysates for Western blots were prepared by lysing cell pellets in
radioimmunoprecipitation assay (RIPA) buffer containing 1 mM EDTA.
Lysates were separated by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes, and then blocked in TBS plus 5% BSA and 0.1% TWEEN for
1 hour, before incubating with anti–␤-catenin (clone BDI480 and clone
BDI770; Abcam, Cambridge, MA), anti-myc (Abcam), or anti–␤-actin at
4°C overnight. Antigen-antibody complexes were detected using secondary
antibodies conjugated to HRP and visualized by enhanced chemiluminescence (GE Healthcare, Piscataway, NJ).
TOPFLASH Wnt reporter assay
To assay for activation of ␤-catenin/TCF target genes, 5TGM1 myeloma
cells were transiently transfected with 2 ␮g Wnt reporter constructs
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
containing wild-type (TOPFLASH) or mutated (FOPFLASH) LEF/TCFbinding sites (Upstate, Charlottesville, VA) by electroporation using
nucleofector technology (solution V, program H023; Amaxa). Cells were
cotransfected with 0.2 ␮g ␤-galactosidase reporter vector as a control for
transfection efficiency. Following transfection, cells were cultured for
24 hours; then luciferase and ␤-galactosidase reporter activities were
assayed using the BrightGlo and ␤-Glo assay kits, respectively (Promega).
TOPFLASH results were normalized to activity of ␤-galactosidase, and
expressed relative to FOPFLASH values. The reporter assay results
represent the average of 3 separate experiments.
5TGM1 myeloma model
Studies were conducted using weight-matched, 8- to 10-week-old female
C57BL/KaLwRijHsd mice (Harlan Netherlands, Horst, The Netherlands).
Studies were approved by the Institute of Animal Care and Use Committees
at the University of Texas Health Science Center at San Antonio and
Vanderbilt University and conducted in accordance with the National
Institutes of Health (NIH) Guide for the Care and Use of Laboratory
Animals.21 Mice were housed in isolator cages where autoclaved chow and
acidified water were provided ad libitum. Disseminated myeloma was
induced by intravenously inoculating 106 5TGM1-GFP cells in 100 ␮L
phosphate-buffered saline (PBS) into C57BL/KaLwRij mice through the
tail vein (n ⫽ 12). Mice were similarly inoculated with PBS alone (n ⫽ 6).
After tumor cell inoculation, mice were randomized to receive either
200 mg/kg per day LiCl in 100 ␮L deionized water (d.H2O) or
100 ␮L d.H2O alone, by oral gavage from time of tumor inoculation until
time of humane killing. Sera were assayed for monoclonal mouse IgG2b␬
paraprotein as described previously,22 and whole-blood ionized calcium
measurements and whole-body radiographs determined as described previously.19 Bone mineral density (BMD) measurements were obtained by
dual-energy X-ray absorptiometry scan using a Lunar PIXImus imager (GE
Medical Systems, Little Chalfont, United Kingdom). Solitary plasmacytomas were induced by injection of 106 5TGM1-GFP cells in 100 ␮L PBS
subcutaneously over the flank, and mice were treated with LiCl (n ⫽ 5) or
d.H2O (n ⫽ 5) for the same duration as described previously for disseminated myeloma. Once tumors were palpable, serial measurements of tumor
diameters in 3 dimensions were made at daily intervals using electronic
calipers and tumor volumes calculated as described by LeBlanc et al.23 In
another series of experiments, 106 5TGM1-pcDNA or 5TGM1-⌬NTCF4
cells were inoculated into C57BL/KaLwRij mice by intravenous tail vein
(n ⫽ 8 per group). Mice inoculated with 100 ␮L PBS served as control
(n ⫽ 6). Mice in either group were randomized to receive 200 mg/kg per
day LiCl or d.H2O, by oral gavage from time of tumor inoculation until time
of humane killing. Sera were assayed for mouse IgG2b␬ as described
previously.22 To examine dynamic changes in bone formation, mice were
injected intraperitoneally with 20 mg/kg calcein green on days 21 and
25 after inoculation. A separate cohort of C57Bl/KaLwRij mice was
inoculated subcutaneously with either 106 5TGM1-pcDNA or 5TGM1⌬NTCF4 cells (n ⫽ 5 per group) to induce plasmacytomas and tumor
volumes calculated as described above.
Bone histologic and cytochemical analyses
Fixed and decalcified long bones and spines were paraffin embedded and
consecutive 4-␮m-thick sections were stained with hematoxylin and eosin
(H&E) and for tartrate-resistant acid phosphatase (TRAP) activity. For
visualization of calcein labeling, spines were fixed in 70% ethanol and
embedded in methylmethacrylate without prior decalcification. Sections
(6-␮m thick) were cut and viewed unstained by epifluorescence microscopy. Histomorphometric analysis was performed as described previously.19,24 Subcutaneous tumors were fixed and paraffin-embedded sections
were stained with H&E. Consecutive sections of long bones and subcutaneous tumors were stained for ␤-catenin using a 1:50 dilution of
2 independent monoclonal antibodies to ␤-catenin or isotype control (clone
14/␤-catenin [BD Biosciences, San Jose, CA]; clone BDI480 [Abcam]).
Sections of subcutaneous tumors were stained for Ki67 using a
1:200 dilution of a polyclonal antibody to Ki67 (Abcam). Antibodies were
detected using a DAKO LSAB visualization system (Glostrup, Denmark),
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
following the manufacturer’s instructions. Following Ki67 immunostaining, sections were scored blind to generate a proliferation index (percentage
of Ki67-positive cells from the total cells). Five fields were assessed per
tumor at 40⫻ magnification. Images were acquired using an Olympus
BX41 microscope, with 40⫻/0.75 NA or 20⫻/0.5 NA objectives and an
Olympus DP71 camera using DP Controller 3.1.1.267 software (Olympus,
Center Valley, PA).
Microcomputed tomography (microCT) analysis
Tibiae were fixed in formalin, and each bone was scanned at an isotropic
voxel size of 12 ␮m using a microCT40 (SCANCO Medical, Bassersdorf,
Switzerland). In analyzing the reconstructed images, contours were drawn
within the cortices around the metaphyses, such that the total volume (TV)
included only trabecular bone at 0.2 mm below the growth plate and
extending 0.12 mm. Bone volume (BV) included all bone tissue that had a
material density greater than 438.7 mgHA/cm3, thereby giving a measure of
BV/TV. The same threshold setting for bone tissue was used for all samples.
For analysis of cortical bone lesions, the region of interest was the entire
metaphysis including the cortices and extending 0.25 mm from the growth
plate. The cross-sectional images of this region were exported in tiff format
and imported into AMIRA 3-D graphics software (Mercury Computer
Systems, Chelmsford, MA). Using a consistent threshold, AMIRA generated 3-D renderings of the metaphyses. Rotating the virtual bone through
360°, the number of osteolytic lesions were counted, which completely
penetrated the cortical bone and were greater than 100 ␮m in diameter.
Flow cytometry
Bone marrow was flushed from the tibia and femur of 5TGM1 myeloma–
bearing mice. Spleens from myeloma-bearing mice were homogenized in
tissue culture media. Cell suspensions were filtered through a 70-␮m filter
and then analyzed for GFP fluorescence using a 3 laser BD LSRII (Becton
Dickinson, San Jose, CA).
Statistical analysis
Statistical significance was determined using a Mann-Whitney U test for
nonparametric data and considered significant given P less than or equal to
.05. Data are presented as means (⫾ SEM) unless otherwise stated.
Results
LiCl inhibits the development of myeloma bone disease in vivo
To test the hypothesis that increasing Wnt signaling in the bone
marrow microenvironment would counteract the effects of myeloma bone disease, we used LiCl to activate the Wnt signaling
pathway in the well-characterized 5TGM1 murine model of
myeloma.19,20,22,25 LiCl is known to increase Wnt signaling by
inhibition of glycogen synthase kinase 3␤ (GSK-3␤).26-28 The
dosing schedule used in the present study has previously been
demonstrated to result in serum LiCl levels within therapeutic
concentrations for inhibition of GSK-3␤ and activation of Wnt
signaling in mice of the same age as used in this investigation.29
Intravenous inoculation of 5TGM1 myeloma cells resulted in the
development of myeloma bone disease, associated with a generalized osteolysis; significant decrease in BMD, trabecular bone
volume, and osteoblast number; and increase in osteoclast number.
Although in some instances, development of 5TGM1 myeloma is
associated with splenomegaly, this was not observed in this study.
Treatment of 5TGM1 myeloma–bearing mice with LiCl resulted in
a significant increase in BMD and trabecular bone volume in the
tibia of myeloma-bearing mice, compared with vehicle-treated
5TGM1 myeloma–bearing mice (Figure 1A,B). Identical results
were found in the femora and spine (data not shown). Histomorpho-
TARGETING Wnt SIGNALING IN MYELOMA BONE DISEASE
2835
metric analyses revealed that LiCl treatment of myeloma-bearing
mice resulted in a significant increase in osteoblasts and a reduction
in TRAP-positive osteoclasts lining the trabecular bone surfaces
(Figure 1C-E). LiCl had no significant effects on these parameters
in non–tumor-bearing mice. LiCl treatment was associated with a
clear increase in ␤-catenin expression in osteoblasts in vivo (Figure
1F). In addition to preventing the development of the characteristic
bone disease, LiCl treatment also resulted in a significant reduction
in tumor burden within bone, assessed histomorphometrically
(79.06% ⫾ 11.1% decrease compared with untreated myelomabearing mice), and a significant reduction in serum IgG2b␬ titers
(Figure 1G).
LiCl increases subcutaneous plasmacytoma growth
To determine whether LiCl has direct effects on myeloma growth
in vivo, independent from the bone microenvironment, mice were
inoculated subcutaneously with 5TGM1 myeloma cells and treated
with LiCl using the same dosing schedule as described above. LiCl
significantly increased subcutaneous tumor volume (Figure 2A)
and whole tumor wet weight at humane killing by 283% (⫾ 60%)
as compared with vehicle-treated subcutaneous tumor-bearing
mice. Treatment with LiCl was also associated with a significant
increase in serum IgG2b␬ titers (Figure 2B). Immunohistochemistry
demonstrated a clear increase in ␤-catenin expression in myeloma
cells in the excised 5TGM1 plasmacytomas from mice treated with
LiCl (Figure 2C). Identical results were found using a different
antibody (clone BDI480, data not shown). Real-time PCR analysis
revealed a significant increase in expression of the ␤-catenin target
gene, axin2, in RNA isolated from LiCl-treated, but not control
(vehicle-treated), subcutaneous plasmacytomas (Figure 2D).
LiCl increases ␤-catenin activity in 5TGM1 myeloma cells
in vitro
To further investigate the effects of LiCl on 5TGM1 myeloma cells,
in vitro studies were performed to study proliferation and ␤-catenin
activity. Although LiCl was found to have no significant effect on
5TGM1 cell proliferation (Figure 3A), the same dose of LiCl was
found to significantly increase expression of axin2 (Figure 3B)
along with expression of unphosphorylated ␤-catenin (Figure 3C).
To confirm the specificity of unphosphorylated ␤-catenin expression, 2 different antibodies were used.30 Results shown are using
clone BDI480, and identical results were found with clone BDI770
(data not shown). To determine the effect of specific blockade of
Wnt signaling in myeloma cells, 5TGM1 myeloma cells were
stably transfected with myc-tagged ⌬NTCF4 or pcDNA3, and
expression of ⌬NTCF4 was confirmed by Western blotting for myc
expression (Figure 3D). Overexpression of ⌬NTCF4 had no
significant effect on the growth rate of 5TGM1 myeloma cells in
vitro, or proliferation in response to LiCl or Wnt 3A (Figure 3E). In
contrast, overexpression of ⌬NTCF4 completely prevented the
increase in ␤-catenin activity as determined by TOPFLASH
activity in response to treatment with both LiCl and Wnt3A (Figure
3F), confirming that Wnt signaling is abrogated in 5TGM1⌬NTCF4 cells.
Inhibition of Wnt signaling prevents the response to LiCl in
subcutaneous 5TGM1 tumors
To determine whether the LiCl-induced increase in the growth of
subcutaneous 5TGM1 tumors was due to direct activation of Wnt
signaling in myeloma cells, we investigated the response in
subcutaneous tumors induced by 5TGM1-⌬NTCF4 cells in which
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2836
EDWARDS et al
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
Figure 1. LiCl inhibits the development of myeloma bone disease in vivo. Bone mineral density is significantly increased in 5TGM1 myeloma–bearing mice following
treatment with LiCl (A). Histomorphometric analysis demonstrated an increase in trabecular bone volume (B), a decrease in osteoclast number (C), and an increase in
osteoblast number (D) in 5TGM1 myeloma–bearing mice following treatment with LiCl. Histologic sections of tibiae from non–tumor-bearing mice, 5TGM1 myeloma–bearing
mice, and 5TGM1 myeloma–bearing mice treated with LiCl demonstrated increased osteoclasts lining the trabecular bone surface in 5TGM1 myeloma–bearing mice, and a
reduction in osteoclast number in myeloma-bearing mice treated with LiCl. Osteoclasts were identified by TRAP staining. Original magnification, ⫻200. (E). Immunohistochemistry demonstrated an increase in ␤-catenin expression in osteoblasts lining the bone surface in myeloma-bearing mice treated with LiCl compared with control. Original
magnification, ⫻400 (F). Treatment with LiCl significantly reduced serum IgG2b␬ concentrations in 5TGM1 myeloma–bearing mice (G). Data are expressed as means (⫾ SEM).
*P ⬍ .05 compared with nontumor control. †P ⬍ .05 compared with 5TGM1 control.
the Wnt signaling pathway is blocked, compared with 5TGM1pcDNA cells in which the Wnt signaling pathway is intact.
Although we found no difference in the in vitro growth rates, there
were significant differences in tumor volume and proliferation
index between 5TGM1-pcDNA and 5TGM1-⌬NTCF4 subcutaneous tumors (Figure 4A,B). Despite the difference in growth rates, it
was possible to determine the response of these subcutaneous
tumors to LiCl as a significant increase in tumor volume was
observed in LiCl-treated 5TGM1-pcDNA plasmacytomas, but
not in LiCl-treated 5TGM1-⌬NTCF4 plasmacytomas (Figure
4A). To investigate further the direct effect of LiCl on proliferation in vivo, we quantitated expression of the Ki67 antigen, which is
expressed by proliferating cells in all phases of the cell cycle. LiCl
significantly increased myeloma cell proliferation by 221% (⫾ 35%) in
5TGM1-pcDNAsubcutaneous tumors, but had no effect on the proliferation index in 5TGM1-⌬NTCF4 subcutaneous tumors (Figure 4B).
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
TARGETING Wnt SIGNALING IN MYELOMA BONE DISEASE
2837
Figure 2. LiCl increases subcutaneous myeloma
growth. Treatment with LiCl significantly increased
subcutaneous tumor volume (A) and serum IgG2b␬
concentrations (B). Immunohistochemistry staining of
sections from subcutaneous tumors demonstrated an
increase in ␤-catenin expression in myeloma cells from
those mice treated with LiCl. Original magnification,
⫻400 (C). Real-time PCR demonstrated an increase in
axin2 expression in mRNA isolated from subcutaneous
tumors treated with LiCl (D). Data are expressed as
means (⫾ SEM). *P ⬍ .05 compared with untreated
control.
Histologic analysis demonstrated no significant difference in the proportion of apoptotic myeloma cells between 5TGM1-pcDNA and 5TGM1⌬NTCF4 tumors in response to LiCl (data not shown).
Increasing Wnt signaling in the bone microenvironment
prevents myeloma bone disease and reduces tumor burden
in bone
Although our data clearly demonstrate that treatment with LiCl
prevents the development of myeloma bone disease in vivo, it is
unclear whether this is mediated by effects on the bone marrow
microenvironment or the tumor cells directly. To address this issue,
we used 5TGM1-⌬NTCF4 myeloma cells in which Wnt signaling
is abrogated. Mice were inoculated intravenously with GFPexpressing 5TGM1-⌬NTCF4 cells or 5TGM1-pcDNA cells and
treated with LiCl from the time of tumor inoculation. Flow
cytometric analysis demonstrated that mice bearing 5TGM1pcDNA or 5TGM1-⌬NTCF4 myeloma accumulated GFP-positive
cells in the bone marrow and spleen (Figure 5A,B). However mice
bearing 5TGM1-⌬NTCF4 cells had a significantly lower proportion of GFP-positive cells in the bone marrow than those mice
bearing 5TGM1-pcDNA myeloma. In contrast, there was a 36%
increase in the proportion of GFP-positive cells in spleens of
5TGM1-⌬NTCF4 myeloma–bearing mice compared with 5TGM1pcDNA myeloma–bearing mice, although this increase was not
statistically significant. Treatment with LiCl resulted in a significant decrease in GFP-positive cells in the bone marrow of both
5TGM1-pcDNA and 5TGM1-⌬NTCF4 myeloma–bearing mice
(Figure 5A), consistent with our previous observations that LiCl
Figure 3. LiCl increases ␤-catenin activity in 5TGM1
myeloma cells in vitro. LiCl (10 mM) had no significant effect on 5TGM1 myeloma cell proliferation (A),
but significantly increased axin2 mRNA expression (B)
and expression of ␤-catenin (C). 5TGM1 myeloma cells
were stably transfected with myc-tagged ⌬NTCF4 or
pcDNA (D). Wnt3A or LiCl treatment had no effect on
proliferation of 5TGM1-pcDNA or 5TGM1-⌬NTCF4 cells
(E). Overexpression of ⌬NTCF4 completely blocked
the increase in ␤-catenin activity as measured by
TOPFLASH reporter activity (F). Data are expressed as
means (⫾ SEM). *P ⬍ .05 compared with control.
**P ⬍ .01 compared with 5TGM1-pcDNA.
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2838
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
EDWARDS et al
LiCl resulted in a significant increase in trabecular bone volume
(Figure 6C) and reduction in the number of osteolytic lesions
(Figure 6D) in both 5TGM1-pcDNA and 5TGM1-⌬NTCF4
myeloma–bearing mice. To account for the differences in tumor
burden within bone between 5TGM1-pcDNA and 5TGM1⌬NTCF4 myeloma–bearing mice, the effect of LiCl was also
expressed as percentage change from control. Although there was
no significant difference in the effect of LiCl on trabecular bone
volume in 5TGM1-pcDNA compared with 5TGM1-⌬NTCF4 (data
not shown), LiCl reduced the number of osteolytic lesions by
43.2% (⫾ 15.6%) in 5TGM1-pcDNA myeloma–bearing mice, compared with 87.5% (⫾ 12.5%) in 5TGM1-⌬NTCF4 myeloma–
bearing mice. MicroCT analysis also revealed a decrease in
trabecular number and thickness and an increase in trabecular
separation in myeloma-bearing mice. LiCl reversed this decrease in
trabecular number and thickness and concomitantly reduced trabecular spacing in both 5TGM1-pcDNA and 5TGM1-⌬NTCF4
myeloma–bearing mice (Table 1). Histomorphometric analysis
revealed that LiCl treatment significantly decreased osteoclast
number by 47.2% (⫾ 11.4%) and 53.1% (⫾ 13.3%; P ⬍ .05 compared with vehicle control), and significantly increased osteoblast
number by 312.5% (⫾ 69.2%) and 233.1% (⫾ 45.9%; P ⬍ .05
compared with vehicle control), in 5TGM1-pcDNA and 5TGM1⌬NTCF4 myeloma–bearing mice, respectively. As in myeloma
patients, the development of myeloma bone disease in the 5TGM1
model was also associated with a significant reduction in the rates
of bone formation, assessed by double calcein labeling of boneforming surfaces. Consistent with an increase in bone volume, LiCl
significantly increased bone formation in both 5TGM1-pcDNA and
5TGM1-⌬NTCF4 myeloma–bearing mice (Figure 6E).
Discussion
Figure 4. Overexpression of ⌬NTCF4 prevents the response to LiCl in subcutaneous 5TGM1 tumors. Mice were inoculated subcutaneously with 5TGM1-pDNA or
5TGM1-⌬NTCF4 cells and treated with 200 mg/kg per day LiCl. LiCl significantly
increased tumor volume in 5TGM1-pcDNA tumors, but not in 5TGM1-⌬NTCF4
tumors (A). Immunohistochemistry for Ki67 demonstrated an increase in the
proliferation index of 5TGM1-pcDNA cells following treatment with LiCl, but not in
5TGM1-⌬NTCF4 cells. Proliferation index was calculated as the percentage of
Ki67-positive cells among the total number of tumor cells (B). Data are expressed as
means (⫾ SEM). *P ⬍ .05 compared with control.
reduces tumor burden within bone. In contrast, LiCl significantly
increased the proportion of GFP-positive cells in the spleen in
5TGM1-pcDNA myeloma–bearing mice, but had no effect in
5TGM1-⌬NTCF4 myeloma–bearing mice (Figure 5B). We also
measured serum IgG2b␬ concentrations, which reflect total myeloma burden in bone marrow and spleen. There was no significant
difference in serum IgG2b␬ concentrations between untreated
5TGM1-pcDNA and 5TGM1-⌬NTCF4 myeloma–bearing mice,
however treatment with LiCl resulted in a significant 34% and 50%
decrease, respectively (Figure 5C).
Mice bearing 5TGM1-pcDNA or 5TGM1-⌬NTCF4 myeloma
developed an osteolytic bone disease, characterized by an increase
in osteolytic bone lesions and osteoclast number and a decrease in
trabecular bone volume, osteoblast number, and bone formation
rates. The reduction in trabecular bone volume and the increase in
osteolytic bone lesions were greater in 5TGM1-pcDNA myeloma–
bearing mice than in 5TGM1-⌬NTCF4 myeloma–bearing mice.
MicroCT analyses of the trabecular (Figure 6A) and cortical
(Figure 6B) bone from the tibia demonstrated that treatment with
The increasing evidence for the role of Wnt signaling in the
regulation of bone formation and for the role of the soluble
antagonist Dkk1 in the pathogenesis of myeloma bone disease
identifies the Wnt signaling pathway as a potential therapeutic
target in multiple myeloma. In the present study, we demonstrate
that increasing Wnt signaling in the bone marrow microenvironment can prevent the development of myeloma bone disease, by
both increasing osteoblast number and bone formation, and concomitantly reducing osteoclast number. Activation of the Wnt
signaling pathway was achieved by a pharmacological approach:
systemic treatment with LiCl that inhibits glycogen synthase kinase
3␤ (GSK-3␤), an enzyme responsible for the phosphorylation and
subsequent proteasomal degradation of ␤-catenin.26-28 The dosing
schedule used in the present study has previously been demonstrated to result in serum LiCl levels within therapeutic concentrations for inhibition of GSK-3␤ and activation of Wnt signaling in
mice of the same age as used in this investigation.29
Wnt signaling is known to increase osteoblast differentiation
and bone formation in vivo.9,11,31 In support of this, we demonstrate
a clear increase in ␤-catenin expression in osteoblasts following
treatment with LiCl, suggesting that LiCl prevents the development
of myeloma bone disease by increasing Wnt signaling in osteoblasts. Lithium treatment of patients with bipolar disease has been
documented to result in an increase in serum parathyroid hormone
and an associated hypercalcemia, however there is little evidence
for a significant effect of lithium on bone mass or bone density.32-36
In our experiments, there was no significant increase in ionized
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
TARGETING Wnt SIGNALING IN MYELOMA BONE DISEASE
2839
Figure 5. Increasing Wnt signaling in the bone
microenvironment reduces myeloma tumor burden
in bone. Mice were inoculated intravenously with
5TGM1-pcDNA or 5TGM1-⌬NTCF4 myeloma cells that
homed to bone marrow and spleen. Single cell suspensions were isolated from bone marrow and spleen, and
the proportion of GFP-positive cells was determined by
flow cytometry. LiCl significantly reduced the proportion
of GFP-positive cells in the bone marrow of both
5TGM1-pcDNA and 5TGM1-⌬NTCF4 myeloma–
bearing mice (A). In contrast, LiCl significantly increased
the proportion of GFP-positive bone marrow cells in the
spleen of 5TGM1-pcDNA myeloma–bearing mice but not
5TGM1-⌬NTCF4 myeloma–bearing mice (B). Treatment
with LiCl significantly reduced serum IgG2b␬ concentrations
in both 5TGM1-pcDNA and 5TGM1-⌬NTCF4 myeloma–
bearing mice (C). Data are expressed as means (⫾ SEM).
*P ⬍ .05 compared with control. **P ⬍ .01 compared with
nontumor. †P ⬍ .05, ††P ⬍ .01 compared with untreated.
calcium in response to LiCl, either in non–tumor- or myelomabearing mice, thus excluding this potential confounding effect from
the present study. LiCl had no significant effect on non–tumorbearing mice, suggesting that the response to increasing Wnt
signaling is enhanced in conditions associated with dysregulation
of this pathway, such as multiple myeloma. This is supported by
studies that demonstrate that the response to LiCl is greater in
LRP5⫺/⫺ mice compared with wild-type mice.29 Wnt signaling in
osteoblasts has also been demonstrated to regulate osteoclast
function, with ␤-catenin in osteoblasts functioning as a negative
regulator of osteoclast function.12,37,38 In support of this, we show
that LiCl treatment significantly reduces osteoclast number in
myeloma-bearing mice. While we found strong expression of
␤-catenin in osteoclasts, there was no change in expression in
response to LiCl treatment, suggesting that the reduction in
osteoclast number may be an indirect response to increasing Wnt
signaling in osteoblasts. The canonical Wnt signaling pathway is
critical for the self-renewal of hematopoietic stem cells, and
therefore increasing Wnt signaling in the bone marrow may
promote the regeneration of the bone marrow microenvironment.39
Thus, the effects of increasing Wnt signaling in hematopoietic stem
cells may also contribute to the protective effects of this approach
in the treatment of myeloma bone disease.
It is well established that the Wnt signaling pathway plays a
key role in bone formation, however its precise role in growth
and survival of myeloma cells remains controversial. Activation
of the Wnt signaling pathway through ␤-catenin plays a critical
oncogenic role in many human malignancies, and expression of
␤-catenin has been demonstrated in myeloma cell lines and in
malignant plasma cells from patients with multiple myeloma.40,41 However, published data are conflicting as to the
function of the Wnt signaling pathway in myeloma cells.
Derksen et al demonstrated that stimulation of the canonical
Wnt signaling pathway increases proliferation of human myeloma cell lines.40 More recently, a small molecule inhibitor of
the ␤-catenin/TCF interaction was found to inhibit myeloma cell
proliferation, both in vitro and in a xenograft model of
myeloma.42 In contrast, Qiang et al have demonstrated that
although activation of Wnt signaling results in an increase in
␤-catenin activity in myeloma cells, this is not associated with a
proliferative effect.43 In addition, they showed that activation of
Wnt signaling with Wnt3A results in morphologic changes and
an increase in invasion and migration of myeloma cells.
Importantly, they also demonstrated that these effects were
mediated through the Wnt/RhoA pathway and were independent
of signaling through ␤-catenin.43,44 The present study combines
both pharmacological and molecular approaches to target the
Wnt signaling pathway in myeloma cells, both in vitro and in
vivo. In agreement with Qiang et al, we found that although
activation of the Wnt signaling pathway in myeloma cells
results in an increase in ␤-catenin activity, there was no
associated proliferative effect in vitro. However, our in vivo
studies suggest that the role of the Wnt signaling pathway in
myeloma cells may be more complex and dependent upon the
microenvironment. In subcutaneous plasmacytoma tumors, increasing Wnt signaling results in an increase in tumor volume
and tumor cell proliferation, associated with an increase in
␤-catenin expression and activation of downstream target genes.
Molecular blockade of ␤-catenin activity by overexpression of
dominant negative TCF4 in myeloma cells completely prevented the increase in subcutaneous tumor burden and proliferation, confirming that this increase was dependent upon active
canonical Wnt signaling in myeloma cells. The difference
between the in vitro and in vivo observations may reflect the
well known limitations of using myeloma cell lines in vitro, but
may also suggest a dependence of the Wnt signaling pathway
upon environmental factors that are not present in the in vitro
cultures and indicate the necessity for in vivo preclinical studies
to fully investigate the pathogenesis of myeloma bone disease.
Although there was no difference in the in vitro growth rates,
there was a difference in the tumor volumes and proliferation
rates between subcutaneous tumors with intact Wnt signaling
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2840
EDWARDS et al
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
Figure 6. Increasing Wnt signaling in the bone microenvironment prevents the development of myeloma bone disease. MicroCT analysis of tibiae of 5TGM1-pcDNA
and 5TGM1-⌬NTCF4 myeloma–bearing mice demonstrated a significant reduction in trabecular bone volume (A,C) and increase in osteolytic bone lesions (B,D) that was
prevented by treatment with LiCl. LiCl also significantly prevented the reduction in bone formation rates associated with the development of myeloma bone disease (E). Data
are expressed as means (⫾ SEM). *P ⬍ .05 compared with nontumor. **P ⬍ .01 compared with nontumor. †P ⬍ .05, ††P ⬍ .01 compared with untreated.
pathway (5TGM1-pcDNA) compared with those with blocked
␤-catenin transcriptional activity (5TGM1-⌬NTCF4). Since we have
clearly demonstrated that increasing Wnt signaling results in a direct
increase in tumor growth in subcutaneous 5TGM1 tumors, it is most
likely that this represents an unexpected difference in the in vivo growth
rates following selection of transfected cells. In support of this, there was
also a trend toward an increase in tumor burden in the spleen in those
mice bearing 5TGM1-⌬NTCF4 myeloma, with a small increase in both
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BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
TARGETING Wnt SIGNALING IN MYELOMA BONE DISEASE
2841
Table 1. Effects of increasing Wnt signaling in the myeloma bone microenvironment on trabecular structure
Nontumor
5TGM1-pcDNA
5TGM1-pcDNA
ⴙ LiCl
5TGM1ⴚ
⌬ NTCF4
5TGM1ⴚ
⌬ NTCF4 ⴙ LiCl
BV/TV, %
5.38 ⫾ 0.26
2.83 ⫾ 0.20*
4.60 ⫾ 0.31†
3.75 ⫾ 0.54*
5.42 ⫾ 0.45†
Tb. no., 1/mm
2.81 ⫾ 0.07
2.34 ⫾ 0.07‡
2.70 ⫾ 0.13†
2.65 ⫾ 0.21
2.93 ⫾ 0.09
Tb. thickness, mm
0.048 ⫾ 0.001
0.045 ⫾ 0.001*
0.048 ⫾ 0.001†
0.043 ⫾ 0.002*
0.047 ⫾ 0.001
Tb. separation, mm
0.36 ⫾ 0.01
0.43 ⫾ 0.01*
0.38 ⫾ 0.02†
0.39 ⫾ 0.03
0.35 ⫾ 0.01
Trabecular bone volume (BV/TV), number, thickness and separation were calculated by microCT analysis of tibiae.
Tb indicates trabecular.
*P ⬍ .05 compared with nontumor.
†P ⬍ .05 compared with vehicle control.
‡P ⬍ .01 compared with nontumor.
spleen weight and the proportion of tumor cells within the spleen,
although this was not statistically significant. In contrast, there was a
significant decrease in tumor burden within bone in those mice bearing
5TGM1-⌬NTCF4 myeloma, compared with 5TGM1-pcDNAmyeloma.
As the results with the subcutaneous tumors suggest that the 5TGM1⌬NTCF4 cells grow faster in vivo, our observations in the bone marrow
microenvironment suggest that myeloma cells may be dependent upon
Wnt signaling to promote growth within this specialized environment,
and so blockade of the Wnt signaling pathway results in a reduction in
tumor burden. The bone marrow microenvironment in multiple myeloma is associated with high concentrations of Dkk1 that are critical in
the pathogenesis of myeloma bone disease, and it is intriguing to
speculate that the Wnt signaling pathway is regulated in myeloma cells
within this specialized microenvironment to compensate for the increase
in Dkk1. These data raise the possibility that response of tumor cells to
the Wnt signaling pathway is highly dependent upon the microenvironment and the local balance of agonists and antagonists of this pathway.
The discovery of Dkk1 as a mediator of the reduction in
osteoblastic bone formation in multiple myeloma, and the
compelling evidence for the critical role of Wnt signaling in
promoting osteoblast differentiation and bone formation, identifies the Wnt signaling pathway as a potential therapeutic target
in multiple myeloma. In support of this, inhibition of Dkk1
using neutralizing antibodies was found to prevent the osteolytic
bone disease and reduce tumor burden in SCID-rab mice
engrafted with primary myeloma cells.16 In this model, myeloma
growth is restricted to the rabbit bone and therefore it is not
possible to discern unequivocally any effects on tumor burden
from effects on the bone microenvironment.45 The 5TGM1
myeloma model is a well-characterized model of myeloma bone
disease that closely reflects the human form of the disease. In
addition, this model enables the study of myeloma growth in
vivo, independent from the bone microenvironment, by inoculating tumor cells subcutaneously. The current study clearly
demonstrates that increasing Wnt signaling within the myeloma
bone microenvironment can inhibit the development of myeloma bone disease. However, our studies also demonstrate that
increasing Wnt signaling can increase myeloma growth at
nonosseous sites, and that this is likely due to direct stimulation
of the Wnt signaling pathway and ␤-catenin activation. Extramedullary disease, while uncommon in patients with myeloma, is
associated with aggressive disease, occurring subsequent to
the development of myeloma bone disease and typically resulting in plasma cell leukemia. The mechanisms involved in
the progression of myeloma beyond the bone marrow cavity
are poorly understood, and current clinical approaches
cannot accurately identify patients at risk of developing extramedullary disease. Although increasing Wnt signaling is
clearly effective to prevent myeloma bone disease in vivo, our
results raise important clinical concerns for targeting Wnt
signaling as a therapeutic approach in myeloma due to the risk
of increasing tumor growth outside the bone marrow microenvironment. Myeloma is primarily a skeletal disease and the
striking difference between the response of myeloma cells
within bone compared with subcutaneous growth raises questions as to the relevance of xenograft models of myeloma that do
not take into consideration the influences of the bone milieu.
The data presented herein further highlight the necessity to
study multiple myeloma in vivo within this specialized host
microenvironment.
Myeloma cells are dependent upon interactions with the cells of
the host bone microenvironment for their growth and survival.46,47
Several studies have demonstrated the potential for indirect antitumor effects by inhibiting osteoclastic bone resorption.48-50 In the
present study, we clearly demonstrate a dramatic effect of directly
targeting the Wnt signaling pathway in osteoblasts in myeloma. In
addition to preventing the development of myeloma bone disease,
the systemic increase in Wnt signaling was shown to decrease
tumor burden within the bone microenvironment, despite increasing the growth of myeloma cells at extraosseous sites. This study
demonstrates the striking potential for directly targeting the bone
microenvironment in multiple myeloma by increasing bone formation and indirectly inhibiting tumor growth.
Acknowledgments
This work was supported by the San Antonio Cancer Institute and
the National Cancer Institute (NCI) through P30 CA-054174-16
(C.M.E.), and by NCI through P01 CA-40035 (G.R.M.). B.O.O. is
supported by KO1 CA-104180 from NCI. C.M.E. is supported by
the International Myeloma Foundation.
Authorship
Contribution: C.M.E. designed, performed, and analyzed research and wrote the paper; J.R.E. performed and analyzed
research and reviewed the paper; S.T.L., J.E., B.M., S.M., and
B.G. performed research; B.O.O. assisted in research design and
reviewed the paper; and G.R.M. discussed the experiments and
reviewed the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Claire M. Edwards, Vanderbilt Center for
Bone Biology, 1235 Medical Research Bldg IV, Nashville, TN
37232-0575; e-mail: [email protected].
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2842
BLOOD, 1 MARCH 2008 䡠 VOLUME 111, NUMBER 5
EDWARDS et al
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2008 111: 2833-2842
doi:10.1182/blood-2007-03-077685 originally published
online December 19, 2007
Increasing Wnt signaling in the bone marrow microenvironment inhibits
the development of myeloma bone disease and reduces tumor burden in
bone in vivo
Claire M. Edwards, James R. Edwards, Seint T. Lwin, Javier Esparza, Babatunde O. Oyajobi,
Brandon McCluskey, Steven Munoz, Barry Grubbs and Gregory R. Mundy
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