Transforming growth factor-beta1 is the predominant isoform required for breast cancer cell outgrowth in bone

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Transforming growth factor-β1 is the predominant isoform required for
breast cancer cell outgrowth in bone
Mourskaia, A. A.; Dong, Z.; Ng, S.; Banville, M.; Zwaagstra, J. C.; O'Connor-
McCourt, M. D.; Siegel, P. M.

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ORIGINAL ARTICLE
Transforming growth factor-b1 is the predominant isoform required for
breast cancer cell outgrowth in bone
AA Mourskaia1, Z Dong1, S Ng2, M Banville3, JC Zwaagstra1,3, MD O’Connor-McCourt2,3,4
and PM Siegel1,2,4
1Department of Medicine, McGill University, Montreal, Quebec, Canada;2Department of Anatomy and Cell Biology, McGill
University, Montreal, Quebec, Canada;3Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec,
Canada and4Department of Biochemistry, McGill University, Montreal, Quebec, Canada
Transforming growth factor (TGF)-b signaling is a potent
modulator of the invasive and metastatic behavior of
breast cancer cells. Indeed, breast tumor responsiveness to
TGF-b is important for the development of osteolytic bone
metastases. However, the specific TGF-b isoforms that
promote breast cancer outgrowth in bone is unknown. We
demonstrate that expression of a TGF-b ligand trap,
which neutralizes TGF-b1 and TGF-b3, in MDA-MB-231
breast cancer cells diminished their outgrowth in bone and
reduced the severity of osteolytic lesion formation when
compared with controls. We further show that a reduction
or loss of TGF-b1 expression within the bone microenvir-
onment of TGF-b1þ/? and TGF-b1?/? mice signifi-
cantly reduced the incidence of breast tumor outgrowth
compared with wild-type animals. Interestingly, those
tumors capable of growing within the tibiae of TGF-b1-
deficient mice had upregulated expression of all three
TGF-b isoforms. Finally, breast cancer cells expressing
the TGF-b ligand trap showed a pronounced reduction in
their ability to form osteolytic lesions when injected into
the tibiae of TGF-b1þ/? mice. Thus, our studies show
that both host- and tumor-derived TGF-b expression plays
a critical role during the establishment and outgrowth of
breast cancer cells in bone.
Oncogene (2009) 28, 1005–1015; doi:10.1038/onc.2008.454;
published online 15 December 2008
Keywords: TGF-b isoforms; breast cancer; ligand trap;
bone microenvironment
Introduction
The bone represents the most common site of metastasis
in breast cancer patients (Mundy, 2002; Coleman, 2006;
Hess et al., 2006). These skeletal metastases are typically
characterized by excessive bone resorption (Kozlow and
Guise, 2005). Breast tumor cells secrete factors that
promote the differentiation and activity of multinu-
cleated osteoclasts, which are the resident bone cells
responsible for bone resorption (Yoneda and Hiraga,
2005; Rose and Siegel, 2006). In addition, the bone
matrix is a reservoir of latent growth factors that can be
released and activated during osteoclast-mediated bone
resorption. These factors then function in a paracrine
manner to promote breast cancer survival and growth
within bone (Mastro et al., 2003; Kozlow and Guise,
2005).
All three transforming growth factor-b isoforms
(TGF-b1, TGF-b2 and TGF-b3) are present in bone;
however, TGF-b1 is the most abundant (Hering et al.,
2001a,b). TGF-b isoforms bind to the TGF-b type II
receptor (TbRII), which in turn recruits and activates
the TGF-b type I receptor (TbRI) (Wrana et al., 1994;
Groppe et al., 2008). TGF-b1 and TGF-b3 directly bind
to the extracellular domain (ECD) of TbRII, whereas
efficient binding of TGF-b2 to TbRII is facilitated by
betaglycan (TbRIII) (Wang et al., 1991; Lopez-Casillas
et al., 1993). The activated TGF-b receptor complex
then initiates signaling through both Smad-dependent
and -independent pathways (Moustakas and Heldin,
2005; Schmierer and Hill, 2007).
Mouse models of experimental breast cancer metas-
tasis to bone show that TGF-b signaling in breast tumor
cells is critical for this process. Expression of a dominant
negative TbRII significantly reduces the ability of
MDA-MB-231 breast cancer cells to form osteolytic
bone metastases, which can be rescued by expression of
constitutively active TbRI (Yin et al., 1999). In vivo
selection approaches to isolate highly aggressive bone
metastatic MDA-MB-231 breast cancer cells have
identified TGF-b targets, such as parathyroid hor-
mone-related protein (PTHrP), interleukin-11 (IL-11)
and connective tissue growth factor, which play
important roles in breast cancer metastasis to bone
(Yoneda et al., 2001; Kang et al., 2003). Activation of
both Smad-dependent and -independent pathways with-
in breast cancer cells is important for the formation of
osteolytic metastases (Kakonen et al., 2002). Stable
knockdown of Smad4 expression in these cells further
reinforces the importance of Smad-mediated TGF-b
signaling for promoting breast cancer metastasis to bone
Received
13 November 2008; published online 15 December 2008
13 August 2008;revised 28October2008;accepted
Correspondence: Dr PM Siegel, Department of Medicine, McGill
University, 740 Dr Penfield Avenue, Room 2201, Montreal, Quebec,
Canada H3A 1A4.
E-mail: peter.siegel@mcgill.ca
Oncogene (2009) 28, 1005–1015
& 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00
www.nature.com/onc

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(Kang et al., 2005; Deckers et al., 2006). Furthermore,
kinase inhibitors targeting TbRI reduce the incidence of
bone metastases following left cardiac ventricle injection
of MDA-MB-435 and MDA-MB-231 breast cancer cells
(Bandyopadhyay et al., 2006; Ehata et al., 2007). These
animal-based studies support a role for TGF-b signaling
within breast tumor cells that enable aggressive metas-
tasis to bone. Finally, it was recently reported that
greater than 50% of breast and prostate cancer patients
with skeletal metastases show elevated serum levels of
TGF-b1, which correlates with increased expression
of markers indicative of enhanced TGF-b signaling
(Baselga et al., 2008).
Although these studies show an important role for
TGF-b signaling in breast cancer metastasis to bone,
they do not identify the specific TGF-b isoforms that are
most important for this process. Notably, they do not
determine whether the primary source of TGF-b
required for the development of osteolytic metastases
originates from the bone matrix, resident bone cells or
the tumor cells themselves. To address these issues, we
have used a selective TGF-b isoform-neutralizing ligand
trap and TGF-b1-deficient mice to examine the relative
importance of tumor cell- or host-derived TGF-b in the
establishment and outgrowth of osteolytic lesions in
bone. Our results show that the removal of TGF-b1 and
TGF-b3 isoforms significantly impairs the progression
of breast cancer lesions in bone. Moreover, we
conclusively show that the bone microenvironment
provides a significant source of TGF-b1 and that its
removal imposes a selective pressure on breast cancer
cells to overexpress TGF-b isoforms, which promote the
formation of osteolytic bone lesions.
Results
TGF-b-neutralizing ligand trap impairs signaling induced
by TGF-b1 and TGF-b3
We first determined the expression pattern of each TGF-
b isoform in parental MDA-MB-231 breast cancer cells
that had been injected into the tibiae of athymic mice.
MDA-MB-231 cells readily form osteolytic lesions
(Figure 1a) that fill the marrow space (Figure 1b). We
readily detected serine phosphorylated Smad2 through-
out the tumor, suggesting that the TGF-b signaling
pathway was active in MDA-MB-231 cancer cells
(Figure 1c). Immunohistocytochemical analysis further
showed that MDA-MB-231 breast cancer cells predomi-
nately expressed TGF-b1, with more moderate levels of
TGF-b2 and very little TGF-b3 detected within
osteolytic lesions (Figures 1d–f).
To assess the importance of the TGF-b isoforms for
breast cancer cell outgrowth in bone, we first used a
soluble ligand trap composed of the ECD of TbRII
fused to the constant region of human IgG (Fc). This
fusion protein has previously been used to study the
impact of impaired TGF-b signaling on breast cancer
progression and metastasis (Mourskaia et al., 2007).
Pooled populations harboring an empty vector control
(VC) or the ligand trap (Fc:TbRII(ECD)) were estab-
lished in parental MDA-MB-231 cells possessing an
H&EPhospho-Smad-2
TGF-β1TGF-β2TGF-β3
X-Ray
Figure 1
the bone. (a) X-ray image illustrating an osteolytic lesion resulting from a tibial injection of MDA-MB-231 cells. Arrows indicate
regions of extensive bone resorption. (b) Hematoxylin and eosin stain of an MDA-MB-231 tumor growing in the marrow space, the
boxed area corresponds to the region displayed for immunohistocytochemical staining of phospho-Smad2 (c), TGF-b1 (d), TGF-b2
(e) and TGF-b3 (f). Scale bars represent 125mm (panel b) and 25mm (panels c–f), respectively.
Transforming growth factor (TGF)-b1 is the predominant isoform expressed in MDA-MB-231-derived osteolytic lesions in
TGF-b isoforms promote breast cancer outgrowth in bone
AA Mourskaia et al
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Oncogene

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imaging reporter system that includes firefly luciferase
(Minn et al., 2005). To test the efficacy of the ligand
trap, VC or Fc:TbRII(ECD)-expressing cells were
allowed to condition media and subsequently challenged
with increasing concentrations of TGF-b1, TGF-b2 or
TGF-b3 (Supplementary
showed a concentration-dependent increase in phos-
pho-Smad2 levels in response to stimulation by all three
TGF-b isoforms (Supplementary Figure S1a–c). Rela-
tive to VC cells, MDA-MB-231 cells expressing the
ligand trap were significantly diminished in their ability
to activate Smad2-mediated signaling in response
to both TGF-b1 and TGF-b3 (Supplementary Figure
S1a–c). However, the ligand trap was incapable of
neutralizing TGF-b2-induced signaling (Supplementary
Figure S1b). Immunoblot analysis further confirmed
that the fusion protein was expressed to similar levels by
trap-producing MDA-MB-231 cells, but was absent in
VC cells (Supplementary Figure S1a–c).
To confirm that the Fc:TbRII(ECD) fusion protein
functioned as a soluble ligand trap, we asked whether
conditioned media (CM) from VC and Fc:TbRII(ECD)-
expressing MDA-MB-231 cells could block TGF-b
isoform signaling in a TGF-b responsive cell line. To
perform this experiment, we used NMuMG cells, an
immortalized normal mouse mammary cell line, which
exhibits numerous TGF-b-induced responses (Northey
et al., 2008). NMuMG cells stimulated with 2ng/ml of
TGF-b1, TGF-b2 or TGF-b3, in the absence or
presence of CM (1:10 dilution) from MDA-MB-231
VC cells, responded with robust Smad2 phosphorylation
(Supplementary Figure S1d–f). However, CM (1:10
dilution) from Fc:TbRII(ECD)-expressing MDA-MB-
231 cells effectively blocked Smad2 activation by TGF-
b1 or TGF-b3, but remained ineffective against TGF-b2
(Supplementary Figure S1d–f). Immunoblot analyses of
whole cell lysates and CM, using an antibody specific to
the ECD of TbRII, confirmed expression of the ligand
trap (Supplementary Figure S1g). These results show
that the Fc:TbRII(ECD) fusion protein effectively
neutralizes both TGF-b1 and TGF-b3 isoforms but
fails to sequester TGF-b2.
FigureS1a–c). VCcells
Expression of the TGF-b ligand trap impairs breast tumor
growth in the mammary fat pad
We first determined whether expression of the ligand
trap altered the growth characteristics of MDA-MB-231
breast cancer cells, relative to VC cells. While VC and
Fc:TbRII(ECD)-expressing MDA-MB-231 cells grew at
the same rate in vitro (Supplementary Figure S2a),
a clear and significant reduction in the growth
of Fc:TbRII(ECD)-expressing tumors was observed in
the mammary fat pad (Supplementary Figure S2b).
Immunohistocytochemical analysis confirmed that the
expression of the ligand trap was absent in VC-derived
mammary tumors and maintained in Fc:TbRII(ECD)-
expressing mammary tumors in situ (Supplementary
Figure S3a). To investigate potential mechanisms for
reduced mammary tumor outgrowth in cells expressing
the ligand trap, we analysed the proliferative, angiogenic
and apoptotic indices of these tumors by Ki67, CD31
and TUNEL staining, respectively. We did not observe
statistically significant differences in any of these
parameters between Fc:TbRII(ECD) and VC-derived
end-stage mammary tumors (Supplementary Figure
S3b–d). However, this does not preclude the possibility
that these parameters are adversely affected by the
ligand trap at earlier stages of tumor outgrowth.
Neutralization of TGF-b1 and TGF-b3 isoforms within
the bone microenvironment impairs the formation of
osteolytic lesions
We next determined the effects of ligand trap expression
on the ability of breast cancer cells to grow in the bone,
following direct tibial injection. The tibial injection model
was specifically chosen to focus our investigations on the
importance of TGF-b isoforms in mediating tumor cell
interactions with the bone microenvironment, thereby
avoiding possible TGF-b effects on tumor cell survival
in circulation, endothelial adhesion and extravasation.
Expression of the ligand trap significantly diminished the
growth of osteolytic lesions within the bone, as measured
by longitudinal bioluminescence imaging and X-ray
imaging at end point (Figures 2a and b). While 100% of
tibiae developed progressively growing lesions following
injection of either VC or Fc:TbRII(ECD)-expressing
MDA-MB-231 cells, the average normalized photon flux
of lesions expressing the ligand trap (4.65?108p/s/cm2/sr)
was approximately one-third the intensity compared with
lesions derived from VC cells (1.45?109p/s/cm2/sr) at 28
days post-injection (Figure 2a). Moreover, analysis of
blinded X-rays showed that the osteolytic lesion area was
reduced threefold in mice injected with Fc:TbRII(ECD)-
expressing MDA-MB-231 cells (7.7% of tibia occupied by
osteolytic lesions) compared with animals receiving VC
cells (21.4% of tibia occupied by osteolytic lesions)
(Figure 2b).
Immunohistocytochemical staining with an antibody
against the ECD of TbRII confirms that breast cancer
lesions retain expression of the ligand trap in vivo
(Supplementary Figure S4a). Examination of breast
tumor cell proliferation, survival and recruitment of
vasculature within osteolytic bone lesions failed to
show statistically significant differences between VC
and Fc:TbRII(ECD)-expressing MDA-MB-231 cells
(Supplementary Figure S4b–d). Although the mechan-
isms remain unclear, these results indicate that neutra-
lization of TGF-b1 and TGF-b3 isoforms significantly
impairs the outgrowth of breast cancer cells in bone.
Transforming growth factor-b is known to induce the
expression of factors important for osteoclast differ-
entiation and function, such as PTHrP and IL-11 (Yin
et al., 1999; Kang et al., 2003, 2005; Deckers et al.,
2006). Thus, we investigated whether expression of the
neutralizing ligand trap could blunt TGF-b-induced
expression of these two factors. Vector control (VC)
or Fc:TbRII(ECD)-expressing MDA-MB-231 breast
cancer cells were allowed to condition serum free media
for 48h and subsequently incubated in the absence or
presence of TGF-b1 for a further 3h. Quantitative
TGF-b isoforms promote breast cancer outgrowth in bone
AA Mourskaia et al
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reverse transcription (RT)–PCR analysis showed that
PTHrP expression was induced 7.7-fold in VC cells,
but only 4.4-fold in ligand trap-producing cells follow-
ing TGF-b1 stimulation (Figure 2c, left panel). Like-
wise, IL-11 mRNA expression was induced 3.9-fold in
MDA-MB-231 VC cells in response to TGF-b1, which
dropped to a 3.1-fold induction in MDA-MB-231 cells
expressing the Fc:TbRII(ECD) fusion (Figure 2c, right
panel). TGF-b-induced expression of both PTHrP and
IL-11
wasrestoredin
MDA-MB-231 cells, to the levels seen in MDA-MB-
231 VC cells, when the ligand trap was removed before
TGF-b1 stimulation (Figure 2c). Thus, the diminished
capacity of MDA-MB-231 cells expressing a TGF-b-
neutralizing ligand trap correlated with a statistically
significant reduction in TGF-b-induced PTHrP and
IL-11 expression.
Fc:TbRII(ECD)-expressing
Loss or reduction of TGF-b1 levels in host-derived tissue
prevents the efficient establishment of breast cancer cells
in bone
To specifically interrogate the importance of bone-
derived TGF-b1 on the ability of breast cancer cells to
form osteolytic lesions, we used TGF-b1-deficient mice
that were bred onto a RAG2-deficient background
(Engle et al., 1999), which rescues the early lethality
observed in TGF-b1-null mice due to widespread
inflammatory disease (Shull et al., 1992; Kulkarni
et al., 1993). To ensure that the RAG2?/? mice
permitted xenografting of human breast cancer cells,
we injected an MDA-MB-231 variant that aggressively
metastasizes to bone (1833-TR cells) into mammary
fat pads of RAG2?/? mice (Kang et al., 2003; Minn
et al., 2005). Primary mammary tumors established
and grew in 100% of TGF-b1þ/þ; RAG2?/?,
MDA-MB-231 –Osteolytic Destruction in Tibia
0
2
4
6
8
10
12
Relative PTHrP
mRNA Expression
7.7X
4.4X
7.1X
**
*
0
1
2
3
4
Relative IL-11
mRNA Expression
-+
VC
Yes
Fc
Yes
Fc
No
-+-+
3.9X
3.1X
5.9X
*
*
-+
VC
Yes
Fc
Yes
Fc
No
TGF-β (3h)
-+-+
CM
VCFc
% Tibia Occupied by
Osteolytic Lesion
VC
Fc
*
0
10
20
30
40
50
VC
Fc:TβRII(ECD)
MDA-MB-231 – Tumor Outgrowth in Tibia
p/sec/cm2/sr (x109)
Days Post-Injection
0.0
0.5
1.0
1.5
2.0
2.5
7142128
*
Figure 2
in bone. (a) 1?105vector control (VC) or Fc:TbRII(ECD)-expressing MDA-MB-231 cells were injected directly into the tibia of
athymic female mice. Breast cancer cell outgrowth in bone was measured by bioluminescence imaging and quantified as the normalized
photon flux (p/s/cm2/sr) in each tibia over time. The averages (±s.d.) from 19 independent tibias are plotted (*Po0.0001) for each
group. (b) Osteolytic destruction was measured in the tibiae of mice injected with VC or Fc:TbRII(ECD)-expressing MDA-MB-231
cells using blinded digital X-ray images. The data are presented as the percentage of the tibia occupied by osteolytic lesions (graph)
(*Po0.0001). Representative X-ray images (day 28) are shown with arrows indicating regions of osteolytic bone resorption (right
panels). (c) Expression of transforming growth factor (TGF)-b ligand trap diminishes TGF-b-induced expression of genes shown to be
important for the formation of osteolytic bone metastasis. Quantitative reverse transcription (RT)–PCR was performed for PTHrP
(left graph) and IL-11 (right graph). VC and Fc:TbRII(ECD)-expressing MDA-MB-231 cells were allowed to condition media for 48h
before a 3h incubation in the absence (?) or presence (þ) of 5ng/ml TGF-b1. Fc:TbRII(ECD)-expressing MDA-MB-231 cultures
were changed to fresh media to remove any accumulated ligand trap and subjected to a 3h incubation in the absence or presence of
TGF-b1. The expression levels of each target gene were first normalized to b-actin and presented at a fold change relative to VC
(?TGF-b1) (±s.d.). The data represents the average of two independent experiments, each performed in triplicate. TGF-b1-induced
fold changes in PTHrP and IL-11 expression were found to be statistically different in the absence of the ligand trap compared with
conditions when the trap is present (*Po0.0001; **P¼0.0008).
MDA-MB-231 breast cancer cells expressing Fc:TbRII(ECD) show reduced outgrowth and formation of osteolytic lesions
TGF-b isoforms promote breast cancer outgrowth in bone
AA Mourskaia et al
1008
Oncogene