Advances in Bioscience and Biotechnology, 2013, 4, 15-30 ABB
http://dx.doi.org/10.4236/abb.2013.410A4003 Published Online October 2013 (http://www.scirp.org/journal/abb/)
Role of TGF-β in breast cancer bone metastases
Antonella Chiechi1, David L. Waning1, Keith R. Stayrook1, Jeroen T. Buijs1,2, Theresa A. Guise1,
Khalid S. Mohammad1
1Division of Endocrinology, Department of Internal Medicine, Indiana University, Indianapolis, USA
2Department of Urology, Medical Center, Leiden University, Leiden, The Netherlands
Email: kmohamma@iu.edu
Received 29 July 2013; revised 29 August 2013; accepted 15 September 2013
Copyright © 2013 Antonella Chiechi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Breast cancer is the most prevalent cancer among fe-
males worldwide leading to approximately 350,000
deaths each year. It has long been known that cancers
preferentially metastasize to particular organs, and
bone metastases occur in ~70% of patients with ad-
vanced breast cancer. Breast cancer bone metastases
are predominantly osteolytic and accompanied by in-
creased fracture risk, pain, nerve compression and
hypercalcemia, causing severe morbidity. In the bone
matrix, transforming growth factor-β (TGF-β) is one
of the most abundant growth factors, which is re-
leased in active form upon tumor-induced osteoclastic
bone resorption. TGF-β, in turn, stimulates bone me-
tastatic tumor cells to secrete factors that further
drive osteolytic bone destruction adjacent to the tu-
mor. Thus, TGF-β is a crucial factor responsible for
driving the feed-forward vicious cycle of cancer growth
in bone. Moreover, TGF-β activates epithelial-to-me-
senchymal transition, increases tumor cell invasive-
ness and angiogenesis and induces immunosuppres-
sion. Blocking the TGF-β signaling pathway to inter-
rupt this vicious cycle between breast cancer and bone
offers a promising target for therapeutic intervention
to decrease skeletal metastasis. This review will de-
scribe the role of TGF-β in breast cancer and bone
metastasis, and pre-clinical and clinical data will be
evaluated for the potential use of TGF-β inhibitors in
clinical practice to treat breast cancer bone metasta-
ses.
Keywords: Transforming Growth Factor-Beta; TGF-β;
Breast Cancer; Bone Metastasis; Bone; Small Molecule
Inhibitors; Antibodies; Bone Resorption
1. INTRODUCTION
Cancer is the leading cause of death in economically de-
veloped countries. Breast cancer is the most frequently
diagnosed cancer in females accounting for 23% (1.38
million) of the total new cancer cases and is the leading
cause of cancer death among females worldwide, [1,2]. It
is estimated that there are nearly 3 million women living
in the United States with a history of invasive breast can-
cer and it is estimated that over 226,870 new cases of in-
vasive breast cancer were diagnosed in 2012 [3]. Breast
cancer frequently metastasizes to the skeleton, and ap-
proximately 70% of patients with advanced breast will
develop bone metastases [4-6].
Patients with bone metastases are at risk of skeletal
complications, including spinal cord compression, pain,
pathological fracture, hypercalcemia, complications due
to surgery to bone, and radiation therapy. These comor-
bidities are known collectively as skeletal-related events
(SREs). SREs are associated with impaired mobility, re-
duced quality of life, increased mortality, and higher health-
care costs [7]. Standard antiresorptive treatments decrease
skeletal morbidity and delay skeletal related events (SRE),
but do not cause regression or cure the disease [6,8].
Cancer patients who develop bone metastases, particular-
ly those with breast and prostate cancer, can survive for
many years after diagnosis, during which they will suffer
significant morbidity. That is why better treatments are
needed to achieve the long-term goal of preventing or cu-
ring bone metastases.
The bone microenvironment is unique and provides
fertile soil for cancers to thrive. Many growth factors and
cytokines are embedded in the mineralized bone matrix
and are released during osteoclastic bone resorption. Tran-
sforming growth factor-β (TGF-β) is the most abundant
of these factors. The TGF-β superfamily also includes
other factors involved in bone homeostasis including: ac-
tivins, inhibins, and bone morphogeneticproteins (BMPs).
TGF-β that is released from bone is activated by either
proteolytic cleavage, interaction with integrins, or pH
changes in the local microenvironment [9]. In addition,
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TGF-β stimulates tumor production of pre-osteolytic and
osteolytic factors that stimulate further bone resorption
[10,11]. This categorizes TGF-β as an important factor
responsible for driving the feed-forward vicious cycle of
tumor growth in bone. Therefore blocking TGF-β release,
its production and/or signaling is a promising strategy to
treat bone metastasis. Over the past several years, several
therapeutic strategies have been developed to inhibit TGF-
β, including, TGF-β receptor kinase inhibitors, TGF-β
neutralizing antibodies, soluble receptor decoys (Fc fu-
sions) and TGF-β antisense oligonucleotides [12]. Many
of these are now in early-stage clinical trials for various
disease indications with particular emphasis as potential
cancer therapies, including bone metastases. In this re-
view, we will focus on the role of TGF-β in breast cancer
and bone metastasis and discuss the potential use of no-
vel TGF-β inhibiting compounds and biologics in clinical
practice to treat bone metastases.
2. TGF-β STRUCTURE AND SIGNALING
2.1. TGF-β Structure
TGF-β was originally named for its ability to induce ma-
lignant behavior of normal fibroblasts. It is ubiquitously
expressed in normal developing and adult tissues. It is a
multifunctional cytokine that controls tissue homeostasis
by regulating cellular processes such as apoptosis, pro-
liferation and differentiation [13]. TGF-β orchestrates the
response to tissue injury and mediates repair by inducing
epithelial-to-mesenchymal transition (EMT) and cell mi-
gration, and it is a critical regulator of the immune res-
ponse. Dysregulation of TGF-β functions have been as-
sociated with many disorders, including chronic fibrosis,
cardiovascular diseases and cancer [14,15].
The TGF-β superfamily includes more than 30 protein
ligands divided into subfamilies based on sequence simi-
larity and function. Members of the TGF-β superfamily
are TGF-βs, bone morphogenetic proteins (BMPs), acti-
vins, inhibins, growth and differentiation factors (GDFs),
NODAL and anti-Müllerian hormone (AMH) [16-18]. The
ligands are all synthesized as precursors with a large N-
terminal pro-domain necessary for the correct protein
folding and dimerization. After cleavage, the mature li-
gands form homodimers or heterodimers held together
by disulfide bonds. In some cases, the prodomain is still
associated with the mature protein after secretion via a
non-covalent association. TGF-β is secreted as a latent
precursor: After secretion the pro-domain (latency asso-
ciated protein, LAP) binds and inactivate the ligand, al-
lowing its association with inhibitory latent TGF binding
proteins (LTBPs) that target the complex to the ECM
where the latent TGF-β is sequestered. In humans, three
isoforms of TGF-β have been described, TGF-β1, TGF-
β2 and TGF-β3. The signaling of these three isoforms is
comparable but their expression level differs across tis-
sue types [19]. Signaling mediated by TGF-β ligands is
transduced through cell surface recaptor complexes of
two distinct types of transmembrane serine-threonine ki-
nases, the type I and type II receptors. Seven type I re-
ceptors (Activin-recaptor like kinases, ALKs, 1-7) and
five type II receptors are known in vertebrates. The li-
gand binds a type II receptor, which phosphorylates a
partner type I receptor, which in turn propagates the sig-
nal inside the cell via phosphorylation of downstream
Smad-dependent and -independent processes [20].
2.2. Smad-Mediated Signaling
In vertebrates, eight Smad proteins are known (Smad 1-
8). Smads 1, 2, 3, 5 and 8 are the receptor-associated
Smads or R-Smads. While Smad1/5/8 are phosphorylat-
ed by ALK1/2/3/6 upon BMP or GDF activation, Smad2/
3 are phosphorylated by ALK4/5/7 following TGF-β,
NODAL or Activin signaling [21]. Active TGF-β binds
TGF-β receptor type II (TβRII), which recruits and acti-
vates ALK5. ALK5 phosphorylates R-Smad2/3, which
form a heterodimeric complex with the common media-
tor Smad (co-Smad or Smad4) and translocate to the nu-
cleus [18,20]. Once in the nucleus, the Smad complex
acts as a transcription factor able to bind chromatin and
modulate its structure. To achieve a high binding affinity
for the Smad-binding elements (SBE) in the TGF-β tar-
get gene promoters, the Smad complex associates with
other transcription factors [22,23]. Various families of
transcription factors, such as forkhead, homeobox, zinc
finger, AP1, Ets and basic helix-loop-helix, are Smad part-
ners [23]. Moreover, the Smad complex recruits co-ac-
tivators, such as p300 and CREB binding protein, or co-
repressors, such as retinoblastoma-like 1 protein, to regu-
late gene transcription [18,20,23]. Therefore, while Smad
proteins are intrinsically transcriptional activators, the
transcriptional outcome of their target genes often de-
pends on the transcriptional partners associated with
Smads [24].
More recently, a novel arm of TGF-β signaling has
been discovered in which ALK5 activates the R-Smads,
Smad1/5, leading to TGF-β-induced anchorage-indepen-
dent growth and cell migration [25,26]. Furthermore,
TGF-β can alternatively activate the R-Smads, Smad1/5/
8 via the TβRI ALK1, which is mainly expressed by en-
dothelial cells [27]. In fact, TGF-β/ALK1 signaling po-
tentiates and TGF-β/ALK5 signaling inhibits endothelial
cell proliferation and migration [28,29].
2.3. Smad-Independent Signaling
In addition to the Smad-mediated signaling, TGFβ can
also activate Smad-independent signaling pathways through
the interaction and association with alternative mediator
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A. Chiechi et al. / Advances in Bioscience and Biotechnology 4 (2013) 15-30 17
proteins [30].
TGF-β can induce mitogen activated protein (MAP)
kinase signaling, including extracellular signal regulated
kinases (Erk1 and 2), p38 and c-Jun amino-terminal ki-
nase (JNK) MAP kinases. The activation of Erk MAP ki-
nase requires the recruitment and phosphorylation of the
adaptor protein Shc, which will in turn associate with the
adaptor protein Grb2 and the GTP exchange factor SOS
[31]. This protein complex activates Ras to its GTP-bound
form, and the kinase cascade consisting of c-Raf, MEK1
or MEK2, and Erk1 or Erk2. TGF-β also induces activa-
tion of p38 and JNK MAP kinase pathway through the
tumor necrosis factor (TNF) receptor-associated factor 6
(TRAF6) and TAK1. TRAF6 interacts with the TGF-β
receptor complex and auto-ubiquitylates and become ac-
tive. Active TRAF6 associates with TAK1, causing poly-
ubiquitylation and phosphorylation of TAK1. Active
TAK in turn activate p38 MAP kinase and JNK [32,33].
Furthermore, TGF-β receptor complexes interact with the
polarity protein Par6 and the tight junction protein oc-
cludin at epithelial cell junctions. Here, Par6 is phospho-
rylated by the receptor complex, and associates with
Smurf1. The Par6-Smurf1 complex confers ubiquityla-
tion of RhoA and the consequent dissociation of tight
junctions. The interaction of occludin with TβRI is re-
quired for the localization of TbRI to tight junctions, a
prerequisite for efficient TGF-β-induced dissolution of
tight junctions during epithelial-mesenchymal transition
[34]. Thus, the dynamic combination of canonical and
non-canonical signaling cascades is responsible for the
cellular responses to TGFβ signaling.
2.4. TGF-β Signaling and
Epithelial-Mesenchymal Transition (EMT)
TGF-β acts as a common and potent inducer of EMT via
Smad-dependent and independent activation of the ex-
pression of the EMT transcription factors Snail, Slug,
ZEB1 and 2, and Twist [35,36]. The Smad3/4 complex
directly binds the regulatory portion of the promoter of
Snail, inducing its transcription. Subsequently a Smad3/
4/Snail complex is formed that binds the regulatory pro-
moter sequences of genes encoding for E-cadherin and
occludin, leading to repression of their expression [37].
Smad signaling also increases the expression of ZEB
transcription factors, which repress miR-200 family ex-
pression, further increasing ZEB protein levels and EMT
[38]. TGF-β also regulates the expression of MMP2 and
9, and ECM components (i.e. fibronectin and collagens)
[39]. Moreover, TGF-β can activate EMT transcription
factor expression via alternative splicing [40].
EMT is also controlled by a group of microRNAs that
define changes in cytoskeleton reorganization and epi-
thetlial polarity, and it is directly activated in response to
TGF-β via the Smad/RhoA pathway [41].
Smad-independent TGF-β signaling pathways, such as
the PI3K/Akt/mTOR pathway, result in increased protein
synthesis and cell motility and invasion during EMT.
TGF-β also induces EMT through ubiquitylation and su-
moylation. Smad3/4 complex regulates the expression of
HDM2, increasing the ubiquitylation and degradation of
p53, inducing EMT progression [42]. TGF-β signaling
downregulates the expression of the SUMO E3 ligase
PIAS1, reducing the levels of sumoylated SnoN, and an-
tagonist of TGF-β mediated EMT [43].
3. TGF-β IN BREAST CANCER
PROGRESSION
TGF-β plays an essential role in maintaining homeostasis
in many tissues through its ability to induce cell cycle ar-
rest, differentiation and apoptosis, thereby preventing un-
controlled proliferation of epithelial, endothelial and he-
matopoietic cells. It is considered the most potent growth
inhibitor for epithelial, hematopoietic and immune cells,
because of its ability to induce cell cycle arrest, differen-
tiation and apoptosis, preventing uncontrolled prolifera-
tion of these cells [44,45]. However, in many cancers
TGF-β signaling is compromised, because of the genetic
loss of some of the pathway components or due to the
downstream influence of other signaling pathways. Hence,
these tumors become refractory to TGF-β growth inhibi-
tion and the pro-tumorigenic actions of TGF-β may pre-
vail, including immunosuppression, induction of angio-
genesis and promotion of the EMT, thus facilitating can-
cer migration and invasion (reviewed in [27,46,47]).
3.1. Dual Role of TGF-β in Breast Cancer
Progression
Transgenic mouse models have been particularly infor-
mative to understand the roles of TGF-β in mammary
gland development and tumor progression. Three inde-
pendent studies tried to inhibit TGF-β signaling in mam-
mary tissue by using the mammary gland selective mouse
mammary tumor virus (MMTV) promoter to drive the
expression of either a soluble TβRII:Fc fusion protein
[48], a dominant negative TβRII (DNTβRII) [49] or full
length TβRII antisense [50]. A proliferative mammary
gland phenotype was observed in all models, consistent
with the homeostatic role of TGF-β, while spontaneous
mammary tumors developed only in the DNTβRII trans-
genic model, but these were mostly carcinoma in situ,
and arose after a prolonged latency [49].
In two additional studies, transgenic mice expressing
the activated neu gene in the mammary gland were cross-
ed with strains that expressed either active TGF-β1 or
constitutively active TβRI/ALK5 [51,52]. A markedly
delayed primary tumor development was observed in
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18
both cases, and tumor growth was slower than in neu
single transgenic mice, underpinning a tumor suppress-
ing role for TGF-β [51,52]. Nevertheless, the carcinomas
that did arise in the double transgenic models were more
invasive and aggressive than those occurring in MMTV-
neu single transgenics. Zakharchenko et al. identified
two novel TGFβ-dependent phosphorylation sites of 14-
3-3σ, Ser69 and Ser74. They found 14-3-3σ phosphory-
lation to be a feed-forward mechanism in TGFβ/Smad3-
dependent transcription, therefore TGFβ-dependent 14-3-
3σ phosphorylation may facilitate the formation of the
protein complexes, including Smad3 and p53, at the
Smad3-specific CAGA element. Also, breast tumor mouse
xenograft and radiobiological assays suggested the in-
volvement of phosphorylation of 14-3-3σ at Ser69 and
Ser74 in the cancer progenitor population regulation and
the radioresistance in breast cancer MCF7 cells. This
study suggests that TGFβ-dependent phosphorylation of
14-3-3σ may play a role in the maintenance of cancer
stem cells [53]. Recently, it was demonstrated that TGF-
β1 down-regulated the junction adhesion molecule A
(JAM-A) expression via its effects on both the transcrip-
tional and post-translational regulations of JAM-A thus
attenuating cell adhesion and promoting cell invasion
and that the effect of TGF-β was achieved via the activa-
tion of Smads [54]. Also, a recent study identifies miR-
155-mediated loss of C/EBPβ as the mechanism that
shifts TGF-β response in breast cancer from growth inhi-
bition to EMT, invasion and metastasis, promoting breast
cancer progression. C/EBPβ seems to work as a tran-
scriptional activator of genes encoding the epithelial
junction proteins E-cadherin and coxsackie virus and
adenovirus receptor [55]. These and other [56,57] studies
have provided strong support for a tumor-suppressive
role for epithelial TGF-β signaling in mammary gland tu-
morigenesis. However, while TGF-β may inhibit the
growth of mammary tumors in the early stages, it also
appears, in these models, to enhance the metastatic po-
tential of those carcinomas that are able to overcome the
TGF-β-dependent growth suppression and develop.
3.2. TGF-β Expression Levels in Human Breast
Cancer
When the TGF-β suppressive effects are lost, TGF-β
overproduction is commonly observed in many solid
tumors. TGF-β expression level is often higher in breast
cancer compared to normal mammary gland tissue and it
appears to increase in the advanced stages of tumor pro-
gression [58-60]. Moreover, TGF-β expression levels
correlate with prognosis and angiogenesis in breast can-
cer patients [61]. Plasma TGF-β1 expression has also
been found increased in breast cancer patients, and its
level correlates with disease stage [62-65]. Plasma TGF-
β1 levels have a prognostic value also after tumor resec-
tion: patients whose plasma TGF-β1 levels normalized
after surgery had a better prognosis than those patients
with persistently elevated levels, who had higher risk of
lymph node metastases and disease progression [64]. These
data may suggest an important causal role for TGF-β in
metastases and disease progression.
Plasma TGF-β1 levels have also been determined in
49 bone metastasis patients, including 23 breast cancer
patients, and were reported to be elevated in more than
half of the cancer patients and positively correlated with
TGF-β signaling related markers, including parathyroid
thyroid hormone-related peptide (PTHrP) and interleukin
10(IL-10) [66]. A recent study shows that elevated circu-
lating levels of TGF-β and CXCL1 are associated with a
poor prognosis, and higher detection of circulating tumor
cells and propensity of these cells to seed lung metasta-
ses in patients with breast cancer [67].
TGF-β plasma levels may be indicative of TGF-β-de-
pendent metastatic disease and may be useful biomarkers
to predict the success of treatment with TGF-β antago-
nists in metastatic disease. Ongoing clinical trials are
trying to answer these questions. In addition, a highly sig-
nificant association between TβRII expression and re-
duced survival has been detected in patients bearing es-
trogen receptor negative breast cancer [68]. Richardsen
et al. recently published immunohistochemical data from
38 cancer patients: high TGF-β levels can be detected in
both primary and metastatic tumors and high stromal
TGF-β expression is associated with increased mortality
[69].
Due to TGF-β dual nature in breast cancer, its use as
single tumor marker that might distinguish patients with
high risk of metastases is unlikely. Molecules involved in
the TGF-β downstream signaling play an important role
in determining TGF-β prognostic implication, as shown
in a retrospective cohort study in patients with invasive
non-metastatic breast cancer. High expression of Smad4
showed a trend for better prognosis while high expres-
sion of pphosphorylated-Smad2 was associated with
poor prognosis [70].
3.3. TGF-β and Breast Cancer Ste m Cells
An increasing body of basic and clinical studies have
provided evidence of self-renewing, stem/progenitor-like
cells within solid tumors, which have also been referred
to as cancer stem cells (CSCs) [71-77]. CSCs are beliv-
ed to constitute a small minority of neoplastic cells with-
in a given tumor and are defined by their ability to pro-
pagate a tumor and potentially seed new metastases [74].
The concept of CSCs remarks the importance of target-
ing the correct cell population in cancer therapy in order
to obtain better results in terms of survival and tumor re-
lapse. Conventional treatments aim to eliminate the rap-
idly dividing cells in a tumor, leaving space and time to
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A. Chiechi et al. / Advances in Bioscience and Biotechnology 4 (2013) 15-30 19
the slower proliferating, less differentiated CSCs to re-
populate the tumor [78].
By sorting breast cancer cells for a normal mammary
stem cell phenotype (CD44+/CD24/low), Al-Hajj et al.
was the first to isolate the breast CSC fraction [71]. More
recently, Shipitsin et al. demonstrated that vimentin, con-
nective tissue growth factor (CTGF), PAI-1, osteonectin,
as well as TβRII were coexpressed with CD44 [79]. In
fact, many of the genes actively transcribed by CD44+
cells were associated with a mesenchymal phenotype and
many were known TGF-β target genes. They were also
able to associate this gene signature with poor prognosis
[79]. Mani et al. demonstrated that EMT generates cells
with properties of stem cells [80]. TGF-β-induced EMT
in immortalized human mammary epithetlial cells (HMEC)
was associated with the acquisition of the CD44+/
CD24/low phenotype and mesenchymal traits, and in-
creased ability to form mammospheres, a property asso-
ciated with mammary epithelial stem cells [81]. Para-
crine and autocrine signals induce and maintain mesen-
chymal and stem cell states in the breast [81]. In addition,
forcing EMT by overexpressing the EMT transcription
factors (and TGF-β target genes) SNAIL1 or TWIST also
resulted in a CD44+/CD24/low phenotype that displayed
enhanced tumorigenic potential when injected in mice. In
a recent study, the loss of DUSP4, a downstream mole-
cule in the TGF-β apoptosis signaling pathway, was shown
to increase mammosphere formation and the expression
of the CSC-promoting cytokines IL-6 and IL-8 in a mo-
del of basal-like breast cancer (BLBC). These effects
were caused in part by loss of control of the MEK and
JNK pathways and involved downstream activation of
the ETS-1 and c-JUN transcription factors. Enforced ex-
pression of DUSP4 reduced the CD44+/CD24 popula-
tion in multiple BLBC cell lines in a MEK-dependent
manner, limiting tumor formation, again underpinning the
dual role of TGF-β in breast cancer [82]. Taken together,
these studies provide evidence that TGF-β is important in
regulating the dynamics of cancer cell populations by
favoring CSC selfrenewal and inhibiting the commitment
to differentiation.
4. TGF-Β, BREAST CANCER AND BONE
METASTASIS
4.1. Normal Bone Physiology
Bone is primarily made of type I collagen that is miner-
alized by hydroxyapatite. By weight, bone is about 60%
mineral, 10% water and 30% organic matrix. Mineral are
made of hydroxyapatite crystals a naturally occurring
calcium phosphate. The organic matrix is 98% type I col-
lagen and 2% noncollagenous protein. The noncollage-
nous proteins include growth factors and cytokines, and
extracellular matrix proteins such as osteonectin, osteo-
pontin, bone sialoprotein, osteocalcin and proteoglycans.
Although noncollangenous components make small con-
tributions to the overall bone volume it represents major
contributions to its biologic function. Growth factors and
cytokines such as transforming growth factor-β (TGF-β),
bone morphogenetic proteins (BMPs) osteoprotegerin
(OPG), insulin-like growth factor (IGF), interferon-γ, the
tumor necrosis factors (TNFs) and the interleukins (ILs),
are present in very small quantities in bone matrix but
have critical effects regulating bone cell differentiation,
activation and growth.
The cellular component of bone that is associated with
the bone homeostasis the bone resorbing osteoclasts, the
bone forming osteoblasts and the cell embedded in the
bone matrix, the osteocytes. Osteoclasts arise from he-
matopoietic progenitors that also give rise to mono-
cyte/macrophages lineage. The osteoclast precursor cells
are recruited to the bone surface where they fuse to form
large multinucleated cell. The interaction of the osteo-
clast precursors with the stromal cell and the osteoblasts,
in the presence of several intermediary factors such as
PTH, Vitamin-D , IL-6, IL-11 and PGE2, induces the
central mediator of osteoclast differentiation the RANKL.
RANKL or receptor activator of NFκB is a membrane
bound member of the TNF receptor family expressed at
the osteoblast surface. RANKL binds to its receptor,
RANK, which is expressed on the surface of osteoclast
precursor. The interaction of the RANKL with its recap-
tor RANK stimulates the osteoclast differentiation.
RANKL/RANK knockout mice develop severe osteo-
petrosis due to total lack of osteoclast [83,84]. OPG is
another TNF superfamliy member, it a soluble factor that
act as a decoy receptor for RANKL. When RANKL binds
to OPG it prevents the interaction of RANKL with RANK
and inhibits osteoclast activation. OPG knockout mice
develop osteoporosis as a result of increase in number of
osteoclast.
The bone forming osteoblasts are derived from mes-
enchymal stem cells, pluripotent cells that can differenti-
ate into a variety of cell types including myoblasts, adi-
pocytes, chondrocytes, osteoblasts, and osteocytes.
Osteoblasts are the bone cells that secrete the organic
matrix within which several growth factors are embed-
ded. Runx2 and Osterix are two transcription factors that
are required for osteoblast formation and differentiation.
The regulatory activity of these central osteoblast regu-
lators is modified by cofactors including members of the
Dlx (distaless), Msx, and Hox homeodomain gene fami-
lies and downstream signal transduction mediators such
as the TGF-β superfamily-related SMADs. As active os-
teoblasts produce bone matrix (osteoid), they become
embedded into their own product and at this stage it is
called an osteocyte.
Osteocytes make up to 95% of all bone cells. Osteo-
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cytes create an interconnected network in bone allowing
for intercellular communications between each other and
the surface-lining osteoblasts [85]. Osteocyte senses me-
chanical load through their canalicular processes and ini-
tiate a series of biochemical signaling events that coor-
dinate and influence the activity of osteoprogenitor cell,
osteoblasts and osteoclasts, which in turn respond by
remodeling bone mass [86,87]. Sclerostin, a secreted pro-
tein expressed by osteocytes, responds to mechanical load.
Sclerostin plays a central role in the anabolic response of
bone to mechanical. Mechanical loads repress Sclerostin
mRNA and protein expression thus, releasing the inhibi-
tion on new bone synthesis [88].
Bone is a dynamic structure and adult bone is con-
tinuously remodeled by the coordinated activities of bone-
resorbing osteoclasts and bone-forming osteoblasts [89].
The continuous remodeling process is necessary to re-
place defective bone as well as to release calcium for va-
rious metabolic processes It is the balance between the
osteoclasts and osteoblast activity is what keep constant
bone mass and the disruption of this balance will result is
significant pathological condition such as osteoporosis or
osteopetrosis.
4.2. TGF-β and Bone Interaction
The involvement of growth factors, cytokines and cell
adhesion molecules in the remodeling process is what
makes bone an attractive site for cancer metastases.
TGF-β1 is one of the most abundant growth factors in
bone matrix [90]. It is an essential factor for bone re-
modeling and can affect both bone formation and resorp-
tion. The effects of TGF-β on osteoblast, osteoclasts and
bone remodeling are complex and are both spatial and
temporal-dependent [91]. Bone is resorbed by osteoclasts
and when the resorption process is completed, a reversal
period follows after which osteoblasts deposit new bone
matrix to fill the resorption cavity, a process known as
coupling. The newly deposited collagenous matrix will
be mineralized following a resting phase.
Evidence is accumulating that TGF-β is a key media-
tor in coupling bone resorption to bone formation [92].
Osteoblasts secrete TGF-β, where it is embedded into the
mineralized bone matrix [93,94]. TGF-β is stored in the
bone matrix in a latent form. Upon bone resorption by
osteoclasts, TGF-β is release and activated which in turn
activates the proliferation of osteoblast precursor which
migrates to the sites of bone resorption [95]. The exposed
bone mineral matrix and release of osteotropic factors,
such as bone morphogenetic proteins (BMPs), insulin
growth factor (IGF)-I and -II, and platelet derived growth
factor (PDGF), may then promote differentiation of the
osteoblast precursor to osteoblasts [96]. It was shown
that TGF-β block osteoblast differentiation and bone mi-
neralization in later phases of osteoblastic differentiation
[97]. In a coculture of osteoclast precursors with osteo-
blast and stromal cells, TGF-β was shown to inhibit re-
sorption factors such as RANKL and M-CSF while acti-
vating the expression of osteoclast inhibitors such as
OPG [98,99].
TGF-β is a major regulator of osteoclast function ei-
ther directly or indirectly through its effect on osteoblast.
The importance of TGF-β on osteoclastogenesis is clear
but the exact mechanism is unclear. During bone resorp-
tion osteoclasts secrete cathepsins, which proteolytically
release activate TGF-β from the latent complex [100,101]
and because osteoclast express both TGF-β and its re-
ceptors they can respond directly to TGF-β signaling.
TGF-β can inhibit the recruitment of osteoclast precur-
sors in fetal bone culture but enhances bone resorption
by stimulating proliferation and differentiation of osteo-
clast precursors. TGF-β also enhances osteoblast lineage
RANKL expression, thus promoting osteoclast precursor
recruitment [102].
It has been recently reported by Nguyen, et al., that
mechanical load rapidly represses the net activity of the
TGF-β pathway in osteocytes. This result in reduced
phosphorylation and activity Smad2 and Smad3 thus com-
promises the anabolic response of bone to mechanical
load, demonstrating that the mechanosensitive regulation
of TGF-β signaling is essential for load-induced bone for-
mation [103].
4.3. TGF-β and Osteolityc Bone Metastases
Bone is a common site of dissemination for breast cancer.
The bone microenvironment consists of a rich store of
multiple growth factors including TGF-β. The metaphy-
seal bone, which is predominantly composed of trabecu-
lar bone and is highly vascular, appears to be the prefer-
red site for bone metastases. Bone metastases develop in
about 70% of patients with advanced breast cancer. This
is usually a late complication of cancer that can lead to
debilitating skeletal related events such as pain, fractures,
hypercalcemia and nerve compression which reduce the
patient’s quality of life [5,6].
Metastasis to bone is a complete multistep process of
events that involves an interaction between the tumor
and the host cells. This multifaceted process consists of a
series of steps whereby cancer cells detach from the pri-
mary tumor, enter into the circulation, disseminate to dis-
tal bone sinusoids, enter the bone marrow by extravasa-
tion, adapt to the new microenvironment, and eventually
grow into lethal tumor which colonies the bone [104].
In bone metastasis biopsies from patients with breast
cancer, 75% show positive nuclear staining for phospho-
rylated-Smad2, as seen on histological sections, indicat-
ing an active TGF-β signaling [105].
It has been well established in the literature that TGF-
Copyright © 2013 SciRes. OPEN ACCESS
A. Chiechi et al. / Advances in Bioscience and Biotechnology 4 (2013) 15-30 21
β signaling pathway play an important role for the de-
velopment of bone metastases. Several studies uncovered
a complicated and context dependent picture regarding
the function and utility of TGF-β. In an animal model of
breast cancer bone metastases, MDA-231 cells were trans-
duced with a retroviral vector expressing a reporter gene
under the control of a TGF-β-sensitive promoter. In this
experiment, it was demonstrated that using this reporter,
active TGF-β-Smad signaling specifically in the bone
was detected and that Knockdown of Smad4 expression
in breast cancer cells reduced the growth of bone metas-
tases [105]. In another bone metastases model the ex-
pression of inhibitory Smad7 dramatically decrease bone
metastases in 1205Lu melanoma models, further impli-
cating role of TGF-β in the bone metastases process
[106].
TGF-β is able to promote and aggravate bone metas-
tases through specific gene inductions. The TGF-β-Smad
signaling pathway induces the production of proosteo-
lytic factors, such as interleukin 11 (IL11), connective
tissue growth factor (CTGF), matrix metalloproteinase-1
(MMP-1), CXCR4 and parathyroid hormone-related pro-
tein (PTHrP) [107]. PTHrP is widely expressed in many
tissues and shares sequence homology with PTH. It is
known to be expressed in most primary breast cancers
tumors as well as in bone metastases. PTHrP plays a
major role in the development of the osteolytic lesions
and is considered to be responsible for the humoral hy-
percalcemia of malignancy [108]. In a large prospective
study it was demonstrated that PTHrP expression in pri-
mary breast cancer was significantly associated with less
bone metastases [109-111]. This study could give the ex-
planation of the observed increase in PTHrP expression
in breast cancer bone metastases, which is, it is the re-
lease of TGF-β from the bone matrix after bone resorp-
tion is what causes the cancer cells to express PTHrP and
not the tumor cells that colonized the bone intrinsically
express higher PTHrP level. In mouse model of bone
metastases, it was first demonstrated by Yin et al. that
blocking TGF-β signaling by stably transfecting a domi-
nant negative TβRII (DNTβRII), in MDA-231 breast can-
cer cells, inhibited TGF-β-induced expression of PTHrP
production in tumor cells. This is in return suppressed the
development of osteolytic lesion area [11]. In another
study, I was reported that stable overexpression of do-
minant-negative Smad 2, 3 and 4 in MDA-231 breast
cancer cells resulted in decrease in PTHrP production
[112]. TGF-β-induced PTHrP stimulated the production
of RANKL and downregulating OPG thus inducing os-
teoclast differentiation and activation and promoting bone
metastases [113]. IL-11 and CTGF both is pro-osteolytic
gene. IL-11 stimulates the expression of osteoclastogenic
factors RANKL and GM-CSF in osteoblasts and stimu-
lating bone resorption. CTGF is an extracellular mediator
of invasion and angiogenesis. Both, IL-11 and CTGF are
shown to be directly regulated by TGF-β via the canoni-
cal TGF-β/Smad pathway in metastatic cells [10] (Fig-
ure 1).
Hypoxia is observed in most solid tumors due to low
oxygen concentration [114]. A major mechanism media-
ting adaptive hypoxia is the regulation of transcription by
hypoxia-inducible factor 1 (HIF-1). The bone microenvi-
ronment is known to be hypoxic with an oxygen level
between 1% and 7% [114]. The enhanced expression and
activation of (HIFs) frequently occur during cancer pro-
gression and is associated with their acquisition of a
more malignant behavior, and hypoxic cells are also con-
sidered to be resistant to most anticancer drugs partially
due to upregulation of genes involved in drug resistance
[115-117].
It was previously shown that HIF-1α promote forma-
tion of osteolytic bone metastases from breast cancer cell,
MDA-MB-231, and that was through stimulating angio-
genesis, osteoclastogenesis and inhibition of differentia-
tion of osteoblasts [118]. Multiple interactions exist be-
tween hypoxia and TGF-β biology. HIF-1a degradation is
inhibited by TGF-β causing it stabilization. In vitro data
showed an additive responses to HIF-1α and TGF- β in
the induction of vascular endothelial growth factor
(VEGF) and CXCR4 [119,120]. In an animal model of
breast cancer bone metastases, inhibition of HIF-1α or
TGF-β by either knock down or DNTβRII causes signi-
ficant reduction in metastases formation with no addi-
tive effect when blocked simultaneously [120]. A com-
bined pharmacological inhibition of both HIF-1α and
TGF-β, which targets both cancer cells and bone micro-
environment had an additive effect more than either
Figure 1. Breast cancer bone metastases. When active TGF-β is
released from the bone matrix upon bone resorption by osteo-
clasts it acts on breast cancer cells to stimulate the production
of osteolytic factors, such as parathyroid hormone-related pro-
tein (PTHrP), connective tissue growth factor (CTGF) and in-
terleukin- (IL) 6 and 11. These factors increase the RANKL/
OPG expression ratio in osteoblasts, which bind to the RANK
receptors expressed on osteoclasts and activate osteoclastoge-
nesis. TGF-β can directly stimulate osteoclast activity and inhi-
biting osteoblast differentiation thus, TGF-β can stimulate tu-
mor growth.
Copyright © 2013 SciRes. OPEN ACCESS
A. Chiechi et al. / Advances in Bioscience and Biotechnology 4 (2013) 15-30
22
treatments alone indicating that hypoxia and TGF-β sig-
naling drive in parallel tumor bone metastases and that
pharmacological inhibitors, by acting on both tumor cells
and the bone microenvironment, can additively decrease
tumor burden [120].
5. TGF-β AS THERAPEUTIC TARGET
As a result of its wide variety of effects, TGF-β signaling
provides many therapeutic opportunities for the treat-
ment of disease. The major classes of TGF-β inhibitors
that have been investigated include: (1) ligand traps, in-
cluding monoclonal neutralizing TGF-β antibodies and
soluble decoy receptor proteins; (2) receptor kinase inhi-
bitors, which inhibit TβRI/ALK5 (and TβRII) kinase ac-
tivity and prevent the downstream signaling; (3) anti-
sense oligonucleotides, which inhibit TGF-β expression
at the transcriptional/translational level.
5.1. Neutralizing Antibodies and Soluble Decoy
Receptor Proteins
TGF-β levels and downstream signaling is often increas-
ed during cancer progression and is correlated with ag-
gressiveness and grade/stage of the tumor [46,50,121,
122]. Reducing the amount of active TGF-β signaling is
achieved either via TGF-β ligand trap, which uses a so-
luble decoy receptor comprised of the TβRII or TβRIII
ectodomain, or via neutralizing TGF-β antibodies. Neu-
tralizing antibodies have been developed to target indi-
vidual ligands as well as all three TGF-β isomers (pan-
neutralizing antibody). The pan-neutralizing mouse mo-
noclonal antibodies, 1D11 and 2G7, bind and reduce bio-
logical activity of all three TGF-β isoforms and have de-
monstrated therapeutic potential in mouse tumor mod-
els. Treatment of mice harboring MCF-7 breast cancer
cells totally abrogated tumor growth [123] and suppress-
ed growth of established MDA-MB-231 sub-cutaneous
tumors and lung metastases in athymic mice [124]. Simi-
larly, treatment of mice with 1D 11 following orthotopic
injections of 4T1 breast cancer cells suppressed metasta-
sis to lungs [125-127]. 1D11 has also been shown to re-
duce skeletal tumor burden and osteolytic bone lesions
and increase bone volume caused by MDA-MB-231 cells
[128].
Another approach to prevent binding of TGF-β to its
receptors is the use of recombinant Fc-fusion proteins
containing the soluble ectodomains of TβRII or TβRIII.
These biologically active compounds have been shown
to reduce lung and breast cancer metastases in animal
models [31,121,129,130].
5.2. Antisense Oligonucleotides (ASO’s)
Antisense oligonucleotides (ASO’s) reduce expression of
specific target proteins. ASO’s are single-stranded poly-
nucleotide molecules 13 - 25 nucleotides in length that are
designed to hybridize to complementary RNA sequenc-
es. ASO’s inhibit mRNA function and protein synthesis
via modulation of splicing and inhibition of translation
[131,132]. ASO’s against TGF-β reduce the bioavailabil-
ity of active ligands in the local tumor microenvironment.
To address the role of autocrine TGF-β in metastasis for-
mation, Muraoka-Cook et al. used an orthotopic model
of PyMT mammary tumors [122]. While PyMT tumors
overexpressing TGF-β resulted in increased metastasis
and survival, overexpression of a TGF-β ASO reduced
metastasis and survival [122].
5.3. Small Molecule Receptor Kinase Inhibitors
TGF-β receptor kinase inhibitors are small molecule in-
hibitors that act via ATP-competitive inhibition of the
kinase catalytic activity of TβRI/ALK5. There are advan-
tages to the development and scalability of small mole-
cule inhibitors but the potential lack of selectivity of ki-
nase inhibitors is problematic. Currently, all known small
molecule TβR1/ALK5 inhibitors described display equi-
potent inhibition against ALK4 kinase activity and less
inhibition against ALK7 [133-137].
Inhibitors of TβRI/ALK5 have been extensively stud-
ied including: SB-431542 [136] (GlaxoSmithKline),
Ki26894 (Kirin Brewery Company) [138], LY364947
(Eli Lilly & Co.), and SD-208 and SD-093 (Scios Inc).
Each of these compounds blocks receptor kinase activity
and inhibits proliferation, invasion or metastasis of tumor
cells in animal models [134-136]. In a xenograft model
of intracardiac inoculated MDA-MB-231 human breast
cancer cells for example, SD-208 significantly inhibited
the size of osteolytic lesions, bone metastatic growth and
survival. Furthermore, SD-208 treatment in mice with al-
ready established bone metastases inhibited further tu-
mor growth and formation of osteolytic lesions [120]. The
same treatment was shown to increase bone mass in non-
tumor model which could be of mutual bebefits for can-
cer patients reduced osteolytic lesion and increasing bone
mass [139].
5.4. Other Molecules that Antagonize TGF-β
Additional biologically active molecules that inactivate
TGF-β or its signaling have also been described. The na-
tural product derivative halofuginone (Hfg) recently com-
pleted phase II clinical trials for the treatment of sarcoma
[140]. We recently published that Hfg inhibits TGF-β
signaling in vitro in several cell types, and that systemic
daily treatment of Hfg in mice significantly inhibits the
formation of osteolytic lesions and bone metastases after
intracardiac inoculation melanoma1205Lu [141]. Although
the exact mechanism remains to be investigated, Hfg
treatment represents a novel agent to inhibit TGF-β sig-
Copyright © 2013 SciRes. OPEN ACCESS
A. Chiechi et al. / Advances in Bioscience and Biotechnology 4 (2013) 15-30 23
naling in bone metastasis.
5.5. Combination Therapy
An attractive approach to increase treatment efficacy for
patients with bone metastases is to combine treatments
that antagonize the effects of TGF-β with other therapies.
For example, targeting TGF-β signaling can enhance the
therapeutic efficacy of various cytotoxic agents as was
recently shown for rapamycin [142] and doxorubicin
[143,144]. Studies in our laboratory show that SD-208
dosed in combination with an inhibitor of bone resorp-
tion, zoledronic acid, reduces the progression of establi-
shed osteolytic metastases from breast cancer more ef-
fectively than either therapy alone [145]. Using the same
bone metastasis model of intracardiac inoculation of MDA-
MB-231 breast cancer cells, we tested the effects of a
combined treatment of SD-208 and 2-methoxyestradiol,
an inhibitor of HIF-1α, the key mediator of hypoxia. Com-
bined treatment with these agents reduces osteolytic le-
sions, tumor burden and improves survival of mice more
effectively than either treatment alone [120].
5.6. Risks, Limitations and Opportunities
As a result of its biological importance and wide variety
of effect, blockade of TGF-β or its signaling provides in-
triguing therapeutic opportunities for the treatment of
many different disease indications. However, potent and/
or chronic inhibition of this wide-spread biologically im-
portant molecule may also potentially result in a variety
of undesirable side effects.
Drug delivery challenges must be overcome for ASO-
based therapies and large biological (neutralizing anti-
bodies). The generation of small molecules such as TGF-
β receptor kinase inhibitors overcomes the necessity of
injectable delivery, loss of efficacy due to neutralizing
antibody generation and/or tissue penetration issues com-
monly observed with biologic-based agents and most are
suitable for oral dosing [12]. However, TGF-β recaptor ki-
nase inhibitors used so far are less selective than the cur-
rent TGF-β ASO’s or biologic-based TGF-β-directed the-
rapies.
Other promising approaches to overcome off-target
tissue toxicity and poor drug exposure to tumor cells in
bone metastatic disease are the use of bisphosphonate to
deliver therapeutics directly to bone. One approach is a
bisphosphonate-coated liposome, which may be useful as
a targeting device to sites of high bone turnover, include-
ing sites with bone metastatic disease [146]. Another pos-
sibility is targeting anti-TGF-β therapies via conjugation
of small molecule inhibitors to bisphosphonates. Poten-
tially, these bone-targeted strategies may allow for a more
prolonged local exposure to higher concentrations of the
compounds thereby enhancing therapeutic efficacy and
minimizing systemic side effects. Additionally, these bi-
oactive compounds of interest could be delivered to bone
metastatic sites in combination with other anticancer agents
with synergistic or mechanistic action.
6. CONCLUSION
TGF-β is a pluripotent cytokine with a prominent role in
breast cancer progression and bone metastasis. TGF-β is
a central mediator in driving a feed-forward vicious cy-
cle of tumor growth in bone. Thus much effort has been
placed on development of agents to inhibit TGF-β activi-
ity. Currently, three therapeutic modalities targeting TGF-
β have been pursued and are presently being tested in
clinical trials in cancer patients (incl. bone metastatses):
TGF-β antibodies, TGF-β receptor kinase inhibitors and
TGF-β antisense oligonucleotides. TGF-β has many other
functions in normal physiology, and may also act as a
tumor suppressor in certain malignancies. Therefore con-
cerns will remain that long-term blockade of this path-
way may have other off-target effects. The next decade
should reveal new and exciting clinical data that will help
determine which TGF-β therapeutic strategies are most
effective for the treatment of patients.
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