Role of TGF-<i>β</i> in breast cancer bone metastases

doi:10.4236/abb.2013.410A4003

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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

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,

OPEN ACCESS

A. Chiechi et al. / Advances in Bioscience and Biotechnology 4 (2013) 15-30

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

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

A. Chiechi et al. / Advances in Bioscience and Biotechnology 4 (2013) 15-30

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

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-

A. Chiechi et al. / Advances in Bioscience and Biotechnology 4 (2013) 15-30

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-

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.

A. Chiechi et al. / Advances in Bioscience and Biotechnology 4 (2013) 15-30

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-

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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