Targeting RANKL in breast cancer: bone metastasis and beyond

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Breast cancer is a frank example of neoplasms that display a high affinity to grow in bone [1]. Unlike other tissues, bone is mainly composed of hard-mineralized tissue; hence it is more resist-ant to invasion and destruction by cancer cells compared with other metastatic sites [2]. Thus, cancer cells must possess the capacity to induce osteoclastic activation, which is the main cellular mechanism for cancer-induced bone destruction [3]. Osteoclastic activation will result in increased bone resorption and consequently the release of a large amount of active forms of TGF-β and IGF-II, along with other cytokines, which are normally stored within the mineralized bone matrix [4,5]. These factors can mediate cellular interaction – in a paracrine fashion – with cancer cells, which is a critical step in the development and progression of bone metastases.

RANKL/RANK pathway & osteoclastogenesisOsteoclasts have been described as the most efficient cells to induce bone resorption [6]. Osteoclastic activation is predominately con-trolled by three members of the TNF family of receptors and ligands known as RANK, RANK ligand (RANKL) and osteoprotegerin

(OPG) [7]. RANK is a surface receptor mainly expressed on mature osteoclasts and their pro-genitors [8]. Further evidence has also shown expression by breast cancer cells [9]. RANK is activated via binding to RANKL, which is expressed on the surface of osteoblasts and bone stromal cells. In the presence of macrophage-colony stimulating factor (M-CSF), RANKL binds to RANK on the pre and mature osteo-clasts and generates osteoclasts from hemat-opoietic cells [10]. Signaling through RANK would then activate transcription factors such as NF-κB, which leads to the differentiation of osteoclast progenitors, fusion of precursor cells and formation of mature activated osteo-clasts. RANK/RANKL interaction would also increase the survival of activated osteoclasts and limits their apoptosis.

Importantly, the stimulatory effects of RANKL on osteoclasts are opposed by OPG, which is also secreted by the osteoblasts and stromal cells, and functions as a soluble decoy receptor for RANKL [10]. OPG competes with RANK for RANKL, thus it prevents the RANKL–RANK interaction on the osteo-clast cell membrane, leading to cessation of osteoclastogenesis and bone resorption.

Hamdy Azim1 and Hatem A Azim Jr2*1Department of Clinical Oncology, Cairo University Hospital, Cairo, Egypt2Breast Cancer Translational Research Laboratory (BCTL) J.C. Heuson, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium*Author for correspondence: Tel.: +32 254 138 54 Fax: +32 541 34 77 [email protected]

In breast cancer, RANK ligand (RANKL) appears to play an important role in the process of chemotaxis between circulating tumor cells and the bone microenvironment, which enables RANK-expressing breast cancer cells to migrate into the bone. Mounting clinical evidence has further demonstrated that the anti-RANKL monoclonal antibody; denosumab is the most effective approach in the prevention of skeletal-related events. On the other hand, inhibiting RANKL in preclinical models, not only reduced breast cancer formation but also decreased the development of lung metastases, suggesting RANKL as a novel target for breast cancer chemoprevention. In addition, recent data have pointed to a potential role of RANKL in the biology of breast cancer arising at a young age. Hence, RANKL emerges as a key molecule, not only in the field of breast cancer bone metastasis but also in the biology of breast cancer as a whole.

Targeting RANKL in breast cancer: bone metastasis and beyondExpert Rev. Anticancer Ther. 13(2), 195–201 (2013)

Expert Review of Anticancer Therapy

© 2013 Expert Reviews Ltd

10.1586/ERA.12.177

1473-7140

1744-8328

Review

Keywords: bone resorption • bone metastasis • breast cancer biology • breast cancer in young women • RANK • RANKL

THEMED ARTICLE ❙ Breast Cancer

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Expert Rev. Anticancer Ther. 13(2), (2013)196

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RANKL/RANK pathway & the vicious cycle of bone destructionMost evidence indicates that breast cancer cells can induce osteo-clastic activation through the release of soluble mediators such as IL-1, IL-6, 1L-8, M-CSF, prostaglandin-E2, TNF-α and, most importantly, the parathyroid hormone-related protein (PTHrP) [11]. Most of these factors contribute to osteolytic lesions via RANKL upregulation by osteoblasts and stromal cells [10]. M-CSF serves as a cofactor for the RANKL-stimulated differ-entiation of hematopoietic precursors into active osteoclasts [10] (Figure 1). The widely accepted soil and seed model of osteolytic bone metastasis in breast cancer is based on the hypothesis that the TGF-β induces PTHrP production by the tumor. This causes stromal cells to secrete RANKL, leading to further increase in bone resorption and more release of TGF-β from bone. This results in ‘a switch on’ of a vicious cycle of bone destruction. Indeed, blocking the TGF-β-signaling inhibits PTHrP secretion by breast cancer cells, which subsequently suppress the develop-ment of bone metastases [12–14]. Moreover, enhanced resorption

of the mineralized bone matrix is also associated with the eleva-tion of the extracellular calcium [15]. It has been shown that increased extracellular calcium stimulates calcium-sensing recep-tors, which are expressed in approximately 30% of human breast cancers [16], to produce PTHrP, which would further enhance the process of PTHrP-induced osteoclastic activation [15]. IGF-I and -II are also released during the process of bone resorption [17,18] and were shown to promote breast cancer cell proliferation, invasion and angiogenesis [19]. Thus, it seems true that several products of bone resorption participate in the process of PTHrP-induced osteolysis, while others support the growth of breast cancer cells within the bone. The reciprocal feedback between tumor cells and the bone microenvironment has been described as the ‘vicious cycle of bone destruction’ [12,14], in which osteo-clasts are the key cellular players, whereas PTHrP and TGF-β are the major molecular co-players. Importantly, RANKL stands as the pivotal and final effector of osteoclastogenesis and hence targeting RANKL seems to be a very rational approach to treat bone metastases.

Targeting RANKL in the treatment of breast cancer bone metastasisEarlier attempts have tried to inhibit RANKL via increasing OPG levels. A Fc-OPG construct was developed by Amgen, however clinical development was stopped. Several limitations appeared from this agent, most importantly was its inhibi-tion to the TNF-related apoptosis inducing ligand (TRAIL) [20]. TRAIL is the princi-pal mediator of tumor cell death induced by the host immune cells [21]. Thus, bind-ing of pharmacological doses of OPG with TRAIL, may protect breast cancer cells from undergoing TRAIL-induced apop-tosis, which may bear a potential risk of tumor growth with the long-term use of the drug [22].

An alternative approach was to develop an antibody against RANKL, which would simulate the beneficial effects of OPG on bone health but avoiding any potential reaction with TRAIL [23]. In a recent large double-blind, randomized Phase III study involving 2049 women with meta-static breast cancer, the administration of a RANKL antibody; denosumab once every 4 weeks was compared with the standard 4-weekly zoledronic acid [24]. Denosumab was shown to significantly delay time to first skeletal-related event (HR: 0.82 [95% CI: 0.71–0.95]; p = 0.01), time to first and subsequent skeletal-related event (p = 0.001) and malignant hypercalcemia (p = 0.007). The apparent higher efficacy

Figure 1. RANKL/RANK pathway and the vicious cycle of bone destruction. When RANKL binds to RANK in the presence of M-CSF, osteoclast (OC) precursors differentiate and fuse together to form mature, multinucleated bone-resorbing OCs. Activated osteoclasts will then attach to the bone, secrete hydrogen ions that dissolve bone minerals, thus releasing calcium ions into the extracellular space .Osteoclasts also secret proteolytic enzymes like MMPs, collagenases, cathepsins and others to induce collagen degradation and digestion of the organic matrix. Large amounts of TGF-b and IGF II and other cytokines are stored within the mineralized bone matrix, and will be released during the process of OC bone resorption. When breast cancer cells colonize within the bone matrix, they start to secrete PTHrP and other osteolytic cytokines, which stimulate osteoblast production of RANKL while OPG levels are reduced, leading to enhanced osteoclastogenesis and increased bone resorption. This reciprocal feedback between tumor cells and the bone microenvironment has been referred to as the ‘vicious cycle’ of bone destruction. CFU-M: Colony-forming unit-macrophage; M-CSF: Macrophage-colony stimulating factor; MMP: Matrix metalloproteinase; OPG: Osteoprotegerin; PTHrP: Parathyroid hormone-related protein; RANKL: RANK ligand.

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of denosumab is probably attributed to the difference in the mechanism of action between RANKL inhibition and bispho-sphonates. Bisphosphonates act only when taken up by mature, actively resorting osteoclasts, and thus residual osteoclasts can still be observed in the bisphosphonate-treated bones [25]. On the other hand, RANKL inhibitors block the activation, survival and differentiation of osteoclasts from their precursors resulting in complete absence of osteoclasts in the treated bones [26].

Targeting RANKL for the prevention of bone metastasisA more intriguing aspect of targeting RANKL may be related to the prevention of bone metastases. Apart from its role in osteo-clastogenesis, RANKL plays also an important role in the pro-cess of chemotaxis between circulating tumor cells and the bone microenvironment. It has been suggested that the rich source of RANKL within the bone microenvironment may act as a chemo-tactic factor for the RANK-expressing cells to migrate to the bone [27]. Current evidence suggests that approximately 20–30% of breast cancers express RANK [27,28]. Indeed, the correlation of high RANK expression with osteotropism in murine models was demonstrated across many tumor cell types, including breast cancer [27,29].

It has been shown that RANKL inhibition by OPG results in marked reduction of the metastatic burden in bones but not in other organs [29]. This suggests that RANKL has an important role in the specific migration of ‘RANK-positive’ cells to bones. More recently, Santini et al. have provided the first clinical evi-dence on the role of RANK expression as a predictive marker of bone metastases in breast cancer [30]. In their study, which included 93 patients with early breast cancer, the ‘RANK-positive’ primary tumors had a significantly higher rate of bone metasta-sis compared with the ‘RANK-negative’ ones. In a multivariate analysis, RANK protein expression was associated with acceler-ated bone metastasis formation (p = 0.029). This finding substan-tiates the novel role of RANKL/RANK pathway in the process of skeletal migration in breast cancer, and suggests that RANKL targeting agents could serve as a rational approach to prevent bone metastases in the subpopulation of RANK-expressing early breast cancer patients. This was also shown to be promising in the preclinical setting. Tometsko et al. treated mice with either RANKL inhibitor (Fc-OPG) or zoledronic acid starting 2 days prior to inoculation with MDA231-RANK tumor cells [31]. Both treatments were associated with less bone metastases compared with no treatment, underscoring that inhibition of bone resorp-tion is able to prevent bone metastases. However, mice treated with Fc-OPG had a significant reduction of bone metastases compared with those treated by zoledronic acid. These obser-vations strongly suggest a potential role for denosumab in the prevention setting. A large Phase III trial evaluating the potential role of denosumab in reducing bone relapse in patients undergo-ing adjuvant therapy has recently completed accrual (D-CARE; ClinicalTrials.gov: NCT01077154 [101]). The study is placebo-controlled, and enrolled around 4500 patients with a primary end point being bone metastasis-free survival. However, we are not

aware whether a subgroup analysis according to RANK expres-sion on the primary tumor is preplanned in this trial, which we believe is highly intriguing.

The RANKL/RANK pathway & postmenopausal women bone lossAlthough remarkable progress has been made in the understand-ing of the impact of estrogen deficiency on bone loss, the mecha-nisms involved remain complex and multifaceted (reviewed in [32]). However, the fact that physiological doses of estrogen are able to decrease the production of bone resorption cytokines like IL-1, TNF-α, IL-6, M-CSF would indicate that the effects of estrogen on bone resorption are partly immune mediated [33].

It has been proposed that estrogen downregulates the produc-tion of these osteoclastogenic cytokines and that estrogen defi-ciency stimulates osteoclastic formation by increasing the T-cell production of RANKL and TNF-α [34–36]. This interaction between T cells and osteoclasts has opened a new field of research known as osteoimmunology, which is dedicated to the relation-ship between the immune processes and the bone metabolism (reviewed in [37,38]).

Similar to the situation in bone metastasis, it appears that RANKL is the final effector of osteoclastogenesis in postmeno-pausal bone loss and all other clinical situations associated with estrogen deficiency, including treatment with aromatase inhibi-tors. Cummings et al. enrolled 7868 postmenopausal women with osteoporosis and randomly assigned them to receive either 60 mg of denosumab or placebo subcutaneously every 6 months for 3 years [39]. Compared with placebo, denosumab reduced the 3-year incidence of new vertebral fractures from 7.2 to 2.3% (a 68% decrease), of hip fractures from 1.2 to 0.7% (a 40% decrease) and of all nonvertebral fractures from 8.0 to 6.5% (a 20% decrease). Similar results were also observed in breast cancer patients receiving aromatase inhibitors [40]. Interestingly, a significant increase in BMD was reported as early as 1 month and was observed at the distal third of the radius suggesting a positive effect on predominantly cortical bone sites, which are known to respond poorly to bisphosphonate treatment. The cortical effect of denosumab, coupled with the effect on trabecular bone, may translate into improved bone quality and strength.

The RANKL/RANK pathway & biology of the mammary glandShortly after the discovery of the bone-resorbing properties of RANKL, studies of genetically engineered mice demonstrated that RANKL/RANK signaling is also required for proper mor-phogenesis of the lactating mammary gland [41,42]. RANKL and its receptor RANK control the proliferation of mammary lobulo-alveolar cells during pregnancy through the inhibitor of NF-κB (IκB) kinase-a (IKK-a), a protein kinase that is needed for the self-renewal of the mammary cancer progenitors [43] (Figure 2). Later studies confirmed that RANKL is a key target gene of progesterone receptor transcription machinery and that RANKL/RANK system is an important molecular link between progesterone and mammary gland stem cell expansion and breast

Targeting RANKL in breast cancer


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carcinogenesis [44,45]. Collectively, these preclinical studies would certainly provide novel avenues for treatment and prevention of breast cancer.

RANKL as a key player in breast cancer biology & possible clinical implicationsThe link between progesterone and breast cancer risk has been emphasized during the last few years. Two large studies; the Women’s Health Initiative study and the Million Women Study, have shown that progestin-containing hormone replace-ment therapy and contraceptives are sig-nificantly associated with an increased the risk of developing breast cancer [46,47]. Importantly, concomitant administration of estrogens and progesterone results in increased breast cancer incidence, whereas administration of estrogen alone did not has significant effect. Preclinical evidence has pointed out that progesterone-induced breast cancer is mediated via its growth stimulatory effects on the mammary stem cell (MaSC) [45,48]. It has been hypothesized that the increased numbers and/or activity of MaSCs may predispose to mammary cancer by providing a greater target pool for malignant transformation. As MaSCs neg-atively express estrogen and progesterone receptors [49,50], the effect that progesterone experts on MaSCS appear to be mediated via indirect paracrine effectors [51].

RANKL has emerged as a progester-one receptor-regulated gene that is criti-cally involved in the expansion of MaSCs induced by progesterone (Figure 3). In vivo administration of medroxyprogesterone acetate resulted in massive induction of RANKL expression by the mammary epi-thelial cells [44]. Signaling through RANK triggers a massive proliferation of mam-mary epithelial cells and MaSC, driving these cells into the cell cycle and protects them from apoptosis. On the other hand, ablation of RANKL in the mammary epithelium blocks progesterone-induced mammary morphogenesis as well as cancer formation. The reduction in tumorigenesis upon RANKL inhibition was preceded by a reduction in preneoplasia as well as rapid and sustained reductions in hormone-induced mammary epithelial proliferation and cyclin-D1 levels.

Figure 2. RANK recruits adaptor molecules; TRAF to transduce the signal after ligand binding. TRAF binds to different regions in the cytoplasmic tail of the TNF family receptors and transduces the signal downstream. Signaling through TRAF will activate intracytoplasmic catalytic subunits, IKK-a and IkB-a and the noncatalytic subunit IKK. RANK-induced IKK-a activation is essential for cyclin D1 expression during mammary gland development. IAP: Inhibitor of apoptosis protein; IkB-a: IkB kinase-b; IKK-a: IkB kinase-a; MaSC: Mammary stem cell; MMP: Matrix metalloproteinase; PR: Progesterone receptor; RANKL: RANK ligand.

Figure 3. The potential role of RANK ligand in progesteronedriven breast cancer. On exposure to progesterone, the luminal cells express RANKL, which binds to RANK on the surface of the mammary stem cells. The increased numbers of mammary stem cells may predispose to mammary cancer by providing a greater target pool for malignant transformation. MaSC: Mammary stem cell; PR: Progesterone receptor; RANKL: RANK ligand.

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Another potential role of RANKL in breast cancer biology is its relation with the Treg lymphocytes. Treg lymphocytes have been shown to correlate with invasion, met-astatic spread and poor prognosis in breast cancer [52,53]. More recently, mice with breast cancer expressing Tregs were more likely to develop lung metastasis due to their enhanced expression of RANKL [54]. Metastatic spread of HER2-transformed carcinoma cells also required Treg cells, whose major prometastatic function was RANKL production [54]. The dependence of pulmonary metastasis on T cells was replaceable by exogenous RANKL, which also stimulated pulmonary metastasis of RANK receptor-positive human breast cancer cells. These results are consistent with the poor prognostic impact of tumor-infiltrating Treg cells in breast cancer and suggest that the targeting RANKL can be used in conjunction with the therapeutic elimination of primary breast tumors to prevent recurrent metastatic disease.

In the clinical setting, RANK and RANKL are expressed in the range of 20–30% and 20%, respectively on primary breast tumors [28]. A large in silico analysis has further shown that the expression of RANKL is apparently higher in breast cancer diagnosed in young women [55]. Using a linear regression model adjusted for tumor size, nodal status, histological grade and breast cancer subtype, decreasing age was significantly associated with higher expression of RANKL, MaSCs and luminal progenitors. This was observed in a training set of 1188 patients (p < 0.0001) and further confirmed in an independent set of 2334 patients (p < 0.0001). In the same study, the expression of RANKL and MaSCs were highly correlated. These results further support the relation between RANKL and MaSCs and provide some hypoth-esis on the aggressive nature of breast cancer arising in young women [55–58]

To put these f indings into context, a proof-of-concept Phase II study (D-BEYOND; EudraCT number 2011-006224-21) is currently starting in which premenopausal women with newly diagnosed breast cancer will receive two injections of denosumab prior to primary surgery (Figure 4). The aim is to understand the effect of denosumab on the different relevant biological processes that can guide tumor progression in young women, including proliferation, apoptosis, stem cells, immune reactions and others. This study is expected to start in March 2013 and would shed light on the potential role of RANKL signaling in managing breast cancer, apart from its role in bone metastases.

Expert commentaryRANKL, a TNF-related molecule, has long been reported to be an essential osteoclastic differentiation and activation factor

through interaction with its receptor RANK. Denosumab, a monoclonal antibody targeted against RANKL, has been shown to be noninferior to zoledronic acid in reducing skeletal-related events in several Phase III trials in advanced solid tumors includ-ing breast cancer. Recent data have further implicated RANK and RANKL in other cancer mechanisms. This includes medi-ating progestin-induced breast cancer and regulation of mam-mary stem cells. In the clinical setting, gene expression data suggest that RANKL expression is associated with breast cancer diagnosed in young women, with the highest expression being observed in pre-menopausal women. This underscores that tar-geting RANKL could open new avenues in managing young breast cancer patients.

Five-year viewThe role of denosumab in the adjuvant setting is currently being explored in the D-CARE trial. This will elucidate the potential role of denosumab in reducing bone metastasis. Also of interest will be the development of denosumab in breast cancer away from the bone metastasis field. The biological evidence strongly favors that this drug could be effective in the early setting and chemo-prevention. The results of the D-BEYOND trial are expected to be available by the end of 2014. This will reveal the relevance of RANKL as a molecular driver of breast cancer diagnosed in young women.

Financial & competing interests disclosureH Azim received honoraria from Amgen. HA Azim Jr received research support from Amgen. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Figure 4. The design of the D-BEYOND trial. ER: Estrogen receptor.



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Key issues

• The activation of the osteoclast is predominately controlled by RANK, RANK ligand (RANKL) and osteoprotegerin.

• RANKL is the main key player in the vicious cycle of bone resorption.

• Denosumab is superior to zoledronic acid in preventing osteoporosis and reducing skeletal-related events secondary to breast cancer-related bone metastases.

• RANKL signaling may be critical in maintenance of the normal mammary gland, breast cancer initiation and progression.

• Inhibition of RANKL was shown to attenuate breast tumor development both in hormonal dependent and independent models.

• RANKL signaling is the key mediator of the increase in the mammary stem cell population.

• RANKL appears to be relevant in the biology of breast cancer arising in young women and the role of denosumab in this patient population is currently under study.

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101 ClinicalTrials.gov. www.clinicaltrials.gov/


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Targeting RANKL in breast cancer: bone metastasis and beyond