Cancer Treatment Reviews 41 (2015) 61–68

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Tumor Review

Crosstalk between bone niche and immune system: Osteoimmunology signaling as a potential target for cancer treatment Carmen Criscitiello a,⇑, Giulia Viale a, Lucia Gelao a, Angela Esposito a, Michele De Laurentiis b, Sabino De Placido c, Michele Santangelo d, Aron Goldhirsch a, Giuseppe Curigliano a a

Division of Experimental Therapeutics, Breast Cancer Program, Istituto Europeo di Oncologia, Via Ripamonti 435, 20133 Milano, Italy Department of Breast Oncology, National Cancer Institute ‘‘Fondazione Pascale’’, Naples, Italy Department of Endocrinology and Molecular and Clinical Oncology, University of Naples Federico II, Napoli, Italy d Department of Advanced Medical Sciences, Operative Unit of General Surgery and Transplants, University of Naples Federico II, Napoli, Italy b c

a r t i c l e

i n f o

Article history: Received 22 July 2014 Received in revised form 26 November 2014 Accepted 1 December 2014

Keywords: Osteoimmunology Cancer Bone Immune system RANK

a b s t r a c t There is a well recognized link between the bone and the immune system and in recent years there has been a major effort to elucidate the multiple functions of the molecules expressed in both bone and immune cells. Several molecules that were initially identified and studied in the immune system have been shown to have essential functions also in the bone. An interdisciplinary field embracing immune and bone biology has been brought together and called ‘‘osteoimmunology’’. The co-regulation of the skeletal and immune systems strikingly exemplifies the extreme complexity of such an interaction. Their interdependency must be considered in designing therapeutic approaches for either of the two systems. In other words, it is necessary to think of the osteoimmune system as a complex physiological unit. Denosumab was originally introduced to specifically target bone resorption, but it is now under evaluation for its effect on the long term immune response. Similarly, our current and still growing knowledge of the intimate link between the immune system and bone will be beneficial for the safety of drugs targeting either of these integrated systems. Given the large number of molecules exerting functions on both the skeletal and immune systems, osteoimmunological understanding is becoming increasingly important. Both bone and immune systems are frequently disrupted in cancer; and they may be crucial in regulating tumor growth and progression. Some therapies – such as bisphosphonates and receptor activator of NF-jB ligand (RANKL) targeted drugs – that aim at reducing pathologic osteolysis in cancer may interact with the immune system, thus providing potential favorable effects on survival. Ó 2014 Elsevier Ltd. All rights reserved.

Introduction Accumulating evidence over the past decade suggests a model of extensive interaction between immune system and bone. It has recently been found that this complex connection plays an important role also in cancer growth and spread to the skeletal sysAbbreviations: RANK, activator of NF-jB; RANKL, RANK ligand; OPG, osteoprotegerin; TRANCE, TNF-related activation-induced cytokine; TNF, tumor necrosis factor; IL, interleukin; DC, dendritic cell; DTCs, disseminated tumor cells; SREs, skeletal-related events; ZOL, zoledronic acid; TRAIL, TNF related apoptosis inducing ligand. ⇑ Corresponding author at: Division of Early Drug Development for Innovative Therapies, Istituto Europeo di Oncologia, Via Ripamonti 435, 20141 Milano, Italy. Tel.: +39 02 57489788; fax: +39 02 57489581. E-mail address: [email protected] (C. Criscitiello). URL: http://www.ieo.it (C. Criscitiello). http://dx.doi.org/10.1016/j.ctrv.2014.12.001 0305-7372/Ó 2014 Elsevier Ltd. All rights reserved.

tem and, in turn, that tumor cells can alter and disrupt both immune and bone systems. Interaction between the bone and the immune systems occurs both in physiologic and pathologic conditions: osteoclastogenesis and hematopoiesis take place in the bone marrow. Immune cells and bone cells have common precursors: cytokines, receptors, adaptor proteins, signaling molecules and transcriptional factors are shared by both systems [1]. Osteoimmunology’’ can be defined as a multidisciplinary area of research focusing on the molecular understanding of the complex interplay between bone and immune system. Many steps forward were made thanks to the discovery of the Receptor Activator of NF-jB (RANK)/RANK Ligand (RANKL)/Osteoprotegerin (OPG) system in the mid-1990s [2]. RANK/RANKL/OPG pathway plays a critical role in regulating osteoclastogenesis; in addition, it greatly influences immune,

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cardiovascular, endocrine, and nervous systems. In particular, RANKL signaling affects the immune system by regulating antigen-specific immune responses and the interaction between T cells and dendritic cells, allowing the immune system to recognize and destroy abnormal cells and non-self antigens [3]. The RANK pathway biological function in osteoclastogenesis and immune system regulation is shown in Fig. 1. Recent studies in hematologic and solid malignancies indicate that targeting the bone microenvironment could have important therapeutic implications. Moreover, currently available drugs – designed to reduce pathologic osteolysis that may occur in cancer – may interact with the immune system, with potential beneficial effects on survival. In this review, we discuss the essential role of signaling transduction pathways and transcription factors in bone and immune system. Also, we focus on the complexity and overlapping cellular and molecular interactions between the immune and bone systems. RANK/RANKL/OPG pathway in bone and immunity Physiological bone turnover requires a continuous interaction between osteoclasts and osteoblasts and the RANK/RANKL/OPG pathway plays a crucial role during the process of bone formation and resorption. RANK receptor is a type I membrane protein, mainly expressed on the surface of mature osteoclasts and their precursors, involved in their activation upon ligand binding, both in physiologic and pathologic conditions [4]. RANKL is a member of the tumor necrosis factor (TNF) cytokine family, a type-2 membrane-bound protein, expressed by stromal cells, osteoblasts, and immune cells or released in soluble form in the bone microenvironment [5]. RANKL, by binding RANK, induces osteoclast maturation, activation and survival. Under physiological conditions, RANKL/RANK pathway is antagonized by OPG, a soluble decoy receptor, member of the TNF superfamily, secreted by osteoblasts and osteogenic stromal stem cells, which competes with RANK for interaction with RANKL [6]. Hence, RANKL/OPG ratio is an

Stromal cell

+

Osteoblast/ osteocyte

+

Osteoblast/ osteocyte

Stromal cell

Acvated T-cell

RANKL

+

important determinant for bone mass and skeletal integrity and enables the continuous remodeling of the bone matrix [7]. The critical role of RANKL/RANK pathway in osteoclastogenesis is demonstrated by experimental studies showing that knockout mice lacking either RANK or RANKL developed osteopetrosis due to the absence of bone resorption [8]. Expression of RANKL by osteoclastogenesis-supporting cells occurs in response to osteoclastogenic factors, such as parathyroid hormone, interleukin (IL)-1, IL6 and TNFa [9]. RANK signal transduction is mediated by TNF receptor-associated factors (TRAFs), in particular TRAF6, which activates PI3K, c-Src, Akt/PKB and mTOR among others, and subsequently different transcription factors including NF-jB [10,11]. TRAF6 seems to play a critical role in osteoclast as well as in dendritic cell (DC) maturation and activation. Moreover, RANKL stimulation activates reactive oxygen species (ROS) production, which are supposed to be important second messengers during osteoclastogenesis [12]. The dysregulation of the physiological equilibrium in the RANK/RANKL/OPG pathway leads to the pathological remodeling associated with cancer and to the development of bone metastasis. Tumor cells release growth factors and/or cytokines into the bone microenvironment, thus stimulating the production of RANKL from osteoblasts. RANK stimulation by RANKL leads to the differentiation of preosteoclasts into active osteoclasts, with resorption of the mineralized bone matrix, which results in releasing factors promoting colonization and tumor growth [13]. Hence, the RANK/RANKL/OPG pathway constitutes an important target for the treatment of cancer bone metastasis. In addition to its biological function in osteoclastogenesis, RANK–RANKL–OPG signaling plays an important role in the development and regulation of the immune system as summarized in Table 1. Indeed it is essential in lymph-node organogenesis, in T and B lymphocyte development and in T cell self tolerance induction (along with CD40L-CD40) [14,15]. In addition, RANKL, mainly expressed on activated CD4+ and CD8+ T cells, may directly stimulate lymphocytes and DC survival, proliferation and function [10,16]. Moreover, autocrine RANKL/RANK signaling seems to play

RANKL OPG

+

-

-

Dendric cell

Osteoclast Differenaon Acvaon

bone resorpon

Survival Maturaon

-

No bone resorpon

Fig. 1. RANKL is found on the surface of stromal cells, osteoblasts, and T cells. The interaction between RANKL and its receptor regulates osteoclast differentiation and activation both normally and in a variety of pathologic conditions associated with increased bone resorption, as rheumatoid arthritis. Indeed activated T lymphocytes expressing RANKL promote osteolysis directly, and indirectly by stimulating stromal cells. OPG, secreted by osteoblasts and osteogenic stromal stem cells, protects the skeleton from excessive bone resorption by binding to RANKL and preventing it from interacting with RANK. RANK is also expressed on dendritic cell surface, facilitating their survival and maturation. RANK = receptor activator of nuclear factor kB; RANKL = RANK ligand; OPG = osteoprotegerin.

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Table 1 Interaction between immune system and bone. Bone and immune system  RANKL system plays an important role in secondary lymphoid organ development [5]  RANKL expressed by T-helper cells is involved in dendritic cell maturation and survival [85]  RANKL regulates T cell-dependent immune response and can modify indirectly the number of regulatory T cells (Treg), thus maintaining peripheral tolerance [85]

an important role in immature DC survival [17] and probably in monocyte/macrophage survival, function and antigen presentation [18]. Similarly to its role in bone turnover, OPG antagonizes RANKL/RANK effects on the immune system, decreasing T cell proliferation [19]. Also, OPG directly influences DC survival and function, reducing cytokine production in response to inflammatory stimuli [20]. Despite the important role of RANK/RANKL/OPG signaling in immune system, the clinical importance of genetic alterations in RANKL/RANK pathway in immunity is to be fully elucidated yet [8,21]. Immune and bone systems display a deep interaction, besides RANKL/RANK/OPG signaling pathway. In preclinical studies, activated T cells (in particular the recently identified Th17 cells) are able to induce osteoclastogenesis directly through RANKL expression, and indirectly through the release of IL-17 that induces RANKL expression on mesenchymal cells [22– 24]. Indeed recent evidence from clinical trials suggests potential therapeutic implications for IL17/Th17 inhibition in a large spectrum of chronic inflammatory diseases [25]. Bone cells, including osteoblasts, osteoclasts and osteocytes participate in the regulation of the immune system. Osteoblasts, as well as perivascular stromal cells and endothelial cells, are components of the hematopoietic stem cell (HSC) niche and regulate HSC maintenance [26,27]. When activated by T cell-derived IFNc and CD40L, osteoblasts effectively suppress T cell proliferation in response to microbial agents, in a feedback loop, thus acting as inducible immunosuppressive cells [28]. Disruption of the skeletal and immune system in cancer Besides the established evidence that cancer frequently alters and disrupts both bone and immune systems, recent studies suggest that bone microenvironment and immune system could also play an active role in promoting tumor growth and progression. Indeed, tumor growth and metastases necessitate evasion of the immune system, in particular of T cells, which are able to identify and destroy cancer cells. Disseminated tumor cells (DTCs), responsible for early metastases of solid tumors, are frequently detected in patients’ bone marrow, where the ‘‘hematopoietic niche’’ hides them from anticancer therapies and possibly also an anticancer immune response [1]. In addition, for the formation of overt metastases, tumor cells need to be edited to reduce their immunogenicity and escape the immune recognition. However, most studies on the mechanisms of breast cancer metastasis to bone utilize immunodeficient mice, thus excluding the regulatory activity of the immune system on the metastatic course [29]. Bidwell and colleagues showed that interferon regulatory factors seem to be suppressed in bone metastases and their restoration leads to suppression of mammary tumors spreading to bone. This phenomenon is caused by activation of tumor immune surveillance mechanisms and elegantly showed that an innate immune pathway in tumor cells is fundamental for the outcome of metastasis [30].

 Bone homeostasis is influenced by the immune system in physiological and particular in pathological conditions, in which infiltrating T cells and other mononuclear cells release factors, including RANKL, leading to an increase of osteoclastogenesis and bone loss [3]  Bone, mainly by factors released by osteoblasts, provides an ideal anatomic microenvironment for hematopoietic stem cells maintenance and differentiation [86]  Long lived memory B and T cells (expressing RANKL) return to specialized niches in the bone marrow, suggesting that bone continues to play a role in adaptive immunity [87]

Cancer cells proliferating in the bone are able to influence the microenvironment, by stimulating osteoclastogenesis and bone remodeling. Increased bone turnover causes the release of different growth factors and cytokines, including RANKL, which attract other cancer cells and promote their proliferation; hence, the recruited tumor cells promote osteoclast-mediated osteolysis, as shown in Fig. 2. This vicious cycle of tumor growth and bone destruction may also lead to localized immunosuppression and recruitment of metastases-supporting tumor-associated macrophages [31]. Moreover it has recently been reported that RANKL is a chemotactic factor for osteoclasts and for RANK-expressing tumor cells, thus facilitating metastatization [32]. On the contrary, in experimental mouse models, OPG was shown to inhibit tumor-induced osteolysis and decrease skeletal tumor burden [33]. In addition to factors released by bone and cancer cells, enhanced osteoclastogenic activity in patients with bone metastases was shown to be directly related to T cell involvement [34,35]. In fact, proliferation and differentiation of osteoclast precursors seems to be stimulated by osteoclastogenic factors, including TNF and RANKL, released by T cells. IL 7, released mainly by B cells in the bone microenvironment seems to play a critical role, since it directly sensitizes T cells to produce pro-osteoclastogenic factors. Moreover T cells seem to modulate tumor growth within bone: their activation decreases bone metastases, whereas their depletion favors cancer cell proliferation [36]. This molecule could be a potential early serum marker for bone metastases development in solid tumors and it is currently under evaluation in non-small cell lung cancer [37]. The important role played by the immune system in cancer metastases may also represent an interesting therapeutic target; for example anti-TGFb therapy, used as anti-bone metastatic treatment, may activate a T cell response against the tumor, since TGFb released in the bone marrow by osteoclast activity inhibits T cells proliferation [38]. Furthermore, immune cells can participate in antitumor responses, for example macrophages mediate the elimination of circulating tumor cells, during monoclonal antibody therapy [39]. The key role of RANKL/RANK/OPG pathway in tumor growth and progression has been recently demonstrated in patients with neuroendocrine tumors (NET) and bone metastases enrolled on a clinical trial [40]. These patients, compared with patients with NET but with no bone metastases, have higher serum levels of RANKL/OPG ratio and lower OPG levels. Thus, specific alterations of RANKL/ RANK/OPG signaling could be used as early markers of bone metastases development [40]. In addition to its role in promoting bone metastases, RANKL/RANK/OPG pathway has been recently identified as prognostic factor in earlier stages of cancer. Indeed, it has been shown that high levels of RANKL and increased RANKL/OPG ratio are independent predictors of early biochemical recurrence in patients undergoing radical prostatectomy for localized prostate cancer [41]. It is still unclear how RANKL might affect the biological behavior of prostate cancer. An activation of NFjB signaling leading to proliferation, tumor cell migration and invasion has been hypothesized. However, an increased activation of osteoclasts

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1

X Tumor cell

Chemotherapy

X Immune cell

2

stromal cell

X

No osteoblast differenaon

Osteoclast precursor

Osteoclast Differenaon and survival

RANK RANKL 3

Fig. 2. Interactions between bone and cancer cells. (1) The ‘‘hematopoietic niche’’ harbors cancer cells against immune system and anticancer therapy. (2) Tumor cells in the bone release different factors such as PTHrP, IL1, IL6, that stimulate osteoblasts to produce RANKL, increasing RANKL/OPG ratio. RANKL binds to its receptor on both precursor and mature cells, thus stimulating osteoclast differentiation and survival. Moreover, in solid tumors, metastatic cancer cells directly interact with osteoclast precursors, activating them. In addition tumor cells produce molecules such as DKK-1 and activin A that inhibit osteoblast differentiation. (3) RANKL can act as a chemotactic factor for RANK expressing cancer cells. DKK-1 = dickkopf-1; IL = interleukin; GM-CSF = granulocyte-macrophage-colony stimulating factor; OPG = osteoprotegerin; PTHrP = parathyroid e hormone-related peptide; RANK = receptor activator of nuclear factor kB; RANKL = RANK ligand.

causing the release of prostate-cancer-promoting cytokines and matrix proteins, including TGFb, platelet-derived growth factor, IGFs, fibroblast growth factors and other factors able to influence prostate cancer progression, might be responsible for this [41]. Moreover, other molecules (e.g. PGE2) are under evaluation as possible regulators in bone metabolism and metastatization in prostate cancer and might hopefully become therapeutic targets. Indeed, high concentration of PGE2 interacting with RANK/ RANKL/OPG system mainly activates osteoclastogenesis, whereas low levels of PGE2 stimulate osteoblasts via activation of the Wnt pathway [42]. Understanding the interaction between tumor and bone might lead to identify new therapeutic opportunities in the bone microenvironment, in order to both prevent and treat bone metastases [43]. When cancer metastasizes to bone, it deregulates bone remodeling and may cause clinical effects known as skeletalrelated events (SREs), such as pathologic fractures, spinal cord compression, hypercalcemia, which greatly affect quality of life. Furthermore, hematological malignancies, such as lymphoma or multiple myeloma, are associated with the development of purely lytic bone lesions, due to increased osteoclast formation and activity of different cytokines, including IL1, TNF and IL6, which directly stimulate bone resorption and inhibit bone formation [44].

Clinical implications: role of bone targeted drugs in cancer therapy Bisphosphonates Bisphosphonates inhibit osteoclast formation (by blocking G-protein signaling), recruitment and adhesion to bone, increase production of OPG by osteoblasts and induce osteoclasts apoptosis.

These drugs prevent physiological and pathological bone resorption and the release of bone-derived growth factors and cytokines, which may enhance both tumor growth and proliferation in the bone microenvironment [45]. In addition, they have direct antitumor effects, as they inhibit tumor cell adhesion, invasion, and proliferation, and they induce cancer cells apoptosis [46]. Moreover, the Nitrogen-containing bisphosphonates, such as zoledronic acid (ZOL) and pamidronate exert indirect anticancer effects through antiangiogenic and immuno-modulatory mechanisms by activating T cells, in particular the cd T cell subset, responsible for tumor surveillance [47,48]. On the contrary, the other non N-containing bisphosphonates, such as clodronate, do not stimulate anticancer immune responses, although they are effective in preventing SREs in different tumors, as shown in Table 2 [49–51]. In addition, it has been suggested that bisphosphonates, in particular third generation bisphosphonates, could be useful as axillary treatment; for example they showed beneficial effects on the prevention of aromatase inhibitor induced bone loss in postmenopausal women with early stage breast cancer [52]. Zoledronic acid is the most used and seems to be – according to a recent meta-analysis – the most effective bisphosphonate for delaying and preventing the risk of SREs in patients with breast or prostate cancer and with multiple myeloma [53] (Table 2). Standard doses of ZOL appear to mediate their antitumor effects by both stimulation of cd T cells and inhibition of osteoclast-mediated bone resorption [54]. Indeed, in a phase IV trial in patients with osteoporosis, treatment with ZOL was associated with a rapid activation of peripheral cd T cells and monocytes in an acute phase response [55]. ZOL could even exert a synergistic effect in combination with cisplatin (but not carboplatin) in a triple negative breast cancer cell line, increasing the antitumor activity of chemotherapy [56]. Similarly, ZOL in combination with serine/threonine phosphatase inhibitors increased efficacy and apoptosis in hor-

C. Criscitiello et al. / Cancer Treatment Reviews 41 (2015) 61–68 Table 2 Efficacy of the main bisphosphonates in patients with bone metastases. Clodronate

 Multiple myeloma [50]  Breast cancer [88–91]  Prostate cancer [51]

Nitrogen- containing bisphosphonates Zolendronic  Multiple myeloma [92] acid  Breast cancer [93]  Lung cancer (NSCLC and small cell lung cancer) [94,95]  Renal cell carcinoma [94]  Prostate cancer [96] Pamidronate  Multiple myeloma [97]  Breast cancer [98] Ibandronate  Breast cancer [99]

mone refractory prostate cancer cell lines [57]. Also, its synergistic effect with chemotherapeutics has been recently studied in Ewing sarcoma [58]. Moreover, ZOL seems to reduce the persistence of DTCs in the bone marrow of patients with early breast cancer [59,60]; hence, some investigators have suggested that it could be used in adjuvant setting, in order to prevent bone metastases from solid tumors [61]. In preclinical studies it has been suggested that ZOL might be used as a potential anti-metastatic agent in different solid tumors, since it greatly affects tumor invasion capacity and dissemination [62]. Finally, ZOL seems to exert antitumor effects outside the bone microenvironment, leading to improved survival. Indeed recent clinical trials showed that ZOL – given as adjuvant therapy – provides a survival benefit over placebo or no treatment in postmenopausal women with early stage breast cancer [63–65]. Also, in patients with multiple myeloma, it has been found that ZOL reduced the risk of death by 16% and prolonged both median overall survival and median progression-free survival [66,67]. Targeting RANK/RANKL/OPG pathway The role of RANK/RANKL in cancer development remains unclear. However, recent studies have demonstrated that RANK/ RANKL signal contributes to progestin-induced mammary epithelial proliferation and carcinogenesis and that inhibition of this pathway reduces breast cancer growth and its spread to the lung [67–69]. Therefore, blocking RANK/RANKL/OPG pathway might exert anticancer activity. Denosumab is a fully human monoclonal antibody, FDA-approved for the treatment and prevention of SREs in bone metastases from multiple myeloma or solid tumors including breast and prostate cancers [14]. By targeting RANKL, Denosumab inhibits osteoclastic function and prevents both bone resorption and destruction [70]. Denosumab has shown to be superior to ZOL in decreasing incidence of SREs, in delaying the onset of SREs and in controlling pain [71–73]; efficacy was demonstrated in all subgroups, regardless prior SRE status or age. Disease progression and overall survival, as well as safety profile were similar between the two treatment arms. Moreover, similarly to third generation bisphosphonates, Denosumab seems to have beneficial effects on bone metabolism, as shown in a recent clinical study, demonstrating the positive association between Denosumab, increased bone mineral density and decreased incidence of vertebral fractures among men receiving androgen deprivation therapy for early prostate cancer [74]. However, the long-term safety of new drugs targeting RANKL has not been assessed yet and longterm treatment surveillance is still ongoing. Indeed the expression of RANKL plays an important role in B cell/T cell differentiation and DC survival; its inhibition to prevent bone destruction could eventually increase the risk of infection [74,75] or new malignancies due to immunosuppression. Then, no carcinogenicity studies have been performed- due to the absence of an appropriate animal

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model [76]. Furthermore there is little clinical evidence supporting a global immune dysregulation due to RANKL inhibition, since the redundancy of pathways may limit immune side effects of denosumab-treatment [77]. Since OPG inhibits the development of osteolytic bone disease and is also associated with a reduction in tumor burden in murine models [78], a genetically engineered recombinant OPG-Fc construct (AMGN-0007) was developed as a potential therapeutic agent and investigated in a clinical trial in patients with multiple myeloma and breast cancer bone metastases [79]. Although it showed to be as effective as pamidronate in reducing bone resorption, it had a short half-life and – above all – it had no specificity for RANKL, as OPG can also block the TNF related apoptosis inducing ligand (TRAIL), which is an important component of natural immunity against cancer [80]. Indeed recent studies suggested that OPG could provide tumor cells with a survival advantage [81–83]. Although the ability of OPG to act as a tumor cell survival factor remains to be proven in vivo, the potential risk of tumor growth and progression with the long-term use of this drug discouraged further clinical investigations.

Conclusion Many potential lines of future investigation are on the horizon in the field of osteoimmunology. So far, research in this context have mostly focused on the interactions between osteoclasts and immune cells, with a few additional studies characterizing the effects on osteoblasts. The effects on bone of many arms of the innate immune system (such as granulocytes, macrophages, or non Toll-like receptor innate immune pathways) remain poorly characterized. Also, it is mostly unknown how bone homeostasis may shape the immune system. Although further work needs to be done to exclude that this is an unrelated epiphenomenon, it seems that bone turnover may influence inflammation, which is an important factor involved in cancerogenesis. Many of the recent studies discussed above demonstrate that lymphocytes are involved in growth factor production, hormone responses, and the pathophysiology of bone loss induced by withdrawal of sex hormones. This is a relevant topic with actual clinical implications. Current clinical data show overall improved outcome with bisphosphonates, although not in all populations. Data suggest that bisphosphonates protect bone and may exert anticancer activity in postmenopausal women with breast cancer treated with adjuvant therapy [84]. Initiating bisphosphonates early and concomitantly with adjuvant therapy generally provided the greatest benefits [64]. However, there are still open key questions regarding the role of adjuvant bisphosphonates in breast cancer, including which patient populations derive benefit, timing/scheduling of therapy, and specific clinical benefits. Further studies are needed to address these controversies in the context of translational research, in order to evolve better treatment paradigms. Osteoimmunology is a field with broad and general relevance to bone metabolism. Undoubtedly, the next decade of osteoimmunology research will lead to a deeper grasp of the intertwined nature of bone metabolism and immune function. A better understanding of the molecular crosstalk between immune system, skeletal system and cancer cells is needed in order to facilitate the design of new therapeutic strategies aimed at disrupting this often pathologic interaction. Indeed, there is emerging evidence for a role of RANK/RANKL/OPG pathway in malignancies, although there are still many unanswered questions. Targeting alterations of this axis and the molecular events leading to early metastatization may permit the development of rational targeted cancer therapies, which will ultimately translate into an improved outcome for cancer patients.

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Conflict of interest Authors have no conflicts of interest to declare. Key issues  Osteoimmunology is the interdisciplinary research field that studies the complex crosstalk between the skeletal and immune systems, in which RANK/RANKL/OPG pathway plays a critical role.  RANK/RANKL/OPG signalling is essential for osteoclastogenesis and bone remodelling, but it plays also an important role in immune system development and regulation.  Cancer cells evade immunosorveillance and the ‘‘hematopoietic niche’’ in bone frequently harbors DTCs against anticancer therapy and anticancer immune response.  The vicious cycle of tumor growth and bone destruction in hematologic and solid malignancies favours cancer metastases and causes SREs.  Besides being effective in preventing and treating SREs, biphosphonates exert direct anti-tumor effects. In addition nitrogen-containing biphosphonates, as ZOL, can activate cd T cells, responsible for tumor surveillance.  ZOL seems to exert antitumor effects beside the bone microenviroment, leading to improved survival in breast cancer and multiple myeloma patients.  Denosumab is a human monoclonal antibody targeting RANKL, effective in SREs treatment in solid and hematologic malignancies. However it might impair immunosorveillance and it has not shown an evident anti-cancer activity.  Targeting RANK pathway with a recombinant OPG-Fc construct might have relevant side effects, since OPG can block also TRAIL, acting as a survival factor for cancer cells.  A better understanding of the molecular crosstalk between immune system, skeletal system and cancer cells could help in the designing of new therapeutic target strategies.

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