Could targeting bone delay cancer progression? Potential mechanisms of action of bisphosphonates

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1 Critical Reviews in Oncology/Hematology 82 (2012) Could targeting bone delay cancer progression? Potential mechanisms of action of bisphosphonates Rebecca Aft a,, Jose-Ricardo Perez b, Noopur Raje c, Vera Hirsh d, Fred Saad e a Department of Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA b Novartis Pharmaceuticals Corporation, Florham Park, NJ, USA c Massachusetts General Hospital Cancer Center, Boston, MA, USA d McGill University Health Centre, Montreal, Quebec, Canada e CRCHUM-Centre Hospitalier de l Université de Montréal, Montreal, Quebec, Canada Accepted 25 May 2011 Contents 1. Introduction Mechanisms of metastasis from solid tumors Seeding of metastases from cancer cells in circulation: role of the bone microenvironment The bone marrow as a sanctuary for cancer cells Prognostic value of disseminated tumor cells in bone marrow Mechanism of action of bisphosphonates: effects on the seed, the soil, or both? Chemistry of bisphosphonates Inhibiting osteolysis modifies the bone microenvironment and may impede tumor progression Bisphosphonate effects on the bone microenvironment may inhibit metastasis: clinical evidence Prevention of breast cancer by bisphosphonates: is this further evidence for soil effects? Effects of bisphosphonates on disseminated/circulating tumor cells and the premetastatic niche in the bone marrow Other potential anticancer effects of bisphosphonates Implications for the future of bisphosphonates as potential anticancer agents Conclusions Modifying the bone microenvironment may be critical for preventing metastasis Ongoing clinical trials of bisphosphonates may offer additional mechanistic insights Conflict of interest statement Reviewers Acknowledgments References Biography Abstract Although dissemination may occur early in the course of many cancers, the development of overt metastases depends upon a variety of factors inherent to the cancer cells and the tissue(s) they colonize. The time lag between initial dissemination and established metastases could be several years, during which period the bone marrow may provide an unwitting sanctuary for disseminated tumor cells (DTCs). Survival in a dormant state within the bone marrow may help DTCs weather the effects of anticancer therapies and seed posttreatment relapses. The importance of the bone marrow for facilitating DTC survival may vary depending on the type of cancer and mechanisms of tumor cell dissemination. By altering the bone microenvironment, bisphosphonates may reduce DTC viability. Moreover, some bisphosphonates have Corresponding author. Tel.: ; fax: address: aftr@wustl.edu (R. Aft) /$ see front matter 2011 Elsevier Ireland Ltd. All rights reserved. doi: /j.critrevonc

2 234 R. Aft et al. / Critical Reviews in Oncology/Hematology 82 (2012) demonstrated multiple anticancer activities. These multiple mechanisms may help explain the improvement in disease outcomes with the use of zoledronic acid in malignancies like breast cancer and multiple myeloma Elsevier Ireland Ltd. All rights reserved. Keywords: Bisphosphonates; Bone metastases; Breast cancer; Disseminated tumor cells; Multiple myeloma; Zoledronic acid 1. Introduction The incidence of cancer is increasing worldwide, with more than 12.5 million new cases and 7.5 million cancerrelated deaths each year [1,2]. In the United States alone, the incidence of new cases of cancer is expected to exceed 1.5 million this year, and more than 550,000 cancer-related deaths are estimated [3]. Lung cancer remains the most common cancer worldwide, as well as a leading cause of cancer-related death, with an estimated 1.6 million new cases and nearly 1.4 million deaths annually [2]. In contrast, although breast and prostate cancers account for 1.38 million and 0.9 million new cases each year, these malignancies were associated with substantially lower mortality rates (0.5 million and 0.26 million deaths, respectively) [2]. In addition to the differences in cancer aggressiveness, the favorable survival for breast and prostate cancers is driven by advances in screening, early detection, and treatment, especially in developed countries. Although more than one-third of patients with solid tumors succumb to their disease, survival rates are excellent for patients whose cancer is diagnosed at an early stage. For example, after a diagnosis of breast cancer, the 5-year survival rates in the United States are approximately 98% for women diagnosed with localized disease, versus 84% for women with regionally advanced disease, and only 23% for those with distant metastases [3]. Preventing metastatic disease development from solid tumors appears to hold the key to improving survival, and understanding the mechanisms involved in metastasis can lead to the identification of additional therapeutic targets. Indeed, factors that influence the development and sites of metastasis from solid tumors have been an area of intensive research for over a century. Cancer progresses via multiple pathways including locoregional expansion, via the lymphatic system, or through hematogenous spread. Regional lymph nodes are frequently the first site of detectable locoregional spread from solid tumors, and highly perfused organs such as the liver, lungs, and skeleton are frequent sites of distant metastases through hematogenous spread of cancer cells, followed by arrest in capillary beds and subsequent metastatic growth [4]. However, not all tumors follow the same patterns of dissemination, and many tumors demonstrate distinct patterns of metastasis to specific organs. For example, breast and prostate cancers have a marked predilection for metastasis to bone, with nearly three-quarters of all patients with advanced disease developing bone metastases [5]. Even within each cancer type, genotypic subtypes exhibit distinct patterns of metastasis for example, brain metastases are more common in HER-2-positive compared with HER-2-negative breast cancers [6]. These observations suggest that although a simple anatomical or mechanical theory of cancer spread may account for regional metastases, the process of metastasis to distant organs is remarkably site-specific [4]. The concept of site-specific metastasis from solid tumors was first introduced by the surgeon Stephen Paget in Based on the frequency of specific organ involvement with different cancers, he proposed the seed and soil hypothesis, wherein certain cancer cells (seeds) have a special affinity for specific organ milieus (soil), resulting in a non-random distribution of cancer metastases in organs best suited to supporting their growth [7]. Thus, although the genotype and phenotype of a tumor are likely to be major determinants of metastatic efficiency, a receptive microenvironment is a prerequisite for establishment of metastatic outgrowth. In addition to explaining the frequency of metastasis to specific organs, the seed and soil concept may also provide insights into avenues for targeting tumor cells and potentially eliminating them before they are able to establish overt metastases. In the case of cancers with a predilection for metastasis to the skeleton, modifying the bone microenvironment (the soil) to render it less conducive to the survival and growth of tumor cells can offer an additional means to limit metastatic spread. Indeed, recent clinical trials demonstrating reduced rates of breast cancer recurrence and improved response rates in multiple myeloma with the use of bisphosphonates support this concept [8 10]. In this review, we will examine tumortype-specific differences in metastatic patterns and how these might correlate with the effectiveness of targeting bone to delay disease recurrence. 2. Mechanisms of metastasis from solid tumors Hematogenous spread and metastasis formation by solid tumors is a multistep process (Fig. 1) [11]. Tumor-induced angiogenesis is necessary for tumor growth beyond a size of approximately 2 mm 3 at the primary site, beyond which the cancer cells cannot be supported by simple diffusion of nutrients, oxygen, and waste products [11,12]. Angiogenesis is followed by local invasion and intravasation through the sub-endothelial basement membrane, leading to hematogenous spread of cancer cells [11,12]. Dissemination can occur early in the process of tumor development. However, in a typical solid tumor comprising approximately cells, fewer than 10 million (<0.1%) of these cells generally reach the interior of blood vessels and become disseminated (Fig. 1) [11]. Of these, fewer than 100 cancer cells typically form overt metastases in distant organs [11]. Therefore, metasta-

3 R. Aft et al. / Critical Reviews in Oncology/Hematology 82 (2012) Reprinted from Timar et al. [11]. Fig. 1. Metastasis is a multi-step process. sis is a highly inefficient process, and a variety of factors can influence the cascade of events between hematogenous dissemination of cancer cells from the primary tumor and final metastasis formation in distant organs. These include factors such as viability and dormancy of the disseminated cells, immune responses against these cells, and genetic and environmental factors that may facilitate their outgrowth into clonal metastases [4,11 13] Seeding of metastases from cancer cells in circulation: role of the bone microenvironment Cancer is often considered a disease of selfseeding cancer cells released into circulation from the primary tumor may recolonize their tissues of origin in a process mediated by tumor-derived cytokines and extracellular enzymes such as matrix metalloproteinases [14], as well as evolve into metastatic foci. Because of the vast surface area and high vascular supply to the skeleton, disseminated cells from primary tumors typically transit through capillary beds in bone regardless of the site of the primary tumor. During their passage through bone, cancer cells can lodge and survive in the bone marrow for prolonged periods of time, possibly acquire increased metastatic competency, and eventually form metastases (Fig. 2) [15]. Although the factors determining which cancer cells take up residence in bone marrow are yet to be fully elucidated, it is becoming evident that genetic predisposition, rather than proximity and physical arrest (in circulation), is responsible for this phenomenon [16 18]. Osteomimetism, or the ability of cancer cells to express genes and proteins typically associated with bone cells, is thought to account for the remarkable ability of breast and prostate cancer cells to transit into, as well as survive and proliferate within, the bone microenvironment [16 18]. Moreover, the release of bone-derived growth factors and cytokines into the microenvironment can attract cancer cells to the bone surface and facilitate their growth and propagation [12], and elevated levels of bone turnover may provide a rich source of these growth factors [19]. In a mouse model of prostate cancer, androgen-deprived (orchiectomized) mice experienced increased bone turnover and accompanying bone loss, together with increased rates of metastasis to bone compared with sham-treated mice; antiresorptive treatment reduced the rate of prostate cancer metastasis to bone in this model [20], providing further support for this hypothesis. Once in the bone microenvironment, cancer cells may secrete factors that can increase rates of bone resorption, thereby setting up a vicious cycle of tumor growth and bone damage (Fig. 2) [12,15]. Multiple myeloma is characterized by malignant plasma cells colonizing bone marrow early in the disease course, and forming osteolytic lesions as the disease progresses. Myeloma cells secrete factors that interact with bone marrowderived growth factors and signaling intermediates, thereby altering the balance of the bone microenvironment and rendering it highly conducive to proliferation of malignant cells [21]. The molecular interactions between myeloma cells and bone are quite similar to the vicious cycle observed in bone metastases from solid tumors [12]; proliferation-stimulating and antiapoptotic effects of bone-derived growth factors and cytokines support myeloma cell survival and growth, which in turn produce high levels of cytokines (e.g., interleukins 6, 1, and 3; tumor necrosis factor-alpha; hepatocyte growth factor; osteopontin; and macrophage inflammatory proteins 1-alpha and 1-beta), which stimulate osteolysis [21]. Myeloma cells also disturb the normal balance between osteolysis and osteogenesis through their effects on production of receptor activator of nuclear factor-kappab ligand (RANKL), which stimulates osteoclast development, and osteoprotegerin, the decoy receptor for RANKL that blocks its function,

4 236 R. Aft et al. / Critical Reviews in Oncology/Hematology 82 (2012) Fig. 2. Interactions between tumor and bone marrow are complex. Abbreviations: BM, bone marrow; BMDC, bone marrow-derived cell; CEP, circulating endothelial progenitor; CTC, circulating tumor cell; DTC, disseminated tumor cell; EMT, epithelial mesenchymal transition; MMP, matrix metalloproteinase. Adapted in part from Coghlin and Murray [15]. by osteoblasts and other stromal cells. Myeloma cells in bone create an imbalance favoring RANKL versus osteoprotegerin, resulting in localized promotion of bone resorption by osteoclasts to levels that exceed the compensatory bone formation by osteoblasts [21] The bone marrow as a sanctuary for cancer cells The last decade has seen an increased focus on understanding how disseminated tumor cells (DTCs) in the bone marrow, and circulating tumor cells (CTCs) in peripheral blood, may correlate with and contribute to clinical outcomes in patients with solid tumors. Because of its unique properties, the bone microenvironment can provide a supportive niche for cancer cell survival and tumor growth [22,23]. The concept of a niche was first proposed to describe the highly specific and physiologically discrete sites where hematopoietic cells reside in the bone marrow. Osteoblasts and endothelial cells are the predominant contributors to the cellular components of the endosteal and vascular niches within the bone marrow, which together provide the microenvironment for maintaining hematopoietic stem cells (HSCs) [23]. Moreover, mesenchymal stem cells (MSCs, the progenitors of osteoblasts) may also reside in niches that are in close proximity to the HSC niche, and interactions between HSCs and MSCs may regulate each other s activities (Fig. 3) [23]. In recent models examining survival of metastatic cancer cells in the bone microenvironment, DTCs are thought to reside in niches created by the overlap of HSC, MSC, endosteal, and vascular niches necessary for normal bone marrow function [23]. Such residence of DTCs within the bone marrow niche results in parasitic behavior wherein DTCs (molecular parasites) commandeer normal host (bone marrow) functions to support their own survival. The inherent genetic instability of cancer cells, coupled with the osteomimetic potential of cells from some malignancies, allows DTCs to adhere within bone marrow niches and exploit the specialized cellular and molecular interactions developed for maintenance of HSCs [22,23]. It is becoming evident that the interaction between the bone marrow and the tumor is complex, with exchange of components that promote the growth of the tumor and contribute to DTC survival. For example, bone marrow stromal cells express ligands that bind cell-surface receptors on DTCs [24,25], and bone marrow-derived cells from the HSC and MSC lineages can contribute to angiogenesis and tumor growth through a variety of mechanisms [26]. Bone

5 R. Aft et al. / Critical Reviews in Oncology/Hematology 82 (2012) Fig. 3. The bone marrow niche can protect disseminated tumor cells from systemic anticancer therapies. Reprinted by permission from Macmillan Publishers Ltd.: [23], copyright marrow-derived endothelial progenitor cells (also termed circulating endothelial progenitors or CEPs) have been implicated in pathologic angiogenesis in cancer and other diseases [27]. Osteoclast activity also results in the secretion of bone-derived factors that can promote tumor growth and angiogenesis [28,29]. Mesenchymal stem cells in the bone marrow have been shown to increase cancer cell motility and invasion by secreting the chemokine RANTES (regulated on activation, normal T-cell expressed and secreted) [30]. Furthermore, macrophages in the bone marrow secrete proangiogenic cytokines [31], and increases in macrophage numbers in the tumor stroma are associated with poorer prognosis [32]. The combination of growth factors and cell cell interactions in bone marrow niches help protect HSCs against immune surveillance [22,23]. Exploitation of this HSC niche by DTCs can, in turn, help protect them from the cytotoxic and proapoptotic effects of systemic anticancer therapies, as well as from anticancer immune surveillance, thereby helping them survive during adjuvant therapy for early stage cancer. It is now evident that CTCs and DTCs are typically dormant or quiescent cells [33,34], which can be activated by external signals to grow into overt tumors [33]. In animal models of cancer, dormant CTCs and DTCs isolated from metastasis-free organs are able to regain tumorigenic properties in response to the appropriate external signals [35]. It is thought that DTCs (or CTCs) may function as cancer stem cells (CSCs) or metastasis-initiating cells, which form the seeds of subsequent metastases. The signals activating quiescent DTCs to form clonal metastases are poorly understood; however, it is becoming evident that interactions between cell surface receptors on tumor cells (e.g., the urokinase receptor, which can influence mitogenic and stress-activated signaling pathways via its crosstalk with integrins) and the microenvironment may be critical determinants in the switch between dormancy and proliferation [33]. Moreover, recent research suggests that multidirectional interactions between cancer and the microenvironment may influence this switch: cancer cells (DTCs) exhibit epithelial plasticity, a phenomenon wherein epithelial cancer cells can transition between epithelial and mesenchymal phenotypes in response to external stimuli as well as to intracellular signals [36]. Epithelial mesenchymal transition (EMT) is associated with the acquisition of stem-cell-like properties and could further contribute to escape from immune surveillance and resistance to systemic anticancer therapies, further reinforcing the potential role of DTCs as CSCs for seeding posttreatment relapses [36]. Furthermore, bone marrow DTCs from patients with high-risk breast cancer have been reported to more frequently express EMT and CSC-like molecular markers compared with patients who had lower-risk disease [37,38]. Bone marrow MSCs have also been shown to regulate CSCs through cytokine networks for example, MSCs regulated breast CSC proliferation and bone metastasis formation in a preclinical mouse

6 238 R. Aft et al. / Critical Reviews in Oncology/Hematology 82 (2012) model through a cytokine loop involving interleukin-6 [39]. Thus, interactions between CSCs/DTCs and the bone microenvironment may play a key role in modulating CSC survival, dormancy, and proliferation into overt metastases Prognostic value of disseminated tumor cells in bone marrow Detection of DTCs in bone marrow is associated with poor clinical outcomes in patients with breast and prostate cancers. In a pooled analysis of 4703 patients with newly diagnosed breast cancer in 9 international treatment centers, DTCs were detected in 31% of patients [40]. In this study, the presence of DTCs at initial diagnosis was associated with reduced disease-free survival (DFS; hazard ratio [HR] = 2.13; p <.001 versus no DTC) and distant DFS (HR = 2.33; p <.001 versus no DTC) [40]. In addition, the presence of DTCs at primary diagnosis was associated with more than 2-fold increased risk of death after 62 months median follow-up (Fig. 4; p <.001) [40]. Similar associations between persistent DTCs and breast cancer-specific survival were also observed in a separate pooled analysis of 726 women with breast cancer [41,42]. In the prostate cancer setting, DTCs have again been consistently associated with poor clinical outcomes in multiple trials (Table 1) [43 52]. In contrast, associations between DTC status in bone marrow and disease outcomes in the lung cancer setting have been inconsistent (Table 1) [43 49], suggesting a less critical role for the bone marrow in harboring dormant tumor cells in lung cancer compared with breast or prostate cancers. 3. Mechanism of action of bisphosphonates: effects on the seed, the soil, or both? 3.1. Chemistry of bisphosphonates Bisphosphonates are bone-targeted antiresorptive agents containing a P C P backbone with 2 covalently bound side chains that vary among different bisphosphonates [53]. These side chains determine the affinities of bisphosphonates for farnesyl diphosphate synthase (FPP synthase; a key enzyme in the mevalonate pathway of posttranslational protein modification), as well as the antiresorptive potency of the compound. Bisphosphonates with nitrogen-containing side chains are several orders of magnitude more potent than non-nitrogen-containing bisphosphonates in preclinical antiresorptive assays [53 56]. All bisphosphonates induce apoptosis in osteoclasts, albeit by different mechanisms [57,58]. Non-nitrogen-containing bisphosphonates (e.g., clodronate) are metabolized into nonhydrolyzable, cytotoxic analogues of adenosine triphosphate (ATP), whose intracellular accumulation results in inhibition of essential metabolic pathways, resulting in cell death [57]. In contrast, nitrogencontaining bisphosphonates (e.g., ibandronate and zoledronic acid) inhibit FPP synthase, thereby blocking prenylation of Fig. 4. The presence of disseminated tumor cells (DTCs) in bone marrow is associated with reduced survival in women with breast cancer. Abbreviations: CI, confidence interval; HR, hazard ratio. Reprinted from Braun et al. [40] Massachusetts Medical Society. All rights reserved. G-proteins, which impedes signal transduction and triggers osteoclast apoptosis [58,59]. Additionally, inhibition of the mevalonate pathway by nitrogen-containing bisphosphonates results in impaired prenylation, subcellular localization, and consequently reduced function of signaling molecules such as the Ras family proteins, which are involved in a range of cellular functions in osteoclasts as well as in cancer cells [54,56] Inhibiting osteolysis modifies the bone microenvironment and may impede tumor progression In the seed and soil theory of metastasis, the bone microenvironment could be considered the fertile soil that supports the growth of cancer cells ( seeds ). Bisphosphonates, by virtue of their antiresorptive activity, can disrupt the vicious cycle between cancer cells and osteoclasts within bone, thereby blocking the release of bone matrix-derived growth factors into the microenvironment surrounding the cancer cells (i.e., making the soil less fertile) [12]. The antiresorptive effects of bisphosphonates are best recognized in the setting of malignant bone disease, wherein bisphosphonate treatment is the standard of care for preventing skeletal morbidity and palliating bone pain [60]. These benefits from bisphosphonate therapy may also translate into indirect survival benefits for patients successful administration of multiple lines of systemic anticancer therapy in the advanced cancer setting is dependent on preserving the patient s performance status, and using bisphosphonates to prevent skeletal morbidity can help in this endeavor. Moreover, the antiresorptive effect of bisphosphonates might also translate into delayed disease progression and improved survival in advanced cancers, as well as prevention of bone metastases when used during adjuvant therapy for early stage disease; however, further clinical investigations are needed to determine the risk benefit profiles of bisphosphonates in all malignancies. Additionally, as discussed in Section 2.2, interactions between cancer cells and bone-derived factors can

7 R. Aft et al. / Critical Reviews in Oncology/Hematology 82 (2012) Table 1 Correlations between CTC/DTC levels and clinical outcomes in BC, PC, and NSCLC. Tumor type/study N Result Breast cancer Braun et al. [40] 4703 DTC status correlated with DFS, distant DFS, and OS Janni et al. [42] 726 DTC status correlated with BC-specific survival Bidard et al. [50] 621 DTC status correlated with risk of locoregional relapse Bidard et al. [51] 138 DTC status correlated with risk of bone metastasis; CTC status correlated with OS Cristofanilli et al. [52] 177 CTC status correlated with PFS and OS Prostate cancer Berg et al. [43] 131 DTC status correlated with long-term risk of DM Garcia et al. [45] 41 CTC status correlated with OS Kollermann et al. [46] 193 DTC status before neoadjuvant endocrine therapy predicted PSA relapse after RP Morgan et al. [47] 98 DTC status post-rp predicted recurrence in patients with no evidence of disease post-rp NSCLC Passlick et al. [48] 139 DTC status predicted OS in node-negative disease, based on 12 representative patients only Sugio et al. [49] 58 DTC status predicted OS in patients with complete surgical resection of primary tumor Brunsvig et al. [44] 196 No correlation between DTC status and outcomes Abbreviations: BC, breast cancer; CTC, circulating tumor cell; DFS, disease-free survival; DTC, disseminated tumor cell; DM, distant metastases; NSCLC, non small-cell lung cancer; OS, overall survival; PC, prostate cancer; PFS, progression-free survival; RP, radical prostatectomy. influence DTC survival and dormancy, thereby potentially affecting seeding of distant metastases. Therefore, by inhibiting the release of bone-derived factors that might facilitate these processes, bisphosphonates may contribute to reducing the risk of skeletal as well as extraskeletal metastases. In patients with bone metastases from solid tumors or bone lesions from multiple myeloma, elevated levels of bone resorption have been associated with increased risks of skeletal morbidity and death [61 63]. Retrospective analyses of the phase III trial database of zoledronic acid, a nitrogencontaining bisphosphonate, revealed that rapid normalization of elevated bone resorption is associated with improved survival compared with persistently elevated levels of osteolysis [64]. Interestingly, elevated levels of osteolysis have been associated with increased risk of relapse in bone in the NCIC CTG MA.14 trial in postmenopausal women receiving adjuvant endocrine therapy (tamoxifen) for early stage breast cancer [65]. Similar to preclinical findings in animal models of prostate cancer [20], these data suggest that bone-derived growth factors released as a result of excessive osteolysis play a key role not only in supporting established metastases but also in facilitating development of metastases earlier in the disease course. Use of bisphosphonates to normalize levels of osteolysis would therefore be expected to help inhibit bone metastases. Consistent with this hypothesis, zoledronic acid treatment was found to significantly reduce the risk of death in patients with bone metastases from lung cancer and elevated levels of osteolysis [66]; moreover, the survival benefit was most pronounced in patients whose osteolysis levels normalized within 3 months of treatment [64,66] Bisphosphonate effects on the bone microenvironment may inhibit metastasis: clinical evidence The first clinical evidence for using bisphosphonates for the prevention of bone metastases was provided by trials of adjuvant clodronate in early breast cancer (Table 2) [67 69]. Although promising, the outcomes from these studies were inconsistent, and a large clinical trial (NSABP B-34) is ongoing to further evaluate the potential anticancer effects of adjuvant clodronate. Adjuvant clodronate has also been evaluated in 2 clinical trials conducted by the Medical Research Council (MRC) in the United Kingdom in men receiving androgen deprivation therapy for hormone-sensitive prostate cancer [70]. In these trials, 3 years of treatment with clodronate improved overall survival in men with metastatic disease (Table 2), but up to 5 years of clodronate treatment had no effect on survival in men with nonmetastatic disease [70]. The survival benefit limited to the metastatic setting argues in favor of a soil effect of the bisphosphonate by virtue of its antiresorptive effect, clodronate might have (at least partially) blocked the high levels of osteolysis induced by androgen deprivation therapy, thereby rendering the bone microenvironment less conducive to progression of metastases from prostate cancer. In contrast, the lack of a survival benefit in nonmetastatic disease might reflect little or no effect of clodronate on the cancer seeds. Additional support for the ability of bisphosphonates to prevent bone metastases by altering the bone marrow microenvironment derives from recent phase III clinical trials in early breast cancer and newly diagnosed multiple myeloma. In the ABCSG-12 (N = 1803 premenopausal women) and ZO-FAST (N = 1065 postmenopausal women) trials in women receiving adjuvant endocrine therapy for early breast cancer, the addition of zoledronic acid significantly improved DFS (by 35% and 41%, respectively, versus endocrine therapy alone) and produced a trend toward lower rates of disease recurrence in bone (Table 2) [8,9]. These effects of zoledronic acid, observed at a dose and schedule shown to prevent bone loss during endocrine therapy [8,71,72], suggest that the bisphosphonate prevents increased osteolysis and alters the bone microenvironment to make it less conducive for colonization and proliferation of cancer cells. This effect of zoledronic acid could potentially also

8 240 R. Aft et al. / Critical Reviews in Oncology/Hematology 82 (2012) Table 2 Clinical evidence for the anticancer activity of bisphosphonates. Agent/study Setting (N; follow-up) Result Clodronate Diel et al. [67] Adj BC (302; 8.5 years) OS (40.7% versus 20.4% PBO; p =.04) Rates of bone mets at 36- and 55-months follow-up Powles et al. [68] Adj BC (1069; 5.6 years) Rates of bone mets (51 versus 73 patients PBO; HR = 0.692; p =.043) OS (HR = 0.768; p =.048) Saarto et al. [69] Adj BC (299; 10 years) Similar rates of bone mets (p =.35) Non-bone mets (50% versus 36% PBO; p =.005) DFS (45% versus 58%; p =.01) MRC PR04 [70] Non-met HSPC (508; 10 years) Similar rates of recurrence and OS MRC PR05 [70] Met HSPC (311; 10 years) OS (HR = 0.77; p =.032) Zoledronic acid ABCSG-12 [9] Premenopausal Adj BC (1803; 48 months) ZO-FAST [8] Postmenopausal Adj BC (1065; 36 months) DFS (HR = 0.64; p =.01) Bone mets (16 versus 23 for no-zol) Locoregional mets (10 versus 20) Contralateral BC (6 versus 10) All distant mets (29 versus 41) DFS (HR = 0.59; p =.0314) Bone mets (9 versus 17 for late-zol) Local mets (2 versus 10) All distant mets (20 versus 30) OS (HR = 0.84; p =.0118) PFS (HR = 0.88; p =.0179) MRC Myeloma IX [10] Newly diagnosed MM (1960; 3.7 years) AZURE [74] Stage II/III BC (3360; 59 months) Similar DFS in ITT population >5 years postmenopausal (n = 1041): DFS (HR = 0.76; p <.05) >5 years postmenopausal or age >60 years (n = 1101): DFS (HR = 0.71; p =.017) Abbreviations: Adj, adjuvant; BC, breast cancer; DFS, disease-free survival; HR, hazard ratio; ITT, intent-to-treat; mets, metastases; MM, multiple myeloma; OS, overall survival; PBO, placebo. reduce the persistence of DTCs in this setting, which would be consistent with reduced rates of cancer recurrence at multiple sites (Table 2) [8,9]. However, DTC studies were not performed in the ABCSG-12 and ZO-FAST trials. Nonetheless, this possibility is further supported by the durability of the anticancer effect of zoledronic acid in the ABCSG-12 trial, wherein zoledronic acid treatment for 3 years was associated with a lower proportion of bone relapses up to 2 years after completion of therapy [73]. Our understanding of the mechanism of action responsible for the potential anticancer activity of bisphosphonates continues to evolve based on clinical trial data. In the AZURE trial, women with stage II/III breast cancer (N = 3360) received standard therapy alone or standard therapy plus zoledronic acid (4 mg every 3 4 weeks 6; 4 mg every 3 months 8; 4 mg every 6 months 5). In this study the primary endpoint, DFS, did not reach statistical significance; however, there was a trend toward improved overall survival (OS) for zoledronic acid compared with placebo in the overall population (HR = 0.85; p =.07) [74]. Data from subgroup analyses may provide further insight into the anticancer potential of bisphosphonates. Protocol-defined secondary endpoints in AZURE included subgroup analyses based on minimization criteria (including menopausal status), and demonstrated significant heterogeneity in the effect of ZOL on disease recurrence in postmenopausal versus pre- and perimenopausal women (p =.02 for heterogeneity) [74]. Using prospectively defined criteria, zoledronic acid was found to significantly improve DFS (HR = 0.76; p <.05) and OS (HR = 0.71; p =.017) in patients who were at least 5 years postmenopausal or older than 60 years at study entry (n = 1101). Additionally, zoledronic acid reduced each type of DFS event and reduced recurrences both in and outside of bone (HR and p value were not reported) compared with placebo in this subset [74]. These findings may suggest that zoledronic acid has the greatest potential for anticancer benefits in a low-estrogen environment (i.e., in this trial patients who have been menopausal for >5 years). At first, this may seem inconsistent with data showing significant DFS benefits from zoledronic acid in premenopausal women in ABCSG- 12. However, ovarian ablation with goserelin therapy plus either anastrozole or tamoxifen resulted in amenorrhea in all patients in ABCSG-12, and residual estrogen levels in these patients were likely similar to those in the AZURE postmenopausal population. Because estrogen levels were not routinely monitored in either the AZURE or the ABCSG-12 trial, this hypothesis needs further experimental validation. Nonetheless, the combined data from these large studies in patients with breast cancer suggest that zoledronic acid may have anticancer activity in postmenopausal women in the adjuvant cancer setting. In the MRC Myeloma IX trial in patients with newly diagnosed multiple myeloma (N = 1960 evaluable patients), the addition of zoledronic acid to standard antimyeloma therapy delayed disease progression by approximately 2 months and produced on overall survival benefit of over 5 months

9 R. Aft et al. / Critical Reviews in Oncology/Hematology 82 (2012) compared with the non-nitrogen-containing bisphosphonate clodronate [10]. Because myeloma cells colonize the bone marrow, resulting in malignant bone lesions in almost all patients with advanced disease, the delayed disease progression and improved survival with zoledronic acid may again be attributable to the bone microenvironment being modified to be less supportive for myeloma cell proliferation Prevention of breast cancer by bisphosphonates: is this further evidence for soil effects? In addition to the effects of zoledronic acid and clodronate on breast cancer recurrence in patients receiving adjuvant therapy for early stage disease, recent case control and population-based studies show that oral bisphosphonates used for the treatment of osteoporosis may prevent the development of invasive breast cancer [75 77]. Although it is impossible to control these retrospective analyses for all factors influencing bone turnover and breast cancer risk, and the mechanisms underlying this breast cancer prevention effect of bisphosphonates are unknown, the effects are maintained in multivariate analyses controlling for established cancer risk factors (e.g., parity, diet, hormone replacement therapy, and smoking) [75 77]. These observations therefore suggest that bisphosphonate treatment may modulate the tissue microenvironment (potentially in bone, and perhaps indirectly in the breast), thereby resulting in reduced incidence of invasive breast cancer. It is intriguing that in one of these studies, the incidence of ductal carcinoma in situ was actually higher in bisphosphonate users versus nonusers, although the incidence of invasive breast cancer was markedly reduced in women who used bisphosphonates [75]. This suggests that bisphosphonates do not affect initial cellular transformation within the breast, but potentially alter downstream steps required for progression to invasive carcinoma, again indicating a possible soil effect (albeit not in bone) rather than a seed effect. Indeed, other (nonbone) effects, including immunomodulation and antiangiogenesis, are possible. Pilot trials in patients with breast cancer and other malignancies demonstrate reductions in circulating levels of angiogenic factors after bisphosphonate treatment [78,79], and preclinical and early clinical evidence support modulation of the immune system by nitrogen-containing bisphosphonates to increase anticancer immune surveillance [80 83]. These studies provide insight into potential mechanisms by which bisphosphonates may alter microenvironments in addition to bone, thereby preventing tumor cell invasion and migration Effects of bisphosphonates on disseminated/circulating tumor cells and the premetastatic niche in the bone marrow Bisphosphonates have a prolonged half-life in the skeleton [56], making them ideally suited in their potential role to target the quiescent or minimally proliferative DTCs surviving in the bone microenvironment. Notably, in preclinical and translational studies, bisphosphonates have been shown to affect practically every component of the premetastatic niche in the bone marrow. In addition to their well-established activity for inducing osteoclast apoptosis, bisphosphonates have also been reported to reduce proliferation of circulating endothelial cells, induce apoptosis in CEPs [84], and decrease proliferation and expression of key adhesion molecules in bone marrow stromal cells [25]. Bisphosphonates can also reduce MSC migration and inhibit their production of RANTES, thereby indirectly inhibiting breast cancer cell motility and invasion [30]. Preclinical data also support a potential role for bisphosphonates in modulating antitumor immune responses through improving the ability of dendritic cells to stimulate innate and adaptive immune responses [85] and through sensitizing CSCs to the cytotoxic effects of gamma-delta T cells [86]. At low doses, bisphosphonates can also inhibit differentiation of circulating endothelial cells and CEPs [84], as well as modulate osteoblast RANKL and osteoprotegerin expression, thereby indirectly influencing osteoclast activity [87]. Furthermore, the potential anticancer effects of bisphosphonates might be cell-cycle independent [88]. In 4 separate trials in patients receiving adjuvant therapy for high-risk breast cancer, the addition of zoledronic acid reduced DTC number and persistence and increased DTC clearance from bone marrow, compared with adjuvant therapy alone (Table 3) [88 91]. In these studies, fewer patients with DTCs at baseline had persistent DTCs detectable after 3 24 months on treatment [88 91]. This effect of zoledronic acid is especially important in view of studies showing that DTC persistence during therapy is strongly correlated with the risk of early relapse in breast cancer [41,42]. It is, however, unclear whether DTC effects of zoledronic acid are direct cytotoxic/proapoptotic effects on the seed (i.e., the DTC), or whether they are mediated indirectly by altering the soil (i.e., the bone marrow microenvironment) to be less conducive to DTC survival. Although the relationship between DTCs and bone metastases is relatively straightforward, it is important to note that the role of DTCs in metastasis is not limited to bone CTCs and DTCs play a role in the development of all types of metastases (including re-seeding the tissue of origin of the primary tumor) (Fig. 2) [14,15]. Therefore, the effects of zoledronic acid on DTCs may also contribute to reduced disease recurrences in extraskeletal sites, as observed in the ABCSG-12 and ZO-FAST trials (Table 2) [8,9]. In these studies, the addition of zoledronic acid to endocrine therapy helped prevent locoregional recurrences, contralateral breast cancers, and skin, brain, and visceral metastases, in addition to preventing bone metastases (Table 2) [8,9]. Moreover, the improved response rates with zoledronic acid in the MRC Myeloma IX trial may be analogous to its effects on DTCs because complete responses (CRs) and very good partial responses (VGPRs) in multiple myeloma are defined as absence of myeloma cells in bone marrow biopsies, zoledronic acid may have exerted direct anticancer effects on the myeloma cells, as well as indirect effects on the bone microenvironment, to elicit additional CRs and VGPRs in this study [10].

10 242 R. Aft et al. / Critical Reviews in Oncology/Hematology 82 (2012) Table 3 Effect of ZOL on DTC levels in clinical trials. Study Patients, N ZOL dose Effect of ZOL on DTCs Aft et al. [89] mg q 3 weeks 1 year % pts DTC + at 3 months (p =.054) versus no ZOL Lin et al. [90] 45 4 mg q 1 month 2 years ZOL DTCs at 24 months (p =.01) versus baseline Rack et al. [88] mg q 1 month 6 months % pts DTC + at 6 months (p =.099) versus no ZOL Solomayer et al. [91] 76 4 mg q 1 month 2 years % pts DTC + at 12 months (p =.009) versus no ZOL Abbreviations: DTC, disseminated tumor cell; ZOL, zoledronic acid Other potential anticancer effects of bisphosphonates A large body of preclinical evidence supports the potential of bisphosphonates to inhibit tumor growth and metastasis in a variety of malignancies [92 100]. Both indirect and direct anticancer mechanisms of action have been observed in preclinical studies of bisphosphonates. Some indirect mechanisms, such as effects on angiogenesis and modulation of the immune system by nitrogen-containing bisphosphonates, have already been discussed. Other indirect effects include inhibition of bone-derived growth factors [101,102], inhibition of matrix metalloproteinase production and macrophage infiltration in tumor stroma (thereby decreasing angiogenesis and increasing tumor necrosis) [31,103], and uptake of bisphosphonates by monocytes (suggesting that tumors could be exposed to bisphosphonates via infiltrating neutrophils) [104]. In addition to indirect anticancer mechanisms, direct mechanisms of action also have been observed in preclinical studies of bisphosphonates. Some direct anticancer mechanisms include direct inhibition of cell proliferation and viability, induction of apoptosis, impaired angiogenesis, interference with cancer cell invasion and adhesion, and cytotoxic or proapoptotic synergy with anticancer therapies [93 100, ]. It is also possible that enhanced anticancer activity in combination therapies involving bisphosphonates might result from alterations in distribution and uptake of these agents. For example, in a recent series of experiments evaluating the effects of sequential treatment with doxorubicin and zoledronic acid in a mouse model of breast cancer, Ottewell et al. showed that treatment with the chemotherapeutic agent followed by zoledronic acid resulted in accumulation of unprenylated Rap1A (a surrogate marker for zoledronic acid) in mammary tumors, an effect not observed in animals receiving bisphosphonate alone [98]. Although a large proportion of the preclinical evidence for anticancer activities of bisphosphonates stems from studies in animal models of breast or prostate cancer (the classic, bone-tropic solid tumors) [95 98,111,112], it should be noted that bisphosphonates (especially zoledronic acid) have also demonstrated promising anticancer activity in preclinical mouse models of multiple myeloma and aggressive solid tumors such as lung cancer [24,107, ]. In the clinical setting, data from the neoadjuvant subset of the ongoing AZURE trial, as well as a smaller phase II trial of neoadjuvant chemotherapy with or without zoledronic acid [89], demonstrate a potential direct anticancer effect of zoledronic acid in breast cancer. In 205 women who received neoadjuvant chemotherapy in the AZURE trial, the addition of zoledronic acid reduced the mean residual invasive tumor size by approximately 44% compared with chemotherapy alone (15.5 mm versus 27.4 mm; p =.006) [119]. Additionally, patients receiving neoadjuvant chemotherapy with zoledronic acid had an approximately 2-fold higher rate of pathologic complete response (pcr) versus neoadjuvant chemotherapy alone (11.7% versus 6.9%) [119]. Similar improvements were observed in a phase II trial in 120 women receiving neoadjuvant chemotherapy pcr rates were higher in patients receiving zoledronic acid (28%) versus standard treatment alone (10%) in the subset of women with ER /HER-2 tumors [89]. However, after a median follow-up of 61.9 months, the OS (p =.92) and DFS (p =.88) in the overall population were similar in the zoledronic acid and no zoledronic acid treatment groups [120]. Although there was no survival benefit with zoledronic acid in the overall population, zoledronic acid may improve OS and DFS in patients with ER /HER-2 tumors, the subset that also showed the largest improvement in pcr rates with the addition of zoledronic acid to neoadjuvant chemotherapy [89]. 4. Implications for the future of bisphosphonates as potential anticancer agents The promising results with zoledronic acid for the prevention of disease recurrence in breast cancer and multiple myeloma are yet to be duplicated on a similar scale in other cancer types or with other bisphosphonates. Ongoing phase III clinical trials are evaluating the anticancer potential of zoledronic acid in patients with prostate cancer and nonsmall cell lung cancer (without skeletal involvement), and the results are awaited. Similarly, a large number of ongoing clinical trials are evaluating the effects of other bisphosphonates (clodronate and ibandronate) in the adjuvant therapy setting for breast cancer [121]. Denosumab, a monoclonal antibody against RANKL, was recently approved in the United States for the reduction of skeletal-related events (SREs) in patients with bone metastases from solid tumors, but not in patients with multiple myeloma [122]. The specificity of denosumab for primate RANKL has resulted in considerable challenges for evaluating the effects of this novel agent in preclinical models (predominantly mouse). Therefore, preclinical data suggesting possible effects of denosumab on the course of

11 R. Aft et al. / Critical Reviews in Oncology/Hematology 82 (2012) Table 4 Effect of ZOL (4 mg q 3 4 weeks) on clinical outcomes in patients with bone metastases from solid tumors. Study Patients, N Setting Result Zaghloul et al. [129] 40 Bladder cancer 1-year OS rate (36 ± 11% versus 0% PBO; p =.004) SRE incidence (54% versus PBO; p =.001) Zarogoulidis et al. [130] 144 Lung cancer OS (median 578 d versus 384 d for no-zol; p <.001) TTP (median 265 d versus 150 d; p <.001) Calderone et al. [131] 168 Lung cancer OS (median 34 versus 19 weeks for no-zol; p =.01) Abbreviations: OS, overall survival; PBO, placebo; SRE, skeletal-related event; TTP, time to progression (of disease). cancer are based on extrapolation from studies evaluating the role of the RANKL pathway in cancer progression rather than any direct evidence for the role of denosumab [ ]. Currently, denosumab is being evaluated for its potential to prevent disease recurrence in early stage breast and prostate cancers. The potential of denosumab in multiple myeloma is uncertain at present in a phase III head-to-head trial versus zoledronic acid (N = 1776), the 2 agents demonstrated similar levels of activity for reducing the risk of skeletal morbidity [126]. However, denosumab was associated with higher rates of on-study mortality compared with zoledronic acid (26% versus 14%, respectively; HR = 2.26; 95% CI: 1.13, 4.50) in the subset of patients with multiple myeloma (n = 180) [122,126]. Further studies are therefore needed to establish the safety and evaluate the therapeutic potential of denosumab in multiple myeloma. In a pilot trial in patients with advanced solid tumors without bone metastases (N = 40), the addition of zoledronic acid to standard antineoplastic therapy resulted in delayed development of bone metastases (only 5% of patients in the control group were bone-metastases-free at 18 months follow-up, versus 20% in the group receiving zoledronic acid; p =.0002) [127]. In contrast, in a phase II trial in patients with advanced lung cancer (n = 98; re-randomized to receive zoledronic acid or not if they responded to treatment with 6 cycles of chemotherapy + zoledronic acid), additional treatment with zoledronic acid post-chemotherapy had no significant effect on disease progression [128]. However, small clinical trials and retrospective database analyses in patients with bone metastases from lung cancer or bladder cancer demonstrate improved survival and delayed disease progression with zoledronic acid treatment (Table 4) [ ]. Taken together with cancer-type-specific differences in the importance of DTCs in bone marrow for disease progression and relapse (Table 1) [40,42 52], these results suggest that the greatest likelihood of an anticancer benefit with bisphosphonates is in malignancies where the bone marrow plays a central role in facilitating the prolonged survival of DTCs, as well as the subsequent activation of proliferation of these cells to form metastases (Fig. 5). In contrast, in the bone metastatic cancer setting, cancer-bone interactions are already established. Therefore, the use of antiresorptives not only helps disrupt the Fig. 5. Zoledronic acid (ZOL) might inhibit metastasis of solid tumors to bone and other organs.