**6. Therapeutic targets in the bone microenvironment**

The bone microenvironment presents multiple targets for developing therapeutic treatments targeting the homing of breast cancer cells to bone, as well as progression of bone metastatic lesions (Figure 3). Molecular mediators of critical events underlying the stimulation of bone resorption and inhibition of bone formation, as well as tumour supportive environmental changes and cellular targets have been explored for their benefits in treatment of osteolytic bone metastases.

Since its discovery, the RANKL pathway has been considered to be of important therapeutic value given its role in osteoclastogenesis mediating osteolysis and subsequently discovered breast cancer cell migration, underlying pre-metastatic homing. Fully human monoclonal antibody against RANKL, Denosumab, was approved for major North American and European markets in 2010 for the prevention of osteoporosis and skeletal related events in patients with bone metastases from solid tumours. Compared to the most potent osteoclasttargeting drug in the market, bisphosphonate zoledronic acid, Denosumab treatment further delayed the occurrence of the first skeletal related event (SRE), and provided a further reduction in bone turnover markers in breast cancer patients (174). In non-metastatic breast cancer patients additionally receiving adjuvant aromatase inhibitors, bone mineral density gains were greater with Denosumab treatment (175). Bisphosphonate-resistant patients with bone metastases from breast or prostate cancer also benefitted from Denosumab treatment, with most having normalized serum markers of bone resorption after 13 weeks of treatment (176). Although Denosumab proves an effective treatment option, long-term use and toxicity data remains unknown.

DKK-1 was identified as a key mediator of myeloma-induced inhibition of bone formation, and was demonstrated to play important role in breast cancer induced inhibition of osteoblastogenesis. Neutralizing anti-DKK-1 antibodies have demonstrated significant benefits in preclinical studies in mouse models of myeloma-induced bone disease, resulting in increased osteoblast numbers, reduced osteoclast numbers and increased bone volume, and stimulating interest in further development of this approach (177). Bortezomib, a proteasome inhibitor that among other proteins affects DKK-1 and BIM (a pro-apoptotic protein that mediates osteoclast apoptosis) (178,179), was shown to inhibit

Breast Cancer Metastases to Bone: Role of the Microenvironment 545

fracture rates by 40-50% (188), and zoledronic acid and risedronate have been shown to significantly reduce non-vertebral fracture risk in pivotal trials (189). Bisphosphonates are widely used in prevention and treatment of breast cancer metastases to bone, resulting in delay and reduction in skeletal related events (190). In addition to their effects on osteoclasts, bisphosphonates have been shown to inhibit tumour growth, induce tumour cell apoptosis, and stimulate the immune response against tumour cells (191). However, some patients do not tolerate bisphosphonates well, and low but significant incidences of osteonecrosis of the jaw have been observed in patients that have undergone dental extraction procedures while treated with bisphosohonates (192). In addition, significant proportion of patients failed to normalize bone resorptive indices in response to bisphosphonate treatment (176), demonstrating the need for new therapeutic approaches.

Breast cancer is the most commonly diagnosed cancer in women, which may lead to bone metastasis resulting in altered mineral homeostasis, the disruption of bone microarchitecture, pain and pathological fractures. Recent studies have demonstrated that breast cancer cells start affecting the bone microenvironment prior to their dissemination from the primary tumour by secreting circulating soluble factors that prepare bone for the future arrival of metastasizing cancer cells, a process that likely involves mediators of the hematopoietic stem cell niche. Multiple mediators of directional migration of breast cancer cells have been identified, as well as mediators of breast cancer cells anti-osteoblastic and pro-osteoclastic actions. Breast cancer-stimulated RANKL, M-CSF, PTHrP, TGF, GPNMB, Runx2 and CXCR4 remain among the most critical mediators of cancer-induced osteoclastic bone resorption. Yet, they are not the whole picture, and new players are being identified, providing more complex and comprehensive description of the events leading from the formation of primary tumour to the establishment of progressive osteolytic bone lesions. However, while considering the multitude of molecular mediators, it is important to remember the heterogeneity of breast cancer disease in patients, suggesting that treatments targeting different molecular mediators should develop in parallel with the testing capabilities able to implicate a particular mediator in disease progression in a specific patient. An alternative approach is to target the processes and cellular targets similarly altered through different molecular mediators. An example of such approach is the clinical success of bisphosphonates, which broadly target osteoclast formation and activity. Nevertheless, both approaches need to be developed to provide clinicians with the set of tools for broad preventive measures, as well as for targeted personalized medicine for non-

[1] Mitchell DM, Juppner H 2010 Regulation of calcium homeostasis and bone metabolism

[2] Kovacs CS, Kronenberg HM 1997 Maternal-fetal calcium and bone metabolism during pregnancy, puerperium, and lactation. Endocrine reviews 18(6):832-72.

in the fetus and neonate. Current opinion in endocrinology, diabetes, and obesity

**7. Conclusion** 

responsive or atypical cases.

17(1):25-30.

**8. References** 

osteoclastogenesis (180) and has been successful in combating the osteolytic effects of multiple myeloma (181), making it an attractive candidate for the prevention and treatment of breast cancer-induced osteolysis.

Fig. 3. Breast cancer cells alter normal bone homeostasis. Breast cancer cells maintain osteoblasts in an immature state and stimulate RANKL production by osteoblasts, while inhibiting OPG. Breast cancer cells stimulate osteoclastogenesis directly through TGF M-CSF and CCN3 production. Increased bone resorption by activated osteoclasts releases matrix-derived growth factors TGF, IGF, FGF, PDGF, which act back on breast cancer cells to stimulate their growth and survival.

VEGF represents an interesting target potentially affecting breast cancer cell homing, development of pre-metastatic niche and new vasculature formation. Many anti-VEGF therapies exist to prevent vascularization of tumours and inhibit their growth (182). There have been several hindrances in the progress of this therapy due to drug resistance and toxicity (183), and the increased incidence of osteonecrosis of the jaw in combined bisphosphonate-antiangiogenic agent therapy (184). Notwithstanding, the use of VEGF-A monoclonal antibody Bevacizumab in combination with chemotherapy has proven beneficial in reducing breast cancer growth (185) and osteolysis (186). Other targets based on the in vitro and in vivo studies, such as TGF, GPNMB, and CXCR4 are being explored in preclinical and clinical studies, providing the basis for the next generation of treatments. Osteoclasts are commonly targeted therapeutically for osteolytic disease, with one of the

most widely used drugs being bisphosphonates. Analogs of mineralization-inhibiting pyrophosphate (187), bisphosphonates are a class of synthetic compounds composed of two phosphate groups covalently linked to carbon with a P-C-P backbone and side groups that vary their properties and pharmacokinetics. Bisphosphonates attach selectively to bone and induce osteoclast apoptosis when they are ingested during resorption. In osteoporosis studies, all bisphosphonates given daily have been shown to reduce osteoporotic vertebral fracture rates by 40-50% (188), and zoledronic acid and risedronate have been shown to significantly reduce non-vertebral fracture risk in pivotal trials (189). Bisphosphonates are widely used in prevention and treatment of breast cancer metastases to bone, resulting in delay and reduction in skeletal related events (190). In addition to their effects on osteoclasts, bisphosphonates have been shown to inhibit tumour growth, induce tumour cell apoptosis, and stimulate the immune response against tumour cells (191). However, some patients do not tolerate bisphosphonates well, and low but significant incidences of osteonecrosis of the jaw have been observed in patients that have undergone dental extraction procedures while treated with bisphosohonates (192). In addition, significant proportion of patients failed to normalize bone resorptive indices in response to bisphosphonate treatment (176), demonstrating the need for new therapeutic approaches.
