**5.1 Interactions of breast cancer cells with osteoblasts**

#### *Inhibition of osteoblasts by breast cancer cells*

Breast cancer metastasis to bone is associated with a reduction in bone formation markers in patients with bone metastases (133). In vitro, breast cancer cells have been shown to produce soluble factors able to inhibit osteoblast differentiation (20,134), the effect that may be mediated at least in part by the dysregulation of Notch and Wnt developmental signalling

Blocking annexin II or its receptor limited the localization of prostate cancer cells to osteoblasts and endothelial cells (111). In keeping, the inhibition of the SDF-1/CXCL12 and annexin II signaling was shown to inhibit breast cancer progression (112,113). Bone matrix proteins, such as bone sialoprotein (BSP) or osteopontin (OPN) have been shown to exhibit a potential to regulate the attachment of breast cancer cells to bone (114). Early reports have argued that BSP inhibits breast cancer cell binding to bone cells (115). However, breast cancer cells have been shown to express both BSP and OPN, and to upregulate BSP expression in pre-osteoblasts through BMP signalling; and OPN was found localized between cancer cells and bone cells at sites of metastasis (116,117). Moreover, the expression of BSP has been found to correlate with bone metastasis development (118), and OPN expression and serum concentrations have been shown to be poor prognosis markers in breast cancer patients (119,120). As osteopontin is also a mediator of the hematopoietic stem cell niche, directing migration and acting as an adhesion molecule to HSC via 1 integrin

(121), it represents a potentially valuable therapeutic target against bone metastases.

**5. Establishing of a metastatic tumour in the bone microenvironment** 

Breast cancer metastasis to bone is associated with a reduction in bone formation markers in patients with bone metastases (133). In vitro, breast cancer cells have been shown to produce soluble factors able to inhibit osteoblast differentiation (20,134), the effect that may be mediated at least in part by the dysregulation of Notch and Wnt developmental signalling

**5.1 Interactions of breast cancer cells with osteoblasts** 

*Inhibition of osteoblasts by breast cancer cells* 

Osteomimicry describes the phenomenon where osteotropic cancer cells express proteins and receptors found on bone cells and the bone matrix. It was speculated that such measures allow cancer cells to evade the immune system and/or establish in the bone microenvironment (122,123). These proteins include but are not limited to osteocalcin, osteopontin, alkaline phopsphatase and Runx2 (124). Osteoblast transcription factor Runx2 is ectopically expressed by breast cancer cells and stimulates their proliferation, motility, and invasion through increased MMP9 expression from both cancer cells and osteoblasts (125,126). Runx2 has also been shown to regulate TGF-influenced PTHrP levels, as well as upregulate Indian hedgehog (127). Breast cancer cells express Hedgehog ligands that activate osteopontin expression in osteoclasts, promoting osteoclast maturation and resorptive activity through upregulated Cathepsin K and MMP9 (128,129). Of interest, expression of anti-resorptive OPG has been demonstrated to correlate with increased bonespecific homing and colonization potential in breast cancer cells (122), and to promote cancer cell survival (130,131). Osteoclastic intergrin v3 (54), has been shown to be upregulated in metastatic versus primary tumour cells, and has been identified as a critical mediator of breast cancer metastasis to bone (107,132). It is unclear whether cells from the primary tumour display osteomimetic features that allow their metastasis to bone, or whether secondary tumour cells established in the bone marrow and matrix receive environmental factors that give them their osteomimetic features. Regardless, the ability of cancer cells to produce many of these factors has been beneficial to thrive in the bone

**4.4 Osteomimicry** 

microenvironment.

pathways. Notch signalling is essential in embryogenesis but has distinct roles in bone homeostasis, regulating the proliferation of immature osteoblasts (135) and suppressing osteoblast differentiation (62,63). Upregulated Notch signalling in breast cancer, through ligand Jagged-1, has been shown to correlate with increased bone metastases (136), and breast cancer cells have been shown to induce Jagged-1 expression and upregulate Notch signalling by osteoblasts (20). Wnt signaling is also a highly conserved developmental pathway, well studied in bone and essential for osteoblast and osteoclast differentiation, as well as for the production of pro-resorptive cytokine RANKL and anti-resorptive OPG (137). Wnt inhibitor DKK-1 has been shown to be upregulated in diseases associated with bone destruction, such as osteoarthritis (138), myeloma (139), and potentially in Paget's disease (140). Blocking DKK-1 in a breast cancer metastasis model has also been shown to reverse breast cancer-mediated suppression of osteoblast differentiation and reinstate OPG expression (141). Breast cancer cells have also been shown to induce osteoblast apoptosis, through increased Bax/Bcl-2 ratio and caspase expression in osteoblasts (142,143). In addition to preventing the formation of new bone, breast cancer-induced inhibition of osteoblast differentiation likely indirectly contributes to the change in production of cytokines regulating osteoclast formation and function.

#### *Contribution of osteoblasts to the creation of an osteolytic environment*

The formation of an osteoclast-supportive microenvironment is critical for the successful establishment of an osteolytic lesion during breast cancer metastasis to bone. It has been previously shown that an increase in the ratio between a pro-resorptive RANKL and antiresorptive OPG is a key change induced by breast cancer cells (reviewed in (144,145)). Since osteoblasts are the primary source of both pro-resorptive and anti-resorptive cytokines, they represent a critical target for cancer-derived factors. Osteoblast production of RANKL is stimulated by tumour-derived PTHrP, Il-8 , Il-6 and Monocyte Chemoattractant Protein (MCP-1) (reviewed in (146)). Moreover, under the influence of breast cancer cells, undifferentiated osteoblasts express higher levels of RANKL and lower OPG, resulting in an increase in osteoblast-mediated osteoclastogenesis (20), an effect that was reversed when osteoblastic cultures were treated with the inhibitors of -secretase – an enzyme implicated in Notch signalling (20,136). One of the mediators of these changes was shown to be the tumour-overexpressed CCN3, that can inhibit osteoblast differentiation and shift the RANKL/OPG ratio to favour osteolysis (147). Another osteoblast-produced osteoclastogenic factor, MCSF, has also been implicated in breast cancer metastases to bone (148).

#### *Role of osteoblasts in supporting breast cancer cells*

An emerging area of interest is the role of osteoblasts in supporting the haematopoietic stem cell niche and how cancer cells parasitize this relationship. Haematopoiesis occurs on the endosteal surface of the bone marrow, where haematopoietic stem cells (HSCs) are maintained by the supporting cells, including osteoblasts. The main functions of the interaction between these cell types are *i*) the maintenance of HSC quiescence through osteoblast-derived osteopontin, and *ii*) modification to expand the progenitor population through Notch signaling (35,121). Several osteoblast-expressed receptors, cytokines and growth factors have been found to regulate an haematopoietic stem cell niche (149,150), including PTH/PTHrP receptors and BMPs acting to expand the osteoblast population, and Notch ligand Jagged-1 to expand the population of HSCs (35,151). Cancer cells disseminated from the primary tumour may also lay dormant for long periods of time before being

Breast Cancer Metastases to Bone: Role of the Microenvironment 543

altering both the growth and phenotype of breast cancer cells (166), and increasing their expression of CTGF, CXCL11 and PTHrP (167) via Smad and MAPK signalling in breast cancer cells (153,168,169). PTHrP increases VEGF production, leading to stimulated osteoclastogenesis through the ERK1/2 and p38 signalling pathways (170). TGF also acts on other cells present in the bone microenvironment, such as osteoclasts themselves by sensitizing them to other breast cancer derived factors (159), through the ERK1/2, p38 and c-Jun-NH2 kinase signalling pathways (160,171). In keeping with a key role of TGF in bone metastases, pharmacological inhibition of TGF signalling through the TβRI kinase inhibitor SD-208 resulted in decreased bone metastasis and tumour burden, and improved bone quality (172). The self-accelerating cycle of osteoclast stimulation by breast cancer cells, resulting in release of matrix growth factors due to osteoclastic resorption, leading to further stimulation of breast cancer cells and further increase in osteoclastic resorption was coined the name of "vicious cycle" (173), underlying the strong rationale for the use of anti-

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

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

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

resorptive drugs for the treatment of cancer metastases to bone.

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

bone metastases.

data remains unknown.

activated to form metastases (152), so it is plausible that cancer cells harvest resources from the HSCs niche to maintain their survival and to induce expansion at the right environmental cues.
