**4. Homing of breast cancer cells to bone**

#### **4.1 Creation of the pre-metastatic niche**

Recent evidence has led to the idea that the bone marrow supports a pre-metastatic niche - a site that receives signals from the primary tumour mass before dissemination, and changes the landscape of the target tissue to be conducive to tumour growth. It has been shown in mice treated with medium conditioned by tumour cells of different origin, the potential to home to different organs of subsequently injected cancer cells can be altered (88). In

Breast Cancer Metastases to Bone: Role of the Microenvironment 539

breast tissue (15), and SDF-1 is strongly expressed in lung, liver, bone marrow and lymph nodes, the primary sites of secondary breast tumours, leading to the identification of the role of the SDF-1/CXCR4 in promoting breast cancer metastasis to bone (91). In addition to directional migration, chemokines have been shown to promote cancer cell survival, proliferation, and adhesion (92). In keeping, the inhibition of CXCR4 limited breast cancer metastases in mice (93), and the overexpression of CXCR4 indicates poor prognosis in both human and murine breast cancer (92,94). Another chemokine implicated in metastases of breast cancer cells expressing high levels of CCL21, is CCR7 that is expressed highly in metastatic sites, such as lymph nodes (15). Since haematopoietic stem cells (HSCs) use these chemokine and receptor interactions to home to the HSC niche in the bone marrow, it has been suggested that cancer cells use this same mechanism to parasitize these microenvironments and harvest the resources of HSCs (95). Another pertinent means of cancer cell migration towards bone relies on the cancer cell expression of RANK (96), which mediates directional migration of breast, melanoma and prostate cancer cells towards

Breast cancer cells may also stimulate the action of matrix metalloproteinases that support cancer cell migration and invasion. The murine orthologue of Glycogen Nonmetastatic Melanoma Protein B (GPNMB) is called osteoactivin and has been identified as a key modulator of osteolysis. Its forced expression leads to increased tumour grade and enhanced bone metastasis by upregulated MMP3 through ERK signaling (99,100). Furthermore, GPNMB was identified as a poor prognostic marker in patients with breast cancer (101). Most recently, this group has identified ADAM10 as a sheddase that releases osteoactivin from the cell, which induces endothelial cell migration and subsequent angiogenesis (102). ADAMTS1 and MMP1 are also tumour-derived metalloproteinases able to degrade the matrix. The stimulated action of these enzymes by breast cancer cells enhances osteoclast differentiation by suppressing OPG expression, and their expression in

Cancer cells express or induce the expression of adhesion molecules that may facilitate their interactions with the bone microenvironment. The best studied family of proteins that bind cancer cells to bone cells are integrins, heterodimeric transmembrane glycoproteins whose and subunits combine to form 24 known combinations with unique specificity for binding, signaling and regulatory mechanisms (104). Integrins have been demonstrated to be involved in several stages of cancer dissemination, with highly metastatic cancer cells displaying a different integrin profile than cells from the primary tumour (105). Several integrins have been shown to interact with extracellular matrix proteins during bone metastasis, with the most important being v3, a receptor for osteopontin, fibronectin and vitronectin (106). Adhesion molecules engaged between breast cancer cells and bone cells may overlap with those that bind haematopoietic stem cells (HSC) to osteoblasts. HSC preferentially home to areas with more fibronectin (88). Breast cancer cells can attach to fibronectin, in an integrin-dependent manner (107). The interaction of cancer cells with fibronectin increases the production of matrix metalloproteinase-2 from fibroblasts to facilitate invasion (108). Another molecule involved the adhesion of HSC to the endosteal niche is annexin II (95). By serving as an anchor for SDF-1/CXCL12, it has been shown to regulate the homing of HSC as well as prostate cancer cells to the HSC niche (109,110).

human samples correlates to a greater incidence of bone metastases (103).

**4.3 Attachment proteins between breast cancer cells and the bone** 

RANKL, produced in bone by osteoblasts (97,98).

particular, in bone, bone marrow derived hematopoietic stem cells have been implicated in mediating the establishment of pre-metastatic niche (19,88). Molecular mediators such as vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) and integrin α4β1 have been implicated in this process. VEGFR1 positive haematopoietic progenitor cells are recruited to sites of future metastasis (88). VEGF receptors are expressed by breast cancer cells as well as osteoclasts and osteoclast precursors, and VEGF expression correlates to increased tumour size and grade in humans (89). In addition, we have shown that breast cancer cells secrete factors that support the subsequent attachment of breast cancer cells acting at least in part through -secretase-mediated Notch signalling (20).

Fig. 2. Cell-cell interactions in the bone microenvironment. Osteoclast differentiation from monocytic precursors is induced by M-CSF, RANKL produced by osteoblastic cells. Osteoblasts are derived from mesenchymal stem cells through Wnt and BMP signalling pathways. Osteoblasts and osteoclasts communicate through osteoblast-derived RANKL/OPG and bidirectional Ephrin-B2/EphB4 signalling. Haematopoietic stem cells (HSC) support osteoblasts in the HSC niche through BMPs, while osteoblasts support HSCs through upregulated Notch signalling through Jagged-1. Osteoclasts cleave SDF-1 to mobilize HSCs from the endosteal niche.

#### **4.2 Migration of breast cancer cells to bone**

Breast cancer cells express receptors that direct their movement towards fertile sites where they may establish into secondary tumors. These proteins are generally expressed in normal cells, and are often involved in developmental pathways. Several chemokines have been suggested to be released from the bone microenvironment, implicating chemoattraction through G-protein-coupled chemokine receptors in driving the movement of tumour cells towards bone (90). Interactions between stromal-derived factor-1 (SDF-1) and CXCR4 are essential for the correct localization of lymphocytes and haematopoietic cells in physiological states. Breast cancer cells express higher levels of CXCR4 compared to normal

particular, in bone, bone marrow derived hematopoietic stem cells have been implicated in mediating the establishment of pre-metastatic niche (19,88). Molecular mediators such as vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) and integrin α4β1 have been implicated in this process. VEGFR1 positive haematopoietic progenitor cells are recruited to sites of future metastasis (88). VEGF receptors are expressed by breast cancer cells as well as osteoclasts and osteoclast precursors, and VEGF expression correlates to increased tumour size and grade in humans (89). In addition, we have shown that breast cancer cells secrete factors that support the subsequent attachment of breast cancer cells

Fig. 2. Cell-cell interactions in the bone microenvironment. Osteoclast differentiation from monocytic precursors is induced by M-CSF, RANKL produced by osteoblastic cells. Osteoblasts are derived from mesenchymal stem cells through Wnt and BMP signalling pathways. Osteoblasts and osteoclasts communicate through osteoblast-derived

RANKL/OPG and bidirectional Ephrin-B2/EphB4 signalling. Haematopoietic stem cells (HSC) support osteoblasts in the HSC niche through BMPs, while osteoblasts support HSCs through upregulated Notch signalling through Jagged-1. Osteoclasts cleave SDF-1 to

Breast cancer cells express receptors that direct their movement towards fertile sites where they may establish into secondary tumors. These proteins are generally expressed in normal cells, and are often involved in developmental pathways. Several chemokines have been suggested to be released from the bone microenvironment, implicating chemoattraction through G-protein-coupled chemokine receptors in driving the movement of tumour cells towards bone (90). Interactions between stromal-derived factor-1 (SDF-1) and CXCR4 are essential for the correct localization of lymphocytes and haematopoietic cells in physiological states. Breast cancer cells express higher levels of CXCR4 compared to normal

mobilize HSCs from the endosteal niche.

**4.2 Migration of breast cancer cells to bone** 

acting at least in part through -secretase-mediated Notch signalling (20).

breast tissue (15), and SDF-1 is strongly expressed in lung, liver, bone marrow and lymph nodes, the primary sites of secondary breast tumours, leading to the identification of the role of the SDF-1/CXCR4 in promoting breast cancer metastasis to bone (91). In addition to directional migration, chemokines have been shown to promote cancer cell survival, proliferation, and adhesion (92). In keeping, the inhibition of CXCR4 limited breast cancer metastases in mice (93), and the overexpression of CXCR4 indicates poor prognosis in both human and murine breast cancer (92,94). Another chemokine implicated in metastases of breast cancer cells expressing high levels of CCL21, is CCR7 that is expressed highly in metastatic sites, such as lymph nodes (15). Since haematopoietic stem cells (HSCs) use these chemokine and receptor interactions to home to the HSC niche in the bone marrow, it has been suggested that cancer cells use this same mechanism to parasitize these microenvironments and harvest the resources of HSCs (95). Another pertinent means of cancer cell migration towards bone relies on the cancer cell expression of RANK (96), which mediates directional migration of breast, melanoma and prostate cancer cells towards RANKL, produced in bone by osteoblasts (97,98).

Breast cancer cells may also stimulate the action of matrix metalloproteinases that support cancer cell migration and invasion. The murine orthologue of Glycogen Nonmetastatic Melanoma Protein B (GPNMB) is called osteoactivin and has been identified as a key modulator of osteolysis. Its forced expression leads to increased tumour grade and enhanced bone metastasis by upregulated MMP3 through ERK signaling (99,100). Furthermore, GPNMB was identified as a poor prognostic marker in patients with breast cancer (101). Most recently, this group has identified ADAM10 as a sheddase that releases osteoactivin from the cell, which induces endothelial cell migration and subsequent angiogenesis (102). ADAMTS1 and MMP1 are also tumour-derived metalloproteinases able to degrade the matrix. The stimulated action of these enzymes by breast cancer cells enhances osteoclast differentiation by suppressing OPG expression, and their expression in human samples correlates to a greater incidence of bone metastases (103).

#### **4.3 Attachment proteins between breast cancer cells and the bone**

Cancer cells express or induce the expression of adhesion molecules that may facilitate their interactions with the bone microenvironment. The best studied family of proteins that bind cancer cells to bone cells are integrins, heterodimeric transmembrane glycoproteins whose and subunits combine to form 24 known combinations with unique specificity for binding, signaling and regulatory mechanisms (104). Integrins have been demonstrated to be involved in several stages of cancer dissemination, with highly metastatic cancer cells displaying a different integrin profile than cells from the primary tumour (105). Several integrins have been shown to interact with extracellular matrix proteins during bone metastasis, with the most important being v3, a receptor for osteopontin, fibronectin and vitronectin (106). Adhesion molecules engaged between breast cancer cells and bone cells may overlap with those that bind haematopoietic stem cells (HSC) to osteoblasts. HSC preferentially home to areas with more fibronectin (88). Breast cancer cells can attach to fibronectin, in an integrin-dependent manner (107). The interaction of cancer cells with fibronectin increases the production of matrix metalloproteinase-2 from fibroblasts to facilitate invasion (108). Another molecule involved the adhesion of HSC to the endosteal niche is annexin II (95). By serving as an anchor for SDF-1/CXCL12, it has been shown to regulate the homing of HSC as well as prostate cancer cells to the HSC niche (109,110).

Breast Cancer Metastases to Bone: Role of the Microenvironment 541

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

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).

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

cytokines regulating osteoclast formation and function.

*Role of osteoblasts in supporting breast cancer cells* 

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

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.
