**5.2 Interactions of breast cancer cells with osteoclasts**

#### *Stimulation of osteoclasts by breast cancer cells*

Breast cancer cells have been found to produce many factors capable of simulating osteoclastogenesis, both by inducing RANKL expression by osteoblasts and stromal cells, and by producing osteoclastogenic factors themselves. PTHrP was one of the first factors identified to be secreted by breast cancer cells and to promote osteolysis through the stimulation of RANKL by stromal cells (153). Although the expression of PTHrP in primary tumours has been associated with a lower incidence of bone metastasis (154,155), it was shown that increased PTHrP expression by cancer cells present in the bone metastatic lesion positively correlates with increasing osteoclast activity and subsequent osteolysis (155), suggesting that the expression pattern of the cancer cells can change during metastasis, and implicating local factors, such as TGF derived from osteoclastic bone resorption in affecting metastasizing breast cancer cells. Osteoclastogenesis may also be stimulated by IL-8 secreted from breast cancer cells and acting both directly on osteoclasts and through osteoblastic RANKL signalling (156,157). Although the mechanisms of IL-8 action are not fully understood, the expression of IL-8 correlated with a higher incidence of bone metastasis in mice *in vivo* (158).

It has also been shown that during differentiation osteoclast precursors may acquire sensitivity to cancer-derived factors that are ineffective in inducing osteoclast formation from naive monocytes (159). Several signalling pathways in osteoclast precursors have been implicated in these effects, including calcium signalling, NFATc1 activation and MAPKs ERK1/2 and p38 (159,160). Tumour-produced CCN3 was demonstrated to stimulate osteoclast formation from RANKL-primed osteoclast precursors (147). These effects can be relevant to the propensity of cancer cells to metastasize to bone sites undergoing active bone remodelling, and thus containing increased numbers of RANKL-primed osteoclast precursors. At such sites, breast cancer cells can promote further osteoclast formation, and can affect the survival of mature osteoclasts, increasing their resorptive capacity. In this regard, M-CSF secreted from breast cancer cells was shown to be responsible for the delayed apoptosis in osteoclasts (146,161). Anti-apoptotic effects of breast cancer-derived factors included PLC-mediated suppression of pro-apoptotic protein BIM, and M-CSF-mediated inhibition of caspase cleavage (146).

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

During osteoclastic resorption, the bone matrix components, including many growth factors stored in the bone, such as TGFβ, BMPs, IGFs, fibroblast growth factors (FGFs), and plateletderived growth factors (PDGF) are released into extracellular space, where they are free to act on surrounding cells, including metastasizing cancer cells (162). Matrix released- TGFβ activated by osteoclastic resorption (163), is one of the most commonly studied matrixderived growth factors, which was shown to stimulate cancer cell growth, modify cell invasion, and affect immune regulation (164,165). Considerable research has linked increased TGF-β in the microenvironment to the progression of metastasis, with TGF

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

Breast cancer cells have been found to produce many factors capable of simulating osteoclastogenesis, both by inducing RANKL expression by osteoblasts and stromal cells, and by producing osteoclastogenic factors themselves. PTHrP was one of the first factors identified to be secreted by breast cancer cells and to promote osteolysis through the stimulation of RANKL by stromal cells (153). Although the expression of PTHrP in primary tumours has been associated with a lower incidence of bone metastasis (154,155), it was shown that increased PTHrP expression by cancer cells present in the bone metastatic lesion positively correlates with increasing osteoclast activity and subsequent osteolysis (155), suggesting that the expression pattern of the cancer cells can change during metastasis, and implicating local factors, such as TGF derived from osteoclastic bone resorption in affecting metastasizing breast cancer cells. Osteoclastogenesis may also be stimulated by IL-8 secreted from breast cancer cells and acting both directly on osteoclasts and through osteoblastic RANKL signalling (156,157). Although the mechanisms of IL-8 action are not fully understood, the expression of IL-8 correlated with a higher incidence of bone metastasis in

It has also been shown that during differentiation osteoclast precursors may acquire sensitivity to cancer-derived factors that are ineffective in inducing osteoclast formation from naive monocytes (159). Several signalling pathways in osteoclast precursors have been implicated in these effects, including calcium signalling, NFATc1 activation and MAPKs ERK1/2 and p38 (159,160). Tumour-produced CCN3 was demonstrated to stimulate osteoclast formation from RANKL-primed osteoclast precursors (147). These effects can be relevant to the propensity of cancer cells to metastasize to bone sites undergoing active bone remodelling, and thus containing increased numbers of RANKL-primed osteoclast precursors. At such sites, breast cancer cells can promote further osteoclast formation, and can affect the survival of mature osteoclasts, increasing their resorptive capacity. In this regard, M-CSF secreted from breast cancer cells was shown to be responsible for the delayed apoptosis in osteoclasts (146,161). Anti-apoptotic effects of breast cancer-derived factors included PLC-mediated suppression of pro-apoptotic protein BIM, and M-CSF-mediated

During osteoclastic resorption, the bone matrix components, including many growth factors stored in the bone, such as TGFβ, BMPs, IGFs, fibroblast growth factors (FGFs), and plateletderived growth factors (PDGF) are released into extracellular space, where they are free to act on surrounding cells, including metastasizing cancer cells (162). Matrix released- TGFβ activated by osteoclastic resorption (163), is one of the most commonly studied matrixderived growth factors, which was shown to stimulate cancer cell growth, modify cell invasion, and affect immune regulation (164,165). Considerable research has linked increased TGF-β in the microenvironment to the progression of metastasis, with TGF

**5.2 Interactions of breast cancer cells with osteoclasts** 

*Stimulation of osteoclasts by breast cancer cells* 

environmental cues.

mice *in vivo* (158).

inhibition of caspase cleavage (146).

*Role of osteoclasts in supporting breast cancer cells* 

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 antiresorptive drugs for the treatment of cancer metastases to bone.
