Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia1. Tumour-secreted proteins play a crucial role in these interactions2-5 and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable6. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality6,7. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in ER-negative breast cancer patients. Global quantitative analysis of the hypoxic secretome identified Lysyl Oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis, independent of RANK Ligand, which disrupts normal bone homeostasis leading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to coloniseCorrespondence and requests for materials should be addressed to janine.erler@bric.ku.dk and a.gartland@shef.ac.uk.

ATP is actively released into the extracellular environment from a variety of cell types in response to mechanical stimuli. This is particularly true in bone where mechanically induced ATP release leads to immediate early gene activation to regulate bone remodelling; however there is no consensus as to which mechanical stimuli stimulate osteoblasts the most. To elucidate which specific type(s) of mechanical stimuli induce ATP release and gene activation in human osteoblasts, we performed an array of experiments using different mechanical stimuli applied to both monolayer and 3D cultures of the same osteoblast cell type, SaOS-2. ATP release from osteoblasts cultured in monolayer significantly increased in response to turbulent fluid flow, laminar fluid flow and substrate strain. No significant change in ATP release could be detected in 3D osteoblast cultures in response to cyclic or static compressive loading of osteoblast-seeded scaffolds, whilst turbulent fluid flow increased ATP release from 3D cultures of osteoblasts to a greater degree than observed in monolayer cultures. Cox-2 expression quantified using real time PCR was significantly lower in cells subjected to turbulent fluid flow whereas c-fos expression was significantly higher in cells subjected to strain. Load-induced signalling via c-fos was further investigated using a SaOS-2 c-fos luciferase reporter cell line and increased in response to substrate strain and turbulent fluid flow in both monolayer and 3D, with no significant change in response to laminar fluid flow or 3D compressive loading. The results of this study demonstrate for the first time strain-induced ATP release from osteoblasts and that turbulent fluid flow in 3D up regulates the signals required for bone remodelling.

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y 13 receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y 13 receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts. To test this hypothesis, wild-type (WT) and P2Y 13 receptor knockout (P2Y 13 R À/À ) mice were subject to non-invasive axial mechanical loading of the left tibiae to induce an osteogenic response. Micro-computed tomography analysis showed mechanical loading induced an osteogenic response in both strains of mice in terms of increased total bone volume and cortical bone volume, with the P2Y 13 R À/À mice having a significantly greater response. The extent of the increased osteogenic response was defined by dynamic histomorphometry data showing dramatically increased bone formation and mineral apposition rates in P2Y 13 R À/À mice compared with controls. In vitro, primary P2Y 13 R À/À osteoblasts had an accumulation of mechanically induced extracellular ATP and reduced levels of hydrolysis. In addition, P2Y 13 R À/À osteoblasts also had a reduction in their maximal alkaline phosphatase (ALP) activity, one of the main ecto-enzymes expressed by osteoblasts, which hydrolyzes extracellular ATP. In conclusion, deletion of the P2Y 13 receptor leads to an enhanced osteogenic response to mechanical loading in vivo, possibly because of the reduced extracellular ATP degradation by ALP. The augmented osteogenic response to mechanical stimulation, combined with suppressed bone remodeling activities and protection from OVXinduced bone loss after P2Y 13 receptor depletion as previously described, suggests a potential role for P2Y 13 receptor antagonist-based therapy, possibly in combination with mechanical loading, for the treatment of osteoporosis.

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The skeleton is maintained throughout life via the finely tuned actions of osteoblasts and osteoclasts, with disruption in this balance eventually leading to bone disease. The exact mechanisms balancing these actions are not fully known, although several regulatory systems are known to be involved. The involvement of purinergic signalling in bone has come to light over the past 20 years or so. This review will highlight the current knowledge of purinergic signalling in osteoblasts - covering expression of P2 receptors, mechanisms of ATP release and degradation, P2 receptor mediated signalling and finally the functional consequences of P2 receptor signalling in bone.