Ankita Agrawal

The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase

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 hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality. 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 patients with oestrogen-receptor negative breast cancer. 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 colonize and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications.

Role of purinergic receptor polymorphisms in human bone.

Osteoporosis is a multifactorial disease with a strong genetic component. Variations in a number of genes have been shown to associate with bone turnover and risk of osteoporosis. P2 purinergic receptors are proteins that have ATP or other nucleotides as their natural ligands. Various P2Y and P2X receptor subtypes have been identified on bone cells. Several cellular functions in bone tissue are coupled to P2-receptor activation, including bone resorption, cytokine release, apoptosis, bone formation, and mineral deposition. Furthermore, ATP release and P2 purinergic signalling is a key pathway in the mechanotransductory process, where mechanical stimulation on bone leads to anabolic responses in the skeleton. A number of single nucleotide polymorphisms have been identified in the P2 receptor genes, where especially the P2X7 subtype has been the focus of extensive investigation where several polymorphisms have been shown to have functional implications on receptor function; moreover, some polymorphisms are associated with alterations in bone turnover and bone mass. This review focuses on variations in P2 receptor genes and the association to bone turnover and -quality.

Reduced Bone Turnover in Mice Lacking the P2Y13 Receptor of ADP

Osteoporosis is a condition of excessive and uncoupled bone turnover, in which osteoclastic resorption exceeds osteoblastic bone formation, resulting in an overall net bone loss, bone fragility, and morbidity. Although numerous treatments have been developed to inhibit bone loss by blocking osteoclastic bone resorption, understanding of the mechanisms behind bone loss is incomplete. The purinergic signaling system is emerging to be a pivotal regulator of bone homeostasis, and extracellular ADP has previously been shown to be a powerful osteolytic agent in vitro. We report here that deletion of the P2Y(13) receptor, a G protein-coupled receptor for extracellular ADP, leads to a 40% reduction in trabecular bone mass, 50% reduction in osteoblast and osteoclast numbers in vivo, as well as activity in vitro, and an overall 50% reduction in the rate of bone remodeling in mice in vivo. Down-regulation of RhoA/ROCK I signaling and a reduced ratio of receptor activator of nuclear factor κB ligand/osteoprotegerin observed in osteoblasts from P2Y(13)R(-/-) mice might explain this bone phenotype. Furthermore, because one of the main causes of osteoporosis in older women is lack of estrogen, we examined the effect of ovariectomy of the P2Y(13)R(-/-) mice and found them to be protected from ovariectomy-induced bone loss by up to 65%. These data confirm a role of purinergic ADP signaling in the skeleton, whereby deletion of the P2Y(13) receptor leads to reduced bone turnover rates, which provide a protective advantage in conditions of accelerated bone turnover such as oestrogen deficiency-induced osteoporosis.

The effects of P2X7 receptor antagonists on the formation and function of human osteoclasts in vitro.

The P2X7 receptor (P2X7R) has been implicated in the process of multinucleation and cell fusion. We have previously demonstrated that blockade of P2X7Rs on osteoclast precursors using a blocking antibody inhibited multinucleated osteoclast formation in vitro, but that P2X7R KO mice maintain the ability to form multinucleated osteoclasts. This apparent contradiction of the role the P2X7R plays in multinucleation has prompted us to examine the effect of the most commonly used and recently available P2X7R antagonists on osteoclast formation and function. When added to recombinant RANKL and M-CSF human blood monocytes cultures, all but one compound, decreased the formation and function of multinucleated TRAP-positive osteoclasts in a concentration-dependent manner. These data provide further evidence for the role of the P2X7R in the formation of functional human multinucleated osteoclasts and highlight the importance of selection of antagonists for use in long-term experiments.

Purinergic signalling in bone

Purinergic signaling in bone was first proposed in the early 1990s with the observation that extracellular ATP could modulate events crucial to the normal functioning of bone cells. Since then the expression of nearly all the P2Y and P2X receptors by osteoblasts and osteoclasts has been reported, mediating multiple processes including cell proliferation, differentiation, function, and death. This review will highlight the most recent developments in the field of purinergic signaling in bone, with a special emphasis on recent work resulting from the European Framework 7 funded collaboration ATPBone, as well as Arthritis Research UK and Bone Research Society supported projects.

P2X7 receptors: role in bone cell formation and function

The role of the P2X7 receptor (P2X7R) is being explored with intensive interest in the context of normal bone physiology, bone-related diseases and, to an extent, bone cancer. In this review, we cover the current understanding of P2X7R regulation of bone cell formation, function and survival. We will discuss how the P2X7R drives lineage commitment of undifferentiated bone cell progenitors, the vital role of P2X7R activation in bone mineralisation and its relatively unexplored role in osteocyte function. We also review how P2X7R activation is imperative for osteoclast formation and its role in bone resorption via orchestrating osteoclast apoptosis. Variations in the gene for the P2X7R (P2RX7) have implications for P2X7R-mediated processes and we review the relevance of these genetic variations in bone physiology. Finally, we highlight how targeting P2X7R may have therapeutic potential in bone disease and cancer.

Human Osteoclast Culture and Phenotypic Characterization

Bone remodelling occurs throughout life via the coupled actions of bone resorption and bone formation. When the balance of bone resorption and formation becomes unequal, bone diseases, such as osteoporosis occur, while the absence of functional osteoclasts leads to diseases such as osteopetrosis and pycnodysostosis. In order to develop effective treatments for bone disease the normal regulatory systems involved in bone resorption need to be fully elucidated. The only cell in the body capable of resorbing bone is the osteoclast - a highly specialized cell of haematopoietic origin. Until relatively recently, the ability to study the formation and function of human osteoclasts in vitro has been limited.In this chapter, we provide an up-to-date detailed guide to isolating and culturing primary osteoclasts from human peripheral blood. In addition, we detail the methodology used to characterize osteoclasts and how to quantify resorption in vitro. In combination these methods provide a powerful tool in the study of human osteoclasts and the development of new novel treatments for bone disease.

P2X7Rs are involved in cell death, growth and cellular signaling in primary human osteoblasts

The ionotropic ATP-gated P2X7 receptor (P2X7R) is involved in the regulation of many physiological functions including bone metabolism. Several studies on osteoblasts from rodents and human osteoblast-like cell lines have addressed the expression and function of P2X7R on these bone-forming cells however; its role in human primary osteoblasts has not yet been reported. The aim of this study was to assess the expression of the P2X7R in bone marrow-derived stromal cells and in primary human trabecular osteoblasts and to determine the function in bone formation and cell signaling. We report that osteoblasts derived from human trabecular explants express a functional P2X7R capable of agonist-induced increase in intracellular calcium concentration and a positive permeability to fluorescent dyes. These osteoblasts are fully differentiated cells with alkaline phosphatase activity and the ability to form mineralized nodules. We show that the transcriptional regulation of osteoblastic markers can be modulated by P2X7R activity or blockade thereby influencing the differentiation, proliferation and bone matrix formation by these primary human osteoblasts. Finally, we demonstrate that the P2X7R is involved in propagation of mechanically-induced intercellular signaling in addition to the known mechanisms involving calcium signaling via P2Y2 receptors and gap junction.

P2X7 receptor regulates osteoclast function and bone loss in a mouse model of osteoporosis

Post-menopausal osteoporosis is a condition that affects millions worldwide and places a huge socio-economic burden on society. Previous research has shown an association of loss of function SNPs in the gene for the purinergic receptor P2X7R with low bone mineral density, increased rates of bone loss and vertebral fractures in post-menopausal women. In this study we use a mouse model of oestrogen deficiency-induced bone loss and the BALB/cJ P2X7R−/− to show that absence of the P2X7R resulted in increased bone loss. Osteoclast precursors were isolated from both BALB/cJ P2X7R−/− and BALB/cJ P2X7R+/+ mice and then cultured in vitro to form mature resorbing osteoclasts. The BALB/cJ P2X7R−/− derived precursors generated slightly more osteoclasts but with a significant reduction in the amount of resorption per osteoclast. Furthermore, when using modified culture conditions osteoclast activity was additionally increased in the absence of the P2X7R suggest that P2X7R may regulate the lifespan and activity of osteoclasts. Finally using mechanical loading as an anabolic stimulus for bone formation, we demonstrated that the increased oestrogen-deficient bone loss could be rescued, even in the absence of P2X7R. This study paves the way for clinical intervention for women with post-menopausal osteoporosis and P2XR7 loss of function polymorphisms.

Bone turnover is altered in transgenic rats overexpressing the P2Y2 purinergic receptor

It is now widely recognized that purinergic signaling plays an important role in the regulation of bone remodeling. One receptor subtype, which has been suggested to be involved in this regulation, is the P2Y2 receptor (P2Y2R). In the present study, we investigated the effect of P2Y2R overexpression on bone status and bone cell function using a transgenic rat. Three-month-old female transgenic Sprague Dawley rats overexpressing P2Y2R (P2Y2R-Tg) showed higher bone strength of the femoral neck. Histomorphometry showed increase in resorptive surfaces and reduction in mineralizing surfaces. Both mineral apposition rate and thickness of the endocortical osteoid layer were higher in the P2Y2R-Tg rats. μCT analysis showed reduced trabecular thickness and structural model index in P2Y2R-Tg rats. Femoral length was increased in the P2Y2R-Tg rats compared to Wt rats. In vitro, there was an increased formation of osteoclasts, but no change in total resorption in cultures from P2Y2R-Tg rats. The formation of mineralized nodules was significantly reduced in the osteoblastic cultures from P2Y2R-Tg rats. In conclusion, our study suggests that P2Y2R is involved in regulation of bone turnover, due to the effects on both osteoblasts and osteoclasts and that these effects might be relevant in the regulation of bone growth.