Alison Gartland

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.

LOXL2-mediated matrix remodeling in metastasis and mammary gland involution

More than 90% of cancer patient mortality is attributed to metastasis. In this study, we investigated a role for the lysyl oxidase-related enzyme lysyl oxidase-like 2 (LOXL2) in breast cancer metastasis, in both patient samples and in vivo models. Analysis of a published microarray data set revealed that LOXL2 expression is correlated with metastasis and decreased survival in patients with aggressive breast cancer. In immunocompetent or immunocompromised orthotopic and transgenic breast cancer models we showed that genetic, chemical or antibody-mediated inhibition of LOXL2 resulted in decreased metastasis. Mechanistic investigations revealed that LOXL2 promotes invasion by regulating the expression and activity of the extracellular proteins tissue inhibitor of metalloproteinase-1 (TIMP1) and matrix metalloproteinase-9 (MMP9). We found that LOXL2, TIMP1, and MMP9 are coexpressed during mammary gland involution, suggesting they function together in glandular remodeling after weaning. Finally, we found that LOXL2 is highly expressed in the basal/myoepithelial mammary cell lineage, like many other genes that are upregulated in basal-like breast cancers. Our findings highlight the importance of LOXL2 in breast cancer progression and support the development of anti-LOXL2 therapeutics for the treatment of metastatic breast cancer.

Expression of a P2X7 Receptor by a Subpopulation of Human Osteoblasts

There is now conclusive evidence that extracellular nucleotides acting via cell surface P2 receptors are important local modulators of bone cell function. Multiple subtypes of P2 receptors have been localized to bone, where their activation modulates multiple processes including osteoblast proliferation, osteoblast‐mediated bone formation, and osteoclast formation and resorptive capacity. Locally released nucleotides also have been shown to sensitize surrounding cells to the action of systemic factors such as parathyroid hormone (PTH). In nonskeletal tissue recent attention has focused on one particular P2 receptor, the P2X7 receptor (previously termed P2Z), and its ability to form nonselective aqueous pores in the plasma membrane on prolonged stimulation. Expression of this receptor originally was thought to be restricted to cells of hemopoietic origin, in which it has been implicated in cell fusion, apoptosis, and release of proinflammatory cytokines. However, recent reports have indicated expression of this receptor in cells of stromal origin. In this study, we investigated the expression of the P2X7 receptor in two human osteosarcoma cell lines, as well as several populations of primary human bone‐derived cells (HBDCs) at the levels of messenger RNA (mRNA) and protein. We found that there is a subpopulation of osteoblasts that expresses the P2X7 receptor and that these receptors are functional as assessed by monitoring ethidium bromide uptake following pore formation. Inhibition of delayed lactate dehydrogenase (LDH) release in response to the specific agonist 2′,3′‐(4‐benzoyl)‐benzoyl‐adenosine triphosphate (BzATP) by the nonspecific P2X receptor antagonist PPADS confirmed a receptor‐mediated event. After treatment with BzATP SaOS‐2 cells exhibited dramatic morphological changes consistent with those observed after P2X7‐mediated apoptosis in hemopoietic cells. Dual staining with terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐biotin nick end labeling (TUNEL) and a P2X7‐specific monoclonal antibody confirmed the induction of apoptosis in osteoblasts expressing the P2X7 receptor. These data show for the first time the expression of functional P2X7 receptors in a subpopulation of osteoblasts, activation of which can result in ATP‐mediated apoptosis.

Extracellular nucleotide signaling : A mechanism for integrating local and systemic responses in the activation of bone remodeling

Bone turnover occurs at discreet sites in the remodeling skeleton. The focal nature of this process indicates that local cues may facilitate the activation of bone cells by systemic factors. Nucleotides such as adenosine triphosphate (ATP) are locally released, short-lived, yet potent extracellular signaling molecules. These ligands act at a large family of receptors-the P2 receptors, which are subdivided into P2Y and P2X subtypes based on mechanism of signal transduction. Nucleotides enter the extracellular milieu via non-lytic and lytic mechanisms where they activate multiple P2 receptor types expressed by both osteoblasts and osteoclasts. In this review the release of ATP by bone cells is discussed in the context of activation of bone remodeling. We provide compelling evidence that nucleotides, acting via P2Y receptors, are potent potentiators of parathyroid hormone-induced signaling and transcriptional activation in osteoblasts. The provision of a mechanism to induce activation of osteoblasts above a threshold attained by systemic factors alone may facilitate focal remodeling and address the paradox of why systemic regulators like PTH exert effects at discreet sites.

Adenosine triphosphate stimulates human osteoclast activity via upregulation of osteoblast-expressed receptor activator of nuclear factor-κB ligand

Nucleotides such as adenosine triphosphate (ATP) and uridine triphosphate (UTP) exist in the extracellular environment where they are agonists at P2 receptors. Both P2Y G-protein-coupled receptors and P2X ligand-gated ion channels are expressed by osteoblasts and osteoclasts, reflected in the diverse nucleotide-induced effects reported to occur in bone. Previous reports have implicated ATP as a proresorptive agent; however, these studies were unable to determine whether ATP mediated its actions directly on osteoclasts, or indirectly via osteoblasts. The development of techniques to generate human osteoclasts in vitro has allowed us to further investigate the intriguing role of extracellular nucleotides with regard to osteoclast activity. This study reports that nearly all P2-receptor-subtype mRNAs were expressed throughout human osteoclast development, and provides evidence for functional P2 receptor expression by these cells. In cultures of human osteoclasts alone, neither ATP nor UTP affected the quantity of resorption by these cells; however, in cocultures of osteoblast-like UMR-106 cells and human osteoclasts, ATP, but not UTP, greatly enhanced resorption, indicating a role for osteoblasts in mediating the proresorptive effects of ATP. Furthermore, ATP, but not UTP, elevated receptor activator of nuclear factor-kappaB ligand (RANKL) mRNA and protein expression by UMR-106 cells. These data are consistent with observations that UMR-106 cells predominantly express P2Y(1) with low expression of P2Y(2), thereby explaining the response to ATP and not UTP, and further substantiating the involvement of osteoblasts in ATP-induced effects on osteoclasts. These results significantly advance our understanding of the role of P2 receptors in bone, and indicate that local-acting ATP may play a pivotal role in osteoclast activation at bone-resorbing sites by inducing elevated expression of RANKL.

Blockade of the pore-forming P2X7 receptor inhibits formation of multinucleated human osteoclasts in vitro

Osteoclasts are large, multinucleated, terminally differentiated cells formed by the fusion of mononuclear hemopoietic precursors. Their function is the resorption of bone, which is an essential part of the growth, modeling and remodeling of the skeleton. Though some osteoclast differentiation factors have recently been identified, the molecular basis for the fusion process that leads to multinucleation is poorly understood. The ATP-gated P2X7 receptor is a plasma membrane receptor belonging to the family of P2X purinergic receptors. It is known to be expressed by cells of hemopoietic origin where its activation leads to multiple downstream events including cytokine release, cell permeabilization and apoptosis. More recently this receptor has been implicated in the generation of multinucleated giant cells and polykaryons. Here we show that human osteoclasts express P2X7 receptors in vitro and in vivo, and that these receptors are functional in vitro, as assessed by pore-formation studies. More importantly, blockade of the P2X7 receptor with the antagonist oxidized ATP or a blocking monoclonal antibody significantly inhibits the fusion of osteoclast precursors to form multinucleated osteoclasts. Taken in combination with previous results from our laboratory demonstrating P2X7 receptor-mediated apoptosis and inhibition of bone resorption in vitro, these data suggest an important role for the P2X7 receptor in the regulation of the osteoclast population. The P2X7 receptor provides a significant new target for modulating osteoclast function in diseases characterized by increased osteoclast number and excessive bone turnover.

Effects of cobalt and chromium ions at clinically equivalent concentrations after metal-on-metal hip replacement on human osteoblasts and osteoclasts: implications for skeletal health

Metal-on-metal hip replacement (MOMHR) using large diameter bearings has become a popular alternative to conventional total hip arthroplasty, but is associated with elevated local tissue and circulating levels of chromium (Cr) and cobalt (Co) ions that may affect bone health. We examined the effects of acute and chronic exposure to these metals on human osteoblast and osteoclast formation and function over a clinically relevant concentration range previously reported in serum and within hip synovial fluid in patients after MOMHR. SaOS-2 cells were cultured with Co(2+), Cr(3+) and Cr(6+) for 3 days after which an MTS assay was used to assess cell viability, for 13 days after which alkaline phosphatase and cell viability were assessed and for 21 days after which nodule formation was assessed. Monocytes were isolated from human peripheral blood and settled onto dentine disks then cultured with M-CSF and RANKL plus either Co(2+), Cr(3+) or Cr(6+) ions for 21 days from day 0 or between days 14 and 21. Cells were fixed and stained for TRAP and osteoclast number and amount of resorption per dentine disk determined. Co(2+) and Cr(3+) did not affect osteoblast survival or function over the clinically equivalent concentration range, whilst Cr(6+) reduced osteoblast survival and function at concentrations within the clinically equivalent serum range after MOMHR (IC(50) =2.2 μM). In contrast, osteoclasts were more sensitive to metal ions exposure. At serum levels a mild stimulatory effect on resorption in forming osteoclasts was found for Co(2+) and Cr(3+), whilst at higher serum and synovial equivalent concentrations, and with Cr(6+), a reduction in cell number and resorption was observed. Co(2+) and Cr(6+) within the clinical range reduced cell number and resorption in mature osteoclasts. Our data suggest that metal ions at equivalent concentrations to those found in MOMHR affect bone cell health and may contribute to the observed bone-related complications of these prostheses.

Single-nucleotide polymorphisms in the P2X7 receptor gene are associated with post-menopausal bone loss and vertebral fractures

The purinergic P2X7 receptor has a major role in the regulation of osteoblast and osteoclast activity and changes in receptor function may therefore affect bone mass in vivo. The aim of this study was to determine the association of non-synonymous single-nucleotide polymorphisms in the P2RX7 gene to bone mass and fracture incidence in post-menopausal women. A total of 1694 women (aged 45–58) participating in the Danish Osteoporosis Prevention Study were genotyped for 12 functional P2X7 receptor variants. Bone mineral density was determined at baseline and after 10 years. In addition, vertebral fracture incidence was documented at 10 years. We found that the rate of bone loss was clearly associated with the Arg307Gln amino acid substitution such that individuals heterozygous for this polymorphism had a 40% increased rate of bone loss. Furthermore, individuals carrying the Ile568Asn variant allele had increased bone loss. In contrast, the Gln460Arg polymorphism was associated with protection against bone loss. The Ala348Thr polymorphism was associated with a lower vertebral fracture incidence 10 years after menopause. Finally, we developed a risk model, which integrated P2RX7 genotypes. Using this model, we found a clear association between the low-risk (high-P2X7 function) alleles and low rate of bone loss. Conversely, high-risk (reduced P2X7 function) alleles were associated with a high rate of bone loss. In conclusion, an association was demonstrated between variants that reduce P2X7 receptor function and increased rate of bone loss. These data support that the P2X7 receptor is important in regulation of bone mass.

Polymorphisms in the P2X7 receptor gene are associated with low lumbar spine bone mineral density and accelerated bone loss in post-menopausal women

The P2X7 receptor gene (P2RX7) is highly polymorphic with five previously described loss-of-function (LOF) single-nucleotide polymorphisms (SNP; c.151+1G>T, c.946G>A, c.1096C>G, c.1513A>C and c.1729T>A) and one gain-of-function SNP (c.489C>T). The purpose of this study was to determine whether the functional P2RX7 SNPs are associated with lumbar spine (LS) bone mineral density (BMD), a key determinant of vertebral fracture risk, in post-menopausal women. We genotyped 506 post-menopausal women from the Aberdeen Prospective Osteoporosis Screening Study (APOSS) for the above SNPs. Lumbar spine BMD was measured at baseline and at 6–7 year follow-up. P2RX7 genotyping was performed by homogeneous mass extension. We found association of c.946A (p.Arg307Gln) with lower LS-BMD at baseline (P=0.004, β=−0.12) and follow-up (P=0.002, β=−0.13). Further analysis showed that a combined group of subjects who had LOF SNPs (n=48) had nearly ninefold greater annualised percent change in LS-BMD than subjects who were wild type at the six SNP positions (n=84; rate of loss=−0.94%/year and −0.11%/year, respectively, P=0.0005, unpaired t-test). This is the first report that describes association of the c.946A (p.Arg307Gln) LOF SNP with low LS-BMD, and that other LOF SNPs, which result in reduced or no function of the P2X7 receptor, may contribute to accelerated bone loss. Certain polymorphic variants of P2RX7 may identify women at greater risk of developing osteoporosis.

Application of multiple forms of mechanical loading to human osteoblasts reveals increased ATP release in response to fluid flow in 3D cultures and differential regulation of immediate early genes

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.