1. Introduction
Primary bone cancers, malignancies that originate directly from bone cells, are quite rare diseases. According to USA National Cancer Institute about 2810 people were diagnosed, and 1500 died of bone and joint cancer in 2011. A similar incidence, one affected individual every 100.000, was reported in the same year by the Italian Association for Cancer Research (AIRC). Primary bone cancers generally originate in long bones of limbs and affect children or young adults accounting for 6% of all new pediatric cancer per year [ 1 ]. Among primary bone cancers osteosarcoma is the most frequent [ 2 ]. Two different osteosarcoma variants are known: a conventional high-grade form intramedullary located at the metaphysis of long tubular bones, frequent in adolescents, and a rarer low-grade variant arising at the surface of long bones, showing a better prognosis, more frequent among adults than young people. Frequency of low-grade is 20 times lower than high-grade ones [ 3 ].
A peculiar primary tumor, causing bone damage without need of spreading from the original site, is multiple myeloma. Multiple myeloma is a clonal B-cell malignancy characterized by an accumulation of mature plasma cells in the bone marrow, leading to bone destruction and failure of normal hematopoiesis [ 4 , 5 ]. The incidence is 3–9 cases/100.000/year; it is more frequent among elderly with a slight male prevalence. Multiple myeloma remains an incurable disease even with the use of proteasome inhibitor bortezomib, immuno-modulatory drugs (thalidomide or lenalidomide), and high-dose chemotherapy associated to autologous stem cell transplantation, as part of first-line therapy [ 6 ]. In multiple myeloma, tumor and stromal cells interact via adhesion molecules and cytokine networks to simultaneously promote tumor cell survival, drug resistance, angiogenesis, and disordered bone metabolism.
Bone metastasis of extra-bone high-grade solid tumors is more frequent than primary bone cancers. The rate of bone metastasis is about 70% in breast, melanoma, lung and prostate cancer and about 15–30% in colon, stomach, bladder, uterus, rectum, thyroid, and kidney carcinomas [ 7 ]. Symptoms related to bone metastasis include pain, fractures, spinal cord compression, and hypercalcemia leading to poor quality of life and reduced life expectancy. It is estimated that the appearance of bone metastasis can reduce the five-years survival rate of breast cancer patients from 98% to 26% and that of prostate cancer patients from 100% to 33% [ 8 ].
Both primary and secondary cancers involving the skeleton can cause osteoblastic (sclerotic) or osteolytic lesions, although generally the final histological picture is a mixture of the two. Osteoblastic lesions originate from proliferation of osteoblasts, while osteolytic lesions are generally due to osteoclast activation caused by factors secreted by cancer cells. Common sites of metastasis in the skeleton are the spine, rib cage, limbs and skull. Once settled into the bone, tumor cells release factors that activate matrix resorption leading to bone destruction; this facilitates cancer spread and proliferation [ 8 , 9 ]. Since bone resorption activity is followed by an increase in bone formation, as it occurs in normal remodeling, these two processes are intimately linked and typically present at sites of bone metastasis. Osteolytic metastasis are common in breast cancer, mainly due to stimulation by tumor cells of osteoclast differentiation and activity [ 10 ]. An associated local bone formation usually occurs, presumably in an attempt to activate repair, but this response is often inefficient, thus leading to final bone loss. In multiple myeloma tumor cells in the bone marrow cause exclusively osteolytic lesions, with almost complete absence of bone formation [ 4 ]. This seems to be due to suppression of osteoblast activity. On the contrary, prostate cancer metastasis are primarily osteoblastic, with possible presence of osteolytic components [ 11 ]. Most of the factors implicated in osteolytic metastatization are also involved in the pathogenesis of osteoporosis to the point that pharmacological treatment of osteolytic metastasis and osteoporosis is similar.
PTHrP (parathormone-related protein) and TGF- β (transforming growth factor beta) are among the most important osteolytic mediators [ 9 ]. PTHrP secreted by breast cancer and other tumors, is a well-known potent stimulator of osteoclast activity in bone metastasis. PTHrP acts through the release of RANKL (receptor activator of nuclear factor kB ligand) which binds to RANK receptor on osteoclasts, the system RANK-RANKL being the main pathway for osteoclast differentiation and activation. Factors released by metastatic cells or by the primary tumor activate bone resorption, this in turn triggers release of TGF- β from bone matrix. TGF- β and tumor-derived PTHrP are believed to act in a vicious cycle of local bone destruction in osteolytic metastasis: TGF- β released in active form during osteoclastic resorption of bone matrix stimulates PTHrP production by tumor cells. In turn, PTHrP mediates bone destruction by stimulating osteoclasts [ 4 , 12 ]. Furthermore, TGF- β released from bone exerts suppressive effects on T lymphocytes and NK cells, thus reducing immune cell response to tumor [ 13 ].
Growth of primary bone tumors and development of bone metastasis are complex processes involving bone-tumor cells crosstalk mediated by several different cytokines and growth factors. In this context the study of molecules moulding tumor micro-environment is of great importance for a better understanding of cancer biology and to find new and efficacious therapies. Here we suggest a possible role of extracellular ATP and its receptor P2X7 in the development of both primary bone cancers and skeletal metastasis.
2. Role of ATP and P2X7 Receptors in Cell Growth and Carcinogenesis
In recent years investigation of tumor microenvironment has gained great importance for the understanding of tumor formation and progression [ 14 ]. Several molecules are released by tumor cells, acting as promoters of proliferation, allowing for immune system escape, helping cell matrix infiltration, neovascularization and distant site invasion. Interestingly, mediators of inflammatory-immune response can also influence cancer progression [ 15 ]. A good candidate molecule for many of these functions is extracellular ATP. This nucleotide has been recently shown to accumulate both at inflammatory sites [ 16 , 17 ] and into the interstitium of solid tumors of different origin [ 18 ]. ATP release by dying tumor cells, following chemotherapy, is also associated to immunogenic cell death [ 19 ]. Immunogenic cell death implies that the host immune system is essential for antitumor effect of certain chemotherapeutic agents such as anthracyclines, and that molecules released from dying tumor cells act as danger signals to activate the host immune response. In this context ATP released from neoplastic cells drives recruitment of dendritic cells and T lymphocytes into the tumor site [ 20 , 21 ]. Effects of ATP are mediated by two families of plasma membrane purinergic receptors: P2Y, metabotropic G coupled receptors, and P2X, ligand gated ion channels [ 22 ]. The P2X7 receptor is involved in many of the tumor-promoting and immune-modulatory effects of extracellular ATP. Like other members of the P2X family, P2X7 mediates cation fluxes across the plasma membrane but thanks to its peculiar C terminal tail it also gates a large nonselective pore [ 23 ]. Opening of this pore is coupled to the well-known P2X7 cytotoxic activity usually triggered by high (i.e. mM) pharmacological ATP concentrations. On the contrary, basal tonic P2X7 activation mediated by endogenous ATP release causes cell proliferation [ 24 , 25 ]. Proliferation and other tumor transformation hallmarks seem to be dependent on the channel activity since they are retained also by cells expressing a C-terminal truncated P2X7 splice variant, which lacks pore-forming activity [ 26 ]. Several reports suggest an association between P2X7 and cancer [ 27 ]. In different cell models P2X7 expression supports organellar calcium increase, NFATc1 activation, matrix infiltration and cell growth [ 24 – 26 , 28 , 29 ]. There is good evidence that P2X7 receptor can influence energy production by increasing mitochondrial potential and intracellular ATP levels [ 25 ], a condition linked to the PI3 K/Akt pathway that positively influences cell proliferation [ 30 ]. Moreover, P2X7 activation alters the biochemical composition of tumor by causing release of microvescicles [ 31 , 32 ] and secretion of cytokines [ 33 ], tissue factor [ 34 ], matrix metalloproteases (MMPs) [ 35 ] and prostaglandins [ 36 ]. Furthermore, P2X7 itself can be a conduit for ATP release [ 37 ]. Direct participation of P2X7 in tumor progression was demonstrated in a recent in vivo study by our laboratory [ 38 ]. We showed that P2X7 inhibition by either pharmacological tools or RNA interference caused a dramatic reduction of tumor masses, and vice-versa that P2X7 overexpression accelerated tumor growth. Interestingly P2X7 expressing tumors showed a thicker vascular network and higher secretion of vascular endothelial growth factor (VEGF). Accordingly, tumor growth was inhibited by administration of the anti-VEGF antibody bevacizumab [ 38 ].
3. Extracellular ATP and P2X7 as Modulators of Osteoblasts and Osteoclasts Responses
The role of P2X7 receptor in bone cells has been extensively studied and was recently appraised in several reviews [ 39 – 42 ]. P2X7 receptor is expressed by both osteoblasts [ 43 ] and osteoclasts [ 44 , 45 ] of different species and plays a central role in mediating osteoblast-osteoclasts crosstalk via calcium oscillations [ 46 ] and other signaling pathways [ 39 ]. One of the main roles attributed to P2X7 receptor in osteoblasts is to promote cell growth and osteodeposition [ 36 ] through a series of different pathways including c-fos [ 47 ], ERK [ 48 ], PI3 K [ 49 ], and COX [ 36 ]. Moreover, P2X7 likely mediates osteoblast ATP release as shown by the inhibitory effect on nucleotide release of P2X7 blockers [ 50 , 51 ].
Skeletal disorders, such as osteoporosis and tumor-induced bone resorption, are caused by increased activity of bone-resorbing osteoclasts. The role of P2X7 in osteoclast biology is still poorly understood. It has been suggested that P2X7 participates in cell fusion, a central step in osteoclastogenesis [ 52 , 53 ], but osteoclasts from P2X7 KO mice are normal in number and size [ 54 , 55 ]. P2X7 receptor might, however exert its activity on osteoclast fusion indirectly by extracellular adenosine generation [ 53 ] or, simply, by increasing survival via RANKL [ 44 ] and NF- κ B [ 56 ] pathways. The effect of P2X7 knock-down on bone phenotype probably depends on the different mice models considered [ 57 ]. Whatever is the role of P2X7 in osteoclast fusion and activation, a reduced activity of the receptor has been associated to increased susceptibility to osteoporosis [ 58 ]. All the known polymorphisms of human P2X7 have now been studied in different postmenopausal women cohorts [ 59 – 61 ]. These studies revealed an association between different complications of osteoporosis and loss of function of P2X7; a lower incidence of vertebral fractures in women expressing a gain of function receptor polymorphism was also evident [ 60 ]. These data suggest that, depending on the P2X7 polymorphism carried, one could be more or less exposed to osteolytic bone cancer complications.
4. P2X7 and Cell Metabolism in Cancer: Warburg Effect and Signaling
Ability to adapt to unfavorable conditions is a key feature of cancer cells, making them more and more aggressive. Tumor-cell survival runs through a reorganization of metabolic pathways to balance energy generation and production of biosynthetic intermediates. Aerobic glycolysis (also known as “Warburg effect”) is known to be the preferred metabolic path adopted by cancer cells, in presence of oxygen. Lactate release, as a consequence of glucose degradation, is observed in many solid tumors and leukemias. Detection of increased glucose uptake in tissues is commonly used for diagnosis of cancer by positron emission tomography (PET) [ 62 ]. In a recent paper, Grol and colleagues showed that in the osteoblast like MC3T3-E1 cell line, P2X7 activation triggers, via PI3 K, release of lactate and increased glucose metabolism [ 49 ]. PI3 K activates the serine threonine kinase Akt, one of the most studied paths involved in tumor progression and aggressiveness [ 63 , 64 ]. Indeed, activation of PI3 K/Akt pathway has been correlated with many cellular critical events such as proliferation, apoptosis, metabolism, adhesion, cytoskeleton modifications, tumorigenesis, metastatization, and drugs resistance [ 65 , 66 ]. A direct effect of P2X7 activation on Akt has been shown in several cell lines [ 30 , 67 – 72 ]. In some models such as neuroblastoma [ 70 ] and non-small-cell lung cancer [ 72 ], P2X7 was reported to reduce Akt phosphorylation while in others such as astrocytes [ 68 ], neurons [ 69 , 73 ], and osteoblasts [ 49 ], the P2X7-Akt axis promoted proliferation and survival. Another Akt-mediated effect during tumor development is induction of HIF-1 α that in turn leads to VEGF production and neovascularization [ 74 , 75 ]. One might speculate that P2X7-mediated VEGF secretion from tumoral masses [ 38 ] could be dependent on PI3 K/Akt also in tumor proliferating osteoblasts; VEGF being a known positive regulator of osteoblastic lesions [ 76 ].
5. P2X7 Receptor in Primary Bone Tumors
A search of EMBL-EBI Atlas database ( http://www.ebi.ac.uk/ ) revealed an association between P2X7 overexpression and different malignancies including blood and bone tumors [ 77 ]. In particular, P2X7 expression was increased in osteosarcoma, Ewing's sarcoma, chondromyxoid fibroma, and multiple myeloma. Moreover, P2X7 receptor was found to be expressed and active in multiple myeloma cell lines where it mediates MMPs activation [ 78 ].
Osteoblasts and osteosarcoma were among the first cell models in which a proliferative activity of P2X7 and ATP was suggested [ 36 ]. Although direct in vivo proof of an oncogenic role of the receptor in osteosarcoma is missing, several experimental findings point to such an involvement. Osteoblast like (MC3T3-E1) and osteosarcoma cells lines (SaOs-2, HOS) generally show high expression of P2X7 at mRNA, protein, and functional level [ 43 , 79 , 80 ]. P2X7 is expressed in MSC osteoblastic precursors [ 81 ] and is highly and constantly detected during osteoblast differentiation [ 82 ]. P2X7 activity has been associated to proliferation and osteodeposition [ 36 ] as well as to upmodulation of the osteosclerotic factor FosB [ 83 ] in osteoblasts [ 84 ]. In a recent study, Liu and Chen demonstrated a trophic effect of ATP on HOS cells that was abolished by suramin, a P2 purinoceptors antagonist. Suramin also inhibited ATP-dependent cytosolic calcium increases.The ATP growth promoting effect was likely mediated via both P2X4 and P2X7 [ 80 ]. A role for P2X7 receptor in osteoblasts proliferation and osteogenesis was also indicated by Panupinthu et al. who reported reduced cell growth and osteodeposition by calvarial cell cultures from P2X7 KO mice [ 36 ]. P2X7 activated pathways in osteoblasts include cyclooxygenase (COX), lysophosphatidic acid, and prostaglandin E2 [ 36 ]. A further study by Gavala et al. showed that P2X7 dependent AP-1/Fos-B activation was responsible for COX-2 expression [ 84 ]. Moreover, mechanical stimulation triggers ATP release and P2X7-dependent activation of several kinases, including ERK [ 48 ] and PI3 K [ 49 ]. It is tempting to speculate that a condition, such as cancer, in which extracellular ATP levels are known to be upregulated [ 18 ], might mimic mechanical loading causing and stimulating osteoblast proliferation. Furthermore, mouse osteoblasts and osteoclasts constitutively release ATP into extracellular microenvironment via P2X7-dependent pathway [ 51 ].
NFAT is one of the main pathways activated through Ca 2+ and calcineurin following P2X7 stimulation [ 29 , 85 – 88 ]. We have shown that NFATc1 activation is central for P2X7 trophic activity as treatment with the NFATc1 inhibitors cyclosporine and VIVIT obliterates P2X7-dependent cell growth [ 29 ]. Moreover, P2X7-positive tumors overexpress NFATc1 [ 38 ]. On the other hand NFAT has a central role also in osteoblast biology. Mice expressing in osteoblasts a constitutively nuclear NFATc1 variant, NFATc1(nuc), develop bone masses characterized by osteoblast overgrowth [ 89 ]. Accordingly, viable NFATc1-deficient mice have defects in bone formation, in addition to impaired osteoclast development. Calcineurin/NFAT-signaling in Osteoblasts controls the expression of chemoattractants for monocytic osteoclast precursors, thereby coupling bone formation and bone resorption, and regulating bone mass [ 89 ]. Elevated levels of NFAT are among factors necessary for in vitro invasiveness of mice metastatic osteosarcoma cell lines [ 90 ]. Finally, calcineurin/NFAT pathway is implicated in prostate cancer bone metastasis. Prostate tumor cells that engraft in the bone stimulate osteoblasts by secreting growth-promoting factors among which endothelin 1 (ET-1). In osteoblasts ET-1 activates calcineurin, causes nuclear translocation of NFAT and, thus, osteoblasts stimulation [ 91 ]. On the other hand, a negative role for NFAT in osteoblasts has been proposed by Choo et al. who demonstrated that constitutively active NFAT inhibits alkaline phosphatase activity and mineralization [ 92 ]. These observations might suggest that P2X7 could cause NFAT activation and osteoblast proliferation both in primary and metastatic osteoblastic lesions.
Moreover, Ca 2+ -NFAT signaling is essential for osteoclast differentiation [ 93 ]. Intriguingly, NFAT activation in osteoclasts has also been related to malignant progression of multiple myeloma. Several studies reported osteoblast NFAT reduction associated with decreased osteoclastogenesis, following myeloma treatment [ 94 – 97 ].
P2X7 was shown to induce ATP secretion from both osteoclasts and osteoblasts [ 51 ]. Increase in extracellular ATP is followed by extracellular adenosine accumulation viaCD39 and CD73 ectonucleotidases [ 98 ]. Adenosine was demonstrated to stimulate proliferation of MC3T3-E1 osteoblastic-like cell line [ 99 ]. Furthermore, HCC1 cells release increased amounts of IL-6 and osteoprotegerin following adenosine receptor stimulation [ 100 ], likely modulating osteoclastogenesis and bone resorption.
VEGF production was found increased in different experimental tumors expressing P2X7 receptor [ 38 ]. The P2X7-VEGF connection in tumors is also supported by the finding that P2X7 activation in rat C6 glioma cells is linked to increased release of proinflammatory factors (MCP-1, IL-8 and VEGF) and to tumor-cell migration [ 101 ]. Accordingly, patients with osteosarcoma showed increased VEGF plasma levels, and this was reduced following tumor removal by surgery [ 102 ]. Moreover, VEGF secretion is known to be central in malignant progression of multiple myeloma; the first lymphohaemopoietic tumor in which increased angiogenesis was detected, and which greatly benefits of the treatment with VEGF-targeted agents [ 103 ].
6. P2X7 Receptor Activity in Bone Metastasizing Cancers
The complications of bone metastasis are thought to be due to the perturbation of the interaction between osteoblasts and osteoclasts. This disruption is thought to be caused by tumor-derived humoral mediators produced by the metastasized cancer cells within the bone marrow. Among factors regulating bone cancer metastasis RANKL plays a central role, as demonstrated by the efficacy of an anti-RANKL antibody in the therapy of such secondary tumors [ 104 ]. During physiological bone remodeling RANKL, produced by proliferating osteoblasts, causes activation of osteoclasts ensuring a balance between osteodeposition and bone resorption. However, the presence of cancer cells can alter bone microenvironment causing an increased production of RANKL, favoring osteoclasts activation and osteolysis. Several factors, such as IL-1, IL-6, COX2/prostaglandins, and VEGF, can cause positive shifts in RANKL production [ 10 , 105 ]. All these mediators are released upon P2X7 stimulation from different cell types, including osteoblasts [ 36 ], immune [ 106 , 107 ], and cancer cells [ 38 , 108 , 109 ]. Moreover, P2X7-mediated ATP secretion [ 26 , 37 ] from tumor cells [ 18 ] could itself upregulate osteoblasts RANKL expression [ 44 ]. Extracellular ATP is rapidly degraded to adenosine, which can directly modulate osteoclasts formation through A2 receptors [ 53 ]. P2X7-dependent Rho-kinase1 (ROCK) activation has been demonstrated in several cell types [ 31 , 110 – 112 ], including osteoblasts [ 113 ]. ROCK is known to activate changes in cell morphology, adhesion, and motility, and is associated to P2X7-dependent cell blebbing, a response that might be related to invasive phenotype of P2X7-expressing cells [ 26 , 38 , 114 ]. Interestingly, an increased ROCK signaling has been shown to contribute to breast cancer invasiveness [ 115 ] as, if overexpressed, the kinase conferred a bone metastatic phenotype to a human breast cancer cell line in an in vivo model. ROCK is also known to mediate activation of the proresorptive factor PTHrP through activation of TGF- β signaling [ 116 ].
Several studies reported an association between primary tumors causing bone metastatization and P2X7 overexpression and function (for a recent review see [ 27 ]). Upregulation of P2X7 in breast cancer was shown for the first time by Slater et al. who demonstrated an association between receptor expression and tumor invasiveness [ 117 ]. Recently, Jelassi et al. also showed an involvement of P2X7 in breast cancer metastasis formation [ 114 ]. P2X7 activation in a highly aggressive breast cancer cell line (MDA-MB-435s) caused increased in vitro cell motility and extracellular matrix infiltration [ 114 ]. Accordingly, P2X7 inhibition significantly reduced in vivo cell migration in a zebra fish embryo metastatic model [ 114 ]. As previously reported in macrophages [ 118 ], P2X7 receptor activation caused release of a broad range of cathepsins, including cathepsin k, also from breast cancer cells [ 114 ]. Another bone-degrading enzyme which is secreted in a P2X7-dependent fashion is MMP9 [ 35 ]. Cathepsin k and MPPs release in resorption pits during bone degradation make these enzymes attractive therapeutic targets to block osteolysis [ 11 ]. Since cathepsin k inhibitors showed adverse effects during clinical trials [ 11 ], pharmacological inhibition of upstream pathways, such as P2X7, could prove useful in therapy.
A peculiar case of tumor causing osteolytic metastasis is neuroblastoma, which is the second most common pediatric malignancy worldwide and is responsible for 15% of childhood cancer deaths [ 119 ]. Patients with high-risk metastatic disease (stages III-IV) show a mere 42% survival rate, despite treatment [ 120 ]. Few years ago Raffaghello et al. showed that P2X7 receptor is expressed in specimens from neuroblastoma patients and in all neuroblastoma cell lines examined [ 109 ]. Interestingly, in these cells P2X7 receptor lacks its well-known cytotoxic activity but rather supports proliferation [ 109 ]. Wu et al. have recently reported expression of P2X7 also in murine neuroblastoma cell lines [ 121 ]. In these cells P2X7 inhibits differentiation [ 121 ], promotes proliferation [ 70 ], and microvesicles release [ 122 ]. P2X7 implication in neuroblastoma progression is further supported from in vivo experiments from our laboratory showing a 2-3 fold reduction in neuroblastoma tumor masses upon P2X7 silencing [ 38 ]. Bone involvement is observed in 55–68% of neuroblastoma patients who present metastatic disease at diagnosis [ 123 ], particularly at the bony orbit. Bone metastasis is generally associated with osteolytic lesions due to either direct action of cancer cells on osteoclasts or to an indirect involvement of osteoblasts finally causing bone resorption [ 123 ]. Although direct evidence of P2X7 involvement in neuroblastoma bone metastatization is still missing all experimental evidence points to such an association [ 38 , 53 , 109 ].
Patients with advanced prostate carcinoma generally develop osteoblastic metastasis due to deregulation of physiological bone remodeling. In these patients prostate cancer cells are adjacent to large numbers of osteoblasts, which are responsible for woven bone deposition [ 76 ]. P2X7 expression by prostate cancer cells has been reported long time ago [ 124 , 125 ]. Furthermore, ATP treatment increases prostate cancer cell invasion [ 126 ]. The cross-talk between prostate cancer cells and osteoblasts contributes to metastatic development. If on one hand cancer cells secrete TGF β , VEGF and other factors promoting osteoblast proliferation and differentiation, on the other osteoblasts can prompt cancer cells to secrete MMP9 and stimulate tumor cell growth [ 76 ]. P2X7 activation has been involved in secretion of almost all the above factors either from osteoblasts [ 44 ] or from cancer cells [ 35 , 38 ].
7. Conclusions and Future Perspectives
Over-expression of a potentially cytotoxic receptor such as P2X7 by cancer cells is puzzling. However, the discovery that this receptor has oncogene-like properties may provide a logical explanation for this finding [ 38 , 114 ]. Due to altered skeletal phenotype of P2X7 KO mice, bone has been one of the tissues in which P2X7 activity has been best characterized. Nevertheless, there is only sporadic evidence linking bone related cancers and P2X7 so far. Here we propose that P2X7 might have a central role in bone cancer development and progression by causing NFATc1, PI3K/Akt, ROCK, VEGF activation, thus driving osteoblast proliferation in primary bone tumors and osteoblastic metastasis. P2X7 might also stimulate bone resorption by causing osteoclast activation [ 53 ] as well as secretion of cathepsins and MMPs [ 35 , 114 ] thus contributing to osteolytic metastasis formation.
The processes of bone resorption and bone formation are tightly coupled and treatments that primarily target the osteoclasts generally exert secondary inhibitory effects on bone formation. The discovery of new pathways occurring in tumorigenic transformation and aggressiveness of bone-related cancers made clinicians pay attention to new targeting drugs: blocking bone lesions acting on osteoblasts/osteoclasts regulation, like bisphosphonates do, is not the only chance anymore. Clinical trials with RANKL blocking antibody (denosumab) produced very encouraging results showing that denosumab was tolerated better than bisphosphonates (zolendronic acid) and even increased patient survival [ 104 ]. Inhibitors of mTOR, an Akt downstream effector are in clinical trials for the treatment of multiple bone related cancers [ 127 ]. In particular, rapamycin is known to arrest cell growth in osteosarcoma [ 128 , 129 ] and breast cancer [ 130 ].
Since P2X7 receptor is an upstream regulator of all the paths inhibited by the RANKL and mTOR blockers, it is an attractive therapeutic target for bone-related diseases too. Several P2X7 antagonists are currently in phase I and II clinical trials for the treatment of chronic inflammatory diseases, showing so far excellent safety profiles [ 131 ]. These drugs are, in principle, available to be used at patient's bed and could be a good therapeutic opportunity for those cancers, such as neuroblastoma and multiple myeloma, which still lack an efficacious cure.
Acknowledgments
The authors are deeply indebted to Professor Francesco Di Virgilio for discussing and revising the manuscript. They also thank Drs. Simonetta Falzoni and Marina Capece for critical reading. The authors are grateful to Drs Alvis Brazma and Margus Lukk for helping them with interpretation of Affimetrix data accessible through the Atlas database. This work was supported by a Grant from the Italian Association for Cancer Research (AIRC) to E. Adinolfi (MFAG 11630). E. Adinolfi and A. L. Giuliani are members of the ATP Bone European Consortium.
1 Hameed M Dorfman H Primary malignant bone tumors-recent developments Seminars in Diagnostic Pathology 2011 28 1 86 101 2-s2.0-79955490402 21675380 10.1053/j.semdp.2011.02.002
2 Ottaviani G Jaffe N The epidemiology of osteosarcoma Cancer Treatment and Research 2009 152 3 13 2-s2.0-77953669219 20213383 10.1007/978-1-4419-0284-9_1
3 Ottaviani G Jaffe N The etiology of osteosarcoma Cancer Treatment and Research 2009 152 15 32 2-s2.0-77953654549 20213384 10.1007/978-1-4419-0284-9_2
4 Roodman GD Osteoblast function in myeloma Bone 2011 48 1 135 140 2-s2.0-78650417743 20601285 10.1016/j.bone.2010.06.016
5 Atanackovic D Cao Y Luetkens T CD4 + CD25 + FOXP3 + T regulatory cells reconstitute and accumulate in the bone marrow of patients with multiple myeloma following allogeneic stem cell transplantation Haematologica 2008 93 3 423 430 2-s2.0-40849141379 18287134 10.3324/haematol.11897
6 Anderson KC New insights into therapeutic targets in myeloma American Society of Hematology Education Program 2011 184 190 10.1182/asheducation-2011.1.184 22160032
7 Kinnane N Burden of bone disease European Journal of Oncology Nursing 2007 11 supplement 2 S28 S31 2-s2.0-34548758051 17768092 10.1016/j.ejon.2007.07.002
8 Mishra A Shiozawa Y Pienta KJ Taichman RS Homing of cancer cells to the bone Cancer Microenvironment 2011 4 3 221 235 2-s2.0-79961101316 21826451 10.1007/s12307-011-0083-6 PMC3234327
9 Suva LJ Washam C Nicholas RW Griffin RJ Bone metastasis: mechanisms and therapeutic opportunities Nature Reviews Endocrinology 2011 7 4 208 218 2-s2.0-79953078905 10.1038/nrendo.2010.227 PMC3134309 21200394
10 Chen YC Sosnoski DM Mastro AM Breast cancer metastasis to the bone: mechanisms of bone loss Breast Cancer Research 2010 12 6 p. 215 10.1186/bcr2781 PMC3046443 21176175
11 Sturge J Caley MP Waxman J Bone metastasis in prostate cancer: emerging therapeutic strategies Nature Reviews Clinical Oncology 2011 8 6 357 368 2-s2.0-79958037582 10.1038/nrclinonc.2011.67 21556025
12 Guise TA Mohammad KS Clines G Basic mechanisms responsible for osteolytic and osteoblastic bone metastases Clinical Cancer Research 2006 12 20, part 2 6213s 6216s 2-s2.0-33750738067 17062703 10.1158/1078-0432.CCR-06-1007
13 Fournier PGJ Chirgwin JM Guise TA New insights into the role of T cells in the vicious cycle of bone metastases Current Opinion in Rheumatology 2006 18 4 396 404 2-s2.0-33748080768 16763461 10.1097/01.bor.0000231909.35043.da
14 Mantovani A Molecular pathways linking inflammation and cancer Current Molecular Medicine 2010 10 4 369 373 2-s2.0-77953485565 20455855 10.2174/156652410791316968
15 Allen M Jones JL Jekyll and Hyde: the role of the microenvironment on the progression of cancer Journal of Pathology 2011 223 2 162 176 2-s2.0-78649997911 21125673 10.1002/path.2803
16 Weber FC Esser PR Müller T Lack of the purinergic receptor P2X7 results in resistance to contact hypersensitivity Journal of Experimental Medicine 2010 207 12 2609 2619 2-s2.0-78649414124 21059855 10.1084/jem.20092489 PMC2989767
17 Wilhelm K Ganesan J Müller T Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R Nature Medicine 2010 16 12 1434 1439 2-s2.0-78649993400 10.1038/nm.2242 21102458
18 Pellegatti P Raffaghello L Bianchi G Piccardi F Pistoia V Di Virgilio F Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase PLoS ONE 2008 3 7 2-s2.0-50249085890 e2599 10.1371/journal.pone.0002599 PMC2440522 18612415
19 Michaud M Martins I Sukkurwala A Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice Science 2011 334 6062 1573 1577 22174255 10.1126/science.1208347
20 Ghiringhelli F Apetoh L Tesniere A Activation of the NLRP3 inflammasome in dendritic cells induces IL-1 β -dependent adaptive immunity against tumors Nature Medicine 2009 15 10 1170 1178 2-s2.0-70350569295 10.1038/nm.2028 19767732
21 Martins I Michaud M Sukkurwala AQ Premortem autophagy determines the immunogenicity of chemotherapy-induced cancer cell death Autophagy . In press 10.4161/auto.19009 22361584
22 Burnstock G Discovery of purinergic signalling, the initial resistance and current explosion of interest British Journal of Pharmacology . In press 10.1111/j.1476-5381.2012.02008.x PMC3481036 22537142
23 Surprenant A Rassendren F Kawashima E North RA Buell G The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7) Science 1996 272 5262 735 738 2-s2.0-0029665112 8614837 10.1126/science.272.5262.735
24 Baricordi OR Melchiorri L Adinolfi E Increased proliferation rate of lymphoid cells transfected with the P2X7 ATP receptor Journal of Biological Chemistry 1999 274 47 33206 33208 2-s2.0-0033584858 10559192 10.1074/jbc.274.47.33206
25 Adinolfi E Callegari MG Ferrari D Basal activation of the P2X7 ATP receptor elevates mitochondrial calcium and potential, increases cellular ATP levels, and promotes serum-independent growth Molecular Biology of the Cell 2005 16 7 3260 3272 2-s2.0-21844450590 15901833 10.1091/mbc.E04-11-1025 PMC1165409
26 Adinolfi E Cirillo M Woltersdorf R Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor FASEB Journal 2010 24 9 3393 3404 2-s2.0-77956626757 20453110 10.1096/fj.09-153601
27 Di Virgilio F Ferrari D Adinolfi E P2X7: a growth-promoting receptor—implications for cancer Purinergic Signalling 2009 5 2 251 256 2-s2.0-67349094026 19263244 10.1007/s11302-009-9145-3 PMC2686832
28 Adinolfi E Pizzirani C Idzko M P2X7 receptor: death or life? Purinergic Signalling 2005 1 3 219 227 2-s2.0-23744489378 18404507 10.1007/s11302-005-6322-x PMC2096546
29 Adinolfi E Callegari MG Cirillo M Expression of the P2X7 receptor increases the Ca2 + content of the endoplasmic reticulum, activates NFATc1, and protects from apoptosis Journal of Biological Chemistry 2009 284 15 10120 10128 2-s2.0-65649141604 19204004 10.1074/jbc.M805805200 PMC2665066
30 Mistafa O Stenius U Statins inhibit Akt/PKB signaling via P2X7 receptor in pancreatic cancer cells Biochemical Pharmacology 2009 78 9 1115 1126 2-s2.0-70249093582 19540829 10.1016/j.bcp.2009.06.016
31 Mackenzie AB Young MT Adinolfi E Surprenant A Pseudoapoptosis induced by brief activation of ATP-gated P2X7 receptors Journal of Biological Chemistry 2005 280 40 33968 33976 2-s2.0-25844484867 15994333 10.1074/jbc.M502705200
32 Pizzirani C Ferrari D Chiozzi P Stimulation of P2 receptors causes release of IL-1 β -loaded microvesicles from human dendritic cells Blood 2007 109 9 3856 3864 2-s2.0-34247353273 17192399 10.1182/blood-2005-06-031377
33 Ferrari D Pizzirani C Adinolfi E The P2X7 receptor: a key player in IL-1 processing and release Journal of Immunology 2006 176 7 3877 3883 2-s2.0-33646072818 10.4049/jimmunol.176.7.3877 16547218
34 Baroni M Pizzirani C Pinotti M Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles FASEB Journal 2007 21 8 1926 1933 2-s2.0-34249821918 17314141 10.1096/fj.06-7238com
35 Gu BJ Wiley JS Rapid ATP-induced release of matrix metalloproteinase 9 is mediated by the P2X7 receptor Blood 2006 107 12 4946 4953 2-s2.0-33745088086 16514055 10.1182/blood-2005-07-2994
36 Panupinthu N Rogers JT Zhao L P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis Journal of Cell Biology 2008 181 5 859 871 2-s2.0-44649092795 18519738 10.1083/jcb.200708037 PMC2396816
37 Pellegatti P Falzoni S Pinton P Rizzuto R Di Virgilio F A novel recombinant plasma membrane-targeted luciferase reveals a new pathway for ATP secretion Molecular Biology of the Cell 2005 16 8 3659 3665 2-s2.0-23044473247 15944221 10.1091/mbc.E05-03-0222 PMC1182305
38 Adinolfi E Raffaghello L Giuliani AL Expression of P2X7 receptor increases in vivo tumor growth Cancer Research 2012 72 12 2957 2969 22505653 10.1158/0008-5472.CAN-11-1947
39 Grol MW Panupinthu N Korcok J Sims SM Dixon SJ Expression, signaling, and function of P2X7 receptors in bone Purinergic Signalling 2009 5 2 205 221 2-s2.0-67349186489 19224395 10.1007/s11302-009-9139-1 PMC2686829
40 Reyes JP Sims SM Dixon SJ P2 receptor expression, signaling and function in osteoclasts Frontiers in Bioscience 2011 3 1101 1118 10.2741/214 21622259
41 Gartland A Orriss IR Rumney RM Bond AP Arnett T Gallagher JA Purinergic signalling in osteoblasts Frontiers in Bioscience 2012 17 16 29 22201729 10.2741/3912
42 Lemaire I Falzoni S Adinolfi E Purinergic signaling in giant cell formation Frontiers in Bioscience 2012 4 41 55 10.2741/359 22201854
43 Gartland A Hipskind RA Gallagher JA Bowler WB Expression of a P2X7 receptor by a subpopulation of human osteoblasts Journal of Bone and Mineral Research 2001 16 5 846 856 2-s2.0-0034744672 11341329 10.1359/jbmr.2001.16.5.846
44 Buckley KA Hipskind RA Gartland A Bowler WB Gallagher JA Adenosine triphosphate stimulates human osteoclast activity via upregulation of osteoblast-expressed receptor activator of nuclear factor- κ B ligand Bone 2002 31 5 582 590 2-s2.0-0036861712 12477572 10.1016/s8756-3282(02)00877-3
45 Gartland A Buckley KA Hipskind RA Bowler WB Gallagher JA P2 receptors in bone—modulation of osteoclast formation and activity via P2X7 activation Critical Reviews in Eukaryotic Gene Expression 2003 13 2-4 237 242 2-s2.0-0345732699 14696970
46 Jørgensen NR Henriksen Z Sørensen OH Eriksen EF Civitelli R Steinberg TH Intercellular calcium signaling occurs between human osteoblasts and osteoclasts and requires activation of osteoclast P2X7 receptors Journal of Biological Chemistry 2002 277 9 7574 7580 2-s2.0-0036510537 11756404 10.1074/jbc.M104608200
47 Okumura H Shiba D Kubo T Yokoyama T P2X7 receptor as sensitive flow sensor for ERK activation in osteoblasts Biochemical and Biophysical Research Communications 2008 372 3 486 490 2-s2.0-44749083240 18501702 10.1016/j.bbrc.2008.05.066
48 Liu D Genetos DC Shao Y Activation of extracellular-signal regulated kinase (ERK1/2) by fluid shear is Ca2 + - and ATP-dependent in MC3T3-E1 osteoblasts Bone 2008 42 4 644 652 2-s2.0-40849111180 18291742 10.1016/j.bone.2007.09.058 PMC2937351
49 Grol MW Zelner I Dixon SJ P2X-mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells American Journal of Physiology 2012 302 5 E561 E575 22185840 10.1152/ajpendo.00209.2011
50 Rumney RM Sunters A Reilly GC Gartland A 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 Journal of Biomechanics 2012 45 3 549 554 22176713 10.1016/j.jbiomech.2011.11.036 PMC3268859
51 Brandao-Burch A Key ML Patel JJ Arnett TR Orriss IR The P2X7 receptor is an important regulator of extracellular ATP levels Frontiers in Endocrinology 2012 3, article 41 10.3389/fendo.2012.00041 PMC3355863 22654865
52 Gartland A Buckley KA Bowler WB Gallagher JA Blockade of the pore-forming P2X7 receptor inhibits formation of multinucleated human osteoclasts in vitro Calcified Tissue International 2003 73 4 361 369 2-s2.0-0042047374 12874700 10.1007/s00223-002-2098-y
53 Pellegatti P Falzoni S Donvito G Lemaire I Di Virgilio F P2X7 receptor drives osteoclast fusion by increasing the extracellular adenosine concentration FASEB Journal 2011 25 4 1264 1274 2-s2.0-79954583662 21233486 10.1096/fj.10-169854
54 Ke HZ Qi H Weidema AF Deletion of the P2X7 nucleotide receptor reveals its regulatory roles in bone formation and resorption Molecular Endocrinology 2003 17 7 1356 1367 2-s2.0-0037634326 12677010 10.1210/me.2003-0021
55 Gartland A Buckley KA Hipskind RA Multinucleated osteoclast formation in vivo and in vitro by P2X 7 receptor-deficient mice Critical Reviews in Eukaryotic Gene Expression 2003 13 2–4 243 253 2-s2.0-0346245902 14696971 10.1615/critreveukaryotgeneexpr.v13.i24.150
56 Korcok J Raimundo LN Ke HZ Sims SM Dixon SJ Extracellular nucleotides act through P2X7 receptors to activate NF- κ B in osteoclasts Journal of Bone and Mineral Research 2004 19 4 642 651 2-s2.0-3242673859 15005852 10.1359/JBMR.040108
57 Orriss I Syberg S Wang N Bone phenotypes of P2 receptor knockout mice Frontiers in Bioscience 2011 3 1038 1046 10.2741/208 21622253
58 Wesselius A Bours MJL Agrawal A Role of purinergic receptor polymorphisms in human bone Frontiers in Bioscience 2011 17 2572 2585 2-s2.0-79959546453 21622196 10.2741/3873
59 Ohlendorff SD Tofteng CL Jensen JEB Single nucleotide polymorphisms in the P2X7 gene are associated to fracture risk and to effect of estrogen treatment Pharmacogenetics and Genomics 2007 17 7 555 567 2-s2.0-34547925564 17558311 10.1097/FPC.0b013e3280951625
60 Gartland A Skarratt KK Hocking LJ Polymorphisms in the P2X7 receptor gene are associated with low lumbar spine bone mineral density and accelerated bone loss in post-menopausal women European Journal of Human Genetics 2012 20 5 559 564 22234152 10.1038/ejhg.2011.245 PMC3330223
61 Jorgensen NR Husted LB Skarratt KK Single-nucleotide polymorphisms in the P2X7 receptor gene are associated with post-menopausal bone loss and vertebral fractures European Journal of Human Genetics 2012 20 6 675 681 22274585 10.1038/ejhg.2011.253 PMC3355253
62 Heiden MGV Cantley LC Thompson CB Understanding the warburg effect: the metabolic requirements of cell proliferation Science 2009 324 5930 1029 1033 2-s2.0-66249108601 19460998 10.1126/science.1160809 PMC2849637
63 Knowles MA Platt FM Ross RL Hurst CD Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer Cancer and Metastasis Reviews 2009 28 3-4 305 316 2-s2.0-76649128715 20013032 10.1007/s10555-009-9198-3 PMC2797439
64 Scrima M De MC Fabiani F Signaling networks associated with AKT activation in non-small cell lung cancer (NSCLC): new insights on the role of phosphatydil-inositol-3 kinase PLoS ONE 2012 7 2 e30427 10.1371/journal.pone.0030427 PMC3281846 22363436
65 Fruman DA Meyers RE Cantley LC Phosphoinositide kinases Annual Review of Biochemistry 1998 67 481 507 2-s2.0-0031663503 10.1146/annurev.biochem.67.1.481 9759495
66 Neri LM Borgatti P Capitani S Martelli AM The nuclear phosphoinositide 3-kinase/AKT pathway: a new second messenger system Biochimica et Biophysica Acta 2002 1584 2-3 73 80 2-s2.0-0037057801 12385889 10.1016/s1388-1981(02)00300-1
67 Liu YP Yang CS Chen MC Sun SH Tzeng SF Ca2 + -dependent reduction of glutamate aspartate transporter GLAST expression in astrocytes by P2X7 receptor-mediated phosphoinositide 3-kinase signaling Journal of Neurochemistry 2010 113 1 213 227 2-s2.0-77749243037 20070863 10.1111/j.1471-4159.2010.06589.x
68 Jacques-Silva MC Rodnight R Lenz G P2X7 receptors stimulate AKT phosphorylation in astrocytes British Journal of Pharmacology 2004 141 7 1106 1117 2-s2.0-2342469938 15023862 10.1038/sj.bjp.0705685 PMC1574879
69 Ortega F Pérez-Sen R Delicado EG Miras-Portugal MT P2X7 nucleotide receptor is coupled to GSK-3 inhibition and neuroprotection in cerebellar granule neurons Neurotoxicity Research 2009 15 3 193 204 2-s2.0-70449530845 19384592 10.1007/s12640-009-9020-6
70 Gómez-Villafuertes R Del Puerto A Díaz-Hernández M Ca2 + /calmodulin-dependent kinase II signalling cascade mediates P2X7 receptor-dependent inhibition of neuritogenesis in neuroblastoma cells FEBS Journal 2009 276 18 5307 5325 2-s2.0-69449095663 19682070 10.1111/j.1742-4658.2009.07228.x
71 Mistafa O Ghalali A Kadekar S Högberg J Stenius U Purinergic receptor-mediated rapid depletion of nuclear phosphorylated Akt depends on pleckstrin homology domain leucine-rich repeat phosphatase, calcineurin, protein phosphatase 2A, and PTEN phosphatases Journal of Biological Chemistry 2010 285 36 27900 27910 2-s2.0-77956236967 20605778 10.1074/jbc.M110.117093 PMC2934657
72 Mistafa O Högberg J Stenius U Statins and ATP regulate nuclear pAkt via the P2X7 purinergic receptor in epithelial cells Biochemical and Biophysical Research Communications 2008 365 1 131 136 2-s2.0-36049032742 17980145 10.1016/j.bbrc.2007.10.148
73 Pérez-Sen R Ortega F Morente V Delicado EG Miras-Portugal MT P2X7, NMDA and BDNF receptors converge on GSK3 phosphorylation and cooperate to promote survival in cerebellar granule neurons Cellular and Molecular Life Sciences 2010 67 10 1723 1733 2-s2.0-77951887802 20146080 10.1007/s00018-010-0278-x PMC2858808
74 Inoki K Corradetti MN Guan KL Dysregulation of the TSC-mTOR pathway in human disease Nature Genetics 2005 37 1 19 24 2-s2.0-11244297916 15624019 10.1038/ng1494
75 Altomare DA Testa JR Perturbations of the AKT signaling pathway in human cancer Oncogene 2005 24 50 7455 7464 2-s2.0-27844445642 16288292 10.1038/sj.onc.1209085
76 Ibrahim T Flamini E Mercatali L Sacanna E Serra P Amadori D Pathogenesis of osteoblastic bone metastases from prostate cancer Cancer 2010 116 6 1406 1418 2-s2.0-77949347731 20108337 10.1002/cncr.24896
77 Lukk M Kapushesky M Nikkilä J A global map of human gene expression Nature Biotechnology 2010 28 4 322 324 2-s2.0-77950644737 10.1038/nbt0410-322 PMC2974261 20379172
78 Farrell AW Gadeock S Pupovac A P2X7 receptor activation induces cell death and CD23 shedding in human RPMI 8226 multiple myeloma cells Biochimica et Biophysica Acta 2010 1800 11 1173 1182 2-s2.0-77956289939 20647033 10.1016/j.bbagen.2010.07.001
79 Alqallaf SM Evans BAJ Kidd EJ Atypical P2X7 receptor pharmacology in two human osteoblast-like cell lines British Journal of Pharmacology 2009 156 7 1124 1135 2-s2.0-67650651202 19226284 10.1111/j.1476-5381.2009.00119.x PMC2697685
80 Liu PS Chen CY Butyl benzyl phthalate suppresses the ATP-induced cell proliferation in human osteosarcoma HOS cells Toxicology and Applied Pharmacology 2010 244 3 308 314 2-s2.0-77950466809 20114058 10.1016/j.taap.2010.01.007
81 Zippel N Limbach CA Ratajski N Purinergic receptors influence the differentiation of human mesenchymal stem cells Stem Cells and Development 2012 21 6 884 900 21740266 10.1089/scd.2010.0576
82 Orriss IR Knight GE Ranasinghe S Burnstock G Arnett TR Osteoblast responses to nucleotides increase during differentiation Bone 2006 39 2 300 309 2-s2.0-33744908812 16616882 10.1016/j.bone.2006.02.063
83 Sabatakos G Sims NA Chen J Overexpression of ΔFosB transcription factor(s) increases bone formation and inhibits adipogenesis Nature Medicine 2000 6 9 985 990 2-s2.0-0033624587 10.1038/79683 10973317
84 Gavala ML Hill LM Lenertz LY Karta MR Bertics PJ Activation of the transcription factor FosB/activating protein-1 (AP-1) is a prominent downstream signal of the extracellular nucleotide receptor P2RX7 in monocytic and osteoblastic cells Journal of Biological Chemistry 2010 285 44 34288 34298 2-s2.0-77958532672 20813842 10.1074/jbc.M110.142091 PMC2962527
85 Ferrari D Stroh C Schulze-Osthoff K P2X7/P2Z purinoreceptor-mediated activation of transcription factor NFAT in microglial cells Journal of Biological Chemistry 1999 274 19 13205 13210 2-s2.0-0033531791 10224077 10.1074/jbc.274.19.13205
86 Shiratori M Tozaki-Saitoh H Yoshitake M Tsuda M Inoue K P2X7 receptor activation induces CXCL2 production in microglia through NFAT and PKC/MAPK pathways Journal of Neurochemistry 2010 114 3 810 819 2-s2.0-77954325416 20477948 10.1111/j.1471-4159.2010.06809.x
87 Yip L Woehrle T Corriden R Autocrine regulation of T-cell activation by ATP release and P2X 7 receptors FASEB Journal 2009 23 6 1685 1693 2-s2.0-67649365966 19211924 10.1096/fj.08-126458 PMC2718802
88 Kataoka A Tozaki-Saitoh H Koga Y Tsuda M Inoue K Activation of P2X7 receptors induces CCL3 production in microglial cells through transcription factor NFAT Journal of Neurochemistry 2009 108 1 115 125 2-s2.0-57249111810 19014371 10.1111/j.1471-4159.2008.05744.x
89 Winslow MM Pan M Starbuck M Calcineurin/NFAT signaling in osteoblasts regulates bone mass Developmental Cell 2006 10 6 771 782 2-s2.0-33646890016 16740479 10.1016/j.devcel.2006.04.006
90 Velupillai P Sung CK Tian Y Polyoma virus-induced osteosarcomas in inbred strains of mice: host determinants of metastasis PLoS Pathogens 2010 6 1 2-s2.0-77649230351 e1000733 10.1371/journal.ppat.1000733 PMC2809769 20107604
91 Van Sant C Wang G Anderson MG Trask OJ Lesniewski R Semizarov D Endothelin signaling in osteoblasts: global genome view and implication of the calcineurin/NFAT pathway Molecular Cancer Therapeutics 2007 6 1 253 261 2-s2.0-33846826885 17237284 10.1158/1535-7163.MCT-06-0574
92 Choo MK Yeo H Zayzafoon M NFATc1 mediates HDAC-dependent transcriptional repression of osteocalcin expression during osteoblast differentiation Bone 2009 45 3 579 589 2-s2.0-68049105392 19463978 10.1016/j.bone.2009.05.009 PMC2732115
93 Negishi-Koga T Takayanagi H Ca2 + -NFATc1 signaling is an essential axis of osteoclast differentiation Immunological Reviews 2009 231 1 241 256 2-s2.0-70349100754 19754901 10.1111/j.1600-065X.2009.00821.x
94 Garcia-Gomez A Ocio EM Crusoe E Dasatinib as a bone-modifying agent: anabolic and anti-resorptive effects PLoS ONE 2012 7 4 e34914 10.1371/journal.pone.0034914 PMC3335111 22539950
95 Oh J Lee MS Yeon JT Inhibitory regulation of osteoclast differentiation by interleukin-3 via regulation of c-Fos and Id protein expression Journal of Cellular Physiology 2012 227 5 1851 1860 21732357 10.1002/jcp.22913
96 Pennisi A Ling W Li X The ephrinB2/EphB4 axis is dysregulated in osteoprogenitors from myeloma patients and its activation affects myeloma bone disease and tumor growth Blood 2009 114 9 1803 1812 2-s2.0-70349263942 19597185 10.1182/blood-2009-01-201954 PMC2738568
97 Yeo H Mcdonald JM Zayzafoon M NFATc1: a novel anabolic therapeutic target for osteoporosis Annals of the New York Academy of Sciences 2006 1068 1 564 567 2-s2.0-33744750147 16831953 10.1196/annals.1346.053
98 Stagg J Smyth MJ Extracellular adenosine triphosphate and adenosine in cancer Oncogene 2010 29 39 5346 5358 2-s2.0-77957564570 20661219 10.1038/onc.2010.292
99 Shimegi S ATP and adenosine act as a mitogen for osteoblast-like cells (MC3T3-E1) Calcified Tissue International 1996 58 2 109 113 2-s2.0-0030044335 8998680 10.1007/BF02529732
100 Evans BAJ Elford C Pexa A Human osteoblast precursors produce extracellular adenosine, which modulates their secretion of IL-6 and osteoprotegerin Journal of Bone and Mineral Research 2006 21 2 228 236 2-s2.0-33144490116 16418778 10.1359/JBMR.051021
101 Wei W Ryu JK Choi HB McLarnon JG Expression and function of the P2X7 receptor in rat C6 glioma cells Cancer Letters 2008 260 1-2 79 87 2-s2.0-38149036646 18039556 10.1016/j.canlet.2007.10.025
102 Chen GL Lin HB Wu XW Li RY Chen XH The correlation between the level of serum VEGF and bFGF with metastasis and prognosis of osteosarcoma Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 2011 27 12 1343 1345 22368795
103 Tzankov A Medinger M Fischer N Vascular endothelial growth factor-related pathways in hemato-lymphoid malignancies Journal of Oncology 2010 2010 13 pages 2-s2.0-77953520737 729725 10.1155/2010/729725 PMC2875768 20508816
104 Coleman RE Bone cancer in 2011: prevention and treatment of bone metastases Nature Reviews Clinical Oncology 2012 9 2 76 78 10.1038/nrclinonc.2011.198 22182971
105 Hanada R Hanada T Sigl V Schramek D Penninger JM RANKL/RANK-beyond bones Journal of Molecular Medicine 2011 89 7 647 656 2-s2.0-79959936606 21445556 10.1007/s00109-011-0749-z
106 Hill LM Gavala ML Lenertz LY Bertics PJ Extracellular ATP may contribute to tissue repair by rapidly stimulating purinergic receptor X7-dependent vascular endothelial growth factor release from primary human monocytes Journal of Immunology 2010 185 5 3028 3034 2-s2.0-78049404838 10.4049/jimmunol.1001298 PMC3156583 20668222
107 Barbera-Cremades M Baroja-Mazo A Gomez AI Machado F Di VF Pelegrin P P2X7 receptor-stimulation causes fever via PGE2 and IL-1beta release The FASEB Journal 2012 26 7 2951 2962 10.1096/fj.12-205765 22490780
108 Chong JH Zheng GG Ma YY The hyposensitive N187D P2X7 mutant promotes malignant progression in nude mice Journal of Biological Chemistry 2010 285 46 36179 36187 2-s2.0-78149263196 20837475 10.1074/jbc.M110.128488 PMC2975240
109 Raffaghello L Chiozzi P Falzoni S Di Virgilio F Pistoia V The P2X7 receptor sustains the growth of human neuroblastoma cells through a substance P-dependent mechanism Cancer Research 2006 66 2 907 914 2-s2.0-31544480053 16424024 10.1158/0008-5472.CAN-05-3185
110 Verhoef PA Estacion M Schilling W Dubyak GR P2X7 receptor-dependent blebbing and the activation of Rho-effector kinases, caspases, and IL-1 β release Journal of Immunology 2003 170 11 5728 5738 2-s2.0-0037851027 10.4049/jimmunol.170.11.5728 12759456
111 Morelli A Chiozzi P Chiesa A Extracellular ATP causes ROCK I-dependent Bleb formation in P2X7-transfected HEK293 cells Molecular Biology of the Cell 2003 14 7 2655 2664 2-s2.0-12444290153 12857854 10.1091/mbc.02-04-0061 PMC165666
112 Pfeiffer ZA Aga M Prabhu U Watters JJ Hall DJ Bertics PJ The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho Journal of Leukocyte Biology 2004 75 6 1173 1182 2-s2.0-2642512455 15075366 10.1189/jlb.1203648
113 Panupinthu N Zhao L Possmayer F Ke HZ Sims SM Dixon SJ P2X7 nucleotide receptors mediate blebbing in osteoblasts through a pathway involving lysophosphatidic acid Journal of Biological Chemistry 2007 282 5 3403 3412 2-s2.0-34047243995 17135244 10.1074/jbc.M605620200
114 Jelassi B Chantme A Alcaraz-Pérez F P2X 7 receptor activation enhances SK3 channels- and cystein cathepsin-dependent cancer cells invasiveness Oncogene 2011 30 18 2108 2122 2-s2.0-79955579562 21242969 10.1038/onc.2010.593
115 Liu S Li S Du Y Polychlorinated biphenyls (PCBs) enhance metastatic properties of breast cancer cells by activating rho-associated kinase (ROCK) PLoS ONE 2010 5 6 2-s2.0-77955283033 e11272 10.1371/journal.pone.0011272 PMC2890589 20585605
116 Ruppender NS Merkel AR Martin TJ Mundy GR Sterling JA Guelcher SA Matrix rigidity induces osteolytic gene expression of metastatic breast cancer cells PLoS ONE 2010 5 11 2-s2.0-78649732806 e15451 10.1371/journal.pone.0015451 PMC2981576 21085597
117 Slater M Danieletto S Pooley M Teh LC Gidley-Baird A Barden JA Differentiation between cancerous and normal hyperplastic lobules in breast lesions Breast Cancer Research and Treatment 2004 83 1 1 10 2-s2.0-0942301260 14997049 10.1023/B:BREA.0000010670.85915.0f
118 Lopez-Castejon G Theaker J Pelegrin P Clifton AD Braddock M Surprenant A P2X7 receptor-mediated release of cathepsins from macrophages is a cytokine-independent mechanism potentially involved in joint diseases Journal of Immunology 2010 185 4 2611 2619 2-s2.0-77956936562 10.4049/jimmunol.1000436 20639492
119 Maris JM Hogarty MD Bagatell R Cohn SL Neuroblastoma The Lancet 2007 369 9579 2106 2120 2-s2.0-34250683435 10.1016/S0140-6736(07)60983-0 17586306
120 Maris JM Recent advances in neuroblastoma The New England Journal of Medicine 2010 362 23 2202 2211 2-s2.0-77953523121 20558371 10.1056/NEJMra0804577 PMC3306838
121 Wu PY Lin YC Chang CL Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells Cellular Signalling 2009 21 6 881 891 2-s2.0-62749172817 19385050 10.1016/j.cellsig.2009.01.036
122 Gutiérrez-Martín Y Bustillo D Gómez-Villafuertes R P2X7 receptors trigger ATP exocytosis and modify secretory vesicle dynamics in neuroblastoma cells Journal of Biological Chemistry 2011 286 13 11370 11381 2-s2.0-79953223696 21292765 10.1074/jbc.M110.139410 PMC3064193
123 Ara T DeClerck YA Mechanisms of invasion and metastasis in human neuroblastoma Cancer and Metastasis Reviews 2006 25 4 645 657 2-s2.0-33846051928 17160711 10.1007/s10555-006-9028-9
124 Calvert RC Shabbir M Thompson CS Mikhailidis DP Morgan RJ Burnstock G Immunocytochemical and pharmacological characterisation of P2-purinoceptor-mediated cell growth and death in PC-3 hormone refractory prostate cancer cells Anticancer Research 2004 24 5 A 2853 2859 2-s2.0-6444231782 15517888
125 Ravenna L Sale P Di Vito M Up-regulation of the inflammatory-reparative phenotype in human prostate carcinoma Prostate 2009 69 11 1245 1255 2-s2.0-67650581533 19444819 10.1002/pros.20966
126 Zhang Y Gong LH Zhang HQ Extracellular ATP enhances in vitro invasion of prostate cancer cells by activating Rho GTPase and upregulating MMPs expression Cancer Letters 2010 293 2 189 197 2-s2.0-77952471484 20202742 10.1016/j.canlet.2010.01.010
127 Ballou LM Lin RZ Rapamycin and mTOR kinase inhibitors Journal of Chemical Biology. 2008 1 1–4 27 36 19568796 10.1007/s12154-008-0003-5 PMC2698317
128 Ogawa T Tokuda M Tomizawa K Osteoblastic differentiation is enhanced by rapamycin in rat osteoblast-like osteosarcoma (ROS 17/2.8) cells Biochemical and Biophysical Research Communications 1998 249 1 226 230 2-s2.0-0032504527 9705862 10.1006/bbrc.1998.9118
129 Vemulapalli S Mita A Alvarado Y Sankhala K Mita M The emerging role of mammalian target of rapamycin inhibitors in the treatment of sarcomas Targeted Oncology 2011 6 1 29 39 2-s2.0-79957509564 21533543 10.1007/s11523-011-0179-4
130 Pang H Faber LE Estrogen and rapamycin effects on cell cycle progression in T47D breast cancer cells Breast Cancer Research and Treatment 2001 70 1 21 26 2-s2.0-0035193323 11767001 10.1023/a:1012570204923
131 Arulkumaran N Unwin RJ Tam FWK A potential therapeutic role for P2X7 receptor (P2X7R) antagonists in the treatment of inflammatory diseases Expert Opinion on Investigational Drugs 2011 20 7 897 915 2-s2.0-79958798010 21510825 10.1517/13543784.2011.578068 PMC3114873