Neuroblastoma (NB) is an aggressive pediatric tumor, responsible for 15% of cancer-related deaths in childhood, lacking an effective treatment in its advanced stages. The P2X7 receptor for extracellular ATP was associated to NB cell proliferation and recently emerged as a promoter of tumor engraftment, growth and vascularization. In an effort to identify new therapeutic options for neuroblastoma, we studied the role of P2X7 receptor in NB biology. We first analyzed the effect of P2X7 activation or down-modulation of the main biochemical ways involved in NB progression: the PI3K/Akt/GSK3β/MYCN and the HIF1α/VEGF pathways. In ACN human NB cells, P2X7 stimulation enhanced PI3K/Akt, while decreasing GSK3β activity. In the same model, P2X7 silencing or antagonist administration reduced the activity of PI3K/Akt and increased that of GSK3β, leading to a decrease in cellular glycogen stores. Similarly, P2X7 downmodulation caused a reduction in HIF1α levels and vascular endothelial growth factor (VEGF) secretion. Systemic administration of two different P2X7 antagonists (AZ10606120 or A740003) in nude/nude mice reduced ACN-derived tumor growth. An even stronger effect of P2X7 blockade was obtained in a syngeneic immune-competent neuroblastoma model: Neuro2A cells injected in AlbinoJ mice. Together with tumor regression, treatment with P2X7 antagonists caused downmodulation of the Akt/HIF1α axis, leading to reduced VEGF content and decreased vessel formation. Interestingly, in both experimental models, P2X7 antagonists strongly reduced the expression of the probably best-accepted oncogene in NB: MYCN. Finally, we associated P2X7 overexpression with poor prognosis in advanced-stage NB patients. Taken together, our data suggest that P2X7 receptor is an upstream regulator of the main signaling pathways involved in NB growth, metabolic activity and angiogenesis, and a promising therapeutic target for neuroblastoma treatment.

The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca2+ mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently coupled to cell growth or activity.

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.

Malignant pleural mesothelioma (MPM) is an aggressive tumor refractory to anti-blastic therapy. MPM cells show several genetic and biochemical defects, e.g. overexpression of oncogenes, downregulation of onco-suppressor genes, dysregulation of microRNA, or alteration of intracellular Ca2+ homeostasis and of apoptosis. No information is as yet available on purinergic signalling in this tumor. Signalling via the P2×7 (P2RX7 or P2×7R) purinergic receptor is attracting increasing attention as a pathway involved in cancer cell death or proliferation. In this report we show that the P2×7R is expressed by three MPM cell lines established from MPM patients but not by mesothelial cells from healthy subjects (healthy mesothelial cells, HMCs). MPM cell proliferation was inhibited by in vitro incubation in the presence of selective P2×7R antagonists, as well as by stimulation with the P2×7R agonist BzATP. Systemic administration of the selective P2×7R blocker AZ10606120 inhibited in vivo growth of MPM tumors whether implanted subcutaneously (s.c.) or intraperitoneally (i.p.). Our findings suggest that the P2×7R might be a novel target for the therapy of mesothelioma.

Lymphocyte growth and differentiation are modulated by extracellular nucleotides and P2 receptors. We previously showed that the P2X7 receptor (P2X7R or P2RX7) is overexpressed in circulating lymphocytes from chronic lymphocytic leukemia (CLL) patients. In the present study we investigated the P2X7R/NLRP3 inflammasome axis in lymphocytes from a cohort of 23 CLL patients. P2X7R, ASC and NLRP3 were investigated by Western blot, PCR and transfection techniques. P2X7R was overexpressed and correlated with chromosome 12 trisomy in CLL patients. ASC mRNA and protein were also overexpressed. On the contrary, NLRP3 was dramatically down-modulated in CLL lymphocytes relative to lymphocytes from healthy donors. To further investigate the correlation between P2X7R, NLRP3 and cell growth, NLRP3 was silenced in THP-1 cells, a leukemic cell line that natively expresses both NLRP3 and P2X7R. NLRP3 silencing enhanced P2X7R expression and promoted growth. On the contrary, NLRP3 overexpression caused accelerated apoptosis. The P2X7R was also up-modulated in hematopoietic cells from NLRP3-KO mice. In conclusion, we show that NLRP3 down-modulation stimulates P2X7R expression and promotes growth, while NLRP3 overexpression inhibits cell proliferation and stimulates apoptosis. These findings suggest that NLRP3 is a negative regulator of growth and point to a role of the P2X7R/NLRP3 axis in CLL.