Study of P2X7 receptor isoforms in the maintenance of cancer stem cells and metastasis

Abstract

ATP plays an essential role in the metastasis of neuroblastoma and lung cancer cells to the bone marrow. It has been observed that neuroblastoma metastasis in murine xenographic model is inhibited by P2X7 receptor antagonism. This receptor induces tumor proliferation and plays an important role in the maintenance of neuroblastoma, but little is known about its role in the maintenance of CSCs in neuroblastoma. It has been demonstrated that in proliferative embryonic stem cells the expression and activity of P2X7 receptor are increased, while under induction of neural differentiation, they are suppressed. Pharmacological inhibition of P2X7 results in rescue of quiescent progenitors in the cell cycle and in promotion of neuronal differentiation, an event that reduces receptor and pluripotency marker expression, Oct-4. Therefore, the P2X7 receptor may be involved in the maintenance of CSCs in neuroblastoma tumor mass. Its overexpression would maintain a higher percentage of cells of the neuroblastoma tumor mass with stem cell characteristics, giving the tumor more aggressiveness, resistance to radio and chemotherapy, and metastatic power. We intend to verify if purinergic signaling increases the percentage of CSCs in the tumor mass of neuroblastoma through its detection by normal stem cells and by the formation of tumors, following treatments with antagonists and agonists of the purinergic system. The larger and better characterized P2X7 receptor isoform (P2X7A) has a C-terminal portion that interacts with panexin-1, causing large pores in the membrane and leading to apoptosis. The truncated isoform B (P2X7B), widely expressed in tissues, has a sequence similar to that of P2X7A, but does not exhibit pro-apoptotic activity. The treatment of neuroblastoma cells with bradykinin by our group resulted in differential levels of purinergic receptor expression, including a significant increase in P2X7 expression and induction of neuroblastoma cells to metastatic behavior. The treatment of these cells with bradykinin increases the expression of both isoforms and favors the upregulation of P2X7B. Since P2X7B retains the cell growth promoting activity but loses apoptotic function, we believe its expression is more required in oncogenesis and maintenance of CSCs. The goal is to investigate the contribution of these isoforms in the maintenance of CSCs in the neuroblastoma tumor mass. At basal levels of ATP, both can be activated and support cell growth. However, increase in the extracellular ATP concentration generates a differentiated cellular response depending on the level of isoforms expression. If P2X7B is predominant in high concentrations of ATP, there will be no pore formation and, consequently, absence of cytotoxicity; on the contrary, there will be promotion of a strong stimulus for growth. On the other hand, if P2X7A is predominant or both subunits are present, pore formation and, consequently, cell death will occur. Therefore, modulation of P2X7A and P2X7B expression would confer a high degree of plasticity to CSCs in cellular responses to extracellular ATP. We will investigate whether bradykinin has the effect of inducing differential expression of isoforms in CSCs to maintain their "stemness" status or lead to the differentiation of cancer cells through quantification of tumors formation and metastatic potential from neuroblastoma cells transfected with P2X7 silencing vectors. The main therapeutic strategies direct CSCs to cell death or promote their differentiation, depleting them from tumor reservoir. Therefore, studies to understand the mechanisms of metastasis and maintenance of CSCs will pave the way for new therapeutic protocols, as well as possibilities for cure, since the elimination of CSCs would prevent tumor recurrence and metastasis. (AU)