Isoform B of P2X7 receptor is crucial for maintenance of cancerstem cells and neuroblastoma progression

Author(s)

Izadora Lorrany Alves Rabelo

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2024-11-11

Examining board members

Claudiana Lameu; Silvia Lima Costa; Evandro Araújo de Souza

• Advisor: Claudiana Lameu

Abstract

Neuroblastoma is a neuroendocrine tumor responsible for 10% of pediatric cancer deaths, with half of the cases already metastatic at diagnosis. Cancer stem cells (CSCs) are responsible for therapy resistance and relapse. The P2X7 receptor, which binds ATP, is involved in tumor proliferation and aggressiveness. Among its isoforms, P2X7A induces apoptosis, while P2X7B, truncated at the C-terminal, promotes cell growth. This study investigated the roles of P2X7A and P2X7B isoforms in CSC maintenance and tumor progression. Human ACN neuroblastoma cells were analyzed for pluripotency marker expression by flow cytometry. CD133 was expressed in 35% of the cells, SOX-2 in 10%, and Oct-4 was not significantly expressed. Among the tested conditions, the medium supplemented with EGF/bFGF/N2 was effective in enriching CSCs, showing high SOX-2 expression and low cell adhesion. Analysis of control ACN neuroblastoma cells (P2X7A+/B+), and cells silenced for isoforms A and B (P2X7A-/B-) or only isoform A (P2X7A-/B+) revealed that isoform B is crucial for CSC self-renewal and proliferation, as determined by tumor sphere formation efficiency (TFE) and colony formation efficiency (CFE). Chemotaxis assays showed that isoform B in cell invasion is relevant for more differentiated cells when isoform A was silenced, tripling the invasive capacity of the cells. In vivo tumorigenesis assays involved injecting ACN neuroblastoma CSCs (P2X7A+/B+, P2X7A-/B-, and P2X7A-/B+) into immunodeficient mice to assess tumor formation and aggressiveness. By day 20, all mice had palpable tumors; however, those receiving P2X7A-/B+ cells lost more weight and had faster-growing tumors (doubling time of 14.63 days) compared to P2X7A+/B+ and P2X7A-/B- tumors (24.95 and 34.98 days, respectively). Mice with tumorsexpressing only isoform B had a worse prognosis, with lower survival rates compared to those with tumors expressing both isoforms or both silenced. The main conclusion of the study is that the P2X7B isoform plays a key in the aggressiveness and growth of neuroblastoma tumors, suggesting that P2X7B is a promising target for therapeutic interventions. Selective inhibition of isoform B could potentially reduce tumor aggressiveness, improve prognosis, and increase survival rates in neuroblastoma patients. (AU)

• FAPESP's process: 17/06132-7 - Study of P2X7 receptor isoforms in the maintenance of cancer stem cells and metastasis
• Grantee: Izadora Lorrany Alves Rabelo
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)