PI3K/Akt signaling pathway triggers P2X7 receptor expression as a pro-survival factor of neuroblastoma cells under limiting growth conditions.

Rosa Gómez-Villafuertes,Mª Teresa Miras-Portugal,Paula García-Huerta,Juan Ignacio Díaz-Hernández

doi:10.1038/srep18417

Abstract

The expression of purinergic P2X7 receptor (P2X7R) in neuroblastoma cells is associated to accelerated growth rate, angiogenesis, metastasis and poor prognosis. Noticeably, P2X7R allows the survival of neuroblastoma cells under restrictive conditions, including serum and glucose deprivation. Previously we identified specificity protein 1 (Sp1) as the main factor involved in the transcriptional regulation of P2rx7 gene, reporting that serum withdrawal triggers the expression of P2X7R in Neuro-2a (N2a) neuroblastoma cell line. Here we demonstrate that PI3K/Akt pathway is crucial for the upregulation of P2X7R expression in serum-deprived neuroblastoma cells, circumstance that facilitates cell proliferation in the absence of trophic support. The effect exerted by PI3K/Akt is independent of both mTOR and GSK3, but requires the activation of EGF receptor (EGFR). Nuclear levels of Sp1 are strongly reduced by inhibition of PI3K/Akt pathway, and blockade of Sp1-dependent transcription with mithramycin A prevents upregulation of P2rx7 gene expression following serum withdrawal. Furthermore, atypical PKCζ plays a key role in the regulation of P2X7R expression by preventing phosphorylation and, consequently, activation of Akt. Altogether, these data indicate that activation of EGFR enhanced the expression of P2X7R in neuroblastoma cells lacking trophic support, being PI3K/Akt/PKCζ signaling pathway and Sp1 mediating this pro-survival outcome.

Highlights

Proteins, and pathways responsible for neuroblastoma tumorigenesis and progression may lead to the development of more effective, less toxic therapies
Quantitative real-time PCR (Q-PCR) studies indicated that SH-SY5Y cells cultured in serum-free medium (SF) suffer an increment in P2X7 transcript comparable to that observed in murine neuroblastoma cells
Based on previous studies reporting that P2X7R supports proliferation of neuroblastoma cells in the absence of serum by triggering the release of trophic factors[4,6], we postulated that the upregulation of P2X7R following serum deprivation could be facilitating neuroblastoma cells survival

Summary

Introduction

Proteins, and pathways responsible for neuroblastoma tumorigenesis and progression may lead to the development of more effective, less toxic therapies. During glucose deprivation P2X7R overexpression correlates with a higher lactate output, overexpression of several glycolytic enzymes and larger intracellular glycogen stores, allowing better adaptability to unfavorable ambient conditions[17]. Based on these findings, a deeper understanding of the relationship between trophic deprivation and P2X7R expression could be biologically and clinically important. Atypical PKCζ is a key component in the regulation of P2rx[7] gene expression through the inactivation of Akt. We demonstrated that the increase in P2X7R expression induced by serum withdrawal in N2a cells is a pro-survival mechanism to compensate the lack of trophic support

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