Characterising the role of PA2G4 and its interaction with MYCN in neuroblastoma progression

Abstract

Neuroblastoma, an embryonal tumour of the sympathetic nervous system, is the most common solid tumour in childhood. MYCN oncogene amplification is found in one third of primary neuroblastoma at diagnosis, and correlates with poor prognosis. We identified Proliferation-associated protein 2G4 (PA2G4) as a binding partner for MYCN using co-immunoprecipitation and mass spectrometry. The long isoform of PA2G4 (p48) has a known oncogenic function, whereas the short isoform (p42) acts as a tumour suppressor. However, the role of PA2G4 in neuroblastoma and its link with MYCN is currently unknown.

Using a panel of human neuroblastoma cell lines and patient tumour samples, we analysed the expression of PA2G4 by real-time PCR and Western blotting. We found that high expression of PA2G4 was a strong independent clinical predictor of poor survival and positively correlated with MYCN expression. Analysis of pre-cancerous ganglia cells from TH-MYCN neuroblastoma mouse model showed PA2G4 mRNA was expressed 8-fold higher in ganglia from TH-MYCN transgenic homozygous, compared to wild-type, mice.

Chromatin immunoprecipitation showed MYCN binds to PA2G4 promoter to activate its transcription. Surprisingly, suppression of PA2G4 in neuroblastoma cell lines markedly reduced MYCN protein level. Cycloheximide-chase assay confirmed that PA2G4 increased MYCN protein stability, thus creating a positive forward feedback loop essential for maintaining mutual high expression. Suppression of PA2G4 down-regulated pMDM2 and pAKT, leading to an increase in p53 expression. Furthermore, PA2G4 knockdown reduced cell migration and colony formation; importantly expression of MYCN was required for the effect of PA2G4 on colony formation.

Most significantly, overexpression of PA2G4 induced tumorigenesis in a non-tumorigenic cell line. Furthermore, a small molecule inhibitor of PA2G4, WS6, significantly decreased neuroblastoma cell growth and PA2G4 and MYCN protein levels in vitro, and delayed tumour growth in vivo. This research highlights the importance and underlying mechanisms of PA2G4 as an onco-factor in MYCN driven neuroblastoma. It identifies PA2G4 as a novel MYCN-binding protein which increases MYCN protein stability and acts as an oncogenic co-factor in a forward feedback expression loop with MYCN. It provides strong evidence that PA2G4 plays a critical role in tumorigenesis and a novel molecular target for the treatment of neuroblastoma.