Study Of ALDH18A1-MYCN Positive Feedback Loop In Neuroblastoma Cells Self-renew

Neuroblastoma(NB)is a neuroendocrine tumor that arises in the developing sympathetic nervous system.Remarkably,NB has a broad spectrum of clinical behavior and heterogeneous biological features ranging from spontaneous regression to progressive disease and metastasis.A number of genetic aberrations have been extensively investigated,among which the amplification of MYCN(encoding N-MYC)was found in approximately 20% of patients and identified as one of the strongest determinants with significant prognostic value,specifically in unfavorable clinical outcomes and poor survival of high-risk NB patients.Accumulating reports have considered N-MYC to be “undruggable” with limited treatment options.Thus,an urgent need to develop alternative therapeutic strategies for targeting MYCN in NB remains and exploring the upstream regulators of MYCN expression at genetic and epigenetic levels is therefore imperative.Tumorigenesis is dependent on reprogramming cellular metabolism during which alterations in intracellular and extracellular metabolites exhibit potentially long-ranging impacts on epigenomic and transcriptomic regulation.Specifically,deregulated glutamine metabolism has become widely accepted to be critical for the establishment and maintenance of the tumorigenic state,resulting in aberrantly activated oncogenes and loss of tumor suppressors,and this,in turn,enables the uncontrolled access of glutamine to cancer cells.Of note,ALDH18A1 is the key enzyme for the synthesis of proline from glutamate that catalyzes the coupled phosphorylation and reduction conversion of glutamate to P5 C and plays a critical role in regulating glutamate metabolism.Recent efforts have shown that ALDH18A1 functions as a critical component in regulating the cell proliferation of melanoma via proline biosynthesis.However,the molecular basis of the effects of ALDH18A1 activation on tumorigenesis,especially on MYCN-amplified NB,remains elusive and warrants further investigation.In the present study,we have identified ALDH18A1 as an unfavorable prognositic factor in the determination of risk in NB patients,especially in those harboring MYCN amplification.Moreover,by incorporating bioinformatic analysis with functional studies,we have identified ALDH18A1 as an important regulator of NB cell proliferation,self-renewal,and tumorigenicity.Notably,mechanistic investigations showed that ALDH18A1 could both transcriptionally and post-transcriptionally regulate MYCN expression,the latter of which was then validated to reciprocally trigger the direct transactivation of ALDH18A1 transcription,thus forming a postive feedback loop between ALDH18A1 and MYCN.The main findings of our study can be described as follows:1.Analysis of NB gene expression(GE)profiles identifies an optimal 6-gene prognostic signature,in which ALDH18A1 is associated with the genetic amplification of MYCN and strongly correlates with the clinico-pathological characteristics of NB patients.2.Multivariate survival analysis based on Cox proportional hazards(CPH)models marks ALDH18A1 as a potential oncogene in NB and defines high ALDH18A1 expression as an unfavorable prognostic marker and risk factor in NB patients,especially in those with MYCN amplification.3.ALHD18A1 exerts profound impacts on cell proliferation,self-renewal potential,and tumor-initiating capacity of NB cells,and high ALDH18A1 expression could possibly lock MYCN-amplified NB cells in a more aggressive or tumorigenic state.4.ALDH18A1 positively modulates MYCN expression at both the mRNA and protein levels in MYCN-amplified NB cells.Specifically,ALDH18A1 could induce significant changes in the reporter activity from the MYCN 5’ promoter via the previously reported miR-29b/SP1 autoregulatory loop,suggesting that ALDH18A1 regulates MYCN at the transcriptional level.More importantly,ALDH18A1 knockdown-induced miR-193 a and miR-29 a expression,together with miR-29 b contribute to the prominent depression of MYCN expression,highlighting a complex interactive miRNAs network that ultimately induced targeted inhibition of MYCN protein translation.5.N-MYC directly targets the ALDH18A1 promoter by specifically associating with N-MYC binding sites and transcriptionally activates ALDH18A1 expression.Collectively,the conclusions of this study can therefore be highlighted as follows:1.ALDH18A1 could serve as an unfavorable prognostic indicator in neuroblastoma patients,especially in those with MYCN amplification.2.A positive feed-back regulatory loop exists between ALDH18A1 and MYCN,which regulates cell proliferation,self-renewal potential,and tumor-initiating capacity of NB cells.3.ALDH18A1 might be a promising therapeutic target to overcome MYCN-amplified NB.Thus,our findings therefore identify ALDH18A1 as an unfavorable prognostic marker in MYCN-amplified NBs and define ALDH18A1-MYCN positive feedback loop as an important regulator of NB cell self-renewal,and support the rationale for circumventing the drawbacks encountered with directly targeting MYCN and provide therapeutic insights into disrupting of the positive feedback loop between ALDH18A1 and MYCN in MYCN-amplified NBs.