MYCN coordinately regulates the entire polyamine pathway to maintain high polyamine levels in neuroblastoma — ASN Events
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MYCN coordinately regulates the entire polyamine pathway to maintain high polyamine levels in neuroblastoma (#116)

Laura Gamble 1 , Stefania Purgato 2 , Jayne Murray 1 , Amanda Russell 1 , Lesley Ashton 1 , Emanuele Valli 2 , Giorgio Milazzo 2 , Daniel Carter 1 , Belamy Cheung 1 , Andre Oberthuer 3 , Wendy London 4 , Mike Hogarty 5 , Matthias Fischer 3 , Glenn Marshall 1 6 , David Ziegler 1 6 , Giovanni Perini 2 , Murray Norris 1 , Michelle Haber 1
  1. Children's Cancer Institute Australia, Randwick, NSW, Australia
  2. Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
  3. Department of Pediatric Oncology and Hematology, University of Cologne, Cologne, Germany
  4. Children's Oncology Group Statistics and Data Center, Dana-Farber Harvard Cancer Care and Children's Hospital Boston, Boston, MA, USA
  5. The Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
  6. Sydney Chilren's Hospital, Sydney, Australia

Background: We have previously shown that the MYCN/MYC target ornithine decarboxylase (ODC1), rate-limiting for polyamine biosynthesis, is a therapeutic target for neuroblastoma (Cancer Res 68:9735, 2008). An international Phase I NANT trial for refractory neuroblastoma has recently opened, based on ODC1 inhibition by high-dose difluoromethylornithine. We have now examined the prognostic impact of all polyamine pathway genes in a large cohort of neuroblastoma.

 

Methods: Gene-expression profiles of 650 primary untreated neuroblastomas were analyzed for all 11 polyamine pathway genes, and related to clinical outcome. Gene expression was evaluated by RT-PCR, while activation and repression of polyamine genes by MYCN were analyzed by chromatin immunoprecipitation (ChIP). Gene promoters were cloned into luciferase reporters and tested as a function of MYCN expression. Histone modifications and DNA methylation of gene promoters were evaluated by ChIP and Methyl-ChIP.

 

Results: High levels of each polyamine biosynthetic gene and low levels of each catabolic gene strongly predicted poor outcome. Multivariate analysis showed 5/11 genes retained independent prognostic significance following adjustment for MYCN, age and stage. RT-PCR analysis demonstrated a direct correlation between MYCN and biosynthetic polyamine gene expression, but an inverse correlation between MYCN and catabolic polyamine gene expression. ChIP assays confirmed that MYCN associates with biosynthetic gene promoters by binding to E-box sites in order to activate transcription. In contrast, MYCN binds the promoters of the catabolic genes by interacting with the Sp1 protein in order to repress transcription of these genes. The findings were confirmed by luciferase reporter assays.

 

Conclusions: These results provide an unprecedented demonstration of an oncogene coordinately regulating the expression of every gene in a metabolic pathway in order to drive cell proliferation. The findings highlight the critical importance of polyamines in neuroblastoma and suggest that targeting polyamine pathway genes in addition to ODC1 will be a valuable therapeutic approach.