Metabolism and neural differentiation in childhood neuroblastoma

Neuroblastoma is the most common and aggressive extracranial solid tumor during childhood. MYCN-amplification is found in approximately 25 % of all neuroblastoma cases, and is defined as high-risk disease. Development of novel therapeutic approaches focused on MYCN targeting are essential for increased survival these children. The MYC family of oncoproteins consists of transcriptional factors involved in many normal cellular processes. Abnormal expression of MYC is associated with 70 % of human cancers and correlates with an aggressive undifferentiated phenotype, chemotherapy resistance and poor clinical prognosis.

Targeting MYCN by small molecular weight molecules remains a challenge. In paper I we established that one known c-MYC targeting compound, the small chemical molecule 10058-F4, is also a potent MYCN inhibitor. 10058-F4 treatment increased cell death and neuronal differentiation in MYCN-amplified neuroblastoma cells and prolonged survival in mice. Interestingly, we found that MYCN inhibition resulted in changes in expression of metabolic proteins, in accumulation of intracellular lipid droplets and demonstrated that this is due to mitochondrial dysfunction. Our data reported in paper I strongly suggests that MYCN regulated metabolic processes may contribute to the aggressiveness of neuroblastoma.

In paper II we applied several approaches to further investigate the MYCN-mediated metabolic alterations in neuroblastoma. The combination of mass spectrometry based proteomics and transcriptome data analysis highlighted key metabolic enzymes involved in energetic pathways of cancer cells. The functional metabolic measurements supported the data analysis and demonstrated that MYCN not only enhanced the glycolytic capacity of neuroblastoma cells, but also increased mitochondrial respiration. The data presented in paper II suggests that MYCN-amplification is associated with a high-energetic metabolic phenotype. Importantly, we demonstrated that targeting of fatty acid oxidation resulted in potentiated neuronal differentiation, decreased viability of MYCN-amplified neuroblastoma as well as decreased tumor burden in vivo in a neuroblastoma xenograft model.

Our previous findings highlighted an important role of fatty acid metabolism in MYCN-amplified neuroblastoma. In paper III we used specific inhibitors and demonstrated that targeting of de novo fatty acid synthesis in MYCN-amplified neuroblastoma cells resulted in increased mitochondrial dysfunction and glycolytic flux. In addition, we observed that MYCN downregulation and neuronal differentiation are consequences of inhibiting de novo synthesis of fatty acids in neuroblastoma cells.

In paper IV we demonstrated that the miR-17~92 cluster, which is upregulated by MYCN, suppresses neuronal differentiation via targeting of the nuclear hormone receptor family in neuroblastoma. Importantly, we showed that MYCN inhibition leads to increased expression of the glucocorticoid receptor, which is accompanied by decreased levels of members of the miR-17~92 clusters and elevated expression of the neural differentiation markers TrkA, SCG2 and TH. Furthermore, increased GR expression followed after MYCN downregulation and decreased tumor burden was observed in a pre-clinical NB model following combined MYC inhibition and activation of glucocorticoid signaling.

Together the data generated in our laboratory and included in the present thesis demonstrates that targeting of MYCN and MYCN-controlled metabolic processes may provide an attractive basis for development of novel therapeutic approaches for childhood neuroblastoma.

List of scientific papers

I. Hanna Zirath, Anna Frenzel, Ganna Oliynyk, Lova Segerström, Ulrica K. Westermark, Karin Larsson, Matilda Munksgaard Persson, Kjell Hultenby, Janne Lehtiö, Christer Einvik, Sven Påhlman, Per Kogner, Per-Johan Jakobsson, and Marie Arsenian Henriksson. MYC inhibition induces metabolic changes leading to accumulation of lipid droplets in tumor cell. Proceeding of the National Academy of Science. 2013, vol. 110, pp. 10258-10263.
https://doi.org/10.1073/pnas.1222404110

II. Ganna Oliynyk, Johanna Dzieran, María Victoria Ruiz-Pérez, Hanna Zirath, Taner Arslan, Henrik Johansson, Erik Fredlund, Janne Lehtiö, and Marie Arsenian Henriksson. Fatty acid-dependent oxidative phosphorylation is the major source of energy production in MYCN-amplified neuroblastoma cells, 2015. [Manuscript]

III. María Victoria Ruiz-Pérez, Ganna Oliynyk, Lourdes Sainero Alcolado, and Marie Arsenian-Henriksson. Induction of neural differentiation in childhood neuroblastoma upon inhibition of de novo fatty acid synthesis. [Manuscript]

IV. Diogo Ribeiro, Marcus D.R. Klarqvist, Ulrica K. Westermark, Ganna Oliynyk, Johanna Dzieran, Anna Kock, Carolina Savatier Banares, Falk Hertwig, John Inge Johnsen, Matthias Fischer, Per Kogner, Jakob Lovén and Marie Arsenian Henriksson. Regulation of nuclear hormone receptors by MYCN driven miRNAs impacts neural differentiation and survival in neuroblastoma patients. Cell Reports. 2016, vol 16, pp. 979-993.
https://doi.org/10.1016/j.celrep.2016.06.052