Citation

  • Authors: Nazio F. et al.
  • Year: 2021
  • Journal: Acta Neuropathol
  • Applications: in vivo / DNA / in vivo-jetPEI

Method

- P0 CD1 mice cerebella were transfected in vivo with the following plasmids: pPBase (encoding a hyperactive form of the piggyBac transposase), together with the piggyBac donor plasmids pPB CAG c-Myc and pPB CAG Otx2-IRES-VenusNLS. The pPB CAG Luc plasmid (encoding the firefly Luciferase) was always co-transfected as a reporter. pPBase and piggyBac donor plasmids were mixed at a 1:4 ratio. - Plasmid DNA and in vivo-jetPEI transfection reagent were mixed according to the manufacturer’s instructions. Newborn CD1 mice were anesthetized on ice for 2 min, placed on a stage in a stereotactic apparatus and medially injected at lambda: −3.6 D/V: −1.6 with 4 µl of transfection mix using a pulled glass capillary and a FemtoJet microinjector. - Mice at P18 were subjected to bioluminescence imaging of luciferase activity to monitor the effective transfection of cerebella. Animals were intraperitoneally administered 150 mg/kg D-Luciferin and anesthetized with 2% isoflurane. Bioluminescent signal was captured using the In-vivo Xtreme system. - Only mice carrying luciferase signals were selected for drug treatment and divided randomly in the two treatment cohorts.

Abstract

Medulloblastoma (MB) is a childhood malignant brain tumour comprising four main subgroups characterized by different genetic alterations and rate of mortality. Among MB subgroups, patients with enhanced levels of the c-MYC oncogene (MBGroup3) have the poorest prognosis. Here we identify a previously unrecognized role of the pro-autophagy factor AMBRA1 in regulating MB. We demonstrate that AMBRA1 expression depends on c-MYC levels and correlates with Group 3 patient poor prognosis; also, knockdown of AMBRA1 reduces MB stem potential, growth and migration of MBGroup3 stem cells. At a molecular level, AMBRA1 mediates these effects by suppressing SOCS3, an inhibitor of STAT3 activation. Importantly, pharmacological inhibition of autophagy profoundly affects both stem and invasion potential of MBGroup3 stem cells, and a combined anti-autophagy and anti-STAT3 approach impacts the MBGroup3 outcome. Taken together, our data support the c-MYC/AMBRA1/STAT3 axis as a strong oncogenic signalling pathway with significance for both patient stratification strategies and targeted treatments of MBGroup3.

Pubmed