MiR-34a Targeting of Notch Ligand Delta-Like 1 Impairs CD15+/CD133+ Tumor-Propagating Cells and Supports Neural Differentiation in Medulloblastoma

Abstract

BackgroundThrough negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies.Methodology/Principal FindingsIn a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133+/CD15+ tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1+/- p53-/-), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo.Conclusions/SignificanceDespite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.

Medulloblastoma Treatment using Gene Therapy

“…Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Infection of adenoviruses carrying the precursor miR-34a induces neurogenesis of tumor spheres and reduces tumor burden in cerebellum xenografts of athymic mice, demonstrating an anti-tumorigenic role of miR-34a in vivo [52]. Interestingly, miR-34a is able to confer chemosensitivity as well through directly targeting the 3' UTRs of MAGE-A genes, reducing MAGE-A and increasing p53 in MB [54].…”

“…miRNAs-mediated inhibition of MB cell proliferation and MB metastasis by targeting the Notch signaling pathway have been tested in xenograft model. Since the Notch signaling pathway regulates a subset of MB cells to enhance the stem-cell-like properties and promotes tumor growth, miRNAs such as miR199b-5p and miR34a that target the Notch signaling pathway may be engineered for MB therapy [48,52]. It has been shown that miR199b-5p level is significantly higher in non-metastatic MB cases than in metastatic cases, suggesting miR199b-5p conferred inhibition of MB metastasis.…”

Micro RNA Regulation of Cancer Stem Cell Phenotypes

“…The miR-34a has further been demonstrated to target Notch signaling in glioblastoma and medulloblastoma stem cells by directly regulating the Notch-1 and Notch-2 pathway components. The miR-34a expression in glioblastoma cells significantly reduced in vivo xenograft growth and inhibits the tumor propagating ability of medulloblastoma cells [58,59].…”

Molecular Biology and Genetics of Medulloblastoma

“…MiRNA 199b-5p was found downregulated in MB patients' tissues compared to healthy control cerebellum tissues. The analysis of 61 patients with MB showed that the expression of miR-199b-5p in the nonmetastatic cases was Targets Notch ligand Delta-like 1(Dll1), impairment of cancer stem cell compartments [ 111 ] signifi cantly higher than in the metastatic cases. The correlation with survival for the patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients.…”