Through negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can 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 cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3–4 years and 8–9 years of age, although it can also occur in adults. Notch regulates a subset of the 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 regulated these phenomena, and can be used in anti-cancer therapies. In a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept. Despite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.

Through 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. In 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. Despite 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.

Stable nucleic acid lipid vesicles (SNALPs) encapsulating miR-34a to treat multiple myeloma (MM) were developed. Wild type or completely 2′-O-methylated (OMet) MiR-34a was used in this study. Moreover, SNALPs were conjugated with transferrin (Tf) in order to target MM cells overexpressing transferrin receptors (TfRs). The type of miR-34a chemical backbone did not significantly affect the characteristics of SNALPs in terms of mean size, polydispersity index, and zeta potential, while the encapsulation of an OMet miR-34a resulted in a significant increase of miRNA encapsulation into the SNALPs. On the other hand, the chemical conjugation of SNALPs with Tf resulted in a significant decrease of the zeta potential, while size characteristics and miR-34a encapsulation into SNALPs were not significantly affected. In an experimental model of MM, all the animals treated with SNALPs encapsulating miR-34a showed a significant inhibition of the tumor growth. However, the use of SNALPs conjugated with Tf and encapsulating OMet miR-34a resulted in the highest increase of mice survival. These results may represent the proof of concept for the use of SNALPs encapsulating miR-34a for the treatment of MM.