MicroRNA-199b-5p Impairs Cancer Stem Cells through Negative Regulation of HES1 in Medulloblastoma

1MicroRNAs (miRNAs) are single-stranded RNAs of ∼22 nucleotides in length, and they constitute a novel class of gene regulators. In animals, miRNAs have regulatory effects through their binding to imperfect complementary sites within the 3′-untranslated regions (3′UTRs) of their mRNA targets. Altered expression of many miRNAs is seen in several tumor types: e.g. B-cell lymphomas (clustered miR-17),, malignant lymphomas (miR-15a, miR-16-1; targeting BCL2), glioblastoma tumors (miR-21up-regulation), colorectal neoplasia (miR-143, miR-145 down-regulated), lung cancer (miR-29), and breast cancer (miR-10b), with several more tumor types under analysis.

2Here, we have focused on the most common of malignant brain tumors, medulloblastomas (MBs). MBs appear to originate from stem cells and from granule neuron precursors in the external granule layer of the cerebellum, or alternatively, from multipotent precursors in the ventricular zone of the cerebellum –. From a clinical point of view, current multimodal treatments include radical surgical resection followed by radiation and chemotherapy. While these treatments can improve the survival rate, MB remains incurable in about one third of these patients. The main cause of death is recurrence associated with tumor dissemination, at which point current therapeutic options have little efficacy. These treatments are also toxic and can lead to long-term disabilities –. Consequently, there is a substantial need for novel, effective, low-toxicity therapies for children with medulloblastoma.

3MB cells can also contain functionally important subsets of cells with stem-like properties that are uniquely able to propagate tumor growth,. Recent studies have demonstrated that both progenitors and stem cells can respond to the Sonic-Hedgehog (Shh) pathway and can serve as cells of origin for MBs.

4Notch activity has been shown to regulate granule-cell progenitors from which MBs arise, with Notch2 gene copy numbers increased in 15% of MBs,. Persistent expression of bHLH HES1, the principal Notch-responsive gene, prevents both migration of neural progenitor cells out of the ventricular zone and expression of neuronal markers. Mice lacking Hes1 show premature neurogenesis, seen as up-regulation of neural bHLH transcription factors, which results in major neural-tube defects; progenitor cells derived from these mice have impaired self-renewing potential, with a commitment towards a neuronal lineage.

5Significantly, expression of HES1 in MB has been associated with worse clinical outcome. Of note, an interplay with Shh in granular cell precursor development has been postulated, as has cross-talk with other pathways, e.g. the polycomb group gene BMI-1.

6Our approach started with a search for miRNAs (miRNA registry) with target genes involved in the Notch pathway, and we identified miR199b-5p as targeting HES1, the Notch effector. We show that miR-199b-5p expression is lost in metastatic patients and postulate a mechanism of regulation following epigenetic silencing through methylation processes occurring during carcinogenesis, identifying a new molecular marker for a poor-risk class in patients with MB. MiR199b-5p expression specifically impairs the cancer-stem-cell (CD133+) population, which results in vivo in impairment of MB tumor development in the cerebellum xenograft mouse model, thus providing proof of concept. As microRNAs have been recently shown to be useful tools to silence cancers, they might be able to fill this gap through their control of multiple target genes. We provide here evidence for the use of miRNAs in the clinic, with our anti-tumour therapy targeting of cancer stem cells by miR-199b-5p.

7Our in-silico analysis of the mirBase targets database was directed towards identification of miRNAs potentially targeting HES1, an effector of the Notch pathway, a fundamental mechanism in the regulation of MB cell proliferation. MiR-199b-5p and miR-199a-5p were the better scoring miRNAs, and were predicted to bind the 3′UTR of human bHLH HES1. We focused on miR-199b-5p due to its ability to decrease HES1 expression under transient over-expression, as compared to miR-199a-5p (Text S1, Fig. S1A, B). MiR-199b-5p is lost in lung tumors and it has been mapped to a genomic region deleted in bladder cancer. MiR-199a* (also known as miR-199a-5p, and with an identical sequence to miR-199b-5p) is also reduced in hepatocellular carcinoma –. The analysis of miR-199b-5p expression in two MB cell lines, human adult tissue and mouse cerebellum, is shown in Text S1, Fig. S1C, D and S2A, B.

8To determine whether HES1 is a target of miR-199b-5p, the HES1 3′UTR was cloned downstream of a luciferase reporter gene vector; pre-miR-199b-5p was also cloned in a mammalian expression vector (see Text S1). HEK-293 cells were then transfected with the relative luciferase activity showing that miR-199b-5p co-transfection decreased reporter gene activity, thus indicating binding with the 3′UTR and destabilisation of productive translation of luciferase mRNA (Text S1, Fig. S1E). As controls, HES1 3′UTR mutated in the miR-199b-5p binding site was not affected by miR-199b-5p (Text S1, Fig. S1E), and when a 2-O′-methyl oligoribonucleotide (2-OM) complementary to mature miR-199b-5p was co-transfected, it counteracted the effects of miR-199b-5p transfection, restoring reporter activity (Text S1, Fig. S1E). Similar results were obtained in Daoy MB cells (Text S1, Fig. S1F).

9To determine the role of miR-199b-5p in MB cell biology, the miR-199b-5p expression construct was transfected into Daoy cells, and several stable clones over-expressing miR-199b-5p were selected. Over-expression was confirmed by real-time PCR (Text S1, Fig. S1G). Three clones demonstrated reduced HES1 protein levels, one of which (199bSC1) showed no detectable HES1. The 199bSC1 and 199bMC1 clones were selected for further investigation (Text S1, Fig. S1H). These effects of miR-199b-5p on HES1 protein expression were not restricted to the stable clones or Daoy cells, as D283MED cells transiently transfected with the expression construct for miR-199b-5p also showed reduced HES1 levels (Text S1, Fig. S1B).

10To strengthen these findings, the 199bSC1 clone was transfected with 2-OM antisense to miR-199b-5p and used as a negative control (Text S1, Fig. S1I). Here, HES1 levels were restored, suggesting 2-OM block of HES1 repression by miR-199b-5p, providing further confirmation that miR-199b-5p targets HES1 directly. Other potential targets of miR-199b-5p were also investigated in this way (Text S1, Fig. S2C, D).

11The 199bSC1 and 199bMC1 clones had reduced proliferation rates under standard culture conditions, when compared to the control clone. Thus, we looked for potential cell-cycle alterations in these clones. FACS analysis on the 199bSC1 clone showed a 31% decrease in S-phase fractions, and an increase in cells in G0-G1 of 15%, as compared to the empty vector clone (Fig. 1A). This suggested that exit from the cell cycle has a role in the reduced proliferation rate of the Daoy cell 199bSC1 clone. In contrast, the 199bMC1 clone did not show significant changes in its cell cycle. We believe these findings to be due to an overall lower efficiency of 199bMC1 for reducing HES1 protein levels. These cell-cycle phenotypes translated into decreased proliferation rates in vitro, as evaluated by in-vitro proliferation assays comparing the 199bSC1 and 199bMC1 clones with an empty-vector clone and a transient transfectant for 2-OM designed against miR-199b-5p (Fig. 1B). Both the 199bMC1 and the 1999SC1 clones showed markedly reduced proliferation rates. Transfection of this antisense 2-OM induced a marked increase in proliferation of the stable 199bSC1 clone, in agreement with restored expression levels of HES1. These results on Daoy cell proliferation were confirmed also with D283 and ONS76 cells (Text S1, Fig. S2F). The effects of miR-199b-specific 2-OM were also confirmed in wild-type Daoy cells, where it potentially acts on the endogenous miR-199b (Text S1, Fig. S2G).

12The effects of the induction of miR-199b-5p were evaluated on molecular markers of proliferation and differentiation by a real-time approach. As illustrated in Fig. 1C, MAP2, which is mostly expressed in mature neurons, was up-regulated in the stable 199bSC1 and 199bMC1 clones. Similarly, Daoy cells have been reported to express GFAP after differentiation with phenylbutyrate, and in the stable 199bSC1 clone, GFAP levels were increased. Overall, the picture of gene expression in our stable cell line over-expressing miR-199b-5p is in agreement with the phenotype that has been seen in the brain of the Hes1−/− mouse. Among the other genes, GABRA6, a marker of cerebellar granule cell differentiation, was also significantly over-expressed in the stable clones (Fig. 1C).

13A fine-tuned cascade of positive and negative bHLH transcription factors is central to neurogenesis, with genes such as MASH1, MATH3 and NGN2 inducing neurogenesis, and HES1-mutant mice showing up-regulation of these activator-type bHLHs. Both miR-199b-5p stable clones showed increases in expression of pro-neural bHLH. In agreement with their reduction in proliferation rate, the proliferation markers c-Myc and cyclin D1 were decreased. Of the two clones analysed, 199bSC1 showed the more consistent phenotype; we explain these results by the more efficient HES1 silencing in this clone.

14Since the miR-199b-5p stable 199bSC1 clone showed a stronger and more consistent phenotype, we next examined it in a standard clonogenic assay, to determine whether anchorage-independent growth was affected by miR-199b-5p. Here, there was an 80% reduction in colony formation potential, compared to the empty-vector clone (Fig. 1D). In agreement with this reduction in proliferation rate, the proliferation markers c-Myc and cyclin D1 were decreased (Fig. 1E). We confirmed also at the protein level that GABRA6 and MATH3 were up-regulated in the 199bSC1 clone (Fig. 1F); the latter is known to be directly repressed by HES1.

15The Notch pathway has been linked to the fraction of MB tumor cells that harbour precursor stem-cell markers, and HES1 has a role in self-renewing of multipotent progenitor cells. This side population (SP) of tumor cells has a role in the engrafting of a tumor in animal models. We thus examined the influence of miR-199b-5p on the population of tumor cells that exclude the Hoechst 33342 dye, a strategy to identify these SP cells. This was determined by flow cytometry in the Daoy cell line, the SP of which accounts for up to 4.9% of the cells, as compared with verapamil-treated cells (the negative control) (Text S1, Fig. S3A, B). This verapamil treatment was based on verapamil inhibition of the ion-pumps responsible for Hoechst 33342 dye exclusion, thus enabling the SP cells to be seen. Staining of 199bSC1 and 199bMC1 cells indicated that the SP was ablated, as there were close to no differences between the Hoechst-treated sample and the Hoechst plus verapamil samples (Text S1, Fig. S3C–F).

16It is also known that central nervous system tumor stem cells express the CD133 antigen, and that these cells are uniquely capable of tumor formation in NOD-SCID mice,. Additionally, the Notch pathway has a central role in the self-renewing process, with its inhibition leading to depletion of CD133-positive (CD133+) Daoy cells via induction of apoptosis of progenitor-like cells. Recently it was shown that CD133+ Daoy cells promote tumor growth in the flank of nude mice, while CD133− cells do not. For these reasons, we evaluated the CD133 positivity of Daoy cells as compared to the stable 199bSC1 and 199bMC1 clones (Fig. 2). Here, the wild-type cells were 14.8% CD133+, while in the stable 199bSC1 and 199bMC1 clones this was reduced to 2.4% and 6.5% CD133+, respectively (Fig. 2C–F). This thus demonstrated a role for miR-199b-5p in negative regulation of this fraction of tumor-initiating cells.

17To confirm that the cellular phenotype is directly correlated with down-regulation of HES1, we performed in vitro cell-rescue experiments. We chose to rescue the more consistent phenotype, shown by the stable 199bSC1 clone. When full-length HES1 cDNA was cloned and transfected into the stable Daoy-cell 199bSC1 clone, HES1 expression was restored, as evaluated by immunoblotting (Fig. 3A). We also noted the re-expression of cyclin D1, which was down-regulated in the stable 199bSC1 clone. Restored HES1 expression also reversed the effects of miR-199b-5p on cell proliferation (Fig. 3B). At the same time, the transfection of HES1 cDNA into the 199bSC1 clone decreased induction of pro-neural bHLHs and differentiation markers, and increased levels of proliferation genes (Fig. 3C). Furthermore, the transient transfection of HES1 cDNA into the stable 199bSC1 clone led to an increase in the percentage of cells in S phase, and a removal of the block on the G0-G1 phase (Fig. 3D). This was opposite to the effects seen in the stable 199bSC1 clone over-expressing miR-199b-5p (see Fig. 1C and Fig. 3C). The effects of restored expression of HES1 on the SP of the stable 199bSC1 clone were also evaluated. When the HES1 transfected cells (GFP positive) were evaluated for their ability to exclude Hoechst dye, they showed a minimum increase in SP, of up to 1.3%. Although higher than that measured for the stable 199b clones (Fig. 3E–H), this was still below the level of SP cells for the wild-type cells (Text S1, Fig. S3). This is probably due to the short time of re-expression of HES1 after transient transfection, which might not be enough to allow for complete phenotype rescue. We then tried to rescue the effects on the CD133 compartment; however, transient transfection of HES1 in the 199bSC1 clone did not result in a significant increase in CD133+ cells. We believe that this is due to the short transient transfection time, which does not allow a re-organization of Daoy cell hierarchy.

18The findings here indicated that miR-199b-5p is a potential inhibitor of tumor formation. Therefore, to investigate the role of miR-199b-5p in an in vivo tumor model, we stabilized the 199bSC1 and control Daoy clones with an expression vector carrying luciferase cDNA. The clones obtained were tested for luciferase expression levels and also validated for retention of the parental phenotype, in terms of both HES1 and miR-199b-5p expression (see Text S1, Fig. S4A–D). These stable 199b-Luc1 and Ctl-Luc-4 Daoy clones were then injected into the left and right flanks, respectively, of five athymic nude/nude mice. Tumor growth was evaluated by weekly in-vivo bioluminescence imaging (BLI) of injected mice. At eight weeks, all of the mice showed visible masses in each control flank, while only three flanks injected with 199b-Luc1 showed tumor engrafting. Overall, a significant difference in tumor volumes between control flanks and miR-199b-5p flanks was seen (Fig. 4A). The bioluminescence measurements showed significant reductions in emission for the miR-199b-5p sides during tumor growth, as compared with the control sides (e.g. mouse #4, Fig. 4B). After nine weeks, four out of the five mice showed statistically significant differences in these bioluminescence signals between control sides and miR-199b-5p counter sides (Fig. 4C). Taken together, these data show that miR-199b-5p can impair tumor formation in vivo in athymic nude/nude mice. The xenograft tumors from mouse #5 and mouse #4 were explanted and analyzed for miR-199b-5p and HES1 expression and evaluated histopathologically (Text S1, Fig. S4E–H).

19To further investigate the ability of miR-199b-5p to regulate MB growth, we injected the 199bSC1 stable clone orthotopically into the fourth ventricle of nude mice of 5 weeks of age (Fig. 4D, E). After four weeks of in vivo non-invasive monitoring tumor growth by BLI, in mice injected with the 199bSC1 clone the tumor growth was considerably lower than that observed in the control (CTL)-cells-injected side. As further confirmation of these effects, we also injected the CTL cells infected with an adenovirus coding for miR-199b-5p: in agreement with the previous findings, these mice also showed reduced BLI after 4 weeks (Fig. 4F). Further BLI acquisitions after 8 weeks from implantation of the cells are shown in Figure 4G. At this time, two mice were sacrificed and further analysed for histopathology. Hematoxylin-eosine staining of frozen tissues showed tumor mass in the cerebellum of AdV5-Mock#2 and AdV5-199b#3 injected animals. Serial parallel frozen histological sections were examined by fluorescence microscopy for endogenous green fluorescence protein (GFP) expressed by adenovirus-infected cells. Then, we evaluated HES1 protein expression by immunohistochemistry staining of other paraffin-embedded tissues, using an anti-HES1 antibody. Overall, we assessed the levels of persistence of adenovirus expression in infected cells, as the down-regulation of HES1 expression due to miR199b carrying the adenovirus expression, thus following tumor growth over time by BLI see Figure 4G–H, and antibodies staining Figure S4L–M (Hes1, Ki67, Gabra6, Nestin, Math-3, see details in Text S1 information). Then, two additional nude mice (AdV5-Mock#7; AdV5-199b#5) underwent PET-CT studies at 12 weeks post-injection, to assess tumor proliferative activity (Fig. 5A, B). Additional BLI data together with two movies (Movie S1 and Movie S2) showing the 3D reconstruction of the orthotopic engraftment are described in the Text S1 information and showed in Figure S6. These analyses showed significant reduction of tumor mass in the AdV5-199b#5 animal, as compared to the AdV5-Mock#7 control mice, with PET-CT analyses also providing tumor volumes (0.024 cm3 versus 0.044 cm3, respectively), as described in Text S1. Overall, these data indicate a beneficial effect of over-expression of miR199b-5p, as a negative regulator of tumor growth of MB cells in this orthotopic xenograft nude-mouse model.

20To determine whether miR-199b-5p is effectively expressed in healthy human pediatric cerebella, we used 13 control samples obtained from the NICHD Brain and Tissue Bank for Developmental Disorders, at the University of Maryland, USA. We measured miR-199b-5p expression, comparing five cerebellum samples obtained from 0–1-year-old children with six from 13–16-year-old children (Fig. 6A). MiR-199b-5p showed greater expression in the explants from the younger healthy controls (Mann-Whitney test, P = 0.006).

21To determine whether miR-199b-5p expression has a role in human MB, samples from a cohort of 61 MB patients were analysed (see Experimental Procedures). Indeed, it has already been shown that HES1 protein levels correlate with negative outcome in MB patients. The whole patient population (n = 61) was then divided into two groups, as low versus high miR-199b-5p expression, based on the overall median. The distribution of miR-199b-5p expression between non-metastatic (M0) and metastatic (M1, M2 and M3) cases showed that miR-199b-5p expression in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001, Pearson Chi-Square test; Fig. 6B). In the subset of patients where follow-up information was available (n = 45), the survival curve for the patients who expressed miR-199b at high levels showed a positive trend, with better overall survival than the low-expressing patients. However, the log-rank test of the Kaplan-Meier curves did not show a significant difference (P = 0.182; Fig. 6C), probably due to the limited number of patients with long-term follow-up.

22These data showing down-regulation of miR-199b-5p in metastatic MBs indicates a mechanism of silencing through epigenetic or genetic alterations. We thus tested expression of miR-199b-5p by real-time PCR in a panel of MB cell lines following induction of de-methylation with 5-aza-deoxycytidine (Fig. 6D). Indeed, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p, thus supporting the hypothesis of epigenetic control of miR-199b-5p expression. Further studies need to be performed to identify this tuned regulation mechanism of action through epigenetic inactivation of miR199b-5p expression during tumor development.

23The interconnections between the signaling pathways altered in MB remain largely unknown, and it is still difficult to define specific patient stratification according to risk classes that would lower therapy-linked mortality and morbidity. In the present study, we have identified a mechanism of HES1 gene regulation via the miRNA miR-199b-5p. HES1 is a key regulator of cerebellum development, as indicated by recent findings that treatment of cerebellar granule cells with Shh increases the levels of HES1. This suggests that HES1 is a transcriptional target of both the Shh and Notch2 signaling cascades.

24We report here a novel level of regulation of HES1 expression that is driven by miR-199b-5p, which binds to the HES1 3′UTR and leads to unproductive translation. Of note, we have also investigated the actions of miR-199b-5p on a reporter construct fused with the 3′UTR of NHLH2, cyclin L1 and Nanog (Text S1, Fig. S2D). Among the predicted targets for miR-199b-5p, which were obtained using two different algorithms (Text S1, Table S1), these genes were possible candidates related to the phenotype obtained after miR-199b over-expression. Nonetheless, we did not obtain down-regulation of reporter activity when tested under the same conditions where we obtained effects on HES1 3′UTR. Furthermore, the stable 199bSC1 clone has a GSK3-β protein level comparable to the control. Of note, when analyzed according to the Pita algorithm, the value of ΔΔG for the binding of miR-199b-5p to the 3′UTR of GSK3-β is very high, indicating poor accessibility to the 3′UTR (see Text S1, Table S1). Recently it has also been shown that miR-199a*, which has the same sequence as miR-199b-5p, is involved in caspase activation by potentially acting through Met proto-oncogene down-regulation. It will be of interest for future studies to also determine the down-regulation of this target by miR-199b, even if it appears unlikely because of the lack of induction of apoptosis after miR-199b over-expression in our cellular systems.

25The analysis of the over-expressing miR-199b-5p clones suggested that miR-199b-5p can impair the proliferation and engrafting potential of MB cells. Indeed loss of Cyclin D1 in over-expressing miR-199b-5p clones are a reminder of the similar effects seen in Ccnd1–/– mice, which have decreased early GNP proliferation and early ataxia as a consequence of a delay in acquiring normal cerebellar function, thus affecting progression of the pre-neoplastic lesions to MBs.

26Our data are also in agreement with the in-vivo data reported from murine cells genetically knocked down for Hes1. While the effects of miR-199b-5p could be explained by triggering of apoptosis, our cytofluorimetric assays suggested that impairment of MB cell proliferation is not linked to extensive apoptosis of cells over-expressing miR-199b-5p (see cell cycle assay on Fig. 1A). This is in agreement with reported data for neural progenitors obtained from Hes1−/− mice, where apoptotic indications were only seen in restricted cell types.

27The correlation that we show between miR-199b-5p and tumor M stage indicates that miR-199b-5p expression levels can be investigated concomitant with tumor resection. This will allow identification of a subset of patients who should develop an aggressive tumor and have future metastasis formation, noting that disseminated disease is the most powerful independent factor associated with poor survival. Despite the correlation with M status, the over-expression of miR-199b-5p did not lead to a decrease in cell motility of Daoy cells (Text S1, Fig. S2E). This may well be due to the extremely high motility of Daoy cells, indicating that to overcome this phenotype, a further step beyond regulation of the Notch pathway needs to take place. However, of note, the expression of PDGFR-B and SPARC, two genes recently correlated with metastatic MB,, is reduced in the presence of over-expression of miR-199b-5p, as we show in Text S1, Fig. S5D and describe in Text S1.

28In the light of our data on the regulation of the Notch pathway via miR-199b-5p modulation, it will be of interest to focus future studies on the role of Notch-regulated genes in metastatic MB patients. Additionally, developing mouse models that better reproduce the human disease is an important task. Within this scenario, the findings that the homozygous Smo/Smo mouse model has leptomeningeal spread confirms involvement of the Notch pathway in regulation of MB growth,. As shown in Fig. 6 and Text S1, Table S2, we show great variability in the miR-199b-5p expression levels, which might be due to genetic chromosomal alterations or epigenetic silencing in a subset of patients with worse outcome. Our data on a panel of MB cell lines (see Fig. 6D) support this last hypothesis. The reason why some of the MB cell lines do not respond to the treatment with increasing miR-199b could be due to genetic loss of the gene itself, or to a strict cell-type regulation of miR-199b expression, reflecting the different origins of the cell lines.

29At present, there is growing interest in the elucidation of the mechanisms that confer unique properties to the ‘cancer stem cells’. Recently, it was shown that glioblastoma CD133+ cells have a better chance of survival after ionizing radiation, through the induction of the repair of damaged DNA. Indeed, Daoy cells expressing the CD133 antigen are radio-resistant, thus supporting the hypothesis that Daoy cells represent a model for the study of a tumor stem-cell compartment. Here miR-199b-5p can influence this side population and the CD133+ population of Daoy cells, namely the cancer stem cells.

30Together with CD133, we also evaluated the expression levels of several cancer stem-cell genes that have recently been shown to be correlated with the aggressiveness of solid tumors; as shown in Text S1, Figure S5 (A–D) and table S3, the expression of these genes is in agreement with an overall reduction in the ‘stemness’ phenotype of Daoy cells treated with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butylester (DAPT). This prevents activation of the Notch response (see Text S1, Materials), thus blocking the presenilin-secretase complex and enhancing miR199b-5p expression,. To date, this is the first report that expression of an miRNA can deplete this tumor cell compartment, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.

31There is a strong indication from published series that children with medulloblastoma presenting at over 3 years of age benefit greatly from radiotherapy, but the treatment of younger children remains a challenge. The absence of significant differences in survival rates between patients with total or subtotal excision of MB supports the view that the total excision of MB can be avoided when the risk for potential neurological deficits is high. Recent trials have demonstrated a beneficial effect of chemotherapy, although this was not universally seen,. Here, we envisage the use and delivery of miR199b-5p in situ into the cerebellum of MB-affected children under 3 years of age (and positive to HES1), to thus impair the maintenance of the tumor-initiating CD133+ cancer cells. Combined with chemotherapy, and with the potential to abrogate radiotherapy and avoid brain damage due to total tumor excision, this should provide an overall improvement in the present treatments for MB. Thus we foresee the possibility of treating MBs with the use of encapsulated stable nucleic acid lipid particles (SNALP). These have been demonstrated to be effective in non-human primates for systemic delivery, and would by-passing the blood-brain barrier using encapsulated nano-particles containing agomir 199b-5p molecules. How these gene-therapy approaches will progress further will be an issue for future pre-clinical animal studies.

32As illustrated in our model (Fig. 6E), we picture two different expression levels of miR-199b-5p in M0 and M+ patients, which might be due to epigenetic regulation during carcinogenesis. In our ‘moderate high’ model, an increase in miR-199b-5p expression represses HES1, which then leads to an increase in pro-neural bHLHs gene expression, driving the cell towards differentiation processes. In the ‘moderate low’ model, miR-199b-5p expression is lowered due to epigenetic control mechanisms, and then HES1 is over-expressed, leading to cell proliferation and induction of the SP and hence an increase in CD133+ cells. As for many other transcription factors, HES1 is a point of integration between and among different signal transduction pathways, and its balance of expression determines fundamental cell decisions, such as whether or not to start a differentiation program. With this scenario, miR-199b-5p can be seen as part of the complex Notch signal-transduction pathway, as a fine tuner of the levels of expression of the HES1 bHLH transcription factor. These phenomena could be considered to occur in a variety of tissues and cancers where an activated Notch pathway is involved.

33All animal work have been conducted according to relevant national and international guidelines. Approval was obtained by Institutional Animal Care and Ethical Committee at CEINGE - University of Naples Federico II, Protocol #13 - 08/01/2007, and the Italian Ministry of Health; Dipartimento Sanità Pubblica Veterinaria D.L. 116/92, confirming that all experiments conform to the relevant regulatory standards.

34The flow cytometry analysis of S-phase fractions and cell-cycle kinetics were carried out using a FACSCalibur (Becton Dickinson, San Jose, CA) using CELL Quest version 3.3 software. The CD133 studies were carried out using the same instrument, with antibodies from Miltenyi Biotec (Auburn, CA), according to the manufacturer instructions. In brief, the cells were blocked in Fc receptor blocking reagent and incubated with an anti-CD133/1 (AC133)-phycoerythrin antibody (Miltenyi Biotec), for 10 min in the dark at 4°C. The cells were then washed and resuspended in PBS. Cells expressing higher levels of CD133 than the immunoglobulin G (IgG) controls were considered positive. For analyses of the side populations, cells (106/mL) were incubated with Hoechst 33342 (5 µmol/L; Sigma) for 90 min at 37°C in EMEM containing 2% FBS. To ensure the correct identification of the side population cells, the cells were incubated as above with the addition of 50 µM verapamil (Sigma).

35Stable Daoy cell clones were generated by transfecting a luciferase-expressing vector (the firefly luciferase gene cloned in the plentiV5 vector; Invitrogen) into the stable 199bSC1 clone or into the control empty-vector clone. Regarding xenograft subcutaneous implantations, 100,000 viable 199b-Luc1 and Ctl-Luc-4 cells were mixed with a 1∶1 PBS∶matrigel solution (BD Biosciences, San Jose, CA). Six-week-old female athymic mice were previously anesthetised using avertin (Sigma) as a 3% solution in tert-amyl alcohol (Fisher), at a dose of 3 mg per 10 g body weight. then injected s.c. with a total volume of 0.1 mL of matrigel PBS cell suspension, into each flank. Regarding xenograft orthotopic delivery into cerebellum a protocol described since by Schabet M. et al.,, was used to deliver MB cells after microsurgical exposure of the atlanto-occipital membrane, 50 µl of cerebrospinal fluid was aspirated through a 27 G needle and replaced by 50 µl of DMEM containing 100,000 DAOY medulloblastoma cells. The wound was sutured with 3-0 polyglactin 910, and 4-0 silk. The mice were imaged after the implantation of the cells, with tumor growth monitored by weekly bioluminescence acquisitions (BLI) using an IVIS 3D Illumina Imaging System (Xenogen Corp. Alameda, CA). For the acquisitions, the mice were isofluorane-anaesthetised i.p., injected with 100 µL D-luciferin (15 mg/mL stock) per 10 g body weight, and imaged for 30 s, 10 min after luciferin injection; four acquisitions per mouse were made (ventral, dorsal and each flank). To quantify the bioluminescence, the integrated fluxes of photons (photons per s) within each area of interest were determined using the Living Images Software Package 3.0 (Xenogen-Caliper). The emission data from the start of tumor growth were collected for at least 6 weeks, and then were normalized to the bioluminescence on the injection day. Calliper measurements of tumor sizes were made weekly, along the long and short axes, and estimations of their volumes were made using the formula: width2×length×0.52.

36Animals were monitored daily by weights and neurological examinations to determine their good standard status of health. Those animals showing sign of health sufferance were promptly sacrificed. On day 63, after cell inoculation, all animals were sacrificed with an overdose of ketanest and xylazin.

37The mice were kept in a ventilated cage (26°C) for 1 h prior to imaging studies. Anesthesia was performed with intraperitoneal administration of a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) (injection volume, 100 µl/10 g). PET was performed 1 h after administration of 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT), a marker of tumor proliferation (50 µL; 7.4 MBq; scan time, 18 min), in a lateral caudal vein, using an animal PET scanner (GE Healthcare eXplore Vista, FWHM 1.6 mm). High resolution CT studies (GE Healtcare eXplore Locus; spatial resolution, 45 µm) were performed within 24 h from the PET.

38Maximum (SUVmax) and mean (SUVmean) standardized uptake values (SUVs) were calculated from the PET studies (SUV = tissue activity (MBq/cc)/[injected dose (MBq)/body weight (g)]). The PET/CT images were post-processed to obtain multiplanar reconstructions (MPRs), maximum intensity projections (MIPs), 3D volume rendering, and fusion images, using Osirix 3.3 (MAC OS 10.5 operating system). Additional 3D reconstructions were obtained using MicroView (GE eXplore Locus).

39Lesion volumes were calculated from PET data using in-house-developed software (based on IDL, ITT Vis Inc), by summarizing all spatially connected voxels with SUV >50% SUVmax. Lesion profiles defined with these procedures were used for ROI-based comparison between AdV5-Mock and AdV5-199b.