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Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity. Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated the expression of the tumor-associated antigen PRAME in biopsies from 60 patients with medulloblastoma. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A*02+ DAOY cells as well as primary HLA-A*02+ medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T-cell–related toxicity, an inducible caspase-9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating patients with HLA-A*02+ medulloblastoma.Significance: These findings identify PRAME as a medulloblastoma tumor-associated antigen that can be targeted using genetically modified T cells. Cancer Res; 78(12); 3337–49. ©2018 AACR.

Primary malignant brain tumors are a major cause of morbidity and mortality in both adults and children, with a dismal prognosis despite multimodal therapeutic approaches. In the last years, a specific subpopulation of cells within the tumor bulk, named cancer stem cells (CSCs) or tumor-initiating cells, have been identified in brain tumors as responsible for cancer growth and disease progression. Stemness features of tumor cells strongly affect treatment response, leading to the escape from conventional therapeutic approaches and subsequently causing tumor relapse. Recent research efforts have focused at identifying new therapeutic strategies capable of specifically targeting CSCs in cancers by taking into consideration their complex nature. Aberrant epigenetic machinery plays a key role in the genesis and progression of brain tumors as well as inducing CSC reprogramming and preserving CSC characteristics. Thus, reverting the cancer epigenome can be considered a promising therapeutic strategy. Three main epigenetic mechanisms have been described: DNA methylation, histone modifications, and non-coding RNA, particularly microRNAs. Each of these mechanisms has been proven to be targetable by chemical compounds, known as epigenetic-based drugs or epidrugs, that specifically target epigenetic marks. We review here recent advances in the study of epigenetic modulators promoting and sustaining brain tumor stem-like cells. We focus on their potential role in cancer therapy.

Medulloblastoma (MB) is the most common malignant childhood brain tumor. The current clinical approach consists of multimodal strategies with debilitating long-term effects and risk of tumor recurrence. Medulloblastoma stem cells (MBSCs) are a fraction of tumor cells with high proliferation potential and the capability to adapt to adverse/restrictive conditions in tumor milieu thus driving the refractoriness to conventional therapies. Recently, high basal levels of Unfolded Protein Response (UPR) molecules have been found in tumors of different tissue-origin and are correlated with poor prognosis and low patient survival. However, little is known about the role of UPR in MB. We investigated the expression and activation of UPR players in MBSCs. Human group 3 MB (G3MB) cell lines, specifically CHLA-01, D283- and D341-Med, were grown in Vitamin A and/or FBS or in stem selective medium (B27™) for 72 h before collection. Cells were fixed, stained with proper primary antibodies and images were acquired by confocal microscopy. The analysis of the transcription factors ATF-4 and CHOP revealed their elevated nuclear expression and co-localization, which resulted to be more marked in G3MB stem-like cells than in the differentiated ones. Also the ATF-6 branch was investigated, in differentiating conditions D283 and D341-Med showed a greater activation of ATF-6, represented by its nuclear localization, in respect to stem cells, while CHLA-01 did not show differences. Conversely XBP1, the transcription factor downstream IRE1 signaling, was not expressed in the three cell lines. Lastly, a Kaplan-Meier analysis on MB patients showed a worse prognosis with a shorter survival rate of patients expressing high ATF4 transcript levels. Our results reveal, even in resting conditions, preferential activation of the PERK branch in G3MB cells grown in stem-like condition suggesting that ATF-4 might be a promising therapeutic and prognostic factor to specifically target the stem compartment in aggressive MB.

Brain tumors are a large and heterogeneous group of neoplasms that affect the central nervous system and include some of the deadliest cancers. Almost all the conventional and new treatments fail to hinder tumoral growth of the most malignant brain tumors. This is due to multiple factors, such as intra-tumor heterogeneity, the microenvironmental properties of the human brain, and the lack of reliable models to test new therapies. Therefore, creating faithful models for each tumor and discovering tailored treatments pose great challenges in the fight against brain cancer. Over the years, different types of models have been generated, and, in this review, we investigated the advantages and disadvantages of the models currently used.

Cancer stem cells (CSCs) represent a sub-population of cancer cells capable of proliferating and generating heterogeneous cancer cell types. Acquisition of stemness features may represent a strong advantage for neoplastic cells to promote tumorigenesis and progression, driving resistance to conventional therapy and promoting disease relapse. CSCs have been discovered and isolated in major pediatric brain tumors, including medulloblastoma (MB), the most common solid malignancy in childhood. The unfolded protein response (UPR) represents an adaptation mechanism to metabolic obstacles in CSCs, able to increase tumor aggressiveness. The initial activation of the UPR is cytoprotective but the acute activation led to cell death. We found that UPR is active in MB stem cells (MBSC) and particularly in group 3 (G3). ONC201 is an imipridone compound that activates p53-independent apoptosis causing changes in gene expression similar to those caused by UPR. Here, we aim to test the in vitro efficacy of ONC201 on G3 MBSC. We selected 4 G3 MBSC (D341-Med, D283-Med, Med411, and CHLA-01-Med), for the in vitro study. Cells were choosen for their “fidelity” to the MB subgroup through the analysis of global methylation profiling, were grown in stemness conditions and expressed stemness markers at high levels. We investigated the efficacy of ONC201 treatment on CSC features, by evaluating cell viability, cell death, protein synthesis, self-renewal, and cell cycle. ONC201 treatment on G3 MB cells led to an upregulation of ATF4, a key molecule of the UPR, and the induction was stronger in MB cultured in a “stem-like” medium. Moreover, in the most MBSC analyzed, ONC201 was effective against CSCs whether by reduced cell viability, protein synthesis, and self-renewal. We also observed a trend of increased cell death. Our results suggest that ONC201 is potentially effective in treating G3 MB by compromising the stem cell compartment, and thus deserving further investigations.

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.

Medulloblastoma (MB) is a malignant pediatric brain tumor, and we reveal a new regulatory molecular axis critical for MB progression. Low expression of miR‐326 and its host gene β‐arrestin1 (ARRB1) promotes tumor growth enhancing the E2F1 pro‐survival function. High levels of EZH2 are responsible for the presence of H3K27me3 that controls miR‐326 and ARRB1 through a bivalent domain. Restoration of miR‐326 and re‐expression of ARRB1 reverse E2F1 function into pro‐apoptotic activity., Persistent mortality rates of medulloblastoma (MB) and severe side effects of the current therapies require the definition of the molecular mechanisms that contribute to tumor progression. Using cultured MB cancer stem cells and xenograft tumors generated in mice, we show that low expression of miR‐326 and its host gene β‐arrestin1 (ARRB1) promotes tumor growth enhancing the E2F1 pro‐survival function. Our models revealed that miR‐326 and ARRB1 are controlled by a bivalent domain, since the H3K27me3 repressive mark is found at their regulatory region together with the activation‐associated H3K4me3 mark. High levels of EZH2, a feature of MB, are responsible for the presence of H3K27me3. Ectopic expression of miR‐326 and ARRB1 provides hints into how their low levels regulate E2F1 activity. MiR‐326 targets E2F1 mRNA, thereby reducing its protein levels; ARRB1, triggering E2F1 acetylation, reverses its function into pro‐apoptotic activity. Similar to miR‐326 and ARRB1 overexpression, we also show that EZH2 inhibition restores miR‐326/ARRB1 expression, limiting E2F1 pro‐proliferative activity. Our results reveal a new regulatory molecular axis critical for MB progression.

Background Colorectal cancer (CRC) is a heterogeneous disease with a complex biology and a wide number of altered genes such as BRAF, KRAS and PIK3CA. Advances with new-targeted therapies have been achieved and available treating options have prolonged patient’s survival. However, BRAF-mutated CRC patients remain unresponsive to available therapies with RAF inhibitors (RAFi) alone or combined with ErbB inhibitors (ErbBi). These unmet needs require further exploitation of oncogenic signaling in order to set up individualized treatments. Methods To this end, we tested the efficacy of single agent or combined treatments using the BRAFi, vemurafenib and two different ErbBi: panitumumab and afatinib in CRC cells characterized by different molecular phenotypes. Results Combination strategies with BRAFi and ErbBi achieved a better response in BRAFV600E mutated cells expressing high levels of ErbB2. Conclusions Our findings support the importance of ErbB2 evaluation in BRAF-mutated CRC patients and its role as a positive predictor factor of response to BRAFi/ErbBi combination.

Exposure to environmental endocrine disruptors has been associated with an increased frequency of thyroid pathology. In this study, we evaluated the effects of various concentrations of methylmercury (MeHg) on immortalized, non-tumorigenic thyroid cells (Nthy-ori-3-1). Exposure to MeHg at 2.5 and 5 &micro, M for 24 h caused a reduction in cell viability with a decrease of the cell population in sub-G0 phase, as detected by MTT and flow cytometry. Conversely, MeHg at the lower concentration of 0.1 &micro, M increased the cell viability with a rise of G2/M phase. An immunoblot analysis showed higher expression levels of phospho-ERK and not of phospho-Akt. Further enhancement of the cell growth rate was observed after a prolonged exposure of the cells up to 18 days to MeHg 0.1 &micro, M. The present findings demonstrate the toxicity of high concentrations of MeHg on thyroid cells, while showing that treatment with lower doses of Hg, as may occur after prolonged exposure to this environmental contaminant, exerts a promoting effect on thyroid cell proliferation, by acting on the ERK-mediated pro-oncogenic signal transduction pathway.

The identification of somatic mutations in cancer specimens enables detection of molecular markers for personalized treatment. We recently developed a novel molecular assay and evaluated its clinical performance as an ancillary molecular method for indeterminate thyroid nodule cytology. Herein we describe the analytical validation of the novel targeted next-generation sequencing (NGS) assay in thyroid samples from different sources. We present validation data of a novel NGS-based panel on 463 thyroid samples, including 310 fine-needle aspiration (FNA) specimens from different sources (FNA collected in preservative solution, liquid-based, and stained smears), 10 fresh frozen, and 143 formalin-fixed paraffin-embedded (FFPE) thyroid tissue specimens. Sequencing performance in the different samples was evaluated along with reproducibility, repeatability, minimum nucleic acid input to detect variants, and analytical sensitivity of the assay. All thyroid samples achieved high sequencing performance, with a mean base coverage depth ranging from 2228 × (in liquid-based FNA) to 3661 × (in FNA stained smears), and coverage uniformity ranging from 86% (in FFPE) to 95% (in FNA collected in preservative solution), with all target regions covered above the minimum depth required to call a variant (500×). The minimum nucleic acid input was 1 ng. Analytic sensitivity for mutation detection was 2–5% mutant allele frequency. This validation study of a novel NGS-based assay for thyroid nodules demonstrated that the assay can be reliably used on multiple thyroid sample types, including FNA from different sources and FF and FFPE thyroid samples, thus providing a robust and reliable assay to genotype thyroid nodules, which may improve thyroid cancer diagnosis and care.

Deciding whether patients with a cytologically indeterminate thyroid nodule should be referred for surgery or for active surveillance is an important challenge for clinicians. The aim of this study was to evaluate the performance of a novel dual-component molecular assay as an ancillary molecular method for resolving indeterminate thyroid nodule cytology. We selected 156 thyroid nodules from those that had undergone fine-needle aspiration processed by liquid-based cytology and surgical resection between June 2016 and December 2017. The sample set included 63 nodules cytologically classified as indeterminate, and 93 other nodules randomly selected from those with non-diagnostic, benign, suspicious, or malignant cytology. Nucleic acids from each nodule were subjected to next-generation sequencing analysis for mutation detection in 23 genes and to digital polymerase chain reaction (PCR) evaluation for miR-146b-5p expression levels. Used alone, mutation analysis in the indeterminate subset (cancer prevalence: 22.5%) displayed high sensitivity (89%) and NPV (96%). In contrast, the miR-146b-5p assay offered high specificity (93%) and PPV (93%). Combined use of both analyses improved panel performance by eliminating false-negative results. These preliminary data suggest that a dual-component molecular test can increase the diagnostic accuracy of thyroid cytology alone by reducing the number of nodules that will be classified as indeterminate and increasing those that can be reliably classified as benign. If these findings are confirmed, this test can be considered for use in clinical practice and is expected to reduce diagnostic surgery and health care costs, and to improve patient quality of life.

Background Development of in vitro models of pediatric brain tumors (pBT) is instrumental for both understanding the contributing oncogenic molecular mechanisms and identifying and testing new therapeutic strategies. Primary cell lines should be established and managed to prevent epigenetic and genetic alterations and thus recapitulating the original tumor. DNA methylation (DM) is a stable epigenetic modification, altered in cancer and recently used to classify tumors. We aim to apply DM and Copy Number Variation (CNV) profiling to characterize pBT primary cell lines and tumors. Methods We investigated 34 pBT tissues from different histology paired to 52 their derived primary cultures in both 2D and 3D conditions, as stem-cells or in serum-supplemented medium, and both short and long-terms in culture. We studied 18 additional pBT-derived cell-lines, 9 organoids, 5 commercial cell-lines, and 122 pBT tissues from the same histological categories, as controls, for a total of 240 genome-wide DM profiles. We analyzed DM and CNV profiles by using Illumina EPIC-arrays. By means of a bump hunting strategy, we identified differentially methylated regions in faithful vs unfaithful cell lines, and performed a functional characterization using over-representation analysis. Results The 69% (25/36) of cells at early passages retained genetic alteration and the same DM patterns of the original tumors, with no differences related to 2D/3D methods or the presence of serum in media. The 70% (24/34) of primary cell lines analyzed at later passages (>5 or >14 days in culture) diverged from the primary tumor, the totality of those cultured with serum. All divergent cells clustered together acquiring common deregulated epigenetic signature induced by serum culture media, 2D methods and longer time in culture. Conclusions We have shown that global DM profiles, along with CNV analysis are useful tools to detect the recapitulation of pBT-derived primary cell-lines from the original tumor. Whatever subgroups tested, our results suggest that in vitro models should be passaged as little as possible to retain the epigenetic and genetic alterations of the tumors and thus to be considered relevant for basic and translational biology.

Redox-responsive nanoparticles were synthesized by desolvation of bovine serum albumin followed by disulfide-bond crosslinking with N , N ʹ-Bis (acryloyl) cystamine. Dynamic light scattering and transmission electron microscopy studies revealed spherical nanoparticles (mean diameter: 83 nm, polydispersity index: 0.3) that were glutathione-responsive. Confocal microscopy revealed rapid, efficient internalization of the nanoparticles by Daoy medulloblastoma cells and healthy controls (HaCaT keratinocytes). Cisplatin-loaded nanoparticles with drug:carrier ratios of 5%, 10%, and 20% were tested in both cell lines. The formulation with the highest drug:carrier ratio reduced Daoy and HaCaT cell viability with IC 50 values of 6.19 and 11.17 μg mL −1 , respectively. The differential cytotoxicity reflects the cancer cells’ higher glutathione content, which triggers more extensive disruption of the disulfide bond-mediated intra-particle cross-links, decreasing particle stability and increasing their cisplatin release. These findings support continuing efforts to improve the safety and efficacy of antineoplastic drug therapy for pediatric brain tumors using selective nanoparticle-based drug delivery systems.

Background: Colorectal cancer (CRC) is a heterogeneous disease with a complex biology and has a wide number of altered genes such as BRAF, KRAS and PIK3CA. Advances with new-targeted therapies have been achieved and available treating options have prolonged patient’s survival. However, BRAF-mutated CRC patients remain unresponsive to available therapies with RAF inhibitors (RAFi) alone or combined with ErbB inhibitors (ErbBi). These unmet needs require further exploitation of oncogenic signaling in order to set up individualized treatments. Methods: To this end, we tested the efficacy of single agent or combined treatments using the BRAFi, vemurafenib and two different ErbBi: panitumumab and afatinib in CRC cells characterized by different molecular phenotypes. Results: Combination strategies with BRAFi and ErbBi achieved a better response in BRAF V600E mutated cells expressing high levels of ErbB2. Conclusions: In conclusion our findings allow us to indicate that high ErbB2 expression levels is a positive predictor factor since those patients are responsive to BRAFi/ErbBi combination.

H&uuml, rthle cell carcinomas (HCC) are rare differentiated thyroid cancers that display low avidity for radioactive iodine and respond poorly to kinase inhibitors. Here, using next-generation sequencing, we analyzed the mutational status of primary tissue and poorly differentiated metastatic tissue from two HCC patients. In both cases, metastatic tissues harbored a mutation of SETD2, each resulting in loss of the SRI and WW domains of SETD2, a methyltransferase that trimethylates H3K36 (H3K36me3) and also interacts with p53 to promote its stability. Functional studies of the novel p.D1890fs6* mutation (case 1) revealed significantly reduced H3K36me3 levels in SETD2-mutated tissue and primary cell cultures and decreased levels of the active form of p53. Restoration of SETD2-wildtype expression in the SETD2-mutant cells significantly reduced the expression of four well-known stemness markers (OCT-4, SOX2, IPF1, Goosecoid). These findings suggest potential roles for SETD2 loss-of-function mutations in HCC progression, possibly involving p53 destabilization and promotion of stemness. Their prevalence and potential treatment implications in thyroid cancer, especially HCC, require further study.

Molecular classification has improved the knowledge of medulloblastoma (MB), the most common malignant brain tumour in children, however current treatments cause severe side effects in patients. Cancer stem cells (CSCs) have been described in MB and represent a sub population characterised by self-renewal and the ability to generate tumour cells, thus representing the reservoir of the tumour. To investigate molecular pathways that characterise this sub population, we isolated CSCs from Sonic Hedgehog Medulloblastoma (SHH MB) arisen in Patched 1 (Ptch1) heterozygous mice, and performed miRNA- and mRNA-sequencing. Comparison of the miRNA-sequencing of SHH MB CSCs with that obtained from cerebellar Neural Stem Cells (NSCs), allowed us to obtain a SHH MB CSC miRNA differential signature. Pathway enrichment analysis in SHH MB CSCs mirnome and transcriptome was performed and revealed a series of enriched pathways. We focused on the putative targets of the SHH MB CSC miRNAs that were involved in the enriched pathways of interest, namely pathways in cancer, PI3k-Akt pathway and protein processing in endoplasmic reticulum pathway. In silico analysis was performed in SHH MB patients and identified several genes, whose expression was associated with worse overall survival of SHH MB patients. This study provides novel candidates whose functional role should be further investigated in SHH MB.

Cerebellar neural stem cells (NSCs) require Hedgehog-Gli (Hh-Gli) signalling for their maintenance and Nanog expression for their self-renewal. To identify novel molecular features of this regulatory pathway, we used next-generation sequencing technology to profile mRNA and microRNA expression in cerebellar NSCs, before and after induced differentiation (Diff-NSCs). Genes with higher transcript levels in NSCs (vs. Diff-NSCs) included Foxm1, which proved to be directly regulated by Gli and Nanog. Foxm1 in turn regulated several microRNAs that were overexpressed in NSCs: miR-130b, miR-301a, and members of the miR-15~16 and miR-17~92 clusters and whose knockdown significantly impaired the neurosphere formation ability. Our results reveal a novel Hh-Gli-Nanog-driven Foxm1-microRNA network that controls the self-renewal capacity of NSCs.

The circulating microRNAs (miRNAs) associated with type 2 diabetes (T2D) in elderly patients are still being defined. To identify novel miRNA biomarker candidates for monitoring responses to sitagliptin in such patients, we prospectively studied 40 T2D patients (age > 65) with HbA1c levels of 7.5–9.0% on metformin. After collection of baseline blood samples (t0), the dipeptidyl peptidase-IV (DPP-IV) inhibitor (DPP-IVi) sitagliptin was added to the metformin regimen, and patients were followed for 15 months. Patients with HbA1c 0.5% after 3 and 15 months of therapy were classified as “responders” (group R, n=34); all others were classified as “nonresponders” (group NR, n=6). Circulating miRNA profiling was performed on plasma collected in each group before and after 15 months of therapy (t0 and t15). Intra- and intergroup comparison of miRNA profiles pinpointed three miRNAs that correlated with responses to sitagliptin: miR-378, which is a candidate biomarker of resistance to this DPP-IVi, and miR-126-3p and miR-223, which are associated with positive responses to the drug. The translational implications are as immediate as evident, with the possibility to develop noninvasive diagnostic tools to predict drug response and development of chronic complications.

Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity. Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated the expression of the tumor-associated antigen PRAME in biopsies from 60 patients with medulloblastoma. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A*02+ DAOY cells as well as primary HLA-A*02+ medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T-cell–related toxicity, an inducible caspase-9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating patients with HLA-A*02+ medulloblastoma. Significance: These findings identify PRAME as a medulloblastoma tumor-associated antigen that can be targeted using genetically modified T cells. Cancer Res; 78(12); 3337–49. ©2018 AACR.

The mechanisms by which microRNAs control pediatric high-grade gliomas (pHGGs) have yet to be fully elucidated. Our studies of patient-derived pHGG tissues and of the pHGG cell line KNS42 revealed down-regulation in these tumors of three microRNAs, specifically miR-107, miR-181c, and miR-29a-3p. This down-regulation increases the proliferation of KNS42 cells by de-repressing expression of the Notch2 receptor (Notch2), a validated target of miR-107 and miR-181c and a putative target of miR-29a-3p. Inhibition (either pharmacologic or genetic) of Notch2 or re-expression of the implicated microRNAs (all three combined but also individually) significantly reduced KNS42 cell proliferation. These findings suggest that Notch2 pathway activation plays a critical role in pHGGs growth and reveal a direct epigenetic mechanism that controls Notch2 expression, which could potentially be targeted by novel forms of therapy for these childhood tumors characterized by high-morbidity and high-mortality.

Background Aberrant Sonic Hedgehog/Gli (Hh/Gli) signaling pathway is a critical regulator of Sonic hedgehog medulloblastoma (SHH-MB). Cancer stem cells (CSCs), thought to be largely responsible for tumor initiation, maintenance, dissemination and relapse, have been identified in SHH-MB. Since we previously demonstrated that Hh/Gli signaling controls CSCs features in SHH-MB and that in these tumors miR-326 is down regulated, here we investigated whether there is a functional link between Hh/Gli signaling and miR-326. Methods We evaluated β-arrestin1 (Arrb1) and its intragenic miR-326 levels in CSCs derived from SHH-MB. Subsequently, we modulated the expression of Arrb1 and miR-326 in CSCs in order to gain insight into their biological role. We also analyzed the mechanism by which Arrb1 and miR-326 control Hh/Gli signaling and self-renewal, using luciferase and protein immunoprecipitation assays. Results Low levels of Arrb1 and miR-326 represent a feature of CSCs derived from SHH-MB. We observed that re-expression of Arrb1 and miR-326 inhibits Hh/Gli signaling pathway at multiple levels, which cause impaired proliferation and self-renewal, accompanied by down regulation of Nanog levels. In detail, miR-326 negatively regulates two components of the Hh/Gli pathway the receptor Smoothened (Smo) and the transcription factor Gli2, whereas Arrb1 suppresses the transcriptional activity of Gli1, by potentiating its p300-mediated acetylation. Conclusions Our results identify a new molecular mechanism involving miR-326 and Arrb1 as regulators of SHH-MB CSCs. Specifically, low levels of Arrb1 and miR-326 trigger and maintain Hh/Gli signaling and self-renewal.

Objective: Microgravity was often shown to cause cell damage and impair cell cycle in a variety of biological systems. Since the effects on the neural system were poorly investigated, we aimed to gain insight into how biological processes such as cell cycle, cell damage, stemness features and metabolic status are involved in neural stem cells (NSC) when they experience simulated microgravity. We also wished to investigate whether these modulations were transient or permanent once cells were returned to normal gravity. Methods: NSC were isolated from mouse cerebella and cultured in the Rotary Cell Culture System (RCCS) to model microgravity. We analyzed cell cycle, stress and apoptotic response. We also performed a 1H NMR-based metabolomic analysis and evaluation of stemness features of NSC in simulated microgravity and once in the returned to normogravity cell culture. Results: Biological processes and metabolic status were modulated by simulated microgravity. Cells were arrested in S-phase together with enhanced apoptosis. Metabolic changes occurred in NSC after simulated microgravity. Interestingly, these modulations were transient. Indeed, stemness features and metabolic footprint returned to basal levels after few days of culture in normal conditions. Moreover NSC clonogenic ability was not impaired. Conclusions: Our data suggest that simulated microgravity impacts on NSC biological processes, including cell cycle and apoptosis. However, NSC does not suffer from permanent damage.

Medulloblastoma is the most common malignant brain tumor in childhood. Four main clinically and molecularly distinct MB subtypes have been identified: WNT, SHH, Group 3, and Group 4. Among them, Group 4 MB (G4MB) appears to have the most skewed age ratio, is predominant in males and shows an intermediate prognosis. In spite of being the most frequent (35%), it is also the least understood of all the subgroups. Diagnosis is based on patients’ clinical symptoms and neuro-imaging techniques with high risk of false positive and/or late tumor discovery. The early detection of the disease is essential to reduce both cancer mortality and long time therapy related toxicity. To date, there is no reliable marker for G4MB that could facilitate the early diagnosis and allow the evaluation of the response to radio and chemotherapy treatment. Therefore, the discovery of new biomarkers as a specific signature of G4MB patients, potentially useful as diagnostic factor, would be extraordinarily worthwhile. MicroRNAs are single-stranded non coding RNA molecules of 19-24 nucleotides in length involved in the post-transcriptional regulation of gene expression, whose expression is strongly deregulated in several types of tumors. Recent studies have shown that microRNAs are released into the bloodstream and are stably detectable thanks to their association with extracellular vescicle, or complex with RNA-binding protein that protect them from RNAse degradation. The minimally invasive liquid biopsy flanked to the reliability and specificity of microRNAs in cancer detection indicates circulating microRNAs not only as promising biomarkers to get early diagnosis, but also to follow treatment response and detect recurrence. Here we report a high-throughput screening of microRNAs in G4MB plasma samples before surgery and during follow-up. Two different approaches (DeepSeq and Low Denstity Array) were used to detect the differentially expressed circulating microRNAs in 4 patients and in 4 age- and sex- matched healthy donors. Baseline microRNA expression profiling allowed us to disclose a signature of 34 miRNAs able to identify patients versus healthy children. Moreover, selected microRNAs were validated by RT-qPCR technology on a wider cohort of G4MB patients (n = 8) and healthy donors (n = 8). Specifically, we identified two important oncogenic microRNA clusters (miR-17/92 and miR-106b/25) significantly upregulated in G4MB plasma patients. Therapy reduced the expression of these microRNAs up to levels of healthy donors. In conclusion we identified circulating microRNA signature in G4MB. Citation Format: Evelina Miele, Vincenzo Alfano, Zein Mersini Besharat, Giuseppina Catanzaro, Angela Mastronuzzi, Andrea Carai, Agnese Po, Luana Abballe, Antonella Cacchione, Franco Locatelli, Elisabetta Ferretti. Circulating microRNA signature in group 4 medulloblastoma patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 970.