ATG7 is a key autophagy-promoting gene that plays a critical role in the regulation of cell death and survival of various cell types. We report here that microRNAs (miRNAs), a class of endogenous 22–24 nucleotide noncoding RNA molecules able to affect stability and translation of mRNA, may represent a novel mechanism for regulating ATG7 expression and therefore autophagy. We demonstrated that ATG7 is a potential target for miR-17, and this miRNA could negatively regulate ATG7 expression, resulting in a modulation of the autophagic status in T98G glioblastoma cells. Treatment of these tumor cells with the miR-17 mimic decreased, and with the antagomir increased, the expression of ATG7 protein. Dual luciferase reporter assay confirmed that a specific miR-17 binding sequence in the 3′-UTR of ATG7 contributed to the modulation of the expression of the gene by miR-17. Interestingly, our results showed that anti-miR-17 administration activated autophagy through autophagosome formation, as resulted by LC3B and A...

PURPOSE: In neuroblastoma (NB), the ALK receptor tyrosine kinase can be constitutively activated through activating point mutations or genomic amplification. We studied ALK genetic alterations in high-risk (HR) patients on the HR-NBL1/SIOPEN trial to determine their frequency, correlation with clinical parameters, and prognostic impact. MATERIALS AND METHODS: Diagnostic tumor samples were available from 1,092 HR-NBL1/SIOPEN patients to determine ALK amplification status (n = 330), ALK mutational profile (n = 191), or both (n = 571). RESULTS: Genomic ALK amplification ( ALKa) was detected in 4.5% of cases (41 out of 901), all except one with MYCN amplification (MNA). ALKa was associated with a significantly poorer overall survival (OS) (5-year OS: ALKa [n = 41] 28% [95% CI, 15 to 42]; no- ALKa [n = 860] 51% [95% CI, 47 to 54], [ P < .001]), particularly in cases with metastatic disease. ALK mutations ( ALKm) were detected at a clonal level (> 20% mutated allele fraction) in 10% of cases (76 out of 762) and at a subclonal level (mutated allele fraction 0.1%-20%) in 3.9% of patients (30 out of 762), with a strong correlation between the presence of ALKm and MNA ( P < .001). Among 571 cases with known ALKa and ALKm status, a statistically significant difference in OS was observed between cases with ALKa or clonal ALKm versus subclonal ALKm or no ALK alterations (5-year OS: ALKa [n = 19], 26% [95% CI, 10 to 47], clonal ALKm [n = 65] 33% [95% CI, 21 to 44], subclonal ALKm (n = 22) 48% [95% CI, 26 to 67], and no alteration [n = 465], 51% [95% CI, 46 to 55], respectively; P = .001). Importantly, in a multivariate model, involvement of more than one metastatic compartment (hazard ratio [HR], 2.87; P < .001), ALKa (HR, 2.38; P = .004), and clonal ALKm (HR, 1.77; P = .001) were independent predictors of poor outcome. CONCLUSION: Genetic alterations of ALK (clonal mutations and amplifications) in HR-NB are independent predictors of poorer survival. These data provide a rationale for integration of ALK inhibitors in upfront treatment of HR-NB with ALK alterations.

The biological and clinical heterogeneity of neuroblastoma (NB) demands novel biomarkers and therapeutic targets in order to drive the most appropriate treatment for each patient. Hypoxia is a condition of low-oxygen tension occurring in poorly vascularized tumor tissues. In this study, we aimed to assess the role of hypoxia in the pathogenesis of NB and at developing a new clinically relevant hypoxia-based predictor of outcome. We analyzed the gene expression profiles of 1882 untreated NB primary tumors collected at diagnosis and belonging to four existing data sets. Analyses took advantage of machine learning methods. We identified NB-hop, a seven-gene hypoxia biomarker, as a predictor of NB patient prognosis, which is able to discriminate between two populations of patients with unfavorable or favorable outcome on a molecular basis. NB-hop retained its prognostic value in a multivariate model adjusted for established risk factors and was able to additionally stratify clinically relevant groups of patients. Tumors with an unfavorable NB-hop expression showed a significant association with telomerase activation and a hypoxic, immunosuppressive, poorly differentiated, and apoptosis-resistant tumor microenvironment. NB-hop defines a new population of NB patients with hypoxic tumors and unfavorable prognosis and it represents a critical factor for the stratification and treatment of NB patients.

Despite intensive treatment, 50% of children with high-risk neuroblastoma (HR-NB) succumb to their disease. Progression through current trials evaluating the efficacy of new treatments for children with HR disease usually depends on an inadequate response to induction chemotherapy, assessed using imaging modalities. In this study, we sought to identify circulating biomarkers that might be detected in a simple blood sample to predict patient response to induction chemotherapy. Since exosomes released by tumor cells can drive tumor growth and chemoresistance, we tested the hypothesis that exosomal microRNA (exo-miRNAs) in blood might predict response to induction chemotherapy. The exo-miRNAs expression profile in plasma samples collected from children treated in HR-NBL-1/SIOPEN before and after induction chemotherapy was compared to identify a three exo-miRs signature that could discriminate between poor and good responders. Exo-miRNAs expression also provided a chemoresistance index predicting the good or poor prognosis of HR-NB patients.

More than fifty percent of neuroblastoma (NB) patients with adverse prognosis do not benefit from treatment making the identification of new potential targets mandatory. Hypoxia is a condition of low oxygen tension, occurring in poorly vascularized tissues, which activates specific genes and contributes to the acquisition of the tumor aggressive phenotype. We defined a gene expression signature (NB-hypo), which measures the hypoxic status of the neuroblastoma tumor. We aimed at developing a classifier predicting neuroblastoma patients’ outcome based on the assessment of the adverse effects of tumor hypoxia on the progression of the disease. Multi-layer perceptron (MLP) was trained on the expression values of the 62 probe sets constituting NB-hypo signature to develop a predictive model for neuroblastoma patients’ outcome. We utilized the expression data of 100 tumors in a leave-one-out analysis to select and construct the classifier and the expression data of the remaining 82 tumors to test the classifier performance in an external dataset. We utilized the Gene set enrichment analysis (GSEA) to evaluate the enrichment of hypoxia related gene sets in patients predicted with “Poor” or “Good” outcome. We utilized the expression of the 62 probe sets of the NB-Hypo signature in 182 neuroblastoma tumors to develop a MLP classifier predicting patients’ outcome (NB-hypo classifier). We trained and validated the classifier in a leave-one-out cross-validation analysis on 100 tumor gene expression profiles. We externally tested the resulting NB-hypo classifier on an independent 82 tumors’ set. The NB-hypo classifier predicted the patients’ outcome with the remarkable accuracy of 87 %. NB-hypo classifier prediction resulted in 2 % classification error when applied to clinically defined low-intermediate risk neuroblastoma patients. The prediction was 100 % accurate in assessing the death of five low/intermediated risk patients. GSEA of tumor gene expression profile demonstrated the hypoxic status of the tumor in patients with poor prognosis. We developed a robust classifier predicting neuroblastoma patients’ outcome with a very low error rate and we provided independent evidence that the poor outcome patients had hypoxic tumors, supporting the potential of using hypoxia as target for neuroblastoma treatment.