UBE4B interacts with the ITCH E3 ubiquitin ligase to induce Ku70 and c-FLIPL polyubiquitination and enhanced neuroblastoma apoptosis.

Expression of the UBE4B ubiquitin ligase is strongly associated with neuroblastoma patient outcomes, but the functional roles of UBE4B in neuroblastoma pathogenesis are not known. We evaluated interactions of UBE4B with the E3 ubiquitin ligase ITCH/AIP4 and the effects of UBE4B expression on Ku70 and c-FLIPL ubiquitination and proteasomal degradation by co-immunoprecipitation and Western blots. We also evaluated the role of UBE4B in apoptosis induced by histone deacetylase (HDAC) inhibition using Western blots. UBE4B binding to ITCH was mediated by WW domains in the ITCH protein. ITCH activation led to ITCH-UBE4B complex formation and recruitment of Ku70 and c-FLIPL via ITCH WW domains, followed by Ku70 and c-FLIPL Lys48/Lys63 branched polyubiquitination and proteasomal degradation. HDAC inhibition induced Ku70 acetylation, leading to release of c-FLIPL and Bax from Ku70, increased Ku70 and c-FLIPL Lys48/Lys63 branched polyubiquitination via the ITCH-UBE4B complex, and induction of apoptosis. UBE4B depletion led to reduced polyubiquitination and increased levels of Ku70 and c-FLIPL and to reduced apoptosis induced by HDAC inhibition via stabilization of c-FLIPL and Ku70 and inhibition of caspase 8 activation. Our results have identified novel interactions and novel targets for UBE4B ubiquitin ligase activity and a direct role for the ITCH-UBE4B complex in responses of neuroblastoma cells to HDAC inhibition, suggesting that the ITCH-UBE4B complex plays a critical role in responses of neuroblastoma to therapy and identifying a potential mechanism underlying the association of UBE4B expression with neuroblastoma patient outcomes.

USP7 Inhibition Suppresses Neuroblastoma Growth via Induction of p53-Mediated Apoptosis and EZH2 and N-Myc Downregulation.

Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.

Identification of a novel gene signature for neuroblastoma differentiation using a Boolean implication network.

Although induction of differentiation represents an effective strategy for neuroblastoma treatment, the mechanisms underlying neuroblastoma differentiation are poorly understood. We generated a computational model of neuroblastoma differentiation consisting of interconnected gene clusters identified based on symmetric and asymmetric gene expression relationships. We identified a differentiation signature consisting of series of gene clusters comprised of 1251 independent genes that predicted neuroblastoma differentiation in independent datasets and in neuroblastoma cell lines treated with agents known to induce differentiation. This differentiation signature was associated with patient outcomes in multiple independent patient cohorts and validated the role of MYCN expression as a marker of neuroblastoma differentiation. Our results further identified novel genes associated with MYCN via asymmetric Boolean implication relationships that would not have been identified using symmetric computational approaches and that were associated with both neuroblastoma differentiation and patient outcomes. Our differentiation signature included a cluster of genes involved in intracellular signaling and growth factor receptor trafficking pathways that is strongly associated with neuroblastoma differentiation, and we validated the associations of UBE4B, a gene within this cluster, with neuroblastoma cell and tumor differentiation. Our findings demonstrate that Boolean network analyses of symmetric and asymmetric gene expression relationships can identify novel genes and pathways relevant for neuroblastoma tumor differentiation that could represent potential therapeutic targets.

Allele-specific expression reveals genes with recurrent cis-regulatory alterations in high-risk neuroblastoma.

BACKGROUND: Neuroblastoma is a pediatric malignancy with a high frequency of metastatic disease at initial diagnosis. Neuroblastoma tumors have few recurrent protein-coding mutations but contain extensive somatic copy number alterations (SCNAs) suggesting that mutations that alter gene dosage are important drivers of tumorigenesis. Here, we analyze allele-specific expression in 96 high-risk neuroblastoma tumors to discover genes impacted by cis-acting mutations that alter dosage. RESULTS: We identify 1043 genes with recurrent, neuroblastoma-specific allele-specific expression. While most of these genes lie within common SCNA regions, many of them exhibit allele-specific expression in copy neutral samples and these samples are enriched for mutations that are predicted to cause nonsense-mediated decay. Thus, both SCNA and non-SCNA mutations frequently alter gene expression in neuroblastoma. We focus on genes with neuroblastoma-specific allele-specific expression in the absence of SCNAs and find 26 such genes that have reduced expression in stage 4 disease. At least two of these genes have evidence for tumor suppressor activity including the transcription factor TFAP2B and the protein tyrosine phosphatase PTPRH. CONCLUSIONS: In summary, our allele-specific expression analysis discovers genes that are recurrently dysregulated by both large SCNAs and other cis-acting mutations in high-risk neuroblastoma.

Characterization of G-CSF receptor expression in medulloblastoma.

BACKGROUND: Identifying mechanisms of medulloblastoma recurrence is a key to improving patient survival, and targeting treatment-resistant subpopulations within tumors could reduce disease recurrence. Expression of the granulocyte colony-stimulating factor receptor (G-CSF-R, CD114) is a potential marker of cancer stem cells, and therefore we hypothesized that a subpopulation of medulloblastoma cells would also express CD114 and would demonstrate chemoresistance and responsiveness to G-CSF. METHODS: Prevalence of CD114-positive (CD114+) cells in medulloblastoma cell lines, patient-derived xenograft (PDX) tumors, and primary patient tumor samples were assessed by flow cytometry. Growth rates, chemoresistance, and responses to G-CSF of CD114+ and CD114-negative (CD114-) cells were characterized in vitro using continuous live cell imaging and flow cytometry. Gene expression profiles were compared between CD114+ and CD114- medulloblastoma cells using quantitative RT-PCR. RESULTS: CD114+ cells were identifiable in medulloblastoma cell lines, PDX tumors, and primary patient tumors and have slower growth rates than CD114- or mixed populations. G-CSF accelerates the growth of CD114+ cells, and CD114+ cells are more chemoresistant. The CD114+ population is enriched when G-CSF treatment follows chemotherapy. The CD114+ population also has higher expression of the CSF3R, NRP-1, TWIST1, and MYCN genes. CONCLUSIONS: Our data demonstrate that a subpopulation of CD114+ medulloblastoma cells exists in cell lines and tumors, which may evade traditional chemotherapy and respond to exogenous G-CSF. These properties invite further investigation into the role of G-CSF in medulloblastoma therapy and methods to specifically target these cells.

Regorafenib is effective against neuroblastoma in vitro and in vivo and inhibits the RAS/MAPK, PI3K/Akt/mTOR and Fos/Jun pathways.

BACKGROUND: Regorafenib is an inhibitor of multiple kinases with aberrant expression and activity in neuroblastoma tumours that have potential roles in neuroblastoma pathogenesis. METHODS: We evaluated neuroblastoma cells treated with regorafenib for cell viability and confluence, and analysed treated cells for apoptosis and cell cycle progression. We evaluated the efficacy of regorafenib in vivo using an orthotopic xenograft model. We evaluated regorafenib-mediated inhibition of kinase targets and performed reverse-phase protein array (RPPA) analysis of neuroblastoma cells treated with regorafenib. Lastly, we evaluated the efficacy and effects of the combination of regorafenib and 13-cis-retinoic acid on intracellular signalling. RESULTS: Regorafenib treatment resulted in reduced neuroblastoma cell viability and confluence, with both induction of apoptosis and of cell cycle arrest. Regorafenib treatment inhibits known receptor tyrosine kinase targets RET and PDGFRß and intracellular signalling through the RAS/MAPK, PI3K/Akt/mTOR and Fos/Jun pathways. Regorafenib is effective against neuroblastoma tumours in vivo, and the combination of regorafenib and 13-cis-retinoic acid demonstrates enhanced efficacy compared with regorafenib alone. CONCLUSIONS: The effects of regorafenib on multiple intracellular signalling pathways and the potential additional efficacy when combined with 13-cis-retinoic acid represent opportunities to develop treatment regimens incorporating regorafenib for children with neuroblastoma.

Stress Conditions Induced by Locoregional Therapies Stimulate Enrichment and Proliferation of Liver Cancer Stem Cells.

PURPOSE: This study tested the hypothesis that stress conditions that simulated percutaneous thermal ablation (PTA), transarterial embolization (TAE), or transarterial chemoembolization stimulated enrichment of hepatocellular carcinoma (HCC) cancer stem cells (hCSCs) and that hCSC inhibitors can suppress this effect. MATERIALS AND METHODS: Human HCC cell lines HepG2 and PLC/PRF/5 were subjected to a 46.5°C heat bath for 10 minutes or to 1% hypoxia for 72 hours without fetal bovine serum and with or without doxorubicin. Cells were then treated with a ß-catenin inhibitor (FH535 or XAV939), a PI3 kinase inhibitor (Ly294002), or niclosamide, a US Food and Drug Administration-approved antihelminthic drug that acts as a mitochondrial decoupler and mixed inhibitor. Surviving cells were analyzed for hCSC markers by flow cytometry, for stemness by colony-forming assay or sphere-forming assay, and for proliferative capacity by MTT assay (where MTT is 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide). Expression of proteins related to CSC renewal and proliferation were analyzed by immunoblotting and immunostaining. RESULTS: Conditions that simulated PTA, TAE, and transarterial chemoembolization resulted in an enrichment of cells bearing hCSC markers (CD133, CD44, and EpCAM). Cells surviving heat stress exhibited higher colony- or sphere-forming capacity and a greater proliferative state. These effects could be suppressed by niclosamide and inhibitors of ß-catenin and PI3 kinase. CONCLUSIONS: Stress conditions induced by locoregional therapies stimulated hCSC enrichment and proliferation, which could be suppressed by niclosamide and inhibitors of pathways important for hCSC renewal. Future studies will determine whether combining locoregional therapies with adjuvant hCSC inhibitors reduces HCC recurrence.