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.

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.

Mechanisms of Efficacy of the FGFR1-3 Inhibitor AZD4547 in Pediatric Solid Tumor Models.

Children with aggressive pediatric solid tumors have poor outcomes and novel treatments are needed. Pediatric solid tumors demonstrate aberrant expression and activity of the fibroblast growth factor receptor (FGFR) family, suggesting FGFR inhibitors may be effective therapeutic agents. AZD4547 is a multikinase inhibitor of the FGFR1-3 kinases, and we hypothesized that AZD4547 would be effective in pediatric solid tumor preclinical models. We evaluated the effects of AZD4547 on neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma cells alone and in combination with STAT3 inhibition. Continuous live cell imaging was used to measure induction of apoptosis and effects on migration. Receptor inhibition and intracellular signaling were examined by western blotting. AZD4547 treatment resulted in decreased cell confluence, increased apoptosis and reduced cell migration in all tested cell lines. AZD4547 treatment led to decreased phosphorylation of signaling proteins involved in cell survival and apoptotic pathways and increased phosphorylation of STAT3, and treatment of cell lines with AZD4547 combined with STAT3 inhibition demonstrated increased efficacy. Sensitivity to AZD4547 appears to be mediated by effects on the Ras/MAPK and JAK/STAT pathways, and AZD4547 represents a potential novel therapeutic agent for children with solid tumors.

Identification of SUMO-2/3-modified proteins associated with mitotic chromosomes.

Sumoylation is essential for progression through mitosis, but the specific protein targets and functions remain poorly understood. In this study, we used chromosome spreads to more precisely define the localization of SUMO-2/3 (small ubiquitin-related modifier) to the inner centromere and protein scaffold of mitotic chromosomes. We also developed methods to immunopurify proteins modified by endogenous, untagged SUMO-2/3 from mitotic chromosomes. Using these methods, we identified 149 chromosome-associated SUMO-2/3 substrates by nLC-ESI-MS/MS. Approximately one-third of the identified proteins have reported functions in mitosis. Consistent with SUMO-2/3 immunolocalization, we identified known centromere- and kinetochore-associated proteins, as well as chromosome scaffold associated proteins. Notably, >30 proteins involved in chromatin modification or remodeling were identified. Our results provide insights into the roles of sumoylation as a regulator of chromatin structure and other diverse processes in mitosis. Furthermore, our purification and fractionation methodologies represent an important compliment to existing approaches to identify sumoylated proteins using exogenously expressed and tagged SUMOs.

Analysis of changes in SUMO-2/3 modification during breast cancer progression and metastasis.

SUMOylation is an essential posttranslational modification and regulates many cellular processes. Dysregulation of SUMOylation plays a critical role in metastasis, yet how its perturbation affects this lethal process of cancer is not well understood. We found that SUMO-2/3 modification is greatly up-regulated in metastatic breast cancer cells compared with nonmetastatic control cells. To identify proteins differentially modified by SUMO-2/3 between metastatic and nonmetastatic cells, we established a method in which endogenous SUMO-2/3 conjugates are labeled by stable isotope labeling by amino acids in cell culture (SILAC), immunopurified by SUMO-2/3 monoclonal antibodies and epitope-peptide elution, and analyzed by quantitative mass spectrometry. We identified 66 putative SUMO-2/3-conjugated proteins, of which 15 proteins show a significant increase/decrease in SUMO-2/3 modification in metastatic cells. Targets with altered SUMOylation are involved in cell cycle, migration, inflammation, glycolysis, gene expression, and SUMO/ubiquitin pathways, suggesting that perturbations of SUMO-2/3 modification might contribute to metastasis by affecting these processes. Consistent with this, up-regulation of PML SUMO-2/3 modification corresponds to an increased number of PML nuclear bodies (PML-NBs) in metastatic cells, whereas up-regulation of global SUMO-2/3 modification promotes 3D cell migration. Our findings provide a foundation for further investigating the effects of SUMOylation on breast cancer progression and metastasis.

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.

Poly-SUMO-2/3 chain modification of Nuf2 facilitates CENP-E kinetochore localization and chromosome congression during mitosis.

SUMO modification is required for the kinetochore localization of the kinesin-like motor protein CENP-E, which subsequently mediates the alignment of chromosomes to the spindle equator during mitosis. However, the underlying mechanisms by which sumoylation regulates CENP-E kinetochore localization are still unclear. In this study, we first elucidate that the kinetochore protein Nuf2 is not only required for CENP-E kinetochore localization but also preferentially modified by poly-SUMO-2/3 chains. In addition, poly-SUMO-2/3 modification of Nuf2 is significantly upregulated during mitosis, which is temporally correlated to the kinetochore localization of CENP-E during mitosis. We further show that the mitotic defects in CENP-E kinetochore localization and chromosome congression caused by global inhibition of sumoylation can be rescued by expressing a fusion protein between Nuf2 and the SUMO-conjugating enzyme Ubc9 for stimulating Nuf2 SUMO-2/3 modification. Moreover, the expression of another fusion protein between Nuf2 and three SUMO-2 moieties (SUMO-2 trimer), which mimics the trimeric SUMO-2/3 chain modification of Nuf2, can also rescue the mitotic defects due to global inhibition of sumoylation. Conversely, expressing the other forms of Nuf2-SUMO fusion proteins, which imitate Nuf2 modifications by SUMO-2/3 monomer, SUMO-2/3 dimer, and SUMO-1 trimer, respectively, cannot rescue the same mitotic defects. Lastly, compared to Nuf2, the fusion protein simulating the trimeric SUMO-2 chain-modified Nuf2 exhibits a significantly higher binding affinity to CENP-E wild type containing a functional SUMO-interacting motif (SIM) but not the CENP-E SIM mutant. Hence, our results support a model that poly-SUMO-2/3 chain modification of Nuf2 facilitates CENP-E kinetochore localization and chromosome congression during mitosis.Abbreviations: CENP-E, centromere-associated protein E; SUMO, small ubiquitin-related modifier; SIM, SUMO-interacting motif.

Molecular Characterization and Functional Analysis of Annulate Lamellae Pore Complexes in Nuclear Transport in Mammalian Cells.

Annulate lamellae are cytoplasmic organelles containing stacked sheets of membranes embedded with pore complexes. These cytoplasmic pore complexes at annulate lamellae are morphologically similar to nuclear pore complexes at the nuclear envelope. Although annulate lamellae has been observed in nearly all types of cells, their biological functions are still largely unknown. Here we show that SUMO1-modification of the Ran GTPase-activating protein RanGAP1 not only target RanGAP1 to its known sites at nuclear pore complexes but also to annulate lamellae pore complexes through interactions with the Ran-binding protein RanBP2 and the SUMO-conjugating enzyme Ubc9 in mammalian cells. Furthermore, upregulation of annulate lamellae, which decreases the number of nuclear pore complexes and concurrently increases that of annulate lamellae pore complexes, causes a redistribution of nuclear transport receptors including importin α/ß and the exportin CRM1 from nuclear pore complexes to annulate lamellae pore complexes and also reduces the rates of nuclear import and export. Moreover, our results reveal that importin α/ß-mediated import complexes initially accumulate at annulate lamellae pore complexes upon the activation of nuclear import and subsequently disassociate for nuclear import through nuclear pore complexes in cells with upregulation of annulate lamellae. Lastly, CRM1-mediated export complexes are concentrated at both nuclear pore complexes and annulate lamellae pore complexes when the disassembly of these export complexes is inhibited by transient expression of a Ran GTPase mutant arrested in its GTP-bound form, suggesting that RanGAP1/RanBP2-activated RanGTP hydrolysis at these pore complexes is required for the dissociation of the export complexes. Hence, our findings provide a foundation for further investigation of how upregulation of annulate lamellae decreases the rates of nuclear transport and also for elucidation of the biological significance of the interaction between annulate lamellae pore complexes and nuclear transport complexes in mammalian cells.

SUMOylation in control of accurate chromosome segregation during mitosis.

Posttranslational protein modification by small ubiquitin-related modifier (SUMO) has emerged as an important regulatory mechanism for chromosome segregation during mitosis. This review focuses on how SUMOylation regulates the centromere and kinetochore activities to achieve accurate chromosome segregation during mitosis. Kinetochores are assembled on the specialized chromatin domains called centromeres and serve as the sites for attaching spindle microtubule to segregate sister chromatids to daughter cells. Many proteins associated with mitotic centromeres and kinetochores have been recently found to be modified by SUMO. Although we are still at the early stage of elucidating how SUMOylation controls chromosome segregation during mitosis, a substantial progress has been achieved over the past decade. Furthermore, a major theme that has emerged from the recent studies of SUMOylation in mitosis is that both SUMO conjugation and deconjugation are critical for kinetochore assembly and disassembly. Lastly, we propose a model that SUMOylation coordinates multiple centromere and kinetochore activities to ensure accurate chromosome segregation.