Amplification of MYCN is a driver mutation in a subset of human neuroendocrine tumors, including neuroblastoma. No small molecules that target N-Myc, the protein encoded by MYCN, are clinically ...available. N-Myc forms a complex with the Aurora-A kinase, which protects N-Myc from proteasomal degradation. Although stabilization of N-Myc does not require the catalytic activity of Aurora-A, we show here that two Aurora-A inhibitors, MLN8054 and MLN8237, disrupt the Aurora-A/N-Myc complex and promote degradation of N-Myc mediated by the Fbxw7 ubiquitin ligase. Disruption of the Aurora-A/N-Myc complex inhibits N-Myc-dependent transcription, correlating with tumor regression and prolonged survival in a mouse model of MYCN-driven neuroblastoma. We conclude that Aurora-A is an accessible target that makes destabilization of N-Myc a viable therapeutic strategy.
•Aurora-A-specific inhibitors disrupt the Aurora-A/N-Myc complex•Inhibitors trigger proteasomal degradation of N-Myc via Fbxw7 ubiquitin ligase•Inhibitors revert N-Myc-dependent gene expression in a mouse model of neuroblastoma•Inhibitors induce tumor regression and extend survival in this model
Small RNA deep sequencing is widely used to characterize non-coding RNAs (ncRNAs) differentially expressed between two conditions, e.g. healthy and diseased individuals and to reveal insights into ...molecular mechanisms underlying condition-specific phenotypic traits. The ncRNAome is composed of a multitude of RNAs, such as transfer RNA, small nucleolar RNA and microRNA (miRNA), to name few. Here we present omiRas, a Web server for the annotation, comparison and visualization of interaction networks of ncRNAs derived from next-generation sequencing experiments of two different conditions. The Web tool allows the user to submit raw sequencing data and results are presented as: (i) static annotation results including length distribution, mapping statistics, alignments and quantification tables for each library as well as lists of differentially expressed ncRNAs between conditions and (ii) an interactive network visualization of user-selected miRNAs and their target genes based on the combination of several miRNA-mRNA interaction databases.
The omiRas Web server is implemented in Python, PostgreSQL, R and can be accessed at: http://tools.genxpro.net/omiras/.
The cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncogenic factor that stabilises the c-Myc protein. CIP2A is overexpressed in several tumours, and expression levels are an independent ...marker for long-term outcome. To determine whether CIP2A expression is elevated in colon cancer and whether it might serve as a prognostic marker for survival, we analysed CIP2A mRNA expression by real-time PCR in 104 colon cancer samples. CIP2A mRNA was overexpressed in colon cancer samples and CIP2A expression levels correlated significantly with tumour stage. We found that CIP2A serves as an independent prognostic marker for disease-free and overall survival. Further, we investigated CIP2A-dependent effects on levels of c-Myc, Akt and on cell proliferation in three colon cancer cell lines by silencing CIP2A using small interfering (si) and short hairpin (sh) RNAs. Depletion of CIP2A substantially inhibited growth of colon cell lines and reduced c-Myc levels without affecting expression or function of the upstream regulatory kinase, Akt. Expression of CIP2A was found to be dependent on MAPK activity, linking elevated c-Myc expression to deregulated signal transduction in colon cancer.
In chicken, the left and right female gonads undergo a completely different program during development. To learn more about the molecular factors underlying side-specific development and to identify ...potential sex- and side-specific genes in developing gonads, we separately performed next-generation sequencing-based deepSuperSAGE transcription profiling from left and right, female and male gonads of 19-day-old chicken embryos. A total of 836 transcript variants were significantly differentially expressed (p < 10(-5)) between combined male and female gonads. Left-right comparison revealed 1,056 and 822 differentially (p < 10(-5)) expressed transcript variants for male and female gonads, respectively, of which 72 are side-specific in both sexes. At least some of these may represent key players for lateral development in birds. Additionally, several genes with laterally differential expression in the ovaries seem to determine female gonads for growth or regression, whereas right-left differences in testes are mostly limited to the differentially expressed genes present in both sexes. With a few exceptions, side-specific genes are not located on the sex chromosomes. The large differences in lateral gene expression in the ovaries in almost all metabolic pathways suggest that the regressing right gonad might have undergone a change of function during evolution.
In mammalian cells, the MYC oncoprotein binds to thousands of promoters. During mitogenic stimulation of primary lymphocytes, MYC promotes an increase in the expression of virtually all genes. In ...contrast, MYC-driven tumour cells differ from normal cells in the expression of specific sets of up- and downregulated genes that have considerable prognostic value. To understand this discrepancy, we studied the consequences of inducible expression and depletion of MYC in human cells and murine tumour models. Changes in MYC levels activate and repress specific sets of direct target genes that are characteristic of MYC-transformed tumour cells. Three factors account for this specificity. First, the magnitude of response parallels the change in occupancy by MYC at each promoter. Functionally distinct classes of target genes differ in the E-box sequence bound by MYC, suggesting that different cellular responses to physiological and oncogenic MYC levels are controlled by promoter affinity. Second, MYC both positively and negatively affects transcription initiation independent of its effect on transcriptional elongation. Third, complex formation with MIZ1 (also known as ZBTB17) mediates repression of multiple target genes by MYC and the ratio of MYC and MIZ1 bound to each promoter correlates with the direction of response.
Deregulated expression of the MYC oncoprotein contributes to the genesis of many human tumours, yet strategies to exploit this for a rational tumour therapy are scarce. MYC promotes cell growth and ...proliferation, and alters cellular metabolism to enhance the provision of precursors for phospholipids and cellular macromolecules. Here we show in human and murine cell lines that oncogenic levels of MYC establish a dependence on AMPK-related kinase 5 (ARK5; also known as NUAK1) for maintaining metabolic homeostasis and for cell survival. ARK5 is an upstream regulator of AMPK and limits protein synthesis via inhibition of the mammalian target of rapamycin 1 (mTORC1) signalling pathway. ARK5 also maintains expression of mitochondrial respiratory chain complexes and respiratory capacity, which is required for efficient glutamine metabolism. Inhibition of ARK5 leads to a collapse of cellular ATP levels in cells expressing deregulated MYC, inducing multiple pro-apoptotic responses as a secondary consequence. Depletion of ARK5 prolongs survival in MYC-driven mouse models of hepatocellular carcinoma, demonstrating that targeting cellular energy homeostasis is a valid therapeutic strategy to eliminate tumour cells that express deregulated MYC.
Alternative polyadenylation (APA) is a widespread mechanism that contributes to the sophisticated dynamics of gene regulation. Approximately 50% of all protein-coding human genes harbor multiple ...polyadenylation (PA) sites; their selective and combinatorial use gives rise to transcript variants with differing length of their 3' untranslated region (3'UTR). Shortened variants escape UTR-mediated regulation by microRNAs (miRNAs), especially in cancer, where global 3'UTR shortening accelerates disease progression, dedifferentiation and proliferation. Here we present APADB, a database of vertebrate PA sites determined by 3' end sequencing, using massive analysis of complementary DNA ends. APADB provides (A)PA sites for coding and non-coding transcripts of human, mouse and chicken genes. For human and mouse, several tissue types, including different cancer specimens, are available. APADB records the loss of predicted miRNA binding sites and visualizes next-generation sequencing reads that support each PA site in a genome browser. The database tables can either be browsed according to organism and tissue or alternatively searched for a gene of interest. APADB is the largest database of APA in human, chicken and mouse. The stored information provides experimental evidence for thousands of PA sites and APA events. APADB combines 3' end sequencing data with prediction algorithms of miRNA binding sites, allowing to further improve prediction algorithms. Current databases lack correct information about 3'UTR lengths, especially for chicken, and APADB provides necessary information to close this gap. Database URL: http://tools.genxpro.net/apadb/.
The unprecedented role of sncRNAs in the regulation of pollen biogenesis on both transcriptional and epigenetic levels has been experimentally proven. However, little is known about their global ...regulation, especially under stress conditions. We used tomato pollen in order to identify pollen stage-specific sncRNAs and their target mRNAs. We further deployed elevated temperatures to discern stress responsive sncRNAs. For this purpose high throughput sncRNA-sequencing as well as Massive Analysis of cDNA Ends (MACE) were performed for three-replicated sncRNAs libraries derived from tomato tetrad, post-meiotic, and mature pollen under control and heat stress conditions.
Using the omiRas analysis pipeline we identified known and predicted novel miRNAs as well as sncRNAs from other classes, responsive or not to heat. Differential expression analysis revealed that post-meiotic and mature pollen react most strongly by regulation of the expression of coding and non-coding genomic regions in response to heat. To gain insight to the function of these miRNAs, we predicted targets and annotated them to Gene Ontology terms. This approach revealed that most of them belong to protein binding, transcription, and Serine/Threonine kinase activity GO categories. Beside miRNAs, we observed differential expression of both tRNAs and snoRNAs in tetrad, post-meiotic, and mature pollen when comparing normal and heat stress conditions.
Thus, we describe a global spectrum of sncRNAs expressed in pollen as well as unveiled those which are regulated at specific time-points during pollen biogenesis. We integrated the small RNAs into the regulatory network of tomato heat stress response in pollen.
Abstract 2461
Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. 50–70 % of adults with AML achieve complete remission ...with induction of chemotherapy - only 20–30 % of patients enjoy long-term disease-free survival. Many AML-patients still die of their disease, most frequently because of drug resistance. We intended to study drug resistance in cancer cells by using a synthetic lethal RNAi screen with the S phase targeting drug cytarabine (Ara-C). Our goal was the identification of siRNAs that overcome drug resistance triggered by cytarabine.
For the chemosensitizer RNAi screen we used lipid-based reverse transfection for transient siRNA-mediated gene silencing in the human osteosarcoma cell line (U2OS). The human osteosarcoma cell line is a good model to study cytarabine resistance: the cells are highly refractory to Ara-C treatment, while activating DNA damage response (indicated by activation of different DNA checkpoint markers (phospho-Chk1, phospho-p53, p21)). Gene silencing was done by using a custom library comprising 437 siRNA probes specific for DNA repair and DNA damage response genes (consisting of a pool of four different siRNA-sequences for one gene). Synthetic lethality of siRNAs with a sub lethal dose of cytarabine (IC20) was measured after 72 hours of incubation with a luminescence-based cell viability assay. Hits were identified after z-score normalization of each plate and by evaluating the cytarabine-sensitivity of siRNAs reducing cell viability in combination with Ara-C vs. cell viability of untreated siRNA transfected cells. Screens were done in duplicate and high confidence hits (with z-scores greater than 2 STD) were validated. For secondary validation screens and further validation experiments we used two different single siRNA-sequences for each gene.
We reproducible identified and validated candidate genes which sensitize cells to cytarabine-treatment when silenced. Our top hits p73 and components of ubiquitine ligase complexes have been implicated in drug resistance and some are also “druggable”. The positively screened p73, a member of the p53 tumor suppressor protein family, is frequently overexpressed in cancer patients and correlates with increased tumor aggressiveness and therapy resistance. Apoptosis of p73-depleted U2OS cells is dramatically increased in the presence of cytarabine (> 50 %) compared to untreated p73-depleted cancer cells. We provide data that p73 and other screen hits enhance the PCNA-coupled post-replicative DNA repair pathway as the basis for cytarabine resistance. Analyzing the genome-wide gene expression profiles of 286 AML-patients with different karyotypes showed that our top screen hits in human osteosarcoma cells have a prognostic impact in AML-patients. Kaplan-Meier survival analysis showed that AML-patients with high-level expression of p73 displayed a significantly reduced overall survival compared to p73 low-expressing AML-patients. Together, these data may lead to a more individualized AML therapy, resulting in better treatment outcome.
Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
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