A G316A Polymorphism in the Ornithine Decarboxylase Gene Promoter Modulates MYCN-Driven Childhood Neuroblastoma.

Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of , results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype -amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays.

Identification and targeting of selective vulnerability rendered by tamoxifen resistance.

Background: The estrogen receptor (ER)-positive breast cancer represents over 80% of all breast cancer cases. Even though adjuvant hormone therapy with tamoxifen (TMX) is saving lives of patients with ER-positive breast cancer, the acquired resistance to TMX anti-estrogen therapy is the main hurdle for successful TMX therapy. Here we address the mechanism for TMX resistance and explore the ways to eradicate TMX-resistant breast cancer in both in vitro and ex vivo experiments.

Thermal Proteome Profiling Identifies Oxidative-Dependent Inhibition of the Transcription of Major Oncogenes as a New Therapeutic Mechanism for Select Anticancer Compounds.

Identification of the molecular mechanism of action (MoA) of bioactive compounds is a crucial step for drug development but remains a challenging task despite recent advances in technology. In this study, we applied multidimensional proteomics, sensitivity correlation analysis, and transcriptomics to identify a common MoA for the anticancer compounds RITA, aminoflavone (AF), and oncrasin-1 (Onc-1). Global thermal proteome profiling revealed that the three compounds target mRNA processing and transcription, thereby attacking a cancer vulnerability, transcriptional addiction.

Cost-effective and robust genotyping using double-mismatch allele-specific quantitative PCR.

For a wide range of diseases, SNPs in the genome are the underlying mechanism of dysfunction. Therefore, targeted detection of these variations is of high importance for early diagnosis and (familial) screenings. While allele-specific PCR has been around for many years, its adoption for SNP genotyping or somatic mutation detection has been hampered by its low discriminating power and high costs.

Expressed repetitive elements are broadly applicable reference targets for normalization of reverse transcription-qPCR data in mice.

Reverse transcription quantitative PCR (RT-qPCR) is the gold standard method for gene expression analysis on mRNA level. To remove experimental variation, expression levels of the gene of interest are typically normalized to the expression level of stably expressed endogenous reference genes. Identifying suitable reference genes and determining the optimal number of reference genes should precede each quantification study.

Inhibition of CDK4/6 as a novel therapeutic option for neuroblastoma.

Background: Neuroblastoma is a neural crest-derived tumor and is the most common cancer in children less than 1 year of age. We hypothesized that aberrations in genes that control the cell cycle could play an important role in the pathogenesis of neuroblastoma and could provide a tractable therapeutic target.

Methods: In this study, we screened 131 genes involved in cell cycle regulation at different levels by analyzing the effect of siRNA-mediated gene silencing on the proliferation of neuroblastoma cells.

Genome wide expression profiling of p53 regulated miRNAs in neuroblastoma.

Restoration of the antitumor activity of p53 could offer a promising approach for the treatment of neuroblastoma. MicroRNAs (miRNAs) are important mediators of p53 activity, but their role in the p53 response has not yet been comprehensively addressed in neuroblastoma. Therefore, we set out to characterize alterations in miRNA expression that are induced by p53 activation in neuroblastoma cells.

CASP8 SNP D302H (rs1045485) is associated with worse survival in MYCN-amplified neuroblastoma patients.

Background: Neuroblastoma is a pediatric cancer that exhibits a wide clinical spectrum ranging from spontaneous regression in low-risk patients to fatal disease in high-risk patients. The identification of single nucleotide polymorphisms (SNPs) may help explain the heterogeneity of neuroblastoma and assist in identifying patients at higher risk for poor survival. SNPs in the TP53 pathway are of special importance, as several studies have reported associations between TP53 pathway SNPs and cancer.

Expressed repeat elements improve RT-qPCR normalization across a wide range of zebrafish gene expression studies.

The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions.

miRBase Tracker: keeping track of microRNA annotation changes.

Unlabelled: Since 2002, information on individual microRNAs (miRNAs), such as reference names and sequences, has been stored in miRBase, the reference database for miRNA annotation. As a result of progressive insights into the miRNome and its complexity, miRBase underwent addition and deletion of miRNA records, changes in annotated miRNA sequences and adoption of more complex naming schemes over time. Unfortunately, miRBase does not allow straightforward assessment of these ongoing miRNA annotation changes, which has resulted in substantial ambiguity regarding miRNA identity and sequence in public literature, in target prediction databases and in content on various commercially available analytical platforms.

Lack of association between MDM2 promoter SNP309 and clinical outcome in patients with neuroblastoma.

While a polymorphism located within the promoter region of the MDM2 proto-oncogene, SNP309 (T > G), has previously been associated with increased risk and aggressiveness of neuroblastoma and other tumor entities, a protective effect has also been reported in certain other cancers. In this study, we evaluated the association of MDM2 SNP309 with outcome in 496 patients with neuroblastoma and its effect on MDM2 expression. No significant difference in overall or event-free survival was observed among patients with neuroblastoma with or without MDM2 SNP309.

Pharmacologic activation of wild-type p53 by nutlin therapy in childhood cancer.

A peculiar feature of several types of childhood cancer is that loss-of-function mutations of the TP53 (p53) tumor suppressor gene are uncommon, in contrast to many adult tumors. As p53 needs to be inactivated in order for tumor cells to survive and thrive, pediatric tumors typically make use of other mechanisms to keep p53 in check. One of the critical negative regulators of p53 is the MDM2 oncoprotein.

The need for transparency and good practices in the qPCR literature.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Effective Alu repeat based RT-Qpcr normalization in cancer cell perturbation experiments.

Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations.

17q24.2 microdeletions: a new syndromal entity with intellectual disability, truncal obesity, mood swings and hallucinations.

Although microdeletions of the long arm of chromosome 17 are being reported with increasing frequency, deletions of chromosome band 17q24.2 are rare. Here we report four patients with a microdeletion encompassing chromosome band 17q24.

Functional analysis of the p53 pathway in neuroblastoma cells using the small-molecule MDM2 antagonist nutlin-3.

Suppression of p53 activity is essential for proliferation and survival of tumor cells. A direct p53-activating compound, nutlin-3, was used in this study, together with p53 mutation analysis, to characterize p53 pathway defects in a set of 34 human neuroblastoma cell lines. We identified 9 cell lines (26%) with a p53 loss-of-function mutation, including 6 missense mutations, 1 nonsense mutation, 1 in-frame deletion, and 1 homozygous deletion of the 3' end of the p53 gene.

Antitumor activity of the selective MDM2 antagonist nutlin-3 against chemoresistant neuroblastoma with wild-type p53.

Background: Restoring p53 function by antagonizing its interaction with the negative regulator MDM2 is an appealing nongenotoxic approach to treating tumors with wild-type p53. Mutational inactivation of p53 is rare in neuroblastoma tumors at diagnosis and occurs in only a subset of multidrug-resistant neuroblastomas.

Methods: The antiproliferative and cytotoxic effect of nutlin-3, a small-molecule MDM2 antagonist, was examined in chemosensitive (UKF-NB-3) and matched chemoresistant neuroblastoma cells with wild-type p53 (UKF-NB-3(r)DOX20) or with mutant p53 (UKF-NB-3(r)VCR10).