The bromodomain protein BRD4 regulates splicing during heat shock.

The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies.

PRMT1-mediated arginine methylation controls ATXN2L localization.

Arginine methylation is a posttranslational modification that is of importance in diverse cellular processes. Recent proteomic mass spectrometry studies reported arginine methylation of ataxin-2-like (ATXN2L), the paralog of ataxin-2, a protein that is implicated in the neurodegenerative disorder spinocerebellar ataxia type 2. Here, we investigated the methylation state of ATXN2L and its significance for ATXN2L localization. We first confirmed that ATXN2L is asymmetrically dimethylated in vivo, and observed that the nuclear localization of ATXN2L is altered under methylation inhibition. We further discovered that ATXN2L associates with the protein arginine-N-methyltransferase 1 (PRMT1). Finally, we showed that neither mutation of the arginine-glycine-rich motifs of ATXN2L nor methylation inhibition alters ATXN2L localization to stress granules, suggesting that methylation of ATXN2L is probably not mandatory.

5-Fluorouracil affects assembly of stress granules based on RNA incorporation.

The antimetabolite 5-fluorouracil is a widely used chemotherapeutic for the treatment of several solid cancers. However, resistance to 5-fluorouracil remains a major drawback in its clinical use. In this study we report that treatment of HeLa cells with 5-fluorouracil resulted in de novo assembly of stress granules. Moreover, we revealed that stress granule assembly under stress conditions as well as disassembly is altered in cells treated with 5-fluorouracil. Notably, we discovered that RACK1, a protein mediating cell survival and apoptosis, is a component of 5-fluorouracil-induced stress granules. To explore the mode of action of 5-fluorouracil accountable for de novo stress granule assembly, we analyzed 5-fluorouracil metabolites and noticed that stress granule assembly is caused by RNA, not DNA incorporating 5-fluorouracil metabolites. Interestingly, we observed that other RNA incorporating drugs also cause assembly of stress granules. Thus, our results suggest that incorporation of chemotherapeutics into RNA may result in stress granule assembly with potential significance in chemoresistance.

High-throughput miRNA and mRNA sequencing of paired colorectal normal, tumor and metastasis tissues and bioinformatic modeling of miRNA-1 therapeutic applications.

MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are, so far, hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We validated six miRNAs in a large tissue screen containing 16 additional tumor entities and identified miRNA-1, miRNA-129, miRNA-497 and miRNA-215 as constantly de-regulated within the majority of cancers. Of these, we investigated miRNA-1 as representative in a systems-biology simulation of cellular cancer models implemented in PyBioS and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system, miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options.

Ataxin-2-like is a regulator of stress granules and processing bodies.

Paralogs for several proteins implicated in neurodegenerative disorders have been identified and explored to further facilitate the identification of molecular mechanisms contributing to disease pathogenesis. For the disease-causing protein in spinocerebellar ataxia type 2, ataxin-2, a paralog of unknown function, termed ataxin-2-like, has been described. We discovered that ataxin-2-like associates with known interaction partners of ataxin-2, the RNA helicase DDX6 and the poly(A)-binding protein, and with ataxin-2 itself. Furthermore, we found that ataxin-2-like is a component of stress granules. Interestingly, sole ataxin-2-like overexpression led to the induction of stress granules, while a reduction of stress granules was detected in case of a low ataxin-2-like level. Finally, we observed that overexpression of ataxin-2-like as well as its reduction has an impact on the presence of microscopically visible processing bodies. Thus, our results imply a functional overlap between ataxin-2-like and ataxin-2, and further indicate a role for ataxin-2-like in the regulation of stress granules and processing bodies.

Targeted high throughput sequencing in clinical cancer settings: formaldehyde fixed-paraffin embedded (FFPE) tumor tissues, input amount and tumor heterogeneity.

BACKGROUND: Massively parallel sequencing technologies have brought an enormous increase in sequencing throughput. However, these technologies need to be further improved with regard to reproducibility and applicability to clinical samples and settings. METHODS: Using identification of genetic variations in prostate cancer as an example we address three crucial challenges in the field of targeted re-sequencing: Small nucleotide variation (SNV) detection in samples of formalin-fixed paraffin embedded (FFPE) tissue material, minimal amount of input sample and sampling in view of tissue heterogeneity. RESULTS: We show that FFPE tissue material can supplement for fresh frozen tissues for the detection of SNVs and that solution-based enrichment experiments can be accomplished with small amounts of DNA with only minimal effects on enrichment uniformity and data variance.Finally, we address the question whether the heterogeneity of a tumor is reflected by different genetic alterations, e.g. different foci of a tumor display different genomic patterns. We show that the tumor heterogeneity plays an important role for the detection of copy number variations. CONCLUSIONS: The application of high throughput sequencing technologies in cancer genomics opens up a new dimension for the identification of disease mechanisms. In particular the ability to use small amounts of FFPE samples available from surgical tumor resections and histopathological examinations facilitates the collection of precious tissue materials. However, care needs to be taken in regard to the locations of the biopsies, which can have an influence on the prediction of copy number variations. Bearing these technological challenges in mind will significantly improve many large-scale sequencing studies and will - in the long term - result in a more reliable prediction of individual cancer therapies.

Effects of EpCAM overexpression on human breast cancer cell lines.

BACKGROUND: Recently, EpCAM has attracted major interest as a target for antibody- and vaccine-based cancer immunotherapies. In breast cancer, the EpCAM antigen is overexpressed in 30-40% of all cases and this increased expression correlates with poor prognosis. The use of EpCAM-specific monoclonal antibodies is a promising treatment approach in these patients. METHODS: In order to explore molecular changes following EpCAM overexpression, we investigated changes of the transcriptome upon EpCAM gene expression in commercially available human breast cancer cells lines Hs578T and MDA-MB-231. To assess cell proliferation, a tetrazolium salt based assay was performed. A TCF/LEF Reporter Kit was used to measure the transcriptional activity of the Wnt/ß-catenin pathway. To evaluate the accumulation of ß-catenin in the nucleus, a subcellular fractionation assay was performed. RESULTS: For the first time we could show that expression profiling data of EpCAM transfected cell lines Hs578TEpCAM and MDA-MB-231EpCAM indicate an association of EpCAM overexpression with the downregulation of the Wnt signaling inhibitors SFRP1 and TCF7L2. Confirmation of increased Wnt signaling was provided by a TCF/LEF reporter kit and by the finding of the nuclear accumulation of ß-catenin for MDA-MB-231 EpCAM but not Hs578T EpCAM cells. In Hs578T cells, an increase of proliferation and chemosensitivity to Docetaxel was associated with EpCAM overexpression. CONCLUSIONS: These data show a cell type dependent modification of Wnt signaling components after EpCAM overexpression in breast cancer cell lines, which results in marginal functional changes. Further investigations on the interaction of EpCAM with SFRP1 and TCF7L2 and on additional factors, which may be causal for changes upon EpCAM overexpression, will help to characterize unique molecular properties of EpCAM-positive breast cancer cells.

The KRAB-containing zinc-finger transcriptional regulator ZBRK1 activates SCA2 gene transcription through direct interaction with its gene product, ataxin-2.

Gene transcription is controlled by transcriptional regulators acting with specific co-regulators to allow gene activation and repression. Here, we report the identification of the KRAB-containing zinc-finger transcriptional regulator, ZBRK1, as an interaction partner of the SCA2 gene product ataxin-2. Furthermore, we discovered that an elevated ZBRK1 level resulted in increased ataxin-2 levels, whereas interference on transcriptional and protein levels of ZBRK1 yielded reduced ataxin-2 levels, suggesting that a complex comprising ZBRK1 and ataxin-2 regulates SCA2 gene transcription. A bioinformatic analysis utilizing the known ZBRK1 consensus DNA-binding motif revealed ZBRK1-binding sites in the SCA2 promoter. These predicted sites were experimentally validated by chromatin-immunoprecipitation experiments along with luciferase-based promoter analyses corroborating that SCA2 gene transcription is controlled by a ZBRK1/ataxin-2 complex. Finally, we demonstrate that SCA2 gene transcription is significantly reduced in colon tumors possessing low ZBRK1 transcripts. Thus, our results provide first evidence that ataxin-2 acts as a co-regulator of ZBRK1 activating its own transcription, thereby representing the first identified ZBRK1 co-activator.

Huntington's disease: From molecular basis to therapeutic advances.

Huntington's disease is an autosomal dominant genetic neurodegenerative disorder, which is characterized by progressive motor dysfunction, emotional disturbances, dementia, and weight loss. The disease is caused by pathological CAG-triplet repeat extension(s), encoding polyglutamines, within the gene product, huntingtin. Huntingtin is ubiquitously expressed through the body and is a protein of uncertain molecular function(s). Mutant huntingtin, containing pathologically extended polyglutamines causes the earliest and most dramatic neuropathologic changes in the neostriatum and cerebral cortex. Extended polyglutamines confer structural conformational changes to huntingtin, which gains novel properties, resulting in aberrant interactions with multiple cellular components. The diverse and variable aberrations mediated by mutant huntingtin perturb many cellular functions essential for neuronal homeostasis and underlie pleiotropic mechanisms of Huntington's disease pathogenesis. The only approved drug for Huntington's disease is a symptomatic treatment, tetrabenazine; thus, novel neuroprotective strategies, slowing, blocking and possibly reversing disease progression, are vital for developing effective therapies.

A catabolic block does not sufficiently explain how 2-deoxy-D-glucose inhibits cell growth.

The glucose analogue 2-deoxy-D-glucose (2-DG) restrains growth of normal and malignant cells, prolongs the lifespan of C. elegans, and is widely used as a glycolytic inhibitor to study metabolic activity with regard to cancer, neurodegeneration, calorie restriction, and aging. Here, we report that separating glycolysis and the pentose phosphate pathway highly increases cellular tolerance to 2-DG. This finding indicates that 2-DG does not block cell growth solely by preventing glucose catabolism. In addition, 2-DG provoked similar concentration changes of sugar-phosphate intermediates in wild-type and 2-DG-resistant yeast strains and in human primary fibroblasts. Finally, a genome-wide analysis revealed 19 2-DG-resistant yeast knockouts of genes implicated in carbohydrate metabolism and mitochondrial homeostasis, as well as ribosome biogenesis, mRNA decay, transcriptional regulation, and cell cycle. Thus, processes beyond the metabolic block are essential for the biological properties of 2-DG.

Ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6 and interferes with P-bodies and stress granules.

Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation.