Androgen receptor phosphorylation at serine 515 by Cdk1 predicts biochemical relapse in prostate cancer patients

<br>Background:Prostate cancer cell growth is dependent upon androgen receptor (AR) activation, which is regulated by specific kinases. The aim of the current study is to establish if AR phosphorylation by Cdk1 or ERK1/2 is of prognostic significance.</br> <br>Methods: Scansite 2.0 was utilised to predict which AR sites are phosphorylated by Cdk1 and ERK1/2. Immunohistochemistry for these sites was then performed on 90 hormone-naive prostate cancer specimens. The interaction between Cdk1/ERK1/2 and AR phosphorylation was investigated in vitro using LNCaP cells.</br><br>Results:Phosphorylation of AR at serine 515 (pAR(S515)) and PSA at diagnosis were independently associated with decreased time to biochemical relapse. Cdk1 and pCdk1(161), but not ERK1/2, correlated with pAR(S515). High expression of pAR(S515) in patients with a PSA at diagnosis of ≤20 ng ml(-1) was associated with shorter time to biochemical relapse (P=0.019). This translated into a reduction in disease-specific survival (10-year survival, 38.1% vs 100%, P<0.001). In vitro studies demonstrated that treatment with Roscovitine (a Cdk inhibitor) caused a reduction in pCdk1(161) expression, pAR(S515)expression and cellular proliferation.</br> <br>Conclusion: In prostate cancer patients with PSA at diagnosis of ≤20 ng ml(-1), phosphorylation of AR at serine 515 by Cdk1 may be an independent prognostic marker.</br&gt

Cell-Cycle Dependence of Transcription Dominates Noise in Gene Expression

The large variability in mRNA and protein levels found from both static and dynamic measurements in single cells has been largely attributed to random periods of transcription, often occurring in bursts. The cell cycle has a pronounced global role in affecting transcriptional and translational output, but how this influences transcriptional statistics from noisy promoters is unknown and generally ignored by current stochastic models. Here we show that variable transcription from the synthetic tetO promoter in S. cerevisiae is dominated by its dependence on the cell cycle. Real-time measurements of fluorescent protein at high expression levels indicate tetO promoters increase transcription rate ~2-fold in S/G2/M similar to constitutive genes. At low expression levels, where tetO promoters are thought to generate infrequent bursts of transcription, we observe random pulses of expression restricted to S/G2/M, which are correlated between homologous promoters present in the same cell. The analysis of static, single-cell mRNA measurements at different points along the cell cycle corroborates these findings. Our results demonstrate that highly variable mRNA distributions in yeast are not solely the result of randomly switching between periods of active and inactive gene expression, but instead largely driven by differences in transcriptional activity between G1 and S/G2/M.GM095733BBBE 103316MIT Startup Fun

ABC-transporter upregulation mediates resistance to the CDK7 inhibitors THZ1 and ICEC0942.

The CDK7 inhibitors (CDK7i) ICEC0942 and THZ1, are promising new cancer therapeutics. Resistance to targeted drugs frequently compromises cancer treatment. We sought to identify mechanisms by which cancer cells may become resistant to CDK7i. Resistant lines were established through continuous drug selection. ABC-transporter copy number, expression and activity were examined using real-time PCR, immunoblotting and flow cytometry. Drug responses were measured using growth assays. ABCB1 was upregulated in ICEC0942-resistant cells and there was cross-resistance to THZ1. THZ1-resistant cells upregulated ABCG2 but remained sensitive to ICEC0942. Drug resistance in both cell lines was reversible upon inhibition of ABC-transporters. CDK7i response was altered in adriamycin- and mitoxantrone-resistant cell lines demonstrating ABC-transporter upregulation. ABCB1 expression correlated with ICEC0942 and THZ1 response, and ABCG2 expression with THZ2 response, in a panel of cancer cell lines. We have identified ABCB1 upregulation as a common mechanism of resistance to ICEC0942 and THZ1, and confirmed that ABCG2 upregulation is a mechanism of resistance to THZ1. The identification of potential mechanisms of CDK7i resistance and differences in susceptibility of ICEC0942 and THZ1 to ABC-transporters, may help guide their future clinical use

Étude de la phosphorylation des récepteurs nucléaires par le facteur de transcription-réparation TFIIH

La transcription des gènes codant les protéines est un mécanisme complexe. Parmi les facteurs impliqués dans ce processus on trouve TFIIH, qui est essentiel à l étape d initiation de la transcription. En plus de ce rôle, TFIIH est indispensable aux mécanismes de réparation de l ADN. Ces derniers maintiennent l intégrité de l information génétique et par conséquent des produits protéiques de l expression des gènes. Des mutations de TFIIH sont d ailleurs à l origine de maladies génétiques dont les symptômes ont d abord été associés à une défaillance de ces mécanismes de réparation : le Xeroderma pigmentosum (XP), la trichothiodystrophie (TTD) et le syndrome de Cockayne (CS). Cependant d autres symptômes développés par ces patients suggèrent plutôt une origine endocrinienne. C est de ce constat qu est née il y a dix ans l hypothèse d une implication de TFIIH dans la transcription activée par les récepteurs nucléaires, médiateurs de l effet des hormones au niveau transcriptionnel. Durant ma thèse, je me suis particulièrement intéressé à la phosphorylation des récepteurs nucléaires par la kinase cdk7 de TFIIH, étape indispensable à l expression des gènes régulés par les hormones. Nous pensions découvrir un rôle à cette phosphorylation qui s appliquerait à tous les récepteurs nucléaires. En fait, il s est avéré qu elle régule plusieurs mécanismes comme les concentrations cellulaires du récepteur aux androgènes ou la fixation à l ADN du récepteur de la vitamine D. Nous avons aussi montré qu XPG, une protéine qu on pensait spécifique de la réparation de l ADN, participe à la régulation hormonale en stabilisant TFIIH et en lui permettant de phosphoryler efficacement le récepteur aux œstrogènes. Ces résultats montrent à quel point la régulation transcriptionnelle de la réponse hormonale est complexe, ce qui explique en partie la diversité des symptômes développés par les patients XP, TTD et CS.The transcription of proteins-coding genes is a complex stepwise mechanism. Among the factors involved in this process, TFIIH is essential for the initiation step of the transcription. In addition to this role, TFIIH is essential for the DNA repair process. These mechanisms maintain the integrity of genetic information and therefore of the protein products of gene expression. Mutations of TFIIH are the causes of genetic diseases whose symptoms were first associated with a failure of DNA repair: the Xeroderma pigmentosum (XP), the trichothiodystrophy (TTD) and the Cockayne syndrome (CS). But other symptoms are more reminiscent of those of endocrine diseases. That rose, ten years ago, the assumption of an involvement of TFIIH in nuclear receptors mediated transcription. During my PhD, I participated in the study of the involvement of TFIIH in the hormonal response. Especially, I was interested in the role played by the phosphorylation of nuclear receptors by the cdk7 kinase of TFIIH, a step that was shown to be essential for the expression of hormone-regulated genes. We thought to discover a single role for this phosphorylation that would apply to all nuclear receptors. Actually, it turned out that it regulates several mechanisms such as cellular concentrations of the Androgen Receptor or the DNA binding capacity of Vitamin D Receptor. We have also shown that XPG, a protein believed to be specific for DNA repair, participates in the hormonal regulation by stabilizing TFIIH and allowing it to efficiently phosphorylate the Estrogen Receptor. These results show how the transcriptional regulation of the hormonal response is complex, which partly explains the wide variety of symptoms developed by XP, TTD and CS patients.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Cell Cycle-Dependent Transcription: The Cyclin Dependent Kinase Cdk1 Is a Direct Regulator of Basal Transcription Machineries

The cyclin-dependent kinase Cdk1 is best known for its function as master regulator of the cell cycle. It phosphorylates several key proteins to control progression through the different phases of the cell cycle. However, studies conducted several decades ago with mammalian cells revealed that Cdk1 also directly regulates the basal transcription machinery, most notably RNA polymerase II. More recent studies in the budding yeast Saccharomyces cerevisiae have revisited this function of Cdk1 and also revealed that Cdk1 directly controls RNA polymerase III activity. These studies have also provided novel insight into the physiological relevance of this process. For instance, cell cycle-stage-dependent activity of these complexes may be important for meeting the increased demand for various proteins involved in housekeeping, metabolism, and protein synthesis. Recent work also indicates that direct regulation of the RNA polymerase II machinery promotes cell cycle entry. Here, we provide an overview of the regulation of basal transcription by Cdk1, and we hypothesize that the original function of the primordial cell-cycle CDK was to regulate RNAPII and that it later evolved into specialized kinases that govern various aspects of the transcription machinery and the cell cycle

Centromeres License the Mitotic Condensation of Yeast Chromosome Arms

During mitosis, chromatin condensation shapes chromosomes as separate, rigid, and compact sister chromatids to facilitate their segregation. Here, we show that, unlike wild-type yeast chromosomes, non-chromosomal DNA circles and chromosomes lacking a centromere fail to condense during mitosis. The centromere promotes chromosome condensation strictly in cis through recruiting the kinases Aurora B and Bub1, which trigger the autonomous condensation of the entire chromosome. Shugoshin and the deacetylase Hst2 facilitated spreading the condensation signal to the chromosome arms. Targeting Aurora B to DNA circles or centromere-ablated chromosomes or releasing Shugoshin from PP2A-dependent inhibition bypassed the centromere requirement for condensation and enhanced the mitotic stability of DNA circles. Our data indicate that yeast cells license the chromosome-autonomous condensation of their chromatin in a centromere-dependent manner, excluding from this process non-centromeric DNA and thereby inhibiting their propagation

Desumoylation of RNA polymerase III lies at the core of the Sumo stress response in yeast

Post-translational modification by small ubiquitin-like modifier (Sumo) regulates many cellular processes, including the adaptive response to various types of stress, referred to as the Sumo stress response (SSR). However, it remains unclear whether the SSR involves a common set of core proteins regardless of the type of stress or whether each particular type of stress induces a stress-specific SSR that targets a unique, largely nonoverlapping set of Sumo substrates. In this study, we used MS and a Gene Ontology approach to identify differentially sumoylated proteins during heat stress, hyperosmotic stress, oxidative stress, nitrogen starvation, and DNA alkylation in Saccharomyces cerevisiae cells. Our results indicate that each stress triggers a specific SSR signature centered on proteins involved in transcription, translation, and chromatin regulation. Strikingly, whereas the various stress-specific SSRs were largely nonoverlapping, all types of stress tested here resulted in desumoylation of subunits of RNA polymerase III, which correlated with a decrease in tRNA synthesis. We conclude that desumoylation and subsequent inhibition of RNA polymerase III constitutes the core of all stress-specific SSRs in yeast

Mapping the synthetic dosage lethality network of CDK1/CDC28

Cdk1 (Cdc28 in yeast) is a cyclin-dependent kinase (CDK) essential for cell cycle progression and cell division in normal cells. However, CDK activity also underpins proliferation of tumor cells, making it a relevant study subject. While numerous targets and processes regulated by Cdc28 have been identified, the exact functions of Cdc28 are only partially understood. To further explore the functions of Cdc28, we systematically overexpressed ∼4800 genes in wild-type (WT) cells and in cells with artificially reduced Cdc28 activity. This screen identified 366 genes that, when overexpressed, specifically compromised cell viability under conditions of reduced Cdc28 activity. Consistent with the crucial functions of Cdc28 in cell cycle regulation and chromosome metabolism, most of these genes have functions in the cell cycle, DNA replication, and transcription. However, a substantial number of genes control processes not directly associated with the cell cycle, indicating that Cdc28 may also regulate these processes. Finally, because the dataset was enriched for direct Cdc28 targets, the results from this screen will aid in identifying novel targets and process regulated by Cdc28