NONO and RALY proteins are required for YB-1 oxaliplatin induced resistance in colon adenocarcinoma cell lines

<p>Abstract</p> <p>Background</p> <p>YB-1 is a multifunctional protein that affects transcription, splicing, and translation. Overexpression of YB-1 in breast cancers causes cisplatin resistance. Recent data have shown that YB-1 is also overexpress in colorectal cancer. In this study, we tested the hypothesis that YB-1 also confers oxaliplatin resistance in colorectal adenocarcinomas.</p> <p>Results</p> <p>We show for the first time that transfection of YB-1 cDNA confers oxaliplatin resistance in two colorectal cancer cell lines (SW480 and HT29 cell lines). Furthermore, we identified by mass spectrometry analyses important YB-1 interactors required for such oxaliplatin resistance in these colorectal cancer cell lines. A tagged YB-1 construct was used to identify proteins interacting directly to YB-1 in such cells. We then focused on proteins that are potentially involved in colorectal cancer progression based on the Oncomine microarray database. Genes encoding for these YB-1 interactors were also examined in the public NCBI comparative genomic hybridization database to determine whether these genes are localized to regions of chromosomes rearranged in colorectal cancer tissues. From these analyses, we obtained a list of proteins interacting with YB-1 and potentially involved in oxaliplatin resistance. Oxaliplatin dose response curves of SW480 and HT29 colorectal cancer cell lines transfected with several siRNAs corresponding to each of these YB-1 interactors were obtained to identify proteins significantly affecting oxaliplatin sensitivity upon gene silencing. Only the depletion of either NONO or RALY sensitized both colorectal cancer cell lines to oxaliplatin. Furthermore, depletion of NONO or RALY sensitized otherwise oxaliplatin resistant overexpressing YB-1 SW480 or HT29 cells.</p> <p>Conclusion</p> <p>These results suggest knocking down NONO or RALY significant counteracts oxaliplatin resistance in colorectal cancers overexpressing the YB-1 protein.</p

Mps1 Phosphorylates Its N-Terminal Extension to Relieve Autoinhibition and Activate the Spindle Assembly Checkpoint

Monopolar spindle 1 (Mps1) is a conserved apical kinase in the spindle assembly checkpoint (SAC) that ensures accurate segregation of chromosomes during mitosis. Mps1 undergoes extensive auto- and transphosphorylation, but the regulatory and functional consequences of these modifications remain unclear. Recent findings highlight the importance of intermolecular interactions between the N-terminal extension (NTE) of Mps1 and the Hec1 subunit of the NDC80 complex, which control Mps1 localization at kinetochores and activation of the SAC. Whether the NTE regulates other mitotic functions of Mps1 remains unknown. Here, we report that phosphorylation within the NTE contributes to Mps1 activation through relief of catalytic autoinhibition that is mediated by the NTE itself. Moreover, we find that this regulatory NTE function is independent of its role in Mps1 kinetochore recruitment. We demonstrate that the NTE autoinhibitory mechanism impinges most strongly on Mps1-dependent SAC functions and propose that Mps1 activation likely occurs sequentially through dimerization of a “prone-to-autophosphorylate” Mps1 conformer followed by autophosphorylation of the NTE prior to maximal kinase activation segment trans-autophosphorylation. Our observations underline the importance of autoregulated Mps1 activity in generation and maintenance of a robust SAC in human cells

The Werner syndrome gene product (WRN): a repressor of hypoxia-inducible factor-1 activity

Hypoxia-inducible factor-1 (HIF-1) is a decisive element for the transcriptional regulation of genes essential for adaptation to low oxygen conditions. HIF-1 is also implicated in the molecular mechanisms of ageing. Here, we show that the cellular depletion of WRN protein (by siRNA targeting) leads to increased HIF-1 complex stabilization and activation. HIF-1 activation in the absence of WRN involves the generation of mitochondrial reactive oxygen species (mtROS) since SkQ1, a mitochondrial-targeted antioxidant, and stigmatellin, an inhibitor of mitochondrial complex III, blocked increased HIF-1 levels. Ascorbate, an essential co-factor involved in HIF-1 stability, was decreased in WRN-depleted cells. Interestingly, expression levels of GLUT1, a known dehydroascorbic acid transporter, were also decreased in WRN-depleted cells. Ascorbate supplementation of WRN-depleted cells led to a dose-dependent inhibition of HIF-1 activation. These results indicate that WRN protein regulates HIF-1 activation by affecting mitochondrial ROS production and intracellular ascorbate levels. This work provides a novel mechanistic link between HIF-1 activity and different age-associated pathologies

Metabolic and Phenotypic Differences between Mice Producing a Werner Syndrome Helicase Mutant Protein and Wrn Null Mice

<div><p>Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter mean life span. In contrast, mice lacking the entire Wrn protein (i.e. Wrn null mice) do not exhibit a premature aging phenotype. In this study, we used a targeted mass spectrometry-based metabolomic approach to identify serum metabolites that are differentially altered in young Wrn helicase mutant and Wrn null mice. An antibody-based quantification of 43 serum cytokines and markers of cardiovascular disease risk complemented this study. We found that Wrn helicase mutants exhibited elevated and decreased levels, respectively, of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine IL-18. Wrn helicase mutants also exhibited an increase in serum hydroxyproline and plasminogen activator inhibitor-1, markers of extracellular matrix remodeling of the vascular system and inflammation in aging. We also observed an abnormal increase in the ratio of very long chain to short chain lysophosphatidylcholines in the Wrn helicase mutants underlying a peroxisome perturbation in these mice. Remarkably, the Wrn mutant helicase protein was mislocalized to the endoplasmic reticulum and the peroxisomal fractions in liver tissues. Additional analyses with mouse embryonic fibroblasts indicated a severe defect of the autophagy flux in cells derived from Wrn helicase mutants compared to wild type and Wrn null animals. These results indicate that the deleterious effects of the helicase-deficient Wrn protein are mediated by the dysfunction of several cellular organelles.</p></div

The Werner syndrome protein affects the expression of genes involved in adipogenesis and inflammation in addition to cell cycle and DNA damage responses.

Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies. The protein deficient in WS (WRN) is a RecQ-type DNA helicase involved in DNA repair, replication, telomere maintenance and transcription. However, precisely how WRN deficiency leads to the numerous WS pathologies is still unknown. Here we use short-term siRNA-based inhibition of WRN to test the direct consequences of its loss on gene expression. Importantly, this short-term knock down of WRN protein level was sufficient to trigger an expression profile resembling fibroblasts established from old donor patients. In addition, this treatment altered sets of genes involved in 14 distinct biological pathways. Besides the already known impact of WRN on DNA replication, DNA repair, the p21/p53 pathway, and cell cycle, gene set enrichment analyses of our microarray data also uncover significant impact on the MYC, E2F, cellular E2A and ETV5 transcription factor pathways as well as adipocyte differentiation, HIF1, NF kappa B and IL-6 pathways. Finally, short-term siRNA-based inhibition of mouse Wrn expression in the pre-adipocyte cell line 3T3-L1 confirmed the impact of WRN on adipogenesis. These results are consistent with the pro-inflammatory status and lipid abnormalities observed in WS patients. This approach thus identified new effectors of WRN activity that might contribute to the WS phenotype

Incidence of each phenotype in old wild type and <i>Wrn</i>^<i>-/-</i> mice.

<p>Incidence of each phenotype in old wild type and <i>Wrn</i>^<i>-/-</i> mice.</p

Fatty acids, sphingomyelins, and eicosanoids showing significant differences between the different <i>Wrn</i> mutants and WT mice.

<p>Values are in μM ± SD except for PGE2 which is in nM.</p><p>Fatty acids, sphingomyelins, and eicosanoids showing significant differences between the different <i>Wrn</i> mutants and WT mice.</p

Serum levels of six serum metabolites significantly altered between <i>Wrn</i>^{<i>Δhel/Δhel</i>} and <i>Wrn</i>^<i>-/-</i> mice.

<p>(A) Hydroxyproline (OH-Pro); (B) Phosphatidylcholine PC aa C30:2; (C) Carnosine; (D) Acetylcarnitine; (E) Sphingomyelin SM C24:1; (F) Sphingomyelin SM (OH) C22:1. Tukey post ANOVA test <i>P</i>-values are shown (*<i>P</i> < 0.05 and **<i>P</i> < 0.01). Bars in all histograms represent SD. N = 6 males for each cohort.</p

Serum levels of three serum cytokines and one cardiovascular risk factor significantly altered in <i>Wrn</i>^{<i>Δhel/Δhel</i>} and <i>Wrn</i>^<i>-/-</i> mice.

<p>(A) IL-10; (B) IL-18; (C) MIP-1α; (D) PAI-1. Tukey post ANOVA test <i>P</i>-values are shown (*<i>P</i> < 0.05 and **<i>P</i> < 0.01). Bars in all histograms represent SD. The number of mice in each cohort is indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140292#pone.0140292.s010" target="_blank">S2 Table</a> for each serum cytokine.</p