Transcription is a major contributor to genome instability. A main cause of transcription‐associated instability relies on the capacity of transcription to stall replication. However, we know little ...of the possible role, if any, of the RNA polymerase (RNAP) in this process. Here, we analyzed 4 specific yeast RNAPII mutants that show different phenotypes of genetic instability including hyper‐recombination, DNA damage sensitivity and/or a strong dependency on double‐strand break repair functions for viability. Three specific alleles of the RNAPII core, rpb1‐1, rpb1‐S751F and rpb9∆, cause a defect in replication fork progression, compensated for by additional origin firing, as the main action responsible for instability. The transcription elongation defects of rpb1‐S751F and rpb9∆ plus our observation that rpb1‐1 causes RNAPII retention on chromatin suggest that RNAPII could participate in facilitating fork progression upon a transcription‐replication encounter. Our results imply that the RNAPII or ancillary factors actively help prevent transcription‐associated genome instability.
Synopsis
Collisions between RNA transcription and DNA replication machineries impair replication fork (RF) progression and cause DNA breaks, demanding homologous recombination for repair and RF restart. Identification and characterization of specific yeast RNA polymerase II mutations that enhance DNA RF stalling and trigger hyper‐recombination suggests that increased RNAP II retention at transcription sites may contribute to genomic instability and that eukaryotic RNAP II is more than a passive entity in the management of transcription–replication encounters.
Specific RNAP II mutants in Saccharomyces cerevisiae, rpb1‐1, rpb1S71F and rbp9Δ show an increase in genetic instability as detected by hyper‐recombination, DNA damage sensitivity, and dependency on DSB repair functions for viability.
Impaired RF progression in these RNAP II mutants can be compensated by additional origin firing.
Under replication stress, checkpoint‐activating DNA lesions accumulate in these mutant cells and are repaired only with considerable delay.
RNase H1 overexpression does not suppress genome instability in rpb1 mutants, indicating that an increase in R‐loops is not a main determinant of RF stalling.
The causative rpb1‐1 mutation increases retention of RNAP II at the site of transcription.
Identification of specific yeast RNA polymerase II mutations that cause genomic instability by enhancing collisions with the DNA replication machinery suggests that eukaryotic RNAP II is more than a passive entity in the management of transcription–replication encounters.
Soft tissue sarcomas (STS) are malignant tumors of mesenchymal origin and represent around 1% of adult cancers, being a very heterogeneous group of tumors with more than 50 different subtypes. The ...Wnt signaling pathway is involved in the development and in the regulation, self-renewal, and differentiation of mesenchymal stem cells, and plays a role in sarcomagenesis. In this study, we have tested pharmacologic inhibition of Wnt signaling mediated by disruption of TCF/β-catenin binding and AXIN stabilization, being the first strategy more efficient in reducing cell viability and downstream effects. We have shown that disruption of TCF/β-catenin binding with PKF118-310 produces
antitumor activity in a panel of prevalent representative STS cell lines and primary cultures. At the molecular level, PKF118-310 treatment reduced β-catenin nuclear localization, reporter activity, and target genes, resulting in an increase in apoptosis. Importantly, combination of PKF118-310 with doxorubicin resulted in enhanced reduction of cell viability, suggesting that Wnt inhibition could be a new combination regime in these patients. Our findings support the usefulness of Wnt inhibitors as new therapeutic strategies for the prevalent STS.
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Upon environmental changes or extracellular signals, cells are subjected to marked changes in gene expression. Dealing with high levels of transcription during replication is critical to prevent ...collisions between the transcription and replication pathways and avoid recombination events. In response to osmostress, hundreds of stress-responsive genes are rapidly induced by the stress-activated protein kinase (SAPK) Hog1 (ref. 6), even during S phase. Here we show in Saccharomyces cerevisae that a single signalling molecule, Hog1, coordinates both replication and transcription upon osmostress. Hog1 interacts with and phosphorylates Mrc1, a component of the replication complex. Phosphorylation occurs at different sites to those targeted by Mec1 upon DNA damage. Mrc1 phosphorylation by Hog1 delays early and late origin firing by preventing Cdc45 loading, as well as slowing down replication-complex progression. Regulation of Mrc1 by Hog1 is completely independent of Mec1 and Rad53. Cells carrying a non-phosphorylatable allele of MRC1 (mrc1(3A)) do not delay replication upon stress and show a marked increase in transcription-associated recombination, genomic instability and Rad52 foci. In contrast, mrc1(3A) induces Rad53 and survival in the presence of hydroxyurea or methyl methanesulphonate. Therefore, Hog1 and Mrc1 define a novel S-phase checkpoint independent of the DNA-damage checkpoint that permits eukaryotic cells to prevent conflicts between DNA replication and transcription, which would otherwise lead to genomic instability when both phenomena are temporally coincident.
MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Selected sarcoma subtypes are being treated with Trabectedin in second line, which promotes DNA damage and p53-dependent ...apoptosis. The aim of this study was to evaluate the improvement of Trabectedin response with MDM2 inhibitors in soft tissue sarcomas. The antitumor effects of Trabectedin, Nutlin-3A and RG7112 as single agents or in combination were examined in vitro. RG7112 significantly synergized with Trabectedin in MDM2-amplified liposarcoma cells, representing a promising new therapeutic strategy for the treatment of sarcomas with MDM2 amplification.
Nilotinib plus doxorubicin showed to be synergistic regarding apoptosis in several sarcoma cell lines. A phase I/II trial was thus designed to explore the feasibility of nilotinib as coadjuvant of ...doxorubicin by inhibiting MRP-1/P-gp efflux activity. The phase I part of the study is presented here.
Nilotinib 400 mg/12 hours was administered in fixed dose from day 1 to 6, and doxorubicin on day 5 of each cycle. Three dose escalation levels for doxorubicin at 60, 65, and 75 mg/m
were planned. Cycles were repeated every 3 weeks for a total of 4 cycles. Eligible subtypes were retroperitoneal liposarcoma, leiomyosarcoma, and unresectable/metastatic high-grade chondrosarcoma.
Thirteen patients were enrolled: 7 chondrosarcoma, 4 liposarcoma, and 2 leiomyosarcoma. In 46 cycles administered, the most relevant grade 3/4 adverse effects per patient were neutropenia 54%, febrile neutropenia 15%, and asthenia 8%. No cardiac toxicity was observed. Only one dose-limiting toxicity (febrile neutropenia) was reported in the third dose level. With regard to efficacy, 1 partial response (1 liposarcoma), 9 stable diseases (5 chondrosarcoma, 2 liposarcoma, 1 leiomyosarcoma), and 3 progressive diseases (2 chondrosarcoma and 1 leiomyosarcoma) were present.
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RNA expression levels decreased by 58.47-fold and 1.47-fold, respectively, on day 5 of the cycle.
Combination of MRP-1/P-gp inhibitor, nilotinib, as coadjuvant with doxorubicin is feasible; it appears not to add substantial toxicity compared with doxorubicin alone. Pharmacodynamic study supports this concept. The recommended dose for the phase II part for doxorubicin was 75 mg/m
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Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most ...sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness, and new treatment approaches are needed. MAP17 is a small non-glycosylated membrane protein overexpressed in carcinomas. The levels of MAP17 could be used as a prognostic marker to predict the response to bortezomib in hematological malignancies and in breast tumors. Therefore, we analyzed the expression of this oncogene in sarcomas and its relationship with clinico-pathological features, as well as tested whether it can be used as a new biomarker to predict the therapeutic response to bortezomib and new therapies for sarcomas. We found that the levels of MAP17 were related to clinical features and poor survival in a cohort of 69 patients with different sarcoma types, not being restricted to any special subtype of tumor. MAP17 expression is associated with poor overall survival (p<0.001) and worse disease-free survival (p=0.002). Cell lines with high levels of MAP17 show a better response to bortezomib in vitro. Furthermore, patient-derived xenografts (PDX) with high levels of MAP17 respond to bortezomib in vivo. Our results showed that this response is due to the lower levels of NFκB and autophagy activation. Therefore, we suggest that MAP17 is a new biomarker to predict the efficacy of bortezomib as a new therapy for sarcomas.
Coordination of DNA replication with DNA-damage sensing, repair, and cell cycle progression ensures with high probability genome integrity during cell divisions, thus preventing mutations and DNA ...rearrangements. Such events are usually associated with pathological disorders, including premature aging, various cancer predispositions, and inherited diseases (Aguilera & Gómez-González, 2008). One important type of genome instability is that associated with transcription. Transcription of a DNA sequence increases its frequency of recombination, a phenomenon referred to as transcription-associated recombination (TAR). We will provide new data on the analysis of protein factors involved in transcription and RNA export that provide evidence that TAR is mediated by replication impairment and that it can be further enhanced by dysfunction of replication and repair factors in yeast and human cells via R-loops. Notably, our study provides a first connection between RNA processing, R-loops, and chromatin structure that will be discussed. In addition, we have analyzed a number of mutants of the yeast RNA polymerase II that are dependent on double-strand repair functions for their viability. We show that in these mutants, replication fork progression and DNA-damage response are impaired by the presence of strong transcription-dependent obstacles. Our data provide evidence that specific alterations of mRNP biogenesis/export machinery lead to different ways by which chromatin structure and progression of replication can be compromised leading to genome instability.
MDM2 is a critical negative regulator of the p53 tumor suppressor protein. Selected sarcoma subtypes are being treated with Trabectedin in second line, which promotes DNA damage and p53-dependent ...apoptosis. The aim of this study was to evaluate the improvement of Trabectedin response with MDM2 inhibitors in soft tissue sarcomas. The antitumor effects of Trabectedin, Nutlin-3A and RG7112 as single agents or in combination were examined in vitro. RG7112 significantly synergized with Trabectedin in MDM2-amplified liposarcoma cells, representing a promising new therapeutic strategy for the treatment of sarcomas with MDM2 amplification.
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