Efficacy of bortezomib in sarcomas with high levels of MAP17 (PDZK1IP1

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.España, Consejeria de Ciencia e innovación CTS-6844 and CTS-1848Andalucia, Consejería de Salud PI-0029-2013Andalucia, Consejería de Salud PI-0096-2014Andalucia, Consejería de Salud PI-0306-201

Impact of Wnt/β-Catenin Inhibition on Cell Proliferation through CDC25A Downregulation in Soft Tissue Sarcomas.

The Wnt signaling pathway is an important cellular mechanism for regulating differentiation processes as well as cell cycle events, and different inhibitors of this pathway, for example, PRI-724, are showing promising results in clinical trials for treatment of advanced pancreatic adenocarcinoma or ovarian cancer. Growing evidence suggests that Wnt signaling may also be crucial for tumorigenesis and progression of soft tissue sarcomas (STS), a malignant neoplasm with few therapeutic options at an advanced state. Our study with several STS cell lines and primary cultures shows that inhibition of Wnt/β-catenin signaling with PRI-724 is able to suppress cell viability/proliferation and to increase cell death rates. TCF/β-catenin-mediated transcriptional activity is decreased in treated cells, leading to downregulation of its target genes CCND1 and CDC25A. The latter was critical because its downregulation via siRNA was able to mimic the effect of PRI-724 on cell cycle arrest and cell death induction. An evaluation of NCBI/GenBank data confirmed that CDC25A mRNA is elevated in STS patients. Importantly, PRI-724 in combination with standard STS chemotherapeutics doxorubicin or trabectedin enhanced their antitumoral effect in a synergistic manner according to isobolographic analysis, suggesting that Wnt inhibition through PRI-724 could be a beneficial combination regime in patients with advanced STS.This study was financed by Grupo Español de Investigación en Sarcomas (GEIS) and Fundación Mari PazJiménez Casado. MPC is supported by Programa Estrategia de Emprendimiento y Empleo Joven,Garantía Juvenil(Ministerio de Trabajo, Migraciones y Seguridad Social-SOIB.S

The S-Phase Checkpoint Is Required To Respond to R-Loops Accumulated in THO Mutants▿ †

Cotranscriptional R-loops are formed in yeast mutants of the THO complex, which functions at the interface between transcription and mRNA export. Despite the relevance of R-loops in transcription-associated recombination, the mechanisms by which they trigger recombination are still elusive. In order to understand how R-loops compromise genome stability, we have analyzed the genetic interaction of THO with 26 genes involved in replication, S-phase checkpoint, DNA repair, and chromatin remodeling. We found a synthetic growth defect in double null mutants of THO and S-phase checkpoint factors, such as the replication factor C- and PCNA-like complexes. Under replicative stress, R-loop-forming THO null mutants require functional S-phase checkpoint functions but not double-strand-break repair functions for survival. Furthermore, R-loop-forming hpr1Δ mutants display replication fork progression impairment at actively transcribed chromosomal regions and trigger Rad53 phosphorylation. We conclude that R-loop-mediated DNA damage activates the S-phase checkpoint, which is required for the cell survival of THO mutants under replicative stress. In light of these results, we propose a model in which R-loop-mediated recombination is explained by template switching

RNA polymerase II contributes to preventing transcription-mediated replication fork stalls

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.. Research was funded by grants from the Spanish Ministry of Economy and Competitiveness (Consolider 2010 CSD2007-0015 and BFU2010-16372), the Junta de Andalucía (CVI4567) and the European Union (FEDER). IF-A and JL-B were recipient of predoctoral training grants from the Spanish Ministry of Economy and Competitiveness and the Instituto Carlos III, respectively.Peer Reviewe

Coordinated control of replication and transcription by a SAPK protects genomic integrity

Upon environmental changes or extracellular signals, cells are subjected to marked changes in gene expression 1,2. Dealing with high levels of transcription during replication is critical to prevent collisions between the transcription and replication pathways and avoid recombination events3–5. 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 phase7. 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 complex8–11. Phosphorylation occurs at different sites to those targeted by Mec1 upon DNA damage8,9. 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 (mrc13A) do not delay replication upon stress and show a marked increase in transcription-associated recombination, genomic instability and Rad52 foci. In contrast, mrc13A induces Rad53 and survivalin the presence of hydroxyurea or methylmethanesulphonate. 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.This work was supported by grants from the Spanish Government (BIO2009-07762 and BFU2012-33503 to F.P., BFU2011-26722 to E.d.N., BFU2010- 16372 to A.A., and Consolider Ingenio 2010 programme CSD2007-0015 to F.P. and A.A.) and FP7 UNICELLSYS grant (no. 201142) and the Fundación Marcelino Botín to F.P. F.P. and E.d.N. are recipients of an ICREA Acadèmia (Generalitat de Catalunya).Peer reviewe

Efficacy of bortezomib in sarcomas with high levels of MAP17 (PDZK1IP1)

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.The authors thank the donors and the Andalusian Public Health System Biobank (ISCIII-Red de Biobancos RD12/0036/0017) for the human specimens used in this study. We also thank the Fundacion Oncologica FERO supported by Fundació Josep Botet. The AC laboratory was supported by grants from the Spanish Ministry of Economy and Competitiveness, Plan Nacional de I+D+I 2008-2011, Plan Estatal de I+D+I 2013-2016, ISCIII (Fis: PI12/00137, PI15/00045, RTICC:RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-6844 and CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0029-2013, PI-0306-2012 and PI-0096-2014). JPS is a beneficiary of a Rio Ortega Fellowship.Peer reviewe

Disruption of TCF/β-Catenin Binding Impairs Wnt Signaling and Induces Apoptosis in Soft Tissue Sarcoma Cells.

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 in vitro 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. Mol Cancer Ther; 16(6); 1166-76. ©2017 AACR

RG7112, a Small-Molecule Inhibitor of MDM2, Enhances Trabectedin Response in Soft Tissue Sarcomas

<div><p>ABSTRACT</p><p>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 <i>in vitro</i>. RG7112 significantly synergized with Trabectedin in MDM2-amplified liposarcoma cells, representing a promising new therapeutic strategy for the treatment of sarcomas with MDM2 amplification.</p></div

Coordinated downregulation of Spinophilin and the catalytic subunits of PP1, PPP1CA/B/C, contributes to a worse prognosis in lung cancer

The scaffold protein Spinophilin (Spinophilin, PPP1R9B) is one of the regulatory subunits of phosphatase-1 (PP1), directing it to distinct subcellular locations and targets. The loss of Spinophilin reduces PP1 targeting to pRb, thereby maintaining higher levels of phosphorylated pRb. Spinophilin is absent or reduced in approximately 40% of human lung tumors, correlating with the malignant grade. However, little is known about the relevance of the coordinated activity or presence of Spinophilin and its reported catalytic partners in the prognosis of lung cancer. In the present work, we show that the downregulation of Spinophilin, either by protein or mRNA, is related to a worse prognosis in lung tumors. This effect is more relevant in squamous cell carcinoma, SCC, than in adenocarcinoma. Downregulation of Spinophilin is related to a decrease in the levels of its partners PPP1CA/B/C, the catalytic subunits of PP1. A decrease in these subunits is also related to prognosis in SCC and, in combination with a decrease in Spinophilin, are markers of a poor prognosis in these tumors. The analysis of the genes that correlate to Spinophilin in lung tumors showed clear enrichment in ATP biosynthesis and protein degradation GO pathways. The analysis of the response to several common and pathway-related drugs indicates a direct correlation between the Spinophilin/PPP1Cs ratio and the response to oxaliplatin and bortezomib. This finding indicates that this ratio may be a good predictive biomarker for the activity of the drugs in these tumors with a poor prognosis.This work was funded by grants from the Spanish Ministry of Economy and Competitivity, Plan Estatal de I+D+I 2013-2016, ISCIII (Fis: PI15/00045) and CIBER de Cáncer (CB16/12/00275, CB16/12/00443, CB16/12/00442), co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0096-2014). Especial thanks to the AECC Foundation for also supporting this work. This work has also been made possible by the Fundacion BBVA.Peer reviewe

Nilotinib as Coadjuvant Treatment with Doxorubicin in Patients with Sarcomas: A Phase I Trial of the Spanish Group for Research on Sarcoma.

Purpose: 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.Patients and Methods: 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/m2 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.Results: 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. ABCB1 and ABCC1 RNA expression levels decreased by 58.47-fold and 1.47-fold, respectively, on day 5 of the cycle.Conclusions: 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/m2Clin Cancer Res; 24(21); 5239-49. ©2018 AACR