A novel regulatory mechanism of MAP kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine phosphatase

Protein tyrosine phosphatase PTP-SL retains mitogen-activated protein (MAP) kinases in the cytoplasm in an inactive form by association through a kinase interaction motif (KIM) and tyrosine dephosphorylation. The related tyrosine phosphatases PTP-SL and STEP were phosphorylated by the cAMP-dependent protein kinase A (PKA). The PKA phosphorylation site on PTP-SL was identified as the Ser231 residue, located within the KIM. Upon phosphorylation of Ser231, PTP-SL binding and tyrosine dephosphorylation of the MAP kinases extracellular signal-regulated kinase (ERK)1/2 and p38α were impaired. Furthermore, treatment of COS-7 cells with PKA activators, or overexpression of the Cα catalytic subunit of PKA, inhibited the cytoplasmic retention of ERK2 and p38α by wild-type PTP-SL, but not by a PTP-SL S231A mutant. These findings support the existence of a novel mechanism by which PKA may regulate the activation and translocation to the nucleus of MAP kinases

Eradication of pseudomonas aeruginosa with inhaled colistin in adults with non-cystic fibrosis bronchiectasis

[Abstract] The persistent isolation of Pseudomonas aeruginosa in the airways of non-cystic fibrosis bronchiectasis (NCFB) patients is associated with a worsening of the symptoms, increase of exacerbations, poor quality of life and functional impairment. The objective of this study was the analysis of the eradication rate of P. aeruginosa in the sputum of patients with NCFB treated with inhaled colistin and the effects of the treatment in the exacerbations. This was a prospective, cohort, study of 67 NCFB patients treated with inhaled colistin at the Hospital of A Corun˜a (Spain).We recorded dyspnoea, exacerbations, lung function and sputum cultures of P. aeruginosa in the patients. The mean age of the patients was 67.25+14.6 years (59.7% male). The percentages of eradication of P. aeruginosa in sputum at 3, 6, 9 and 12 months were 61.2%, 50.7%, 43.3% and 40.3%, respectively.We observed a significant decrease in exacerbations after 1 year of colistin treatment (1.98+3.62) versus the previous year (3.40 + 4.21, p < 0.001). We conclude that treatment with inhaled colistin in patients with NCFB and P. aeruginosa in sputum can achieve high rates of eradication even in patients with several previous positive cultures, as well as a significant decrease of exacerbations and hospital admissions

Multiple cancer pathways regulate telomere protection

Telomeres are considered as universal anti-cancer targets, as telomere maintenance is essential to sustain indefinite cancer growth. Mutations in telomerase, the enzyme that maintains telomeres, are among the most frequently found in cancer. In addition, mutations in components of the telomere protective complex, or shelterin, are also found in familial and sporadic cancers. Most efforts to target telomeres have focused in telomerase inhibition; however, recent studies suggest that direct targeting of the shelterin complex could represent a more effective strategy. In particular, we recently showed that genetic deletion of the TRF1 essential shelterin protein impairs tumor growth in aggressive lung cancer and glioblastoma (GBM) mouse models by direct induction of telomere damage independently of telomere length. Here, we screen for TRF1 inhibitory drugs using a collection of FDA-approved drugs and drugs in clinical trials, which cover the majority of pathways included in the Reactome database. Among other targets, we find that inhibition of several kinases of the Ras pathway, including ERK and MEK, recapitulates the effects of Trf1 genetic deletion, including induction of telomeric DNA damage, telomere fragility, and inhibition of cancer stemness. We further show that both bRAF and ERK2 kinases phosphorylate TRF1 in vitro and that these modifications are essential for TRF1 location to telomeres in vivo Finally, we use these new TRF1 regulatory pathways as the basis to discover novel drug combinations based on TRF1 inhibition, with the goal of effectively blocking potential resistance to individual drugs in patient-derived glioblastoma xenograft models.We thank the Confocal Microscopy, Protein Engineering, Mass Spectrometry,Comparative Pathology, and Mouse Facility Units at CNIO. MAB laboratory is funded by SAF 2013-45111-R from MINECO,Fundación Botín and Banco Santander, Worldwide Cancer Research 16-1177. LB is a fellow of the La Caixa-Severo Ochoa International PhD Programme.S

Anti-tumor effects of PIM/PI3K/mTOR triple kinase inhibitor IBL-302 in neuroblastoma

The PI3K pathway is a major driver of cancer progression. However, clinical resistance to PI3K inhibition is common. IBL-302 is a novel highly specific triple PIM, PI3K, and mTOR inhibitor. Screening IBL-302 in over 700 cell lines representing 47 tumor types identified neuroblastoma as a strong candidate for PIM/PI3K/mTOR inhibition. IBL-302 was more effective than single PI3K inhibition in vitro, and IBL-302 treatment of neuroblastoma patient-derived xenograft (PDX) cells induced apoptosis, differentiated tumor cells, and decreased N-Myc protein levels. IBL-302 further enhanced the effect of the common cytotoxic chemotherapies cisplatin, doxorubicin, and etoposide. Global genome, proteome, and phospho-proteome analyses identified crucial biological processes, including cell motility and apoptosis, targeted by IBL-302 treatment. While IBL-302 treatment alone reduced tumor growth in vivo, combination therapy with low-dose cisplatin inhibited neuroblastoma PDX growth. Complementing conventional chemotherapy treatment with PIM/PI3K/mTOR inhibition has the potential to improve clinical outcomes and reduce severe late effects in children with high-risk neuroblastoma.This work was supported by funding from the Swedish Cancer Society (to SM, DB), the Swedish Research Council (to DB), the Swedish Childhood Cancer Fund (to SM, KvS, DB), Region Skåne and the research funds of Skåne University Hospital (to DB), the Mary Bevé Foundation (to SM, KvS, DB), Magnus Bergvalls stiftelse (to SM, DB), the Thelma Zoéga Foundation (to SM), Hans von Kantzow Foundation (to SM), Crafoord Foundation (to DB), Åke Wiberg Foundation (to DB), Jeanssons Stiftelser (to DB), Ollie och Elof Ericssons stiftelser (to DB), Berth von Kantzows stiftelse (to DB), the Royal Physiographic Society of Lund (to SM, DB), and the Spanish Ministry of Health and Social Policy (ADE 08 / 90038 ) and the Spanish Ministry of Science and Innovation (CIT- 090000 - 2008 - 14 ) (to JP, SMa, CBA). We would like to thank the Local MS Support at Medical Faculty, Lund University. The authors would like to acknowledge support of the National Genomics Infrastructure (NGI)/Uppsala Genome Center and UPPMAX for providing assistance in massive parallel sequencing and computational infrastructure. Work performed at NGI/Uppsala Genome Center has been funded by RFI/VR and Science for Life Laboratory, SwedenS

Screening health-promoting compounds for their capacity to induce the activity of FOXO3

Several chemical compounds including natural products have been suggested as being effective against age-related diseases or as beneficial for a healthy life. On the other hand, forkhead box O (FOXO) proteins are emerging as key cellular components associated with extreme human longevity. FOXO proteins are mainly regulated by posttranslational modifications and as these modifications are reversible, activation and inactivation of FOXO are attainable through pharmacological treatment. Here, we questioned whether a panel of compounds with known health-beneficial properties has the capacity to induce the activity of FOXO factors. We show that resveratrol, a phytoalexin present in grapes and other food products, the amide alkaloid piperlongumine found in the fruit of the long pepper, and the plant-derived beta-carboline compound harmine induced nuclear translocation of FOXO3. We also show that piperlongumine and harmine but not resveratrol activate FOXO-dependent transcription. We determined the half maximal effective concentration (EC50) values for resveratrol, piperlongumine, and harmine for FOXO translocation, and analyzed their inhibitory impact on chromosomal maintenance 1 (CRM1)-mediated nuclear export and the production of reactive oxygen species (ROS). We also used chemical biology approach and Western blot analysis to explore the underlying molecular mechanisms. We show that harmine, piperlongumine, and resveratrol activate FOXO3 independently of phosphoinositide 3-kinase (PI3K)/AKT signaling and the CRM1-mediated nuclear export. The effect of harmine on FOXO3 activity is at least partially mediated through the inhibition of dual-specificity tyrosine (Y) phosphorylationregulated kinase 1A (DYRK1A) and can be reverted by the inhibition of sirtuins (SIRTs).Spanish Government RTI2018-094629-B-I00, 2 LPCC-NRS/Terry Fox grants 2016/2017, 2017/2018, German Research Foundation (DFG) BR1034/6-1info:eu-repo/semantics/publishedVersio

Screening health-promoting compounds for their capacity to induce the activity of FOXO3

Several chemical compounds including natural products have been suggested as being effective against age-related diseases or as beneficial for a healthy life. On the other hand, forkhead box O (FOXO) proteins are emerging as key cellular components associated with extreme human longevity. FOXO proteins are mainly regulated by posttranslational modifications and as these modifications are reversible, activation and inactivation of FOXO are attainable through pharmacological treatment. Here, we questioned whether a panel of compounds with known health-beneficial properties has the capacity to induce the activity of FOXO factors. We show that resveratrol, a phytoalexin present in grapes and other food products, the amide alkaloid piperlongumine found in the fruit of the long pepper, and the plant-derived β-carboline compound harmine induced nuclear translocation of FOXO3. We also show that piperlongumine and harmine but not resveratrol activate FOXO-dependent transcription. We determined the half maximal effective concentration (EC50) values for resveratrol, piperlongumine, and harmine for FOXO translocation, and analyzed their inhibitory impact on chromosomal maintenance 1 (CRM1)-mediated nuclear export and the production of reactive oxygen species (ROS). We also used chemical biology approach and Western blot analysis to explore the underlying molecular mechanisms. We show that harmine, piperlongumine, and resveratrol activate FOXO3 independently of phosphoinositide 3-kinase (PI3K)/AKT signaling and the CRM1-mediated nuclear export. The effect of harmine on FOXO3 activity is at least partially mediated through the inhibition of dual-specificity tyrosine (Y) phosphorylationregulated kinase 1A (DYRK1A) and can be reverted by the inhibition of sirtuins (SIRTs).This work was supported by Fundação para a Ciência e a Tecnologia (FCT) Research Center Grant UID/BIM/04773/2013 Centre for Biomedical Research (CBMR) and by the Spanish Ministry of Science, Innovation and Universities through grant RTI2018-094629-B-I00 to W.L. B.I.F. was supported by FCTSFRH/BPD/100434/2014 and Marie Curie Individual Fellowship project TRIBBLES (#748585). This work was also supported by 2 LPCC-NRS/Terry Fox grants (2016/2017 and 2017/2018). F.B. was supported by Deutsche Forschungsgemeinschaft (grant BR1034/6-1)

Modulation of telomere protection by the PI3K/AKT pathway

Telomeres and the insulin/PI3K pathway are considered hallmarks of aging and cancer. Here, we describe a role for PI3K/AKT in the regulation of TRF1, an essential component of the shelterin complex. PI3K and AKT chemical inhibitors reduce TRF1 telomeric foci and lead to increased telomeric DNA damage and fragility. We identify the PI3Kα isoform as responsible for this TRF1 inhibition. TRF1 is phosphorylated at different residues by AKT and these modifications regulate TRF1 protein stability and TRF1 binding to telomeric DNA in vitro and are important for in vivo TRF1 telomere location and cell viability. Patient-derived breast cancer PDX mouse models that effectively respond to a PI3Kα specific inhibitor, BYL719, show decreased TRF1 levels and increased DNA damage. These findings functionally connect two of the major pathways for cancer and aging, telomeres and the PI3K pathway, and pinpoint PI3K and AKT as novel targets for chemical modulation of telomere protection.We are indebted to D. Megias for microscopy analysis, to D. Calvo for protein purification as well as to J. Muñoz and F. García for LC/MS/MS analysis. The research was funded by project SAF2013-45111-R of Societal Changes Program of the Spanish Ministry of Economics and Competitiveness (MINECO) co-financed through the European Fund of Regional Development (FEDER), Fundación Botín, Banco Santander (Santander Universities Global Division) and Worldwide Cancer Research (WCR 16-1177).S

mTORC2 Is the major second layer kinase negatively regulating FOXO3 activity

Forkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.info:eu-repo/semantics/publishedVersio

The current role of radiologists in a multidisciplinary team treating breast cancer

In the last decade, advances in the diagnosis and treatment of breast cancer have achieved a significant increase in the general and disease-free survival of affected women but have also increased the complexity of therapeutic decisions. The decision-making process requires agreement between the physicians involved in the management of these patients. Radiologists must understand what other physicians expect and inform them about the usefulness of imaging modalities. This review attempts to provide an update on these subjects

Induction of Lysosome Membrane Permeabilization as a Therapeutic Strategy to Target Pancreatic Cancer Stem Cells

Despite significant efforts to improve pancreatic ductal adenocarcinoma (PDAC) clinical outcomes, overall survival remains dismal. The poor response to current therapies is partly due to the existence of pancreatic cancer stem cells (PaCSCs), which are efficient drivers of PDAC tumorigenesis, metastasis and relapse. To find new therapeutic agents that could efficiently kill PaCSCs, we screened a chemical library of 680 compounds for candidate small molecules with anti-CSC activity, and identified two compounds of a specific chemical series with potent activity in vitro and in vivo against patient-derived xenograft (PDX) cultures. The anti-CSC mechanism of action of this specific chemical series was found to rely on induction of lysosomal membrane permeabilization (LMP), which is likely associated with the increased lysosomal mass observed in PaCSCs. Using the well characterized LMP-inducer siramesine as a tool molecule, we show elimination of the PaCSC population in mice implanted with tumors from two PDX models. Collectively, our approach identified lysosomal disruption as a promising anti-CSC therapeutic strategy for PDAC