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

Compromised Blood-Brain Barrier Junctions Enhance Melanoma Cell Intercalation and Extravasation

Melanoma frequently metastasises to the brain, and a detailed understanding of the molecular and cellular mechanisms underlying melanoma cell extravasation across the blood-brain barrier (BBB) is important for preventing brain metastasis formation. Making use of primary mouse brain microvascular endothelial cells (pMBMECs) as an in vitro BBB model, we imaged the interaction of melanoma cells into pMBMEC monolayers. We observed exclusive junctional intercalation of melanoma cells and confirmed that melanoma-induced pMBMEC barrier disruption can be rescued by protease inhibition. Interleukin (IL)-1β stimulated pMBMECs or PECAM-1-knockout (-ko) pMBMECs were employed to model compromised BBB barrier properties in vitro and to determine increased melanoma cell intercalation compared to pMBMECs with intact junctions. The newly generated brain-homing melanoma cell line YUMM1.1-BrM4 was used to reveal increased in vivo extravasation of melanoma cells across the BBB of barrier-compromised PECAM-1-deficient mice compared to controls. Taken together, our data indicate that preserving BBB integrity is an important measure to limit the formation of melanoma-brain metastasis

GPR56/ADGRG1 Inhibits Mesenchymal Differentiation and Radioresistance in Glioblastoma

A mesenchymal transition occurs both during the natural evolution of glioblastoma (GBM) and in response to therapy. Here, we report that the adhesion G-protein-coupled receptor, GPR56/ADGRG1, inhibits GBM mesenchymal differentiation and radioresistance. GPR56 is enriched in proneural and classical GBMs and is lost during their transition toward a mesenchymal subtype. GPR56 loss of function promotes mesenchymal differentiation and radioresistance of glioma initiating cells both in vitro and in vivo. Accordingly, a low GPR56-associated signature is prognostic of a poor outcome in GBM patients even within non-G-CIMP GBMs. Mechanistically, we reveal GPR56 as an inhibitor of the nuclear factor kappa B (NF-κB) signaling pathway, thereby providing the rationale by which this receptor prevents mesenchymal differentiation and radioresistance. A pan-cancer analysis suggests that GPR56 might be an inhibitor of the mesenchymal transition across multiple tumor types beyond GBM

GPR56/ADGRG1 inhibits mesenchymal differentiation and radioresistance in glioblastoma

A mesenchymal transition occurs both during the natural evolution of glioblastoma (GBM) and in response to therapy. Here, we report that the adhesion G-protein-coupled receptor, GPR56/ADGRG1, inhibits GBM mesenchymal differentiation and radioresistance. GPR56 is enriched in proneural and classical GBMs and is lost during their transition toward a mesenchymal subtype. GPR56 loss of function promotes mesenchymal differentiation and radioresistance of glioma initiating cells both in vitro and in vivo. Accordingly, a low GPR56-associated signature is prognostic of a poor outcome in GBM patients even within non-G-CIMP GBMs. Mechanistically, we reveal GPR56 as an inhibitor of the nuclear factor kappa B (NF-κB) signaling pathway, thereby providing the rationale by which this receptor prevents mesenchymal differentiation and radioresistance. A pan-cancer analysis suggests that GPR56 might be an inhibitor of the mesenchymal transition across multiple tumor types beyond GBM

Study of the TRF1 telomeric protein as therapeutic target in Glioblastoma

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 14-09-2018Esta tesis tiene embargado el acceso al texto completo hasta el 14-09-2021Glioblastoma multiforme (GBM) is the deadliest and more common type of brain tumor in the adult, with a mean survival of only 14-16 months. This poor prognosis is linked to its high proliferation index and to tumor heterogeneity, including the existence of tumor cells with stem-like properties, also known as glioma stem-like cells (GSCs). Interestingly, the vast majority of GBM show mutations in telomere maintenance genes. Telomeres are nucleo-protein structures located at the end of the chromosomes and are essential for chromosome stability. They are protected by a six-protein complex, also termed the shelterin complex. The shelterin complex constitutes the so-called “capping” of the telomeres, which is essential for their protection, preventing telomeres from degradation and fusion to other chromosomes. TRF1 is an essential component of shelterin, which binds to the doublestrand telomeric DNA. TRF1 inhibition results in telomere uncapping and effectively blocks the growth rapidly growing, p53-null, lung tumors independently of telomere length. TRF1 also marks and it is essential for both adult and pluripotent stem cells. Based on this, the principal aim of this thesis is to address whether targeting TRF1 could be an effective therapeutic strategy for GBM. To this end, we generated mice with GBM by targeting neural stem cells (NSCs) with different oncogenic insults. In particular, we generated three different GBM subtypes by overexpressing PDGFA and PDGFB in Cdkn2a deficient background or knocking down both Nf1 and p53. Interestingly, we found that the three GBM subtypes overexpressed TRF1 in a telomere length independent manner. In addition, Trf1 genetic ablation effectively inhibited both GBM initiation and progression leading to a significant increase in mouse survival. This was accompanied by increased telomeric DNA damage, inhibition of proliferation and reduced stemness. Of note, brainspecific or whole-body Trf1 deletion in healthy mice did not compromise organism viability or cognitive functions. To address whether our findings could be translated to human patients, we first checked whether TRF1 was upregulated in human samples. Using both human cells lines and patient-derived tissue we demonstrated that GBM presented higher TRF1 levels compared to normal brain. Next, we test the effect of TRF1 chemical modulators in human GBM cells and patient-derived GSCs. Interestingly, TRF1 chemical modulators also induced DNA damage, and decreased proliferation and stemness in GBM cells. Moreover, TRF1 chemical downregulation blocked tumor-sphere formation and tumor growth in xenografts from patient-derived primary GSCs. Taken together, these results suggest that targeting telomeres throughout TRF1 inhibition may be a novel therapeutic strategy for GBM.El Glioblastoma multiforme (GBM) es uno de los tumores cerebrales más comunes y agresivos en adultos, con una supervivencia media de 14 a 16 meses. La esperanza de vida tan reducida se debe a son tumores muy proliferativos y heterogéneos, así como a la existencia de células madre tumorales, también conocidas como “células madre de glioma”. La mayoría de los casos de GBM presentan mutaciones en genes asociados con el mantenimiento telómerico. Los telómeros con unas estructuras nucleo-proteícas localizadas al final de los cromosomas, y son esenciales para su estabilidad. Los telómeros están protegidos por un complejo de seis proteínas, también conocido como complejo shelterina. Este complejo evita que los cromosomas se degraden o se fusionen entre ellos. Uno de los componentes de este complejo de proteínas es TRF1, que se une al ADN telomérico de doble cadena. La inhibición de TRF1 causa una “desprotección” telomérica y bloquea así el crecimiento de tumores de pulmón muy agresivos y deficientes de p53, independientemente de la longitud telomérica. Además, TRF1 es esencial para las células madre adultas y las células madre inducidas. El principal objetivo de esta tesis es determinar si TRF1 podría usarse como diana terapéutica en GBM. Para ello, generamos ratones con GBM introduciendo diferentes estímulos oncogénicos en células madre neuronales. En particular, generamos tres subtipos diferentes de GBM, sobrexpresando PDGFA o PDGFB en un fondo genético deficiente para Cdkn2a o inhibiendo los supresores tumorales Nf1 y p53 al mismo tiempo. Observamos que en los tres subtipos la expresión de TRF1 era más alta en comparación con cerebro normal, de forma independiente a la longitud telomérica. Además, la deleción genética de Trf1 bloquea tanto la iniciación como la progresión del GBM, mediante un mecanismo que incluye la inducción de daño en el DNA y la inhibición de la proliferación y las capacidades de células madre. También observamos que la pérdida genética de Trf1 en ratones sanos no afecta a la supervivencia ni causa deterioro cognitivo. Con el fin de comprobar si la inhibición de TRF1 podría ser efectiva en pacientes de GBM, comprobamos si estos también exhibían altos niveles de TRF1. Para ellos, usamos células y tejido de paciente y demostramos que el GBM humano tenia niveles más altos de TRF1 que el cerebro normal. Además, comprobamos que la inhibición química de TRF1 en células humanas de GBM también induce daño en el ADN y reduce la proliferación y las capacidades de células madre. Por último, también vimos que la inhibición química de TRF1 afectaba a la formación de esferas y al crecimiento de xenoinjertos con células madre de tumor. En conclusión, estos resultados sugieren que atacar los telómeros mediante la inhibición de TRF1 podría ser una estrategia prometedora para el tratamiento de GB

Inhibition of TRF1 Telomere Protein Impairs Tumor Initiation and Progression in Glioblastoma Mouse Models and Patient-Derived Xenografts.

Glioblastoma multiforme (GBM) is a deadly and common brain tumor. Poor prognosis is linked to high proliferation and cell heterogeneity, including glioma stem cells (GSCs). Telomere genes are frequently mutated. The telomere binding protein TRF1 is essential for telomere protection, and for adult and pluripotent stem cells. Here, we find TRF1 upregulation in mouse and human GBM. Brain-specific Trf1 genetic deletion in GBM mouse models inhibited GBM initiation and progression, increasing survival. Trf1 deletion increased telomeric DNA damage and reduced proliferation and stemness. TRF1 chemical inhibitors mimicked these effects in human GBM cells and also blocked tumor sphere formation and tumor growth in xenografts from patient-derived primary GSCs. Thus, targeting telomeres throughout TRF1 inhibition is an effective therapeutic strategy for GBM.We thank R. Serrano for mice handling, J.M. Flores for histopathology, M. Valiente for the human astrocyte (HA) cell line, and the Comparative Pathology and Biobank Units at CNIO. MAB laboratory is funded by SAF2013-45111-R from MINECO, Fundacion Botin, and Banco Santander, Worldwide Cancer Research 16-1177. L.B. is a fellow of the La Caixa-Severo Ochoa International PhD Program.S

Safety of Whole-Body Abrogation of the TRF1 Shelterin Protein in Wild-Type and Cancer-Prone Mouse Models

Telomeres are considered potential anti-cancer targets. Most studies have focused on telomerase inhibition, but this strategy has largely failed in clinical trials. Direct disruption of the shelterin complex through TRF1 inhibition can block tumorigenesis in cancer mouse models by a mechanism that involves DNA damage induction and reduction of proliferation and stemness. Any anti-cancer target, however, must fulfill the requisite of not showing deleterious effects in healthy tissues. Here, we show that Trf1 genetic deletion in wild-type and cancer-prone p53- and Ink4Arf-deficient mice does not affect organismal viability and only induces mild phenotypes like decreased body weight and hair graying or hair loss, the skin being the most affected tissue. Importantly, we found that Trf1 is essential for tumorigenesis in p53- and Ink4Arf-deficient mice, as we did not find a single tumor originating from Trf1-deleted cells. These findings indicate a therapeutic window for targeting Trf1 in cancer treatment.We thank R. Serrano for mice handling and the Comparative Pathology and Mouse Facility Units at CNIO. M.A.B. laboratory is funded by SAF2013-45111-R from MINECO,Fundación Botın, and Banco Santanderand Worldwide Cancer Research 16-1177. L.B. is a fellow of the La Caixa-Severo Ochoa International PhD Program.S

On-line Training and Monitoring of Robot Tasks through Virtual Reality

Currently, the implementation of virtual, augmented and mixed realities-based solutions is one of the megatrends in the Industrial Automation domain. In this context, Virtual Reality (VR) permits the development of virtual environments that can be used for different purposes, such as designing, monitoring and/or training industrial machinery. Moreover, the access to such environments can be remote, facilitating the interaction of humans with cyber models of real-world systems without the need of being at the system facilities. This article presents a virtual environment that has been developed within VR technologies not only for training and monitoring robot tasks but also to be done at robot operation runtime within an on-line mode. In this manner, the user of the presented environment is able to train and monitor de tasks at the same time that the robot is operating. The research work is validated within the on-line training and monitoring tasks of an ABB IRB 14000 industrial robot.acceptedVersionPeer reviewe

Splicing machinery dysregulation drives glioblastoma development/aggressiveness: oncogenic role of SRSF3.

Glioblastomas remain the deadliest brain tumour, with a dismal ∼12-16-month survival from diagnosis. Therefore, identification of new diagnostic, prognostic and therapeutic tools to tackle glioblastomas is urgently needed. Emerging evidence indicates that the cellular machinery controlling the splicing process (spliceosome) is altered in tumours, leading to oncogenic splicing events associated with tumour progression and aggressiveness. Here, we identify for the first time a profound dysregulation in the expression of relevant spliceosome components and splicing factors (at mRNA and protein levels) in well characterized cohorts of human high-grade astrocytomas, mostly glioblastomas, compared to healthy brain control samples, being SRSF3, RBM22, PTBP1 and RBM3 able to perfectly discriminate between tumours and control samples, and between proneural-like or mesenchymal-like tumours versus control samples from different mouse models with gliomas. Results were confirmed in four additional and independent human cohorts. Silencing of SRSF3, RBM22, PTBP1 and RBM3 decreased aggressiveness parameters in vitro (e.g. proliferation, migration, tumorsphere-formation, etc.) and induced apoptosis, especially SRSF3. Remarkably, SRSF3 was correlated with patient survival and relevant tumour markers, and its silencing in vivo drastically decreased tumour development and progression, likely through a molecular/cellular mechanism involving PDGFRB and associated oncogenic signalling pathways (PI3K-AKT/ERK), which may also involve the distinct alteration of alternative splicing events of specific transcription factors controlling PDGFRB (i.e. TP73). Altogether, our results demonstrate a drastic splicing machinery-associated molecular dysregulation in glioblastomas, which could potentially be considered as a source of novel diagnostic and prognostic biomarkers as well as therapeutic targets for glioblastomas. Remarkably, SRSF3 is directly associated with glioblastoma development, progression, aggressiveness and patient survival and represents a novel potential therapeutic target to tackle this devastating pathology