Targeting mTOR as a Therapeutic Approach in Medulloblastoma

Mechanistic target of rapamycin (mTOR) is a master signaling pathway that regulates organismal growth and homeostasis, because of its implication in protein and lipid synthesis, and in the control of the cell cycle and the cellular metabolism. Moreover, it is necessary in cerebellar development and stem cell pluripotency maintenance. Its deregulation has been implicated in the medulloblastoma and in medulloblastoma stem cells (MBSCs). Medulloblastoma is the most common malignant solid tumor in childhood. The current therapies have improved the overall survival but they carry serious side effects, such as permanent neurological sequelae and disability. Recent studies have given rise to a new molecular classification of the subgroups of medulloblastoma, specifying 12 different subtypes containing novel potential therapeutic targets. In this review we propose the targeting of mTOR, in combination with current therapies, as a promising novel therapeutic approach.J.A. is recipient of a predoctoral fellowship from the Department of Education of the Basque Government. This work was supported by grants from the Department of Industry of the Basque Government (SAIO13-PC13BN011), and the European Regional Developmental Fund, Institute of Health Carlos III (ISCIII) (PI16/01730) to I.G

Targeting Myotonic Dystrophy Type 1 with Metformin

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder of genetic origin. Progressive muscular weakness, atrophy and myotonia are its most prominent neuromuscular features, while additional clinical manifestations in multiple organs are also common. Overall, DM1 features resemble accelerated aging. There is currently no cure or specific treatment for myotonic dystrophy patients. However, in recent years a great effort has been made to identify potential new therapeutic strategies for DM1 patients. Metformin is a biguanide antidiabetic drug, with potential to delay aging at cellular and organismal levels. In DM1, different studies revealed that metformin rescues multiple phenotypes of the disease. This review provides an overview of recent findings describing metformin as a novel therapy to combat DM1 and their link with aging.M.G.-P. and A.S.-A. are recipient of predoctoral fellowships from the University of the Basque Country (PIF 15/245) and Carlos III Institute (FI17/00250), respectively. This work is supported by grants from the Carlos III Institute and FEDER funds (PI17/01841, DTS18/00181, PI19/01355, PI21/00557), Health Department from Basque Country (2017222021, 2018222021, 2020333008) and CIBERNED funds

Myotonic Dystrophy type 1 cells display impaired metabolism and mitochondrial dysfunction that are reversed by metformin

Myotonic dystrophy type 1 (DM1; MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in patients with DM1 resemble the appearance of a multisystem accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. In this study, we characterized the impact of metabolism and mitochondria on fibroblasts and peripheral blood mononuclear cells (PBMCs) derived from patients with DM1 and healthy individuals. Our results revealed a decrease in oxidative phosphorylation system (OXPHOS) activity, oxygen consumption rate (OCR), ATP production, energy metabolism, and mitochondria! dynamics in DM1 fibroblasts, as well as increased accumulation of reactive oxygen species (ROS). PBMCs of DM1 patients also displayed reduced mitochondria! dynamics and energy metabolism. Moreover, treatment with metformin reversed the metabolic and mitochondria! defects as well as additional accelerated aging phenotypes, such as impaired proliferation, in DM1-derived fibroblasts. Our results identify impaired cell metabolism and mitochondria! dysfunction as important drivers of DM1 pathophysiology and, therefore, reveal the efficacy of metformin treatment in a pre-clinical setting.This work was supported by grants from the Instituto Salud Carlos III and FEDER funds (CP16/00039, PI16/01580, PI17/01841) and Health department from Basque Country (2017 and 2018-2017222021)

In Vitro P38MAPK Inhibition in Aged Astrocytes Decreases Reactive Astrocytes, Inflammation and Increases Nutritive Capacity After Oxygen-Glucose Deprivation

Proper astroglial functioning is essential for the development and survival of neurons and oligodendroglia under physiologic and pathological circumstances. Indeed, malfunctioning of astrocytes represents an important factor contributing to brain injury. However, the molecular pathways of this astroglial dysfunction are poorly defined. In this work we show that aging itself can drastically perturb astrocyte viability with an increase of inflammation, cell death and astrogliosis. Moreover, we demonstrate that oxygen glucose deprivation (OGD) has a higher impact on nutritive loss in aged astrocytes compared to young ones, whereas aged astrocytes have a higher activity of the anti-oxidant systems. P38MAPK signaling has been identified to be upregulated in neurons, astrocytes and microglia after ischemic stroke. By using a pharmacological p38 alpha specific inhibitor (PH-797804), we show that p38MAPK pathway has an important role in aged astrocytes for inflammatory and oxidative stress responses with the subsequent cell death that occurs after OGD.Deutsche Forschungsgemeinschaft (SCHE 2078/2-1). Forderverein fur fruhgeborene Kinder an der Charite e.V. Basque Government Postdoc (2017_1_0095

Senescence plays a role in myotonic dystrophy type 1 br

Myotonic dystrophy type 1 (DM1; MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in patients with DM1 resemble the appearance of an accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. Transcriptomic analysis of fibroblasts derived from patients with DM1 and healthy individuals revealed a decrease in cell cycle activity, cell division, and DNA damage response in DM1, all of which related to the accumulation of cellular senescence. The data from transcriptome analyses were corroborated in human myoblasts and blood samples, as well as in mouse and Drosophila models of the disease. Serial passage studies in vitro confirmed the accelerated increase in senescence and the acquisition of a senescence-associated secretory phenotype in DM1 fibroblasts, whereas the DM1 Drosophila model showed reduced longevity and impaired locomotor activity. Moreover, functional studies highlighted the impact of BMI1 and downstream p16INK4A/ RB and ARF/p53/p21CIP pathways in DM1-associated cellular phenotypes. Importantly, treatment with the senolytic compounds Quercetin, Dasatinib, or Navitoclax reversed the accelerated aging phenotypes in both DM1 fibroblasts in vitro and in Drosophila in vivo. Our results identify the accumulation of senescence as part of DM1 pathophysiology and, therefore, demonstrate the efficacy of senolytic compounds in the preclinical setting.MGP and ASA are recipient of predoctoral fellowships from the University of the Basque Country (PIF 15/245) and Carlos III Institute (FI17/00250), respectively. We thank the methodological support service of Biodonostia Institute for help with statistical analysis. This work is supported by grants from the Instituto Salud Carlos III and FEDER funds (PI16/01580, PI17/01841, DTS18/00181, PI19/01355, CPII19/00021, and DTS20/00179), La Caixa, and Health department from Basque Country (2017222021, 2018222021, and 2020333008)

High SOX9 Maintains Glioma Stem Cell Activity through a Regulatory Loop Involving STAT3 and PML

Glioma stem cells (GSCs) are critical targets for glioma therapy. SOX9 is a transcription factor with critical roles during neurodevelopment, particularly within neural stem cells. Previous studies showed that high levels of SOX9 are associated with poor glioma patient survival. SOX9 knockdown impairs GSCs proliferation, confirming its potential as a target for glioma therapy. In this study, we characterized the function of SOX9 directly in patient-derived glioma stem cells. Notably, transcriptome analysis of GSCs with SOX9 knockdown revealed STAT3 and PML as downstream targets. Functional studies demonstrated that SOX9, STAT3, and PML form a regulatory loop that is key for GSC activity and self-renewal. Analysis of glioma clinical biopsies confirmed a positive correlation between SOX9/STAT3/PML and poor patient survival among the cases with the highest SOX9 expression levels. Importantly, direct STAT3 or PML inhibitors reduced the expression of SOX9, STAT3, and PML proteins, which significantly reduced GSCs tumorigenicity. In summary, our study reveals a novel role for SOX9 upstream of STAT3, as a GSC pathway regulator, and presents pharmacological inhibitors of the signaling cascade.P.A. and A.S.-A. were recipients of predoctoral fellowships from the AECC foundation and Carlos III Institute (ISCIII), respectively. M.a.-S. holds a Sara Borrell postdoctoral contract from the ISCIII (CD19/00154). E.C.-G. was a recipient of a Stop Fuga de Cerebros postdoctoral fellowship and holds a Miguel Servet contract from the ISCIII (CP19/00085). We thank the Histology Platform of the Biodonostia Health Research Institute, The Neuro-Oncology Committee of Donostia University Hospital, and Basque Biobank for their help. This research was supported by grants from ISCIII and FEDER Funds (CP16/00039, DTS16/00184, PI16/01580, DTS18/00181, PI18/01612, CP19/00085), and the Industry and Health Departments of the Basque Country

PR-LncRNA Signature Regulates Glioma Cell Activity Through Expression of SOX Factors

Long non-coding RNAs (LncRNAs) have emerged as a relevant class of genome regulators involved in a broad range of biological processes and with important roles in tumor initiation and malignant progression. We have previously identified a p53-regulated tumor suppressor signature of LncRNAs (PR-LncRNAs) in colorectal cancer. Our aim was to identify the expression and function of this signature in gliomas. We found that the expression of the four PR-LncRNAs tested was high in human low-grade glioma samples and diminished with increasing grade of disease, being the lowest in glioblastoma samples. Functional assays demonstrated that PR-LncRNA silencing increased glioma cell proliferation and oncosphere formation. Mechanistically, we found an inverse correlation between PR-LncRNA expression and SOX1, SOX2 and SOX9 stem cell factors in human glioma biopsies and in glioma cells in vitro. Moreover, knock-down of SOX activity abolished the effect of PR-LncRNA silencing in glioma cell activity. In conclusion, our results demonstrate that the expression and function of PR-LncRNAs are significantly altered in gliomagenesis and that their activity is mediated by SOX factors. These results may provide important insights into the mechanisms responsible for glioblastoma pathogenesis.PA, JA-I and AS-A were recipients of a predoctoral fellowship from the Spanish Association Against Cancer (AECC Gipuzkoa), Basque Government and Instituto Salud Carlos III. This work was supported by grants from the Carlos III Institute of Health and the European Regional Development Fund (PI13/02277, CP16/00039, DTS16/084, and PI16/01580) and Industry and Health Departments of the Basque Country

Human exceptional longevity: transcriptome from centenarians is distinct from septuagenarians and reveals a role of Bcl‐xL in successful aging

Centenarians not only enjoy an extraordinary aging, but also show a compression of morbidity. Using functional transcriptomic analysis of peripheral blood mononuclear cells (PMBC) we identified 1721 mRNAs differentially expressed by centenarians when compared with septuagenarians and young people. Sub‐network analysis led us to identify Bcl ‐ xL as an important gene up‐regulated in centenarians. It is involved in the control of apoptosis, cellular damage protection and also in modulation of immune response, all associated to healthy aging. Indeed, centenarians display lower plasma cytochrome C levels, higher mitochondrial membrane potential and also less cellular damage accumulation than septuagenarians. Leukocyte chemotaxis and NK cell activity are significantly impaired in septuagenarians compared with young people whereas centenarians maintain them. To further ascertain the functional role of Bcl‐ xL in cellular aging, we found that lymphocytes from septuagenarians transduced with Bcl‐xL display a reduction in senescent‐related markers. Finally, to demonstrate the role of BcL‐xL in longevity at the organism level, C. elegans bearing a gain of function mutation in the BcL‐xL ortholog ced‐9, showed a significant increase in mean and maximal life span. These results show that mRNA expression in centenarians is unique and reveals that BcL‐ xL plays an important role in exceptional aging

Stratification and therapeutic potential of PML in metastatic breast cancer.

Patient stratification has been instrumental for the success of targeted therapies in breast cancer. However, the molecular basis of metastatic breast cancer and its therapeutic vulnerabilities remain poorly understood. Here we show that PML is a novel target in aggressive breast cancer. The acquisition of aggressiveness and metastatic features in breast tumours is accompanied by the elevated PML expression and enhanced sensitivity to its inhibition. Interestingly, we find that STAT3 is responsible, at least in part, for the transcriptional upregulation of PML in breast cancer. Moreover, PML targeting hampers breast cancer initiation and metastatic seeding. Mechanistically, this biological activity relies on the regulation of the stem cell gene SOX9 through interaction of PML with its promoter region. Altogether, we identify a novel pathway sustaining breast cancer aggressiveness that can be therapeutically exploited in combination with PML-based stratification.The work of A.C. is supported by the Ramón y Cajal award, the Basque Department of Industry, Tourism and Trade (Etortek), Health (2012111086) and Education (PI2012-03), Marie Curie (277043), Movember Foundation (GAP1), ISCIII (PI10/01484, PI13/00031), FERO (VIII Fellowship) and ERC (336343). N.M.-M. and P.A. are supported by the Spanish Association Against Cancer (AECC), AECC JP Vizcaya and Guipuzcoa, respectively. J.U. and F.S. are Juan de la Cierva Researchers (MINECO). L.A., A.A.-A. and L.V.-J. are supported by the Basque Government of education. M.L.-M.C. acknowledges SAF2014-54658-R and Asociación Española contra el Cancer. R.B. acknowledges Spanish MINECO (BFU2014-52282-P, Consolider BFU2014-57703-REDC), the Departments of Education and Industry of the Basque Government (PI2012/42) and the Bizkaia County. M.S., V.S. and J.B. acknowledge Banco Bilbao Vizcaya Argentaria (BBVA) Foundation (Tumour Biomarker Research Program). M.S. and J.B. are supported by NIH grant P30 CA008748. M.dM.V. is supported by the Institute of Health Carlos III (PI11/02251, PI14/01328) and Basque Government, Health Department (2014111145). A.M. is supported by ISCIII (CP10/00539, PI13/02277) and Marie Curie CIG 2012/712404. V.S. is supported by the SCIII (PI13/01714, CP14/00228), the FERO Foundation and the Catalan Agency AGAUR (2014 SGR 1331). R.R.G. research support is provided by the Spanish Ministry of Science and Innovation grant SAF2013-46196, BBVA Foundation, the Generalitat de Catalunya (2014 SGR 535), Institució Catalana de Recerca i Estudis Avançats, the Spanish Ministerio de Economia y Competitividad (MINECO) and FEDER funds (SAF2013-46196).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms1259

Regulación del locus Ink4a/Arf : acción de los inhibidores de histonas y generación de ratones modificados genéticamente

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 27-05-2005The scientific community has largely investigated the relevance of Ink4a/Arf locus. The impact in cancer resistance of the Ink4a/Arf locus is well known. In this work, we have characterized in depth the tumor suppressor activity of Ink4a/Arf locus. Our studies have opened two different lines of research: First, we have investigated, and finally identified new regulatory elements of Ink4a and Arf expression. Histone deacetylase inhibitors (HDACIs) control the expression of numerous genes and present a potent cytostatic effect inducing cell cycle arrest. We have observed that HDACIs are able to modulate transcriptionally the expression of the members of the Ink4a/Arf locus. HDACIs repressed the activity of Ink4a and increased the expression of Arf. We have found that binding of Sp-1 to the Ink4a promoter is necessary for the repressive effect of the HDACIs on Ink4a. On the other hand, we have found that Arf activation is essential for the HDACIs induced growth arrest. Finally, we have unraveled that the antitumoral effect induced by HDACIs is mediated by the activation of Arf The negative consequences of loosing Ink4a and Arf function have been extensively characterized. Different Ink4a and/or Arf null mice have been generated that developed tumors at early age. We were interested in understanding the beneficia! effects in cancer resistance obtained by increasing the gene dosage of Ink4a and Arf. For this, we have generated a mouse strain carrying an extra copy of the entire Ink4b/Ink4a/Arf locus. Previously, we had generated mouse strains with extra gene copies of p53. These "Super p53" mice have an increased resistance to develop cancer and, importantly, are normal with regard to aging. We have first analyzed the behavior of the ectopic Ink4a/Arf copy in a genetic background lacking the endogenous Ink4a/Arf alleles. We have observed that the transgenic allele retains the main attributes found in the endogenous allele as a sensor of stress and rescues the phenotypes associated to Ink4a/Arf deficiency. Together, we conclude that the transgenic allele is active and tunctional in providing tumor suppression. We have then turned our attention to the phenotype of mice with an extra copy of the Ink4a/Arf locus. "Super Ink4a/Arf" mice were significantly more resistant to chemically induced carcinogenesis than their wild-type littermates. Regarding fitness, "Super Ink4a/Arf" mice had a normal fertility and they do not manifest accelerated aging. We conclude that it is possible to enhance the function of Ink4a and Arf, providing an increased resistance to cancer and, at the same time, without noticeable detrimental effects on fitness or aging. These results are in line with our previous data on the "Super p53" mice, and on double transgenic mice, "Super p53" /"Super Ink4a/Arf", also generated in this work. Together, our results suggest that tumor suppression can be enhanced in mammalian organisms without incurring in overt deleterious consequences