Orchestration of the neural stem cell fate by NRF2 and TAZ

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 11-10-2019Neurogenesis is a multiple step process that must be tightly regulated or otherwise results in pathological events. Therefore, a deep characterization of the molecular mechanisms that control the biology of neural stem/progenitor cells (NSPCs) will provide a better understanding of the role of neurogenic niches and new therapeutic strategies for neurodegenerative diseases and brain tumours. In this thesis we have analyzed the regulation of NSCs fate by the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (NRF2), which is considered a master regulator of cellular homeostasis, and the Transcriptional co-activator with PDZ-binding motif (TAZ), a major effector of the Hippo pathway. NRF2 controls the expression of a wide battery of cytoprotective genes that have a tremendous impact on physiological responses such as inflammation, senescence or metabolism. However, its relevance in neurogenesis is just starting to be unveiled. On the other hand, TAZ is a major effector of the Hippo pathway, which plays a key role in tissue homeostasis and organ size control by regulating tissue-specific stem cells. However, the implication of TAZ in neurogenesis has not been analyzed. In this study, we have identified NRF2 as a regulator of hippocampal NSCs self-renewal and differentiation. We show that genetic manipulation of NRF2 results in the modulation of NSPCs differentiation and proliferation capacity. To assess the functional relevance of NRF2 in neurogenesis under pathological conditions, we analyzed the impact of NRF2 deficiency in neurogenesis of the subgranular zone (SGZ) of the hippocampus in a mouse model of Alzheimer´s Disease (AD). We found that NRF2 deficiency results in an accelerated loss of NSCs, loss of synaptic plasticity measured as long term potentiation (LTP) and impaired the execution of cognitive tasks. At the molecular level, we have identified NRF2 enhancer sequences, termed Antioxidant Response Elements (AREs), in the promoter region of the TAZ coding gene. Consequently, we show that genetic and pharmacological manipulation of NRF2 results in the modulation of TAZ gene expression in NSPCs. These findings open a new window to understand the molecular mechanisms underlying NRF2 function in stemness. We have also established a novel role of TAZ as repressor of neuronal differentiation, based on the transcriptional repression of SOX2 and the basic helix-loop-helix (bHLH) factors ASCL1, NEUROG2 and NEUROD1. Data mining of The Cancer Genome Atlas showed a negative correlation between TAZ and the expression of these proneurogenic factors in lower grade gliomas and glioblastomas. We found that TAZ favours glioblastoma CSCs tumorigenic capacity and that genetic modulation of TAZ in these cells inversely correlated with proneurogenic genes expression. Due to the relevance of these proneurogenic factors in the ablation of glioblastoma cancer stem cells (CSCs), this novel TAZ/proneurogenic factors axis may have important implications in the development of this type of brain tumours. The characterization of molecular mechanism governing NSPCs fate provides new insights to harness these cells for brain repair. Overall, this thesis describes a novel role of NRF2 and TAZ in the control of neural stem cell fate, suggesting a new strategy to combat brain pathology

NRF2-dependent gene expression promotes ciliogenesis and Hedgehog signaling

The transcription factor NRF2 is a master regulator of cellular antioxidant and detoxification responses, but it also regulates other processes such as autophagy and pluripotency. In human embryonic stem cells (hESCs), NRF2 antagonizes neuroectoderm differentiation, which only occurs after NRF2 is repressed via a Primary Cilia-Autophagy-NRF2 (PAN) axis. However, the functional connections between NRF2 and primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae, remain poorly understood. For instance, nothing is known about whether NRF2 affects cilia, or whether cilia regulation of NRF2 extends beyond hESCs. Here, we show that NRF2 and primary cilia reciprocally regulate each other. First, we demonstrate that fibroblasts lacking primary cilia have higher NRF2 activity, which is rescued by autophagy-activating mTOR inhibitors, indicating that the PAN axis also operates in differentiated cells. Furthermore, NRF2 controls cilia formation and function. NRF2-null cells grow fewer and shorter cilia and display impaired Hedgehog signaling, a cilia-dependent pathway. These defects are not due to increased oxidative stress or ciliophagy, but rather to NRF2 promoting expression of multiple ciliogenic and Hedgehog pathway genes. Among these, we focused on GLI2 and GLI3, the transcription factors controlling Hh pathway output. Both their mRNA and protein levels are reduced in NRF2-null cells, consistent with their gene promoters containing consensus ARE sequences predicted to bind NRF2. Moreover, GLI2 and GLI3 fail to accumulate at the ciliary tip of NRF2-null cells upon Hh pathway activation. Given the importance of NRF2 and ciliary signaling in human disease, our data may have important biomedical implicationsThis work was supported by European Regional Development Fund (ERDF)-cofunded grants from the Spanish Ministry of Economy and Competitiveness (MINECO) to FRGG (SAF2015-66568-R and RYC2013-14887) and to A.C. and I.L.B. (SAF2016-76520-R

Transcription factor NRF2 uses the Hippo pathway effector TAZ to induce tumorigenesis in glioblastomas

Transcription factor NRF2 orchestrates a cellular defense against oxidative stress and, so far, has been involved in tumor progression by providing a metabolic adaptation to tumorigenic demands and resistance to chemotherapeutics. In this study, we discover that NRF2 also propels tumorigenesis in gliomas and glioblastomas by inducing the expression of the transcriptional co-activator TAZ, a protein of the Hippo signaling pathway that promotes tumor growth. The expression of the genes encoding NRF2 (NFE2L2) and TAZ (WWTR1) showed a positive correlation in 721 gliomas from The Cancer Genome Atlas database. Moreover, NRF2 and TAZ protein levels also correlated in immunohistochemical tissue arrays of glioblastomas. Genetic knock-down of NRF2 decreased, while NRF2 overexpression or chemical activation with sulforaphane, increased TAZ transcript and protein levels. Mechanistically, we identified several NRF2-regulated functional enhancers in the regulatory region of WWTR1. The relevance of the new NRF2/TAZ axis in tumorigenesis was demonstrated in subcutaneous and intracranial grafts. Thus, intracranial inoculation of NRF2-depleted glioma stem cells did not develop tumors as determined by magnetic resonance imaging. Forced TAZ overexpression partly rescued both stem cell growth in neurospheres and tumorigenicity. Hence, NRF2 not only enables tumor cells to be competent to proliferate but it also propels tumorigenesis by activating the TAZ-mediated Hippo transcriptional program.This study was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) under the grant SAF2016-76520-R. ME is recipient of a postdoctoral contract Juan de la Cierva; DL and NRA of a FPU contract of MINECO; MP and RFG of a FPI contracts of Autonomous University of Madrid. RG has been funded by the AECC Scientific Foundation

Transcription factor NRF2 as a therapeutic target for chronic diseases: a systems medicine approach

Systems medicine has a mechanism-based rather than a symptom- or organ-based approach to disease and identifies therapeutic targets in a nonhypothesis-driven manner. In this work, we apply this to transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) by cross-validating its position in a protein-protein interaction network (the NRF2 interactome) functionally linked to cytoprotection in low-grade stress, chronic inflammation, metabolic alterations, and reactive oxygen species formation. Multiscale network analysis of these molecular profiles suggests alterations of NRF2 expression and activity as a common mechanism in a subnetwork of diseases (the NRF2 diseasome). This network joins apparently heterogeneous phenotypes such as autoimmune, respiratory, digestive, cardiovascular, metabolic, and neurodegenerative diseases, along with cancer. Importantly, this approach matches and confirms in silico several applications for NRF2-modulating drugs validated in vivo at different phases of clinical development. Pharmacologically, their profile is as diverse as electrophilic dimethyl fumarate, synthetic triterpenoids like bardoxolone methyl and sulforaphane, protein-protein or DNA-protein interaction inhibitors, and even registered drugs such as metformin and statins, which activate NRF2 and may be repurposed for indications within the NRF2 cluster of disease phenotypes. Thus, NRF2 represents one of the first targets fully embraced by classic and systems medicine approaches to facilitate both drug development and drug repurposing by focusing on a set of disease phenotypes that appear to be mechanistically linked. The resulting NRF2 drugome may therefore rapidly advance several surprising clinical options for this subset of chronic diseases

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies

TAZ Represses the Neuronal Commitment of Neural Stem Cells

© 2020 by the authors.The mechanisms involved in regulation of quiescence, proliferation, and reprogramming of Neural Stem Progenitor Cells (NSPCs) of the mammalian brain are still poorly defined. Here, we studied the role of the transcriptional co-factor TAZ, regulated by the WNT and Hippo pathways, in the homeostasis of NSPCs. We found that, in the murine neurogenic niches of the striatal subventricular zone and the dentate gyrus granular zone, TAZ is highly expressed in NSPCs and declines with ageing. Moreover, TAZ expression is lost in immature neurons of both neurogenic regions. To characterize mechanistically the role of TAZ in neuronal differentiation, we used the midbrain-derived NSPC line ReNcell VM to replicate in a non-animal model the factors influencing NSPC differentiation to the neuronal lineage. TAZ knock-down and forced expression in NSPCs led to increased and reduced neuronal differentiation, respectively. TEADs-knockdown indicated that these TAZ co-partners are required for the suppression of NSPCs commitment to neuronal differentiation. Genetic manipulation of the TAZ/TEAD system showed its participation in transcriptional repression of SOX2 and the proneuronal genes ASCL1, NEUROG2, and NEUROD1, leading to impediment of neurogenesis. TAZ is usually considered a transcriptional co-activator promoting stem cell proliferation, but our study indicates an additional function as a repressor of neuronal differentiation.This study was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (Grant SAF2016-76520-R) and The Autonomous Community of Madrid (grant B2017/BMD-3827). N.R.A was recipient of an FPU contract of MINECO; M.E was the recipient of a postdoctoral contract Juan de la Cierv

Activators and Inhibitors of NRF2: A Review of Their Potential for Clinical Development

© 2019 Natalia Robledinos-Antón et al.The transcription factor NRF2 (nuclear factor erythroid 2-related factor 2) triggers the first line of homeostatic responses against a plethora of environmental or endogenous deviations in redox metabolism, proteostasis, inflammation, etc. Therefore, pharmacological activation of NRF2 is a promising therapeutic approach for several chronic diseases that are underlined by oxidative stress and inflammation, such as neurodegenerative, cardiovascular, and metabolic diseases. A particular case is cancer, where NRF2 confers a survival advantage to constituted tumors, and therefore, NRF2 inhibition is desired. This review describes the electrophilic and nonelectrophilic NRF2 activators with clinical projection in various chronic diseases. We also analyze the status of NRF2 inhibitors, which at this time provide proof of concept for blocking NRF2 activity in cancer therapy.This work was supported by grants SAF2016-76520-R of the Spanish Ministry of Economy and Competitiveness, B2017/BMD-3827 of the Autonomous Community of Madrid, and P_37_732/2016 REDBRAIN of the European Regional Development Fund; Competitiveness Operational Program 2014-2020. NRA and RFG are recipients of FPU and FPI contracts, respectively, of the Spanish Ministry of Economy and Competitiveness

WIP modulates oxidative stress through NRF2/KEAP1 in glioblastoma cells

Due to their high metabolic rate, tumor cells produce exacerbated levels of reactive oxygen species that need to be under control. Wiskott–Aldrich syndrome protein (WASP)-interacting protein (WIP) is a scaffold protein with multiple yet poorly understood functions that participates in tumor progression and promotes cancer cell survival. However, its participation in the control of oxidative stress has not been addressed yet. We show that WIP depletion increases the levels of reactive oxygen species and reduces the levels of transcription factor NRF2, the master regulator of redox homeostasis. We found that WIP stabilizes NRF2 by restraining the activity of its main NRF2 repressor, the E3 ligase adapter KEAP1, because the overexpression of a NRF2ΔETGE mutant that is resistant to targeted proteasome degradation by KEAP1 or the knock-down of KEAP1 maintains NRF2 levels in the absence of WIP. Mechanistically, we show that the increased KEAP1 activity in WIP-depleted cells is not due to the protection of KEAP1 from autophagic degradation, but is dependent on the organization of the Actin cytoskeleton, probably through binding between KEAP1 and F-Actin. Our study provides a new role of WIP in maintaining the oxidant tolerance of cancer cells that may have therapeutic implications.This work was supported by PID2019-110061RB-I00, RTI2018-096303-B-C31 and SAF2017-82436R of the Spanish Ministry of Economy and Competiveness; and by the P_37_732/2016 grant (REDBRAIN) financed by the European Regional Development Fund, Competitiveness Operational Program 2014–2020, and Comunidad Autónoma de Madrid (grant B2017/BMD-3827). M.E. was recipient of a postdoctoral contract Juan de la Cierva; D.L. and N.R.-A. enjoied a FPU contract of MINECO.Peer reviewe

Treatment with Brusatol inhibits oncogenic transcription factors NRF2 and TAZ, reducing glioma stem cells survival

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018.Glioblastomas are nervous system solid tumours with poor prognosis and hard treatment, since they present glioma stem cells (GSCs), a subpopulation of cells responsible for resistance to chemotherapy and radiotherapy, which lead to tumour recurrence. NRF2 and TAZ transcription factors are involved in tumour development, but their role in regulation of cancer stem cells (CSCs) and their possible crosstalk have not been explored. Our studies demonstrate a correlation between NRF2 and TAZ expression and the prognosis of patients with glioma. Knock-down of NRF2 in human glioblastoma explants and glioblastoma cell lines, decreased messenger RNA and protein levels of TAZ and its transcriptional signature. In addition, we identified functional NRF2- binding sites (Antioxidant Response Element, ARE) in the promoter region of the TAZ encoding gene (WWTR1). Besides, NRF2 knock-down reduces cell growth both in vitro and in vivo, being rescued with TAZ overexpression. Consequently, we conclude that at least part of the tumorigenic capacity of NRF2 is TAZ-dependent. Following this evidence, we have used NRF2 inhibitor Brusatol. This treatment reduced TAZ levels and GSCs growth in the same way as NRF2 knock-down. Because of this, we propose NRF2 and TAZ pharmacological inhibition as a novel glioblastoma therapy.Peer reviewe