WIP Drives Tumor Progression through YAP/TAZ-Dependent Autonomous Cell Growth

In cancer, the deregulation of growth signaling pathways drives changes in the cell¿s architecture and its environment that allow autonomous growth of tumors. These cells then acquire a tumor-initiating ¿stemness¿ phenotype responsible for disease advancement to more aggressive stages. Here, we show that high levels of the actin cytoskeleton-associated protein WIP (WASP-interacting protein) correlates with tumor growth, both of which are linked to the tumor-initiating cell phenotype. We find that WIP controls tumor growth by boosting signals that stabilize the YAP/TAZ complex via a mechanism mediated by the endocytic/endosomal system. When WIP levels are high, the ß-catenin Adenomatous polyposis coli (APC)-axin-GSK3 destruction complex is sequestered to the multi-vesicular body compartment, where its capacity to degrade YAP/TAZ is inhibited. YAP/TAZ stability is dependent on Rac, p21-activated kinase (PAK) and mammalian diaphanous-related formin (mDia), and is Hippo independent. This close biochemical relationship indicates an oncogenic role for WIP in the physiology of cancer pathology by increasing YAP/TAZ stability.MINECO (SAF2013-45937-R to I.M.A.) and MINECO/FEDER (SAF2015-70368-R to I.M.A. and F.W.), the European Union (EU-FP7-2009-CT222887 to F.W.), the Instituto de Salud Carlos III Centro de Investigación Biomédica en Red (CIBERNED to F.W.) and by a grant from ISCIII-RETIC (RD12/0036/0009 to R.G.E.)Peer Reviewe

Transcription factor NFE2L2/NRF2 is a regulator of macroautophagy genes

Autophagy is a highly coordinated process that is controlled at several levels including transcriptional regulation. Here, we identify the transcription factor NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) as a regulator of autophagy gene expression and its relevance in a mouse model of Alzheimer disease (AD) that reproduces impaired APP (amyloid β precursor protein) and human (Hs)MAPT/TAU processing, clearance and aggregation. We screened the chromatin immunoprecipitation database ENCODE for 2 proteins, MAFK and BACH1, that bind the NFE2L2-regulated enhancer antioxidant response element (ARE). Using a script generated from the JASPAR's consensus ARE sequence, we identified 27 putative AREs in 16 autophagy-related genes. Twelve of these sequences were validated as NFE2L2 regulated AREs in 9 autophagy genes by additional ChIP assays and quantitative RT-PCR on human and mouse cells after NFE2L2 activation with sulforaphane. Mouse embryo fibroblasts of nfe2l2-knockout mice exhibited reduced expression of autophagy genes, which was rescued by an NFE2L2 expressing lentivirus, and impaired autophagy flux when exposed to hydrogen peroxide. NFE2L2-deficient mice co-expressing HsAPP(V717I) and HsMAPT(P301L), exhibited more intracellular aggregates of these proteins and reduced neuronal levels of SQSTM1/p62, CALCOCO2/NDP52, ULK1, ATG5 and GABARAPL1. Also, colocalization of HsAPP(V717I) and HsMAPT(P301L) with the NFE2L2-regulated autophagy marker SQSTM1/p62 was reduced in the absence of NFE2L2. In AD patients, neurons expressing high levels of APP or MAPT also expressed SQSTM1/p62 and nuclear NFE2L2, suggesting their attempt to degrade intraneuronal aggregates through autophagy. This study shows that NFE2L2 modulates autophagy gene expression and suggests a new strategy to combat proteinopathies

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

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

Transcription factor NRF2 participates in cell cycle progression at the level of G1/S and mitotic checkpoints

Transcription factor NRF2 is a master regulator of the multiple cytoprotective responses that confer growth advantages on a cell. However, its participation in the mechanisms that govern the cell division cycle has not been explored in detail. In this study, we used several standard methods of synchronization of proliferating cells together with flow cytometry and monitored the participation of NRF2 along the cell cycle by the knockdown of its gene expression. We found that the NRF2 levels were highest at S phase entry, and lowest at mitosis. NRF2 depletion promoted both G1 and M arrest. Targeted transcriptomics analysis of cell cycle regulators showed that NRF2 depletion leads to changes in key cell cycle regulators, such as CDK2, TFDP1, CDK6, CDKN1A (p21), CDKN1B (p27), CCNG1, and RAD51. This study gives a new dimension to NRF2 effects, showing their implication in cell cycle progression.This research was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (grants SAF2016-76520-R and PDC2021-121421-I00) and The Autonomous Community of Madrid (grant B2017/BMD-3827). D.L. enjoyed a FPU contract of MINECO

Mutant p53 oncogenic functions in cancer stem cells are regulated by WIP through YAP/TAZ

Wild-type p53 (wtp53) is described as a tumour suppressor gene; mutations in this gene occur in many human cancers and promote oncogenic capacity. Here, we establish that the oncogenic activity of mutant p53 (mtp53) is driven by the WASP-interacting protein (WIP). WIP knockdown from mtp53-expressing glioblastoma and breast cancer cells (BCC) greatly reduced proliferation and growth capacity of cancer stem cell (CSC)-like cells and decreased CSC-like markers (CD133, CD44 or YAP/TAZ). mtp53 overexpression in human astrocytes enhanced their proliferative capacity in suspension culture and increased expression of CSC markers and WIP. WIP knockdown compromised tumour glioblastoma and BCC growth capacity in vivo. We show that WIP is phosphorylated by AKT2 and is regulated by mtp53/p63 through enhancement of PI3K/AKT2-mediated integrin/receptor recycling pathways. WIP regulates this oncogenic pathway by controlling YAP/TAZ stability. We thus establish a new CSC signalling pathway downstream of mtp53 in which AKT2 regulates WIP and controls YAP/TAZ stability.This work was supported by grants from the MICINN, the Plan Nacional of the Dirección General de Ciencia y Tecnología (SAF2012-39148-C03-01 to FW, SAF2013-45937-R to IMA and SAF2015-70368-R to both and SAF2013-43271-R to AC), the European Union (EU-FP7-2009-CT222887 to FW) and the Instituto de Salud Carlos III Centro de Investigación Biomédica en Red (CIBERNED).Peer reviewe

NRF2 drives glioma malignancy through transcriptional activation of an important oncogenic mechanism

Resumen del póster presentado al 1st Joint Meeting of the French-Portuguese-Spanish Biochemical and Molecular Biology Societies y al XL Spanish Society of Biochemistry and Molecular Biology (SEBBM) Congress, celebrado en Barcelona (España) del 23 al 26 de octubre de 2017.Gliomas are nervous system solid tumors with poor prognosis and hard treatment, since they present cancer stem cells (CSCs), a subpopulation of tumor cells responsible of tumor initiation, treatment resistance, metastasis and recurrence, producing highly aggressive phenotypes. The transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2), a master regulator of homeostasis, has been widely described as a tumor growth promoter, implicated in tumor survival and drug resistance. However, despite it has also been implied in CSCs’ metabolism, its importance remains unclear. Our study has identified for the first time how NRF2 is able of promoting CSCs maintenance and survival stimulating the transcription of an important oncogenic effector through novel identified ARE elements (Antioxidant Response Elements, functional NRF2-binding sites). In addition, we have identified a clear correlation between levels of expression of NRF2 and the prognosis of glioma patients. Overall, our studies show a new oncogenic mechanism through which NRF2 produces tumorogenesis and CSCs maintenance. Consequently, we propose NRF2 as a new therapeutic target and prognosis marker in gliomas.Peer reviewe

Oestradiol signalling through the Akt-mTORC1-S6K1

The oestradiol plays an important role in normal brain development and exerts neuroprotective actions. Oestradiol is mainly produced in the ovary and in addition is locally synthesised in the brain. Most of the oestradiol functions have been associated with its capacity to directly bind and dimerize >classical oestrogen receptors> (ERs), alpha and beta. The ERs' actions have been classified as >genomic> and >non-genomic> depending on whether protein synthesis occurs through ER driven transcription or not. Indeed, recent evidence suggests that oestrogen may also act as a more general >trophic factor>. Hence, we have studied the capacity of oestradiol to activate the PI3K/Akt pathway and its implication in axonal growth and neuronal morphogenesis. Our data show that when oestrogen receptors are blocked the axonal and dendritic lengths are reduced in mouse primary neurons. We found that Akt/Rheb/mTORC1 responds to ER activation in neurons and that some elements of this pathway are able to restore a normal neuronal morphology even in the presence of oestrogen receptor antagonist. All these data demonstrate a new mechanism regulated by oestradiol, at least in neuronal morphogenesis. © 2013 Elsevier B.V.CIBERNED; Plan Nacional DGCYT; SAF2006-12782-C03-01; DGCYT (SAF2009-12249-C02-01 and SAF2009-12249-C02-02); EU-FP7-2009-CT222887; Fundación Ramón ArecesPeer Reviewe

Cancer stem cell-like phenotype and survival are coordinately regulated by Akt/FoxO/Bim pathway

Many solid tumors contain a subpopulation of cells with stem characteristics and these are known as cancer stem cells (CSCs) or tumor-initiating cells (TICs). These cells drive tumor growth and appear to be regulated by molecular pathway different from other cells in the tumor bulk. Here, we set out to determine whether elements of the PI3K-AKT pathway are necessary to maintain the CSC-like phenotype in breast tumor cells and for these cells to survive, bearing in mind that the identification of such elements is likely to be relevant to define future therapeutic targets. Our results demonstrate a close relationship between the maintenance of the CSC-like phenotype and the survival of these TICs. Inhibiting PI3K activity, or eliminating AKT activity, mostly that of the AKT1 isoform, produces a clear drop in TICs survival, and a reduction in the generation and growth of CD44(High) /CD24(Low) mammospheres. Surprisingly, the apoptosis of these TICs that is triggered by AKT1 deficiency is also associated with a loss of the stem cell/mesenchymal phenotype and a recovery of epithelial-like markers. Finally, we define downstream effectors that are responsible for controlling the CSC-phenotype, such as FoxO-Bim, and the death of these cells in the absence of AKT1. In summary, these data closely link the maintenance of the stem cell-like phenotype and the survival of these cells to the AKT-FoxO-Bim pathway.Fil: Gargini, Ricardo. Universidad Autonoma de Madrid. Centro de Biologia Molecular; EspañaFil: Cerliani, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Escoll, Maribel. Universidad Autonoma de Madrid. Centro de Biologia Molecular; España. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; EspañaFil: Anton, Ines M.. Consejo Superior de Investigaciones Cientificas. Centro Nacional de Biotecnologia; España. Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas; EspañaFil: Wandosell, Francisco. Universidad Autonoma de Madrid. Centro de Biologia Molecular; Españ