Residues K128, 132, and 134 in the thyroid hormone receptor-α are essential for receptor acetylation and activity

The thyroid hormone receptor (TR)-α is a nuclear receptor that mediates both transrepression and ligand-dependent transactivation. Here we show that TRα is posttranslationally modified by acetylation in response to its own ligand (T3). Acetylation increases binding to DNA. Using mutagenesis, we identified three conserved lysine residues in the carboxi-terminal extension (CTE) of the DNA binding domain that are targets of the cAMP-response element-binding protein acetyltransferase. Substitution of these lysines by arginines in TRα decreased ligand binding affinity and precluded ligand-dependent release of corepressors and recruitment of coactivators. The acetylation TRα mutant lost the ability to transactivate even at high T3 concentrations and acts as a dominant-negative inhibitor of wild-type TR activity. In addition, whereas native TRα interferes with AP-1 function, the mutant is unable to mediate transrepression. Finally, TRα suppresses NIH-3T3 fibroblast transformation by the Ras oncogene both in a ligand-dependent and -independent manner, but the CTE mutant is unable to mediate ligand-dependent repression of transformation. These results reveal a key role for the CTE region on acetylation, ligand affinity, transactivation, transrepression, and antitransforming properties of TRα. Copyright © 2009 by The Endocrine Society.This work was supported by Grant BFU2006-13497/2007-62402 from the Ministerio de Educación y Ciencia and from the Fundación Médica Mutua Madrileña, Red Temática de Investigacion Cooperativa en Cancer from Fondo de Investigaciones Sanitarias, and Consortium for Research into Nuclear Development and Aging from the European Union.Peer Reviewe

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

Antagonismo transcripcional entre el receptor de hormonas tiroideas y el factor de transcripción CREB

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 26-10-2001La hormona tiroidea, T3, ejerce sus acciones a través de la unión a receptores nucleares (TRs). Estos receptores son factores de transcripción que reconocen secuencias dianas en el DNA (TREs) para modular la transcripción. Sin embargo, los receptores nucleares también pueden regular la expresión de genes que no contienen TREs a través de mecanismos de "cross-talk" con otros factores de transcripción y vías de transducción de señales. En nuestro laboratorio se ha descrito que la T3 regula negativamente la transcripción del gen del factor de transcripción hipofisario Pit-1/GHF-1, que no contienen sitios TRE, a través de interferencia transcripcional con los elementos de respuesta al cAMP (CREs) presentes en su promotor. En esta tesis hemos demostrado que la T3 regula negativamente no sólo la actividad de este promotor, sino la de otros promotores con elementos CRE, tanto a nivel basal, como en respuesta a la forskolina, un agente estimulador de la adenilato ciclasa, y al éster de forbol TPA, que en estas células sinergizan para activar la transcripción mediada tanto por sitios CRE como por sitios AP-1. Hemos demostrado que la T3 reprime de forma muy potente la activación transcripcional mediada por CREB, el principal factor de transcripción que se une a los sitios CRE en células hipofisarias GH4C1 que expresan altos niveles de TR ende/geno. Resultados similares se observan en células HeLa tras la expresión de TR. La actividad transcripcional de CREB se induce por fosforilación en la serina 133. En células GH4C1 el tratamiento con T3 inhibe significativamente tanto la fosforilación basal de CREB como la inducida por forskolina, TPA o los factores de crecimiento FGF, IGF-1 y EGF, los cuales estimulan la transcripción mediada por los elementos CRE en este tipo celular. Existe una interacción directa entre TR y CREB, demostrada tanto "in vitro" como "in vivo", que podría estar implicada en el antagonismo transcripcional observado. La interacción de estas proteínas afecta la capacidad de ambas para unirse a sus respectivos elementos de respuesta, reprime la fosforilación de CREB por proteína kinasa A "in vitro", y disminuye la capacidad de TR para reclutar coactivadores. Hemos comprobado que la competición por cantidades limitantes de CBP (CREB-binding protein), un coactivador común para los receptores nucleares y CREB, no es responsable del antagonismo transcripcional. También hemos demostrado que el antagonismo entre TR y CREB es mutuo, y que CREB reprime la transactivación de los elementos TRE por T3. La interacción entre estos factores mapea en el dominio de unión a DNA de TR y en el dominio b-Zip de CREB, que media la dimerización y unión a DNA. Los mutantes en estos dominios no median efectos transrepresores, demostrando que la interacción entre ambos factores de transcripción se requiere para mediar este "cross-tal" transcripcional. Por último, se han estudiado las vías de señalización que intervienen en la estimulación de los promotores de la somatostatina y colagenasa por el cAMP y los ésteres de forbol, y que podrían por tanto ser antagonizadas por la T3. En esta regulación parecen intervenir las vías de la proteína kinasa A, la proteína kinasa C y las kinasas ERKs. En conjunto, nuestros resultados demuestran la existencia de un mecanismo no convencional, diferente de la unión a elementos respuesta en el DNA, por los que los receptores nucleares de hormonas tiroideas pueden regular negativamente la expresión de genes que contienen elementos de respuesta al cAMP. Este mecanismo parece implicar interacciones proteína-proteína entre los receptores y el factor de transcripción CREB. Debido a que este factor es una importante diana de diferentes vías de señalización que median la acción de diferentes hormonas y factores de crecimiento, este efecto de las hormonas tiroideas podría jugar un importante papel en la regulación transcripcional de los tipos celulares que expresan niveles elevados de receptores para estas hormonas

The thyroid hormone receptor antagonizes CREB-mediated transcription

Combinatorial regulation of transcription involves binding of transcription factors to DNA as well as protein-protein interactions between them. In this paper, we demonstrate the existence of a mutual transcriptional antagonism between the thyroid hormone receptor (TR) and the cyclic AMP response element binding protein (CREB), which involves a direct association of both transcription factors. TR inhibits transcriptional activity of CREB and represses activation of cAMP response element (CRE)-containing promoters. TR does not bind to the CRE in vitro, but in vivo the liganded receptor is tethered to the promoter through protein-protein interactions. In turn, expression of CREB reduces TR-dependent transcriptional responses. The association of TR with CREB inhibits the ability of protein kinase A to phosphorylate CREB at Ser133, and leads to a reduction in the ligand-dependent recruitment of the p160 coactivators by TR. These results indicate the existence of a transcriptional cross-talk between CREB and TR signalling pathways, which can have important functional consequences.This work was supported by grants BMC2001-2275 from the Ministerio de Ciencia y Tecnología, 08.1/0047.1/2001 from the Comunidad de Madrid and 02-101 from the AICR

The thyroid hormone receptor antagonizes CREB-mediated transcription

Combinatorial regulation of transcription involves binding of transcription factors to DNA as well as protein–protein interactions between them. In this paper, we demonstrate the existence of a mutual transcriptional antagonism between the thyroid hormone receptor (TR) and the cyclic AMP response element binding protein (CREB), which involves a direct association of both transcription factors. TR inhibits transcriptional activity of CREB and represses activation of cAMP response element (CRE)-containing promoters. TR does not bind to the CRE in vitro, but in vivo the liganded receptor is tethered to the promoter through protein–protein interactions. In turn, expression of CREB reduces TR-dependent transcriptional responses. The association of TR with CREB inhibits the ability of protein kinase A to phosphorylate CREB at Ser133, and leads to a reduction in the ligand-dependent recruitment of the p160 coactivators by TR. These results indicate the existence of a transcriptional cross-talk between CREB and TR signalling pathways, which can have important functional consequences

Therapeutic inhibition of TRF1 impairs the growth of p53-deficient K-RasG12V-induced lung cancer by induction of telomeric DNA damage

We are indebted to R. Serrano for animal care. We thank C. Guerra, R. Blasco, and D. Santamaria for scientific and technical advice. M.A.B.'s laboratory is funded with the Spanish Ministry of Science and Innovation, projects SAF2008-05384 and 2007-A-200950 (TELOMARKER), European Research Council Advanced grant GA#232854, the Korber Foundation, Fundacion Botin, and Fundacion Lilly.Telomeres are considered anti-cancer targets, as telomere maintenance above a minimum length is necessary for cancer growth. Telomerase abrogation in cancer-prone mouse models, however, only decreased tumor growth after several mouse generations when telomeres reach a critically short length, and this effect was lost upon p53 mutation. Here, we address whether induction of telomere uncapping by inhibition of the TRF1 shelterin protein can effectively block cancer growth independently of telomere length. We show that genetic Trf1 ablation impairs the growth of p53-null K-Ras(G12V)-induced lung carcinomas and increases mouse survival independently of telomere length. This is accompanied by induction of telomeric DNA damage, apoptosis, decreased proliferation, and G2 arrest. Long-term whole-body Trf1 deletion in adult mice did not impact on mouse survival and viability, although some mice showed a moderately decreased cellularity in bone marrow and blood. Importantly, inhibition of TRF1 binding to telomeres by small molecules blocks the growth of already established lung carcinomas without affecting mouse survival or tissue function. Thus, induction of acute telomere uncapping emerges as a potential new therapeutic target for lung cancer.S

The UBC-40 urothelial bladder cancer cell line index: a genomic resource for functional studies

Background. Urothelial bladder cancer is a highly heterogeneous disease. Cancer cell lines are useful tools for its study. This is a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines including information on origin, mutation status of genes implicated in bladder cancer (FGFR3, PIK3CA, TP53, and RAS), copy number alterations assessed using high density SNP arrays, uniparental disomy (UPD) events, and gene expression./nResults. Based on gene mutation patterns and genomic changes we identify lines representative of the FGFR3-driven tumor pathway and of the TP53/RB tumor suppressor-driven pathway. High-density array copy number analysis identified significant focal gains (1q32, 5p13.1-12, 7q11, and 7q33) and losses (i.e. 6p22.1) in regions altered in tumors but not previously described as affected in bladder cell lines. We also identify new evidence for frequent regions of UPD, often coinciding with regions reported to be lost in tumors. Previously undescribed chromosome X losses found in UBC lines also point to potential tumor suppressor genes. Cell lines representative of the FGFR3-driven pathway showed a lower number of UPD events./nConclusions. Overall, there is a predominance of more aggressive tumor subtypes among the cell lines. We provide a cell line classification that establishes their relatedness to the major molecularly-defined bladder tumor subtypes. The compiled information should serve as a useful reference to the bladder cancer research community and should help to select cell lines appropriate for the functional analysis of bladder cancer genes, for example those being identified through massive parallel sequencing.This work was supported, in part, by grants Consolider ONCOBIO, SAF2011-15934-E, Red Temática de Investigación Cooperativa en Cáncer (RTICC)]; Asociación Española Contra el Cáncer, EU-FP7-201663, and NIH RO-1 (CA089715); and National Institutes of Health grants CA075115 and CA104106 (to D.T.)

Erratum to: The UBC-40 urothelial bladder cancer cell line index: a genomic resource for functional studies

Following the publication of our recent article in BMC Genomics [1] a number of aspects were called to our attention. We have carefully reviewed the experiments reported in this manuscript, as well as additional data from our laboratories, and would like to make the following points

Erratum to: The UBC-40 urothelial bladder cancer cell line index: a genomic resource for functional studies

Following the publication of our recent article in BMC Genomics [1] a number of aspects were called to our attention. We have carefully reviewed the experiments reported in this manuscript, as well as additional data from our laboratories, and would like to make the following points