Dissection of two routes to naïve pluripotency using different kinase inhibitors

Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors

Inactivation of EMILIN-1 by Proteolysis and Secretion in Small Extracellular Vesicles Favors Melanoma Progression and Metastasis

EMILIN-1; Melanoma; MetàstasiEMILIN-1; Melanoma; MetástasisEMILIN-1; Melanoma; MetastasisSeveral studies have demonstrated that melanoma-derived extracellular vesicles (EVs) are involved in lymph node metastasis; however, the molecular mechanisms involved are not completely defined. Here, we found that EMILIN-1 is proteolyzed and secreted in small EVs (sEVs) as a novel mechanism to reduce its intracellular levels favoring metastasis in mouse melanoma lymph node metastatic cells. Interestingly, we observed that EMILIN-1 has intrinsic tumor and metastasis suppressive-like properties reducing effective migration, cell viability, primary tumor growth, and metastasis. Overall, our analysis suggests that the inactivation of EMILIN-1 by proteolysis and secretion in sEVs reduce its intrinsic tumor suppressive activities in melanoma favoring tumor progression and metastasis.The authors gratefully acknowledge the support of the following sources of funding: Fundación Ramon Areces, MINECO (SAF2014-54541-R), Ramón y Cajal Programme, Asociación Española Contra el Cáncer, Constantes y Vitales (ATRES MEDIA/AXA Foundation) and FERO Foundation. We are also grateful for the support of the support of the Translational NeTwork for the CLinical application of Extracellular VesicleS, TeNTaCLES. RED2018-102411-T (AEI/10.13039/501100011033) and MINECO-Severo Ochoa predoctoral program to support A.A.L thesis and short term stay in Italy to perform this study. The CNIO, certified as Severo Ochoa Excellence Centre, is supported by the Spanish Government through the Instituto de Salud Carlos III (ISCIII)

Identificación de los mecanismos moleculares asociados con la toxicidad inducida por MeHg mediante el análisis de la expresión proteica diferencial

Comunicaciones a congreso

Understanding the first Neolithic occupation of Cova d’En Pardo (Planes, Alicante): preliminary results of the multidisciplinary analysis of levels VIII and VIIIb

Se presentan los resultados de las excavaciones llevadas a cabo en la Cova d’En Pardo (Planes, Alicante), concretamente los niveles VIII y VIIIb. El desarrollo de un proyecto multidisciplinar ha permitido caracterizar la ocupación de una pequeña cavidad por parte de las primeras comunidades campesinas asociadas al inicio del proceso de neolitización del levante de la península Ibérica.We present the results of excavations carried out in the Cova d’En Pardo (Planes, Alicante), specifically the levels VIII and VIIIb. The development of a multidisciplinary project has allowed characterize the occupation of a small cavity by the first farming communities associated with the Neolithization process of the Levant of Iberian Peninsula.Este trabajo se ha realizado en el marco del proyecto Origins and Spread of Agriculture in the western Mediterranean región (ERC-2008-AdG 230561)

La primera ocupación neolítica de la Cova d’En Pardo (Planes, Alicante). Avance de estudio pluridisciplinar de los niveles VIII y VIIIb

Se presentan los resultados de las excavaciones llevadas a cabo en la Cova d’En Pardo (Planes, Alicante), concretamente los niveles VIII y VIIIb. El desarrollo de un proyecto multidisciplinar ha permitido caracterizar la ocupación de una pequeña cavidad por parte de las primeras comunidades campesinas asociadas al inicio del proceso de neolitización del levante de la Península Ibérica

pTINCR microprotein promotes epithelial differentiation and suppresses tumor growth through CDC42 SUMOylation and activation

The human transcriptome contains thousands of small open reading frames (sORFs) that encode microproteins whose functions remain largely unexplored. Here, we show that TINCR lncRNA encodes pTINCR, an evolutionary conserved ubiquitin-like protein (UBL) expressed in many epithelia and upregulated upon differentiation and under cellular stress. By gain- and loss-of-function studies, we demonstrate that pTINCR is a key inducer of epithelial differentiation in vitro and in vivo. Interestingly, low expression of TINCR associates with worse prognosis in several epithelial cancers, and pTINCR overexpression reduces malignancy in patient-derived xenografts. At the molecular level, pTINCR binds to SUMO through its SUMO interacting motif (SIM) and to CDC42, a Rho-GTPase critical for actin cytoskeleton remodeling and epithelial differentiation. Moreover, pTINCR increases CDC42 SUMOylation and promotes its activation, triggering a pro-differentiation cascade. Our findings suggest that the microproteome is a source of new regulators of cell identity relevant for cancer.Acknowledgements: The authors thank VHIO Proteomics, Molecular Oncology and Genomics Core Facilities for technical assistance. We are grateful to Manuel Serrano for providing several reagents, advice and critical discussion on the manuscript. We also thank Alonso García and Raquel Pérez for their help in processing and analyzing digital images, Gemma Serra and Sandra Peiró for their assistance with subcellular fractionation and immunoprecipitation experiments, Sara Arce and Joaquín Mateo for providing several reagents during the development of critical experiments of this manuscript, and Juan Angel Recio for his help with the cSCC cohort. We are immensely grateful to all the members of the Abad lab for generating the know-how for the identification of novel sORFs, for the critical reading on the manuscript and in general for their constant support to this project. Work in the Abad lab is supported by VHIO, Fero Foundation, La Caixa Foundation, Asociación Española Contra el Cancer (AECC), La Mutua Foundation and by grants from the Spanish Ministry of Science and Innovation (SAF2015-69413-R; RTI2018-102046-B-I00). M.A. was recipient of a Ramón y Cajal contract from the Spanish Ministry of Science and Innovation (RYC-2013-14747). O.B. is recipient of a FPIAGAUR fellowship from Generalitat de Catalunya. We also acknowledge funding from grant PGC2018-094091-B-I00 from the Spanish Government

Phosphoproteomic analysis of neoadjuvant breast cancer suggests that increased sensitivity to paclitaxel is driven by CDK4 and filamin A

Precision oncology research is challenging outside the contexts of oncogenic addiction and/or targeted therapies. We previously showed that phosphoproteomics is a powerful approach to reveal patient subsets of interest characterized by the activity of a few kinases where the underlying genomics is complex. Here, we conduct a phosphoproteomic screening of samples from HER2-negative female breast cancer receiving neoadjuvant paclitaxel (N = 130), aiming to find candidate biomarkers of paclitaxel sensitivity. Filtering 11 candidate biomarkers through 2 independent patient sets (N= 218) allowed the identification of a subgroup of patients characterized by high levels of CDK4 and filamin-A who had a 90% chance of achieving a pCR in response to paclitaxel. Mechanistically, CDK4 regulates filamin-A transcription, which in turn forms a complex with tubulin and CLIP-170, which elicits increased binding of paclitaxel to microtubules, microtubule acetylation and stabilization, and mitotic catastrophe. Thus, phosphoproteomics allows the identification of explainable factors for predicting response to paclitaxel

pTINCR microprotein promotes epithelial differentiation and suppresses tumor growth through CDC42 SUMOylation and activation

The human transcriptome contains thousands of small open reading frames (sORFs) that encode microproteins whose functions remain largely unexplored. Here, we show that TINCR lncRNA encodes pTINCR, an evolutionary conserved ubiquitin-like protein (UBL) expressed in many epithelia and upregulated upon differentiation and under cellular stress. By gain- and loss-of-function studies, we demonstrate that pTINCR is a key inducer of epithelial differentiation in vitro and in vivo. Interestingly, low expression of TINCR associates with worse prognosis in several epithelial cancers, and pTINCR overexpression reduces malignancy in patient-derived xenografts. At the molecular level, pTINCR binds to SUMO through its SUMO interacting motif (SIM) and to CDC42, a Rho-GTPase critical for actin cytoskeleton remodeling and epithelial differentiation. Moreover, pTINCR increases CDC42 SUMOylation and promotes its activation, triggering a pro-differentiation cascade. Our findings suggest that the microproteome is a source of new regulators of cell identity relevant for cancer

Inhibition of de novo NAD(+) synthesis by oncogenic URI causes liver tumorigenesis through DNA damage.

Molecular mechanisms responsible for hepatocellular carcinoma (HCC) remain largely unknown. Using genetically engineered mouse models, we show that hepatocyte-specific expression of unconventional prefoldin RPB5 interactor (URI) leads to a multistep process of HCC development, whereas its genetic reduction in hepatocytes protects against diethylnitrosamine (DEN)-induced HCC. URI inhibits aryl hydrocarbon (AhR)- and estrogen receptor (ER)-mediated transcription of enzymes implicated in L-tryptophan/kynurenine/nicotinamide adenine dinucleotide (NAD(+)) metabolism, thereby causing DNA damage at early stages of tumorigenesis. Restoring NAD(+) pools with nicotinamide riboside (NR) prevents DNA damage and tumor formation. Consistently, URI expression in human HCC is associated with poor survival and correlates negatively with L-tryptophan catabolism pathway. Our results suggest that boosting NAD(+) can be prophylactic or therapeutic in HCC.We are thankful to F. Real and M. Barbacid for providing the Ela-1-myc and K-Ras512v pancreatic models, respectively. We thank R. Ricci, M. Serrano, R. Hamacher, G. Gomes, S. Wurm, F. Diaz, and S. Anderson for.support and advice. K.S.T. is a recipient of La Caixa predoctoral fellowship. A.L.G. is a recipient of the Caja Navarra postdoctoral fellowship. P.X.E. is a recipient of the Fondo de Investigaciones Sanitarias grant (CA10/01231). M.R.J. is supported by UCL Hospitals Biomedical Research Centre. The E.F.W. lab is supported by F-BBVA, the Spanish Ministry of Economy and Competitiveness (BFU201240230) and the European Research Council (ERC)-Advanced grant (ERC-FCK2008/37). N.D. is a recipient of the Spanish Ramon y Cajal fellowship. This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2010 - 18518), the Association for International Cancer Research AICR-UK (11-0242), CNIO (BC1104-08), and the European Foundation for the Study of Diabetes./S