Contribution of the R-Ras2 GTP-binding protein to primary breast tumorigenesis and late-stage metastatic disease

R-Ras2 is a transforming GTPase that shares downstream effectors with Ras subfamily proteins. However, little information exists about the function of this protein in tumorigenesis and its signalling overlap with classical Ras GTPases. Here we show, by combining loss- and gain-of-function studies in breast cancer cells, mammary epithelial cells and mouse models, that endogenous R-Ras2 has a role in both primary breast tumorigenesis and the late metastatic steps of cancer cells in the lung parenchyma. R-Ras2 drives tumorigenesis in a phosphatidylinostiol-3 kinase (PI3K)-dependent and signalling autonomous manner. By contrast, its prometastatic role requires other priming oncogenic signals and the engagement of several downstream elements. R-Ras2 function is required even in cancer cells exhibiting constitutive activation of classical Ras proteins, indicating that these GTPases are not functionally redundant. Our results also suggest that application of long-term R-Ras2 therapies will result in the development of compensatory mechanisms in breast tumoursFil: Larive, Ramon. Universidad de Salamanca; España. University of Montpellier I and II; FranciaFil: Moriggi, Giulia. Universidad de Salamanca; EspañaFil: Menacho Márquez, Mauricio Ariel. Universidad de Salamanca; España. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Cañamero, Marta. Centro Nacional de Investigaciones Oncológicas; EspañaFil: de Alava, Enrique. Universidad de Salamanca; España. Hospital Universitario Virgen del Rocío. Sevilla; EspañaFil: Alarcón, Balbino. Centro de Biología Molecular ‘‘Severo Ochoa’. Madrid; EspañaFil: Dosil, Mercedes. Universidad de Salamanca; EspañaFil: Bustelo, Xosé R.. Universidad de Salamanca; Españ

The APC/C cofactor Cdh1 prevents replicative stress and p53-dependent cell death in neural progenitors

The E3-ubiquitin ligase APC/C-Cdh1 is essential for endoreduplication but its relevance in the mammalian mitotic cell cycle is still unclear. Here we show that genetic ablation of Cdh1 in the developing nervous system results in hypoplastic brain and hydrocephalus. These defects correlate with enhanced levels of Cdh1 substrates and increased entry into the S phase in neural progenitors. However, cell division is prevented in the absence of Cdh1 due to hyperactivation of cyclin-dependent kinases, replicative stress, induction of p53, G2 arrest and apoptotic death of these progenitor cells. Concomitant ablation of p53 rescues apoptosis but not replicative stress, resulting in the presence of damaged neurons throughout the adult brain. These data indicate that the inactivation of Cdh1 in vivo results in replicative stress, cell cycle arrest and cell death, supporting recent therapeutic proposals aimed to inhibit the APC/C in tumours.M.E. was supported by the Spanish Ministry of Economy and Competitiveness (MINECO). This work was funded by grants from the Foundation Ramón Areces and MINECO SAF2012-38215 to M.M.). The Cell Division and Cancer Group of the CNIO are supported by the OncoCycle Programme (S2010/BMD-2470) from the Comunidad de Madrid, the OncoBIO Consolider-Ingenio 2010 Programme (MINECO, CSD2007-00017) and the European Union Seventh Framework Programme (MitoSys project; HEALTH-F5-2010-241548).Peer Reviewe

Betunes modificados con azufre: aditivos estabilizadores

En el presente trabajo se estudia la influencia de aditivos encaminados a conseguir la estabilidad de dichos betunes modificados. Mediante medidas de viscosidad y penetración se ha comprobado que la presencia combinada de diciclopentadieno + aminas, favorece la interacción del azufre con el betún por formación de polisulfuros. Asimismo, se ha analizado la influencia de inhibidores de radicales libre

Thermal behaviour of bitumen modified by sulphur addition

The most frequent use of bitumen is as binder for pavement applications. The effect of sulphur addition on the properties of the bitumen has been extensively studied several decades ago. Recently, there is a renewed interest in researching the behaviour of sulphur-bitumen combination, because off 1.The future availability of bitumen may be limited and 2. The beneficial consumption of great amounts of sulphur compounds from petroleum refining is advisable. The addition of sulphur to bitumen provokes the beginning of chemical reactions depending on the sulphur content and heating temperature. At heating temperatures T< 140 ºC liquid sulphur reacts with naphthenic-aromatic fraction forming polysulphides. At temperatures above 150 ºC dehydrogenization reactions with emission of hydrogen sulfide take place and naphthenic-aromatic molecules are transformed into asphaltenes. Therefore, the addition of sulphur to bitumen provokes changes in the chemical structure of the bitumen. The objective of this work is to analyze, the thermal behaviour of sulphur-bitumen mixtures of different composition (0-35 %wt sulphur content) prepared at 130 and 140 ºC, by means of differential scanning calorimetry (DSC). Besides, the volatile emissions of the mixtures at high temperature have been estimated from loss weight measurements as a function of stored tim

Targeting Mitotic Exit Leads to Tumor Regression In Vivo: Modulation by Cdk1, Mastl, and the PP2A/B55α,δ Phosphatase

SummaryTargeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55α or B55δ regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells

Replication stress caused by low MCM expression limits fetal erythropoiesis and hematopoietic stem cell functionality

Replicative stress during embryonic development influences ageing and predisposition to disease in adults. A protective mechanism against replicative stress is provided by the licensing of thousands of origins in G1 that are not necessarily activated in the subsequent S-phase. These `dormant' origins provide a backup in the presence of stalled forks and may confer flexibility to the replication program in specific cell types during differentiation, a role that has remained unexplored. Here we show, using a mouse strain with hypomorphic expression of the origin licensing factor mini-chromosome maintenance (MCM)3 that limiting origin licensing in vivo affects the functionality of hematopoietic stem cells and the differentiation of rapidly-dividing erythrocyte precursors. Mcm3-deficient erythroblasts display aberrant DNA replication patterns and fail to complete maturation, causing lethal anemia. Our results indicate that hematopoietic progenitors are particularly sensitive to replication stress, and full origin licensing ensures their correct differentiation and functionality.We thank members of our laboratories for helpful discussions, Marcos Malumbres (CNIO) for advice on the design of the Mcm3-Lox allele, Isabel Blanco for her administrative help with mouse work and Soraya Ruiz for excellent handling of the mouse colony in J.M.'s group. Research was supported by the Spanish Ministry of Economy and Competitiveness (grants BFU2013-49153-P and Consolider-Ingenio CSD2007-00015 to J.M., SAF2011-23753 to O.F.-C., BFU2012-35892 to J.I.) and RO1 HL092471 to E.P., S.A. was the recipient of an EMBO short-term fellowship to visit E.P.'s laboratory at UCSF. We are grateful to Manuel Serrano (CNIO), Almudena Ramiro (CNIC) and Arkaitz Ibarra (The Salk Institute, USA) for useful comments on the manuscript.S

P27Kip1 directly represses Sox2 during embryonic stem cell differentiation

The mechanisms responsible for the transcriptional silencing of pluripotency genes in differentiated cells are poorly understood. We have observed that cells lacking the tumor suppressor p27 can be reprogrammed into induced pluripotent stem cells (iPSCs) in the absence of ectopic Sox2. Interestingly, cells and tissues from p27 null mice, including brain, lung, and retina, present an elevated basal expression of Sox2, suggesting that p27 contributes to the repression of Sox2. Furthermore, p27 null iPSCs fail to fully repress Sox2 upon differentiation. Mechanistically, we have found that upon differentiation p27 associates to the SRR2 enhancer of the Sox2 gene together with a p130-E2F4-SIN3A repressive complex. Finally, Sox2 haploinsufficiency genetically rescues some of the phenotypes characteristic of p27 null mice, including gigantism, pituitary hyperplasia, pituitary tumors, and retinal defects. Collectively, these results demonstrate an unprecedented connection between p27 and Sox2 relevant for reprogramming and cancer and for understanding human pathologies associated with p27 germline mutations

A new role of SNAI2 in postlactational involution of the mammary gland links it to luminal breast cancer development

PMCID: PMC4560637Breast cancer is a major cause of mortality in women. The transcription factor SNAI2 has been implicated in the pathogenesis of several types of cancer, including breast cancer of basal origin. Here we show that SNAI2 is also important in the development of breast cancer of luminal origin in MMTV-ErbB2 mice. SNAI2 deficiency leads to longer latency and fewer luminal tumors, both of these being characteristics of pretumoral origin. These effects were associated with reduced proliferation and a decreased ability to generate mammospheres in normal mammary glands. However, the capacity to metastasize was not modified. Under conditions of increased ERBB2 oncogenic activity after pregnancy plus SNAI2 deficiency, both pretumoral defects - latency and tumor load - were compensated. However, the incidence of lung metastases was dramatically reduced. Furthermore, SNAI2 was required for proper postlactational involution of the breast. At 3 days post lactational involution, the mammary glands of Snai2-deficient mice exhibited lower levels of pSTAT3 and higher levels of pAKT1, resulting in decreased apoptosis. Abundant noninvoluted ducts were still present at 30 days post lactation, with a greater number of residual ERBB2+ cells. These results suggest that this defect in involution leads to an increase in the number of susceptible target cells for transformation, to the recovery of the capacity to generate mammospheres and to an increase in the number of tumors. Our work demonstrates the participation of SNAI2 in the pathogenesis of luminal breast cancer, and reveals an unexpected connection between the processes of postlactational involution and breast tumorigenesis in Snai2-null mutant mice.JPL was partially supported by FEDER and MICINN (PLE2009-119, SAF2014-56989-R), Instituto de Salud Carlos III (PI07/0057, PI10/00328, PIE14/00066), Junta de Castilla y León (SAN673/SA26/08, SAN126/SA66/09, SA078A09, CSI034U13), the “Fundación Eugenio Rodríguez Pascual”, the “Fundación Inbiomed” (Instituto Oncológico Obra Social de la Caja Guipozcoa-San Sebastian, Kutxa), and the “Fundación Sandra Ibarra de Solidaridad frente al Cáncer”. AC was supported by FIS (PI07/0057) and MICINN (PLE2009-119). SCLL was funded by a JAEdoc Fellowship (CSIC)/FSE. MMSF and ABG are funded by fellowships from the Junta de Castilla y Leon. JHM was supported by the National Institutes of Health, a National Cancer Institute grant (R01 CA116481), and the Low-Dose Scientific Focus Area, Office of Biological & Environmental Research, US Department of Energy (DE-AC02-05CH11231).Peer Reviewe

Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment

This is the peer reviewed version of the following article: Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment. Gut 64.12 (2015): 1921-1935 and which has been published in final form at http://dx.doi.org/10.1136/gutjnl-2014-308935OBJECTIVES: The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance. DESIGN: Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology. RESULTS: We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-β1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R. CONCLUSIONS: Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis.CH: ERC Advanced Investigator Grant (Pa-CSC 233460), European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 256974 (EPC-TM-NET) and n° 602783 (CAM-PaC), the Subdirección General de Evaluación y Fomento de la Investigación, Fondo de Investigación Sanitaria (PS09/02129 & PI12/02643) and the Programa Nacional de Internacionalización de la I+D, Subprogramma: FCCI 2009 (PLE2009-0105; both Ministerio de Economía y Competitividad (es), Spain), BSJr: Rámon y Cajal Merit Award from the Ministerio de Economía y Competitividad, Spain and Clinic and Laboratory Integration Program (CLIP) grant from the Cancer Research Institute, NY, NY. MC: La Caixa Predoctoral Fellowshi