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News Article | May 10, 2017
Site: www.eurekalert.org

A technique designed by researchers at the Spanish National Cancer Research Centre makes it possible to generate translocations in the genome in an efficient and accurate manner; the model facilitates the study of mechanisms underlying certain pathologies A team from the Spanish National Cancer Research Centre (CNIO) has optimized a system capable of generating a cellular model of Ewing sarcoma. The technique, based on CRISPR and described in the pages of Stem Cell Reports, makes it possible to generate cellular models to analyse the mechanisms underlying the origin and progression of this and other diseases, as well as the search for new treatments. CRISPR, the famous genomic editing technique, not only serves to cure diseases, also to recreate them in cellular models to study the molecular events that give rise to them. These models are crucial to study new diagnostic and therapeutic pathways. In the paper published in the journal Stem Cell Reports, the authors present a significant technological development capable of recreating Ewing sarcoma in adult and embryonic human stem cells. "The idea is to have a system that enables us to generate a model that is as accurate as possible to what is happening in a tumour," said Sandra Rodríguez Perales, from the Molecular Cytogenetics and Genomic Engineering Unit and leader of the research project. In this way, with a model that reproduces the origins of the disease, it will be possible to analyse the underlying mechanisms and molecular bases of each pathology. In the case of Ewing sarcoma, the trigger of the disease is a translocation between chromosomes 11 and 22, which gives rise to the fusion of two genes, resulting in a new oncogene. The authors had already used CRISPR to induce this alteration and generate a model of this disease, but they had encountered a low level of efficacy and other methodological difficulties in applying the technique to human stem cells. "When we were working with cell lines, everything went smoothly, but when we applied it to stem cells, we came across a lot of problems," explains Raúl Torres Ruiz, co-author of the paper. To improve the results and to refine the technique, they compared three strategies to generate this translocation in the most efficient way possible using CRISPR. After several experiments, they noted that by combining the use of a sgRNA-Cas9 ribonucleoprotein complex generated in the laboratory (in place of a plasmid expression) and of a DNA "staple" - a short sequence that connects the ends of two chromosomes that breaks the CRISPR system and therefore facilitates translocation-? the success rate increased by up to a multiple of seven. This suggests, according to the authors, that we are faced with "a solid tool to induce targeted translocations". All the improvements implemented during the study have enabled the authors to generate this model in induced pluripotent stem cells (iPSC), which have enormous potential from a scientific point of view, since they constitute an ideal cellular model for the study of the development of various pathologies, among them the initial stages of oncogenic processes. All this will allow the study of the mechanistic bases of pathologies such as Ewing sarcoma. In addition, it may not only be useful for this sarcoma, it is also "a valid approximation for other pathologies," said Rodríguez Perales. "This strategy - the authors conclude- will facilitate the creation of cancer models from human stem cells and accurate genome editing to search for new drugs or cellular therapies, thus accelerating the advance from the laboratory to the clinic."


The project, lead by Manuel Valiente, focuses in blocking melanoma brain metastasis by targeting the microenvironment. Valiente in the only Spanish researcher awarded this year by the prestigious institution Manuel Valiente, head of the Brain Metastasis Group at the Spanish National Cancer Research Centre (CNIO), has been funded in the 2017 grant cycle by the Melanoma Research Alliance (MRA), the largest private funder of melanoma research. The research, titled 'Blocking melanoma brain metastasis by targeting the microenvironment', will focus on investigate actionable targets that will lead to more efficient design of anticancer treatments for patients with metastatic melanoma to the brain. MRA's 2017 awards portfolio totals over $8.5 million and funds 34 scientists at 28 leading academic institutions in six countries. Valiente is the only Spanish researcher awarded this year. The 34 funded programs aim to accelerate research into novel prevention and treatment strategies to drive better outcomes for melanoma patients and those at risk. "We are thrilled to be funding Dr. Valiente at the CNIO," says Louise M. Perkins, PhD, Chief Science Officer at the Melanoma Research Alliance. "Fresh perspectives from senior and young melanoma investigators as well as insights from astrophysicists, materials scientists and others new to the field are converging to drive pivotal advances in the prevention and diagnosis of melanoma and continue to build on our momentum of unlocking the most favorable treatments." The aim of Valiente and the Brain Metastasis Group is to uncover the mechanisms that allow cancer cells to spread, colonize and grow in the brain. "We believe that developing a research program devoted to this unmet clinical need is the only way to change the historical endpoint stage assigned to the diagnosis of brain metastasis", explains Valiente. The poor prognosis of this advanced stage of cancer is due to the poor efficacy of the limited therapeutic options available. "I have been always interested in working in Melanoma given the remarkable number of patients that develop brain metastases, however until know I did not have the chance", says Valiente. "We are trying to develop a new concept of treating metastasis in the context of personalized medicine, which is based on the possibility that new drugs can be developed to treat patients according to where metastases are located." MRA will allow Valiente and his group to explore this biology and its therapeutic implications in melanoma. This grant also reinforces CNIO's research in the melanoma field where Marisol Soengas, head of the Melanoma Group and also an MRA grantee, and Héctor Peinado, head of the Metastasis and Microenvironment Group, have ongoing projects.


News Article | April 18, 2017
Site: www.eurekalert.org

Cross-talk between malignant hepatocytes and neighboring progenitor cells signals progenitor cells to become activated, to expand and to maintain them in an undifferentiated state; thus, progenitor cells become oncogenic and contribute to tumourigenesis The malignant transformation of hepatocytes is the origin of most hepatocellular carcinomas, an aggressive type of liver cancer with high mortality rates. But these cells do not act alone. Research conducted by scientists at the Spanish National Cancer Research Centre (CNIO) reveals how hepatocytes "recruit" and "instruct" liver progenitor cells to contribute to the hepatic lesions. "The cellular origin of liver cancer, as well as the origin of tumour heterogeneity, are not clear yet and may be context-dependent," say the authors in the journal Cell Reports. Hepatocytes -the main liver cells- have been considered the main source of hepatocellular carcinomas but the results of this research provide various novel perspectives. "What we are showing here is that hepatic progenitor cells expand during tumorigenesis and, at one point, they become transformed because of oncogenic hepatocyte cross-talk; this makes them participate in liver tumour development in general, and in hepatocellular carcinomas in particular," explains Nabil Djouder, head of the Growth Factors, Nutrients and Cancer Group at the CNIO and main author of the paper. Thanks to an animal model -generated by Djouder and his team- that fairly reproduces the tumour formation process seen in human hepatocarcinogenesis, and to other genetic experiments, the authors have tried to follow the formation of the lesions and to define the histopathology of the various tumours that develop in this organ, whether benign (regenerative nodules, adenomas) or malignant (hepatocellular carcinoma or HCC). "This is what we've seen: oncogenic hepatocytes lead to hepatocellular carcinoma but, in a model that mimics human hepatocarcinogenesis, progenitor cells also participate in liver tumour heterogeneity. They mostly lead to benign tumours but sometimes they can lead to aggressive carcinomas", points out Djouder. In other words, progenitor cells become oncogenic even though they were not initially transformed. Malignant hepatocytes crosstalk and instruct neighbouring progenitor cells to become activated, maintaining them in an undifferentiated state while at the same time proliferating, becoming oncogenic themselves, and contributing to the lesions. This activation occurs, as shown by this study, when the hepatocytes secrete two substances (?-ketoglutarate and galectin-3) that act and transform progenitor cells. "Blocking galectin-3 can block the cross-talk between these cells thereby reducing tumourigenesis", a finding that could have therapeutic implications, explains Djouder.


News Article | May 4, 2017
Site: www.biosciencetechnology.com

DNA over-replication is a phenomenon that can have devastating consequences for proliferating cells. When parts of the genome are duplicated more than once, cells suffer from 'genomic instability' (alterations to the structure, composition and/or number of chromosomes), and this process gives rise to aberrant cells as those detected in many carcinomas. The cooperation of two proteins called CDC6 and CDT1 is essential for normal DNA replication but when they are not properly regulated, the genetic material replicates in excess. A paper published in Cell Reports by the DNA Replication Group of the Spanish National Cancer Research Centre (CNIO) sets out the fatal consequences of in vivo re-replication for the first time in mammalian organisms. Genome stability depends, to a great extent, on the accuracy of the DNA replication process. Exposure to UV light or to certain toxic chemicals increase the frequency of errors in the copy that may cause the death or the malignant transformation of the cell. Recent epidemiological studies indicate, for example, that two-thirds of cancerous mutations occur due to replication errors. "Broadly, there are three things that can go wrong in genome replication," explained Juan Méndez, head of the DNA Replication Group at the CNIO and leader of the study. "There may be too many mutations, the cell may replicate prematurely, without being prepared to do so and, finally, it may replicate too far." There are control mechanisms throughout all the key points of the process to prevent these situations. Two of these crucial links are the CDC6 and CDT1 proteins, which assemble the replicating machinery responsible for copying the 2 metres of DNA contained in each cell. Once the process is over, these proteins are inhibited biochemically because if they stay active, they can restart the replication process. In unicellular organisms such as yeast, DNA re-replication can lead to gene amplification, a genetic alteration common in cancer cells. Méndez and his group have used genetically modified mice to demonstrate that when CDC6 and CDT1 accumulate at abnormally high levels, DNA re-replication occurs in some cell types, affecting tissue functionality. Animals that overexpress one or another protein do not present replication issues but those with excessive levels of CDC6 and CDT1 do not survive more than two weeks, affected mainly by the loss of progenitor cells required for the regeneration of gastrointestinal tissue. "Previous cellular studies pointed in the direction that CDT1 deregulation was sufficient to induce over-replication," explained Méndez. However, "in the in vivo studies, we have found that most tissues need the combination of both proteins." What are the implications of this finding? "Cancer cells frequently have a very high basal level of CDC6", said Méndez-, which is related to their high rate of proliferation." Therefore, in these cells, it would be relatively easy to induce re-replication by simply increasing CDT1 levels, which would not affect normal cells. That is, precisely, what Méndez and his group are working on now. Using drugs to increase the levels of this protein, they are trying to determine whether, in the light of the results obtained so far, lethal DNA re-replication can be induced selectively in cancer cells in order to eliminate them from the body.


News Article | May 3, 2017
Site: www.eurekalert.org

Simultaneous over-expression of two proteins provokes DNA re-replication in some cell types. This phenomenon can lead to cell malignancy but also might be used to attack cancer cells DNA over-replication is a phenomenon that can have devastating consequences for proliferating cells. When parts of the genome are duplicated more than once, cells suffer from 'genomic instability' (alterations to the structure, composition and/or number of chromosomes), and this process gives rise to aberrant cells as those detected in many carcinomas. The cooperation of two proteins called CDC6 and CDT1 is essential for normal DNA replication but when they are not properly regulated, the genetic material replicates in excess. A paper published in Cell Reports by the DNA Replication Group of the Spanish National Cancer Research Centre (CNIO) sets out the fatal consequences of in vivo re-replication for the first time in mammalian organisms. Genome stability depends, to a great extent, on the accuracy of the DNA replication process. Exposure to UV light or to certain toxic chemicals increase the frequency of errors in the copy that may cause the death or the malignant transformation of the cell. Recent epidemiological studies indicate, for example, that two-thirds of cancerous mutations occur due to replication errors. "Broadly, there are three things that can go wrong in genome replication," explains Juan Méndez, head of the DNA Replication Group at the CNIO and leader of the study. "There may be too many mutations, the cell may replicate prematurely, without being prepared to do so and, finally, it may replicate too far." There are control mechanisms throughout all the key points of the process to prevent these situations. Two of these crucial links are the CDC6 and CDT1 proteins, which assemble the replicating machinery responsible for copying the 2 metres of DNA contained in each cell. Once the process is over, these proteins are inhibited biochemically because if they stay active, they can restart the replication process. In unicellular organisms such as yeast, DNA re-replication can lead to gene amplification, a genetic alteration common in cancer cells. Méndez and his group have used genetically modified mice to demonstrate that when CDC6 and CDT1 accumulate at abnormally high levels, DNA re-replication occurs in some cell types, affecting tissue functionality. Animals that overexpress one or another protein do not present replication issues but those with excessive levels of CDC6 and CDT1 do not survive more than two weeks, affected mainly by the loss of progenitor cells required for the regeneration of gastrointestinal tissue. "Previous cellular studies pointed in the direction that CDT1 deregulation was sufficient to induce over-replication," explains Méndez. However, "in the in vivo studies, we have found that most tissues need the combination of both proteins." What are the implications of this finding? "Cancer cells frequently have a very high basal level of CDC6", says Méndez-, which is related to their high rate of proliferation." Therefore, in these cells, it would be relatively easy to induce re-replication by simply increasing CDT1 levels, which would not affect normal cells. That is, precisely, what Méndez and his group are working on now. Using drugs to increase the levels of this protein, they are trying to determine whether, in the light of the results obtained so far, lethal DNA re-replication can be induced selectively in cancer cells in order to eliminate them from the body.


News Article | May 3, 2017
Site: phys.org

DNA over-replication is a phenomenon that can have devastating consequences for proliferating cells. When parts of the genome are duplicated more than once, cells suffer from 'genomic instability' (alterations to the structure, composition and/or number of chromosomes), and this process gives rise to aberrant cells as those detected in many carcinomas. The cooperation of two proteins called CDC6 and CDT1 is essential for normal DNA replication but when they are not properly regulated, the genetic material replicates in excess. A paper published in Cell Reports by the DNA Replication Group of the Spanish National Cancer Research Centre (CNIO) sets out the fatal consequences of in vivo re-replication for the first time in mammalian organisms. Genome stability depends, to a great extent, on the accuracy of the DNA replication process. Exposure to UV light or to certain toxic chemicals increase the frequency of errors in the copy that may cause the death or the malignant transformation of the cell. Recent epidemiological studies indicate, for example, that two-thirds of cancerous mutations occur due to replication errors. "Broadly, there are three things that can go wrong in genome replication," explains Juan Méndez, head of the DNA Replication Group at the CNIO and leader of the study. "There may be too many mutations, the cell may replicate prematurely, without being prepared to do so and, finally, it may replicate too far." There are control mechanisms throughout all the key points of the process to prevent these situations. Two of these crucial links are the CDC6 and CDT1 proteins, which assemble the replicating machinery responsible for copying the 2 metres of DNA contained in each cell. Once the process is over, these proteins are inhibited biochemically because if they stay active, they can restart the replication process. In unicellular organisms such as yeast, DNA re-replication can lead to gene amplification, a genetic alteration common in cancer cells. Méndez and his group have used genetically modified mice to demonstrate that when CDC6 and CDT1 accumulate at abnormally high levels, DNA re-replication occurs in some cell types, affecting tissue functionality. Animals that overexpress one or another protein do not present replication issues but those with excessive levels of CDC6 and CDT1 do not survive more than two weeks, affected mainly by the loss of progenitor cells required for the regeneration of gastrointestinal tissue. "Previous cellular studies pointed in the direction that CDT1 deregulation was sufficient to induce over-replication," explains Méndez. However, "in the in vivo studies, we have found that most tissues need the combination of both proteins." What are the implications of this finding? "Cancer cells frequently have a very high basal level of CDC6", says Méndez-, which is related to their high rate of proliferation." Therefore, in these cells, it would be relatively easy to induce re-replication by simply increasing CDT1 levels, which would not affect normal cells. That is, precisely, what Méndez and his group are working on now. Using drugs to increase the levels of this protein, they are trying to determine whether, in the light of the results obtained so far, lethal DNA re-replication can be induced selectively in cancer cells in order to eliminate them from the body. Explore further: DNA replication protein Cdt1 also has a role in mitosis, cancer


In contrast to the cells in the rest of the body, sex cells hold half the number of chromosomes (they are haploid) as a result of this special kind of cell division. In meiosis, a precursor cell —primordial germ cell— produces four spermatozoids during spermatogenesis, while only one oocyte is formed during oogenesis (the other three cells die during the process). Mice deficient in RingoA, generated in Nebreda's Signalling and Cell Cycling Laboratory, are apparently healthy but both sexes are completely sterile. After three years of experiments, IRB Barcelona postdoctoral researchers Petra Mikolcevic and Michitaka Isoda describe the molecular imbalances that occur during meiosis as a result of the absence of this protein. This study sheds new light on a key process for all forms of life that engage in sexual reproduction. "We all start life through meiosis so understanding how this process works is intellectually interesting," says Nebreda. Although meiosis was first described in the late 19th century, "many questions remain unanswered," explains this scientist, holder of a European Research Council grant. "There are no good in vitro models available to study meiosis. It is difficult to extract spermatocytes and to perform studies in plates; they have to be studied in the testicles. And oocytes are even worse because ovules are formed in early stages of development and working with embryos is technically complex." The scientists have discovered that RingoA is a key activator of Cdk2, the protein kinase with which it forms a complex required for meiosis. In fact, the genetic mouse model deficient in Cdk2, which was reported 12 years ago by Mariano Barbacid's group at CNIO, is also viable but sterile and shows the exact same alterations in meiosis as those observed by the researchers at IRB Barcelona. "In biology, if two practically indistinguishable phenotypes are obtained, it is an indication that the proteins have the same function and that they may work together." What was not known until now was that RingoA is the key partner for Cdk2 in meiosis, as Cdk2 normally forms complexes with another family of proteins called cyclins. The study demonstrates that RingoA is active at telomeres—structures that protect the ends of chromosomes and where Cdk2 is also found.  During meiosis, telomeres allow chromosomes to attach to the nuclear membrane, thus allowing them to exchange DNA fragments. This recombination of chromosomes is an essential feature of meiosis. Without the RingoA-Cdk2 complex, the telomeres of the chromosomes do not tether to the membrane but rather float in the nucleus, leading to chaotic recombination. The breaks in DNA needed for fragment exchange are not repaired and thus meiosis is not completed. Consequently, sex cells are not formed. "It would not be unreasonable to consider the development of a male contraceptive based on RingoA-Cdk2 inhibitors," proposes Nebreda. In the same way that women produce oocytes during embryo development, men can produce spermatozoids throughout adulthood. "If the pharmaceutical industry wanted to invest in this field, we have the biochemical techniques set up for the identification of inhibitors." Explore further: Researchers identify first sex chromosome gene involved in meiosis and male infertility More information: Essential role of the Cdk2 activator RingoA in meiotic telomere tethering to the nuclear envelope. Nature Comms. (2016, 30 March): DOI: 10.1038/NCOMMS11084


News Article | February 6, 2017
Site: www.medicalnewstoday.com

During the 'in vivo' reprogramming process, cellular telomeres are extended due to an increase in endogenous telomerase. This is the main conclusion of a paper published in Stem Cell Reports by a team from the Spanish National Cancer Research Centre (CNIO). Their observations show, for the first time, that the reprogramming of living tissue results in telomerase activation and telomere elongation; thus reversing one of the hallmarks of aging: 'the presence of short telomeres'. "We have found that when you induce cell dedifferentiation in an adult organism, the telomeres become longer, which is consistent with cellular rejuvenation", explains María A. Blasco, head of the CNIO Telomeres and Telomerase Group and leader of this research. This lengthening of the telomeres is an unequivocal sign of cell rejuvenation, which has been quantified for the first time here in a living organism. Blasco and her colleagues have worked with the so-called "reprogrammable mice" - created by Manuel Serrano, also a CNIO researcher, whose group is also involved in this project. Broadly speaking, the cells of these transgenic animals carry the four Yamanaka factors (OSKM) whose expression is turned on when an antibiotic is administered. In doing so, the cells regress to an embryonic-like state, a condition known as known as pluripotency. In light of the importance of telomeres in tissue regeneration, ageing and cancer, the authors decided to analyse the changes that occur in these protective structures of the chromosomes during the 'in vivo' reprogramming process, which leads to dedifferentiation of the tissues. Their observations indicate that this process entails a lengthening of the telomeres, a marker of cellular rejuvenation. This elongation occurs, according to this research, due to the action of telomerase. "What we have seen for the first time is the induction of telomerase 'in vivo'," explained Blasco and Rosa M. Marion, the leading authors of the paper. "To date, we do not know of any study that describes the induction of endogenous telomerase by defined transcription factors in the context of adult tissues," say the authors. The loss of cell differentiation is a phenomenon that takes place in a physiological context. It occurs during tissue regeneration and also in tumourigenesis. In fact, "this is increasingly considered one of the critical initial steps in cancer development," as stated in the paper. Dedifferentiation induced by 'in vivo' reprogramming, as well as the dedifferentiation associated with the initiation of cancer, involve "similar changes in the telomeres", say the authors. In both of these processes, we see the activation of telomerase and the subsequent elongation of the telomeres. A better knowledge of the changes described in the telomeres during 'in vivo' reprogramming and in pathological processes, such as cancer, will improve our understanding about the molecular events associated with cellular differentiation and, most likely, with other processes that involve cell plasticity. This work has been funded funded by the Spanish Ministry of Economy and Competiveness (PLAN RETOS) and by the Fundación Botín. Article: Common Telomere Changes during In Vivo Reprogramming and Early Stages of Tumorigenesis, Rosa M. Marión, Isabel López de Silanes, Lluc Mosteiro, Benjamin Gamache, Maria Abad, Carmen Guerra, Diego Megías, Manuel Serrano and Maria A. Blasco, Stem Cell Reports, doi: 10.1016/j.stemcr.2017.01.001, published online 2 February 2017.


Millions of human cells are constantly dividing to repair tissue damage and ensure continuity. This is one of the most complex cellular processes, and in order for it to be successful, cells must produce a copy of their genetic material (DNA). Researchers from the Spanish National Cancer Research Centre (CNIO) have discovered the critical role of the POLD3 protein in this DNA replication process; without POLD3, cells do not divide, and even the embryonic development process may be curtailed.


SUMO protein with the areas marked with ubiquitin in green. Credit: CNIO Three years ago, the research team directed by Óscar Fernández-Capetillo, head of the Genomic Instability Group at the Spanish National Cancer Research Centre (CNIO), obtained, for the first time, a panoramic view of the proteins that intervene in one of the most important and delicate cellular processes: the copying of genetic material during cellular division. They observed that the parts of the genome where the DNA was copied were also very rich in the modification by some very particular proteins, SUMOylations, and poor in others, ubiquitinations, but they were unable to understand why. The continuation of the paper, published today in the Nature Structural & Molecular Biology journal, reveals how the balance between these chemical markers in these regions is, in fact, key for the division of genetic material. During this process, the USP7 protein travels with an entourage of molecules that form part of the replisome—a set of proteins involved in copying the DNA—and eliminates the ubiquitination marks of proteins in the complex, thus explaining the low concentration of ubiquitin in these areas. "USP7 acts as an traffic officer that regulates the marks or traffic signs near the replisome. Eliminating ubiquitin prevents the proteins at the replisome from being expelled, thus favouring their concentration and the DNA copying process," explains Fernández-Capetillo. As the team described in the previous paper, replisomes contain up to 50 different proteins that participate in the delicate process of copying genetic material. Some proteins open DNA's double helix, others twist it to favour copying, others stabilise it, etc. They all move together through the genome to ensure a complete copy. In order to understand the role of USP7 and its cutting action on ubiquitin marks during the DNA copying process, the researchers used advanced protein tools. "We knew that replisome proteins could present both modifications simultaneously [ubiquitinations and SUMOylations], but we did not know how they worked," explains Fernández-Capetillo. "We now know that USP7 eliminates the ubiquitin marks on proteins that are also SUMOylated in replication areas, which explains why there is a low concentration of ubiquitin and high levels of SUMO." This balance between SUMO and ubiquitin establishes a code that regulates the concentration of proteins in the replisome. "If a protein is SUMOylated, it becomes enriched in the replisome, but if it is also ubiquitinated, it is expelled. This is a code of signals or flags that regulates the concentration of factors in the DNA replication area," say the researchers. Apart from being of academic interest, these studies are relevant for chemotherapy. USP7 inhibitors are currently being studied as possible anti-cancer agents in pre-clinical tests. "The model that had been proposed is that the compounds increase p53 levels, resulting in the suicide of tumour cells. Our data indicate that USP7 is essential for genome replication in cells with or without p53." With these data, the authors of the paper warn that these molecules may not be specifically anti-tumour agents. "We believe they inhibit the cell division process regardless of whether the cells are cancerous or healthy and, therefore, their use for treating cancer in the future will have to be reconsidered." Explore further: Researchers 'capture' the replication of the human genome for the first time More information: Emilio Lecona et al. USP7 is a SUMO deubiquitinase essential for DNA replication, Nature Structural & Molecular Biology (2016). DOI: 10.1038/nsmb.3185

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