Publications by authors named "Rocío Sotillo"

24 Publications

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Methrix: an R/bioconductor package for systematic aggregation and analysis of bisulfite sequencing data.

Bioinformatics 2020 Dec 21. Epub 2020 Dec 21.

Division of Cancer Epigenomics, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Motivation: Whole genome bisulfite sequencing (WGBS), measures DNA methylation at base pair resolution resulting in large bedGraph like coverage files. Current options for processing such files are hindered by discrepancies in file format specification, speed and memory requirements.

Results: We developed methrix, an R package, which provides a toolset for systematic analysis of large datasets. Core functionality of the package includes a comprehensive bedGraph or similar tab-separated text file reader - which summarizes methylation calls based on annotated reference indices, infers and collapses strands, and handles uncovered reference CpG sites while facilitating a flexible input file format specification. Additional optimized functions for quality control filtering, sub-setting, and visualization allow user-friendly and effective processing of WGBS results. Easy integration with tools for differentially methylated region (DMR) calling and annotation further eases the analysis of genome-wide methylation data. Overall, methrix enriches established WGBS workflows by bringing together computational efficiency and versatile functionality.

Availability And Implementation: Methrix is implemented as an R package, made available under MIT license at https://github.com/CompEpigen/methrix and can be installed from the Bioconductor repository.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa1048DOI Listing
December 2020

Targeting alveolar macrophages shows better treatment response than deletion of interstitial macrophages in EGFR mutant lung adenocarcinoma.

Immun Inflamm Dis 2020 06 3;8(2):181-187. Epub 2020 Mar 3.

Department of Molecular Thoracic Oncology, German Cancer Research Center, Heidelberg, Germany.

Introduction: Alveolar macrophages (AMs) are critical in the development of lung adenocarcinoma driven by epidermal growth factor receptor (EGFR) mutations. Whether interstitial macrophages (IMs) are also involved in lung tumorigenesis is still unclear. Thus, the aim of this study is to evaluate the role of both AM and IM in the development of EGFR mutant driven lung adenocarcinoma.

Methods: We used the EGFR mutant doxycycline-inducible mouse model of lung adenocarcinoma to deplete interstitial or AMs by clodronate-encapsulated liposomes administered intravenously (IV) and intratracheally (IT), respectively. Tumor burden, AMs, and the tumor microenvironment were examined by immunohistochemistry, bronchoalveolar lavage fluid or flow cytometry.

Results: Clodronate treatment resulted in a significant reduction of tumor burden compared with vehicle liposomes alone. Elimination of AMs resulted in a significant reduction of proliferation compared with IV treatment. However, both treatments resulted in a significantly higher number of Ki67 positive cells compared with control mice, suggesting that tumor cells still proliferate despite the treatment. The number of natural killer cells decreased during tumor development, and it remained low even after the elimination of AMs. We also observed that IT instillation of clodronate significantly increased the number of CD8+ T cells, which was higher compared with vehicle-treated mice and mice where only IMs were depleted. The similar trend was observed in immunohistological analyses of CD8+ T cells.

Conclusions: These results suggest that the reduction of AMs has a stronger impact on restricting tumor progression compared with targeting IMs. The depletion of AMs leads to an elevated infiltration of CD8+ T cells into the lung that might be responsible for tumor growth impairment. Altogether, elimination of AMs is a better strategy to reduce EGFR mutant tumor growth and is less toxic, suggesting the selectively targeting of AMs to complement established therapies.
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http://dx.doi.org/10.1002/iid3.293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212197PMC
June 2020

Acquisition of chromosome instability is a mechanism to evade oncogene addiction.

EMBO Mol Med 2020 03 6;12(3):e10941. Epub 2020 Feb 6.

Division of Molecular Thoracic Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Chromosome instability (CIN) has been associated with therapeutic resistance in many cancers. However, whether tumours become genomically unstable as an evolutionary mechanism to overcome the bottleneck exerted by therapy is not clear. Using a CIN model of Kras-driven breast cancer, we demonstrate that aneuploid tumours acquire genetic modifications that facilitate the development of resistance to targeted therapy faster than euploid tumours. We further show that the few initially chromosomally stable cancers that manage to persist during treatment do so concomitantly with the acquisition of CIN. Whole-genome sequencing analysis revealed that the most predominant genetic alteration in resistant tumours, originated from either euploid or aneuploid primary tumours, was an amplification on chromosome 6 containing the cMet oncogene. We further show that these tumours are dependent on cMet since its pharmacological inhibition leads to reduced growth and increased cell death. Our results highlight that irrespective of the initial CIN levels, cancer genomes are dynamic and the acquisition of a certain level of CIN, either induced or spontaneous, is a mechanism to circumvent oncogene addiction.
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http://dx.doi.org/10.15252/emmm.201910941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059010PMC
March 2020

Rare, functional, somatic variants in gene families linked to cancer genes: GPCR signaling as a paradigm.

Oncogene 2019 09 23;38(38):6491-6506. Epub 2019 Jul 23.

BioQuant, Heidelberg University, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany.

Oncodriver genes are usually identified when mutations recur in multiple tumours. Different drivers often converge in the activation or repression of key cancer-relevant pathways. However, as many pathways contain multiple members of the same gene family, individual mutations might be overlooked, as each family member would necessarily have a lower mutation frequency and thus not identified as significant in any one-gene-at-a-time analysis. Here, we looked for mutated, functional sequence positions in gene families that were mutually exclusive (in patients) with another gene in the same pathway, which identified both known and new candidate oncodrivers. For instance, many inactivating mutations in multiple G-protein (particularly G) coupled receptors, are mutually exclusive with Gα oncogenic activating mutations, both of which ultimately enhance cAMP signalling. By integrating transcriptomics and interaction data, we show that the G pathway is upregulated in multiple cancer types, even those lacking known GNAS activating mutations. This suggests that cancer cells may develop alternative strategies to activate adenylate cyclase signalling in multiple cancer types. Our study provides a mechanistic interpretation for several rare somatic mutations in multi-gene oncodrivers, and offers possible explanations for known and potential off-label cancer treatments, suggesting new therapeutic opportunities.
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http://dx.doi.org/10.1038/s41388-019-0895-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756116PMC
September 2019

Club cells form lung adenocarcinomas and maintain the alveoli of adult mice.

Elife 2019 05 29;8. Epub 2019 May 29.

Laboratory for Molecular Respiratory Carcinogenesis, Department of Physiology, Faculty of Medicine, University of Patras, Rio, Greece.

Lung cancer and chronic lung diseases impose major disease burdens worldwide and are caused by inhaled noxious agents including tobacco smoke. The cellular origins of environmental-induced lung tumors and of the dysfunctional airway and alveolar epithelial turnover observed with chronic lung diseases are unknown. To address this, we combined mouse models of genetic labeling and ablation of airway (club) and alveolar cells with exposure to environmental noxious and carcinogenic agents. Club cells are shown to survive mutations and to form lung tumors after tobacco carcinogen exposure. Increasing numbers of club cells are found in the alveoli with aging and after lung injury, but go undetected since they express alveolar proteins. Ablation of club cells prevents chemical lung tumors and causes alveolar destruction in adult mice. Hence club cells are important in alveolar maintenance and carcinogenesis and may be a therapeutic target against premalignancy and chronic lung disease.
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http://dx.doi.org/10.7554/eLife.45571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606035PMC
May 2019

Plk1 overexpression induces chromosomal instability and suppresses tumor development.

Nat Commun 2018 08 1;9(1):3012. Epub 2018 Aug 1.

Division of Molecular Thoracic Oncology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.

Polo-like kinase 1 (Plk1) is overexpressed in a wide spectrum of human tumors, being frequently considered as an oncogene and an attractive cancer target. However, its contribution to tumor development is unclear. Using a new inducible knock-in mouse model we report here that Plk1 overexpression results in abnormal chromosome segregation and cytokinesis, generating polyploid cells with reduced proliferative potential. Mechanistically, these cytokinesis defects correlate with defective loading of Cep55 and ESCRT complexes to the abscission bridge, in a Plk1 kinase-dependent manner. In vivo, Plk1 overexpression prevents the development of Kras-induced and Her2-induced mammary gland tumors, in the presence of increased rates of chromosome instability. In patients, Plk1 overexpression correlates with improved survival in specific breast cancer subtypes. Therefore, despite the therapeutic benefits of inhibiting Plk1 due to its essential role in tumor cell cycles, Plk1 overexpression has tumor-suppressive properties by perturbing mitotic progression and cytokinesis.
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http://dx.doi.org/10.1038/s41467-018-05429-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070485PMC
August 2018

Metabolic shifts in residual breast cancer drive tumor recurrence.

J Clin Invest 2017 Jun 15;127(6):2091-2105. Epub 2017 May 15.

EMBL Monterotondo, Adriano Buzzati-Traverso Campus, Monterotondo, Italy.

Tumor recurrence is the leading cause of breast cancer-related death. Recurrences are largely driven by cancer cells that survive therapeutic intervention. This poorly understood population is referred to as minimal residual disease. Here, using mouse models that faithfully recapitulate human disease together with organoid cultures, we have demonstrated that residual cells acquire a transcriptionally distinct state from normal epithelium and primary tumors. Gene expression changes and functional characterization revealed altered lipid metabolism and elevated ROS as hallmarks of the cells that survive tumor regression. These residual cells exhibited increased oxidative DNA damage, potentiating the acquisition of somatic mutations during hormonal-induced expansion of the mammary cell population. Inhibition of either cellular fatty acid synthesis or fatty acid transport into mitochondria reduced cellular ROS levels and DNA damage, linking these features to lipid metabolism. Direct perturbation of these hallmarks in vivo, either by scavenging ROS or by halting the cyclic mammary cell population expansion, attenuated tumor recurrence. Finally, these observations were mirrored in transcriptomic and histological signatures of residual cancer cells from neoadjuvant-treated breast cancer patients. These results highlight the potential of lipid metabolism and ROS as therapeutic targets for reducing tumor recurrence in breast cancer patients.
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http://dx.doi.org/10.1172/JCI89914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451224PMC
June 2017

Cellular Prion Protein PrP and Ecto-5'-Nucleotidase Are Markers of the Cellular Stress Response to Aneuploidy.

Cancer Res 2017 06 4;77(11):2914-2926. Epub 2017 Apr 4.

Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.

Aneuploidy is a hallmark of most human tumors, but the molecular physiology of aneuploid cells is not well characterized. In this study, we screened cell surface biomarkers of approximately 300 proteins by multiparameter flow cytometry using multiple aneuploid model systems such as cell lines, patient samples, and mouse models. Several new biomarkers were identified with altered expression in aneuploid cells, including overexpression of the cellular prion protein CD230/PrP and the immunosuppressive cell surface enzyme ecto-5'-nucleotidase CD73. Functional analyses associated these alterations with increased cellular stress. An increased number of CD73 cells was observed in confluent cultures in aneuploid cells relative to their diploid counterparts. An elevated expression in CD230/PrP was observed in serum-deprived cells in association with increased generation of reactive oxygen species. Overall, our work identified biomarkers of aneuploid karyotypes, which suggest insights into the underlying molecular physiology of aneuploid cells. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-3052DOI Listing
June 2017

Pan-cancer analysis distinguishes transcriptional changes of aneuploidy from proliferation.

Genome Res 2017 04 20;27(4):501-511. Epub 2017 Mar 20.

European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg 69117, Germany.

Patterns of gene expression in tumors can arise as a consequence of or result in genomic instability, characterized by the accumulation of somatic copy number alterations (SCNAs) and point mutations (PMs). Expression signatures have been widely used as markers for genomic instability, and both SCNAs and PMs could be thought to associate with distinct signatures given their different formation mechanisms. Here we test this notion by systematically investigating SCNA, PM, and transcriptome data from 2660 cancer patients representing 11 tumor types. Notably, our data indicate that similar expression signatures can be derived from correlating gene expression with either SCNA or PM load. Gene sets related to cell growth and proliferation generally associated positively, and immunoregulatory gene sets negatively, with variant burden. In-depth analyses revealed several genes whose de-regulation correlates with SCNA but not with PM burden, yielding downstream effectors of and signaling unique to high-SCNA tumors. We compared our findings to expression changes observed in two different cancer mouse models with persistent mitotic chromosomal instability, observing a decrease in proliferative expression signatures. Our results suggest that overexpression of cell-cycle-related genes are a characteristic of proliferation, and likely tumor evolution, rather than ongoing genomic instability.
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http://dx.doi.org/10.1101/gr.212225.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378169PMC
April 2017

Pan-cancer analysis of somatic copy-number alterations implicates IRS4 and IGF2 in enhancer hijacking.

Nat Genet 2017 01 21;49(1):65-74. Epub 2016 Nov 21.

European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.

Extensive prior research focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearrangement of cis-regulatory elements (CREs) remains unclear. Here we present a framework for inferring cancer-related gene overexpression resulting from CRE reorganization (e.g., enhancer hijacking) by integrating SCNAs, gene expression data and information on topologically associating domains (TADs). Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer is associated with recurrent deletions in cis, and we present evidence supporting a tumor-promoting role. We additionally pursued cancer-type-specific analyses and uncovered IGF2 as a target for enhancer hijacking in colorectal cancer. Recurrent tandem duplications intersecting with a TAD boundary mediate de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, resulting in high-level gene activation. Our framework enables systematic inference of CRE rearrangements mediating dysregulation in cancer.
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http://dx.doi.org/10.1038/ng.3722DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5791882PMC
January 2017

Negative Selection and Chromosome Instability Induced by Mad2 Overexpression Delay Breast Cancer but Facilitate Oncogene-Independent Outgrowth.

Cell Rep 2016 06 9;15(12):2679-91. Epub 2016 Jun 9.

Mouse Biology Unit, European Molecular Biology Laboratory, Via Ramarini 32, 00015 Monterotondo, Italy; Division of Molecular Thoracic Oncology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Electronic address:

Chromosome instability (CIN) is associated with poor survival and therapeutic outcome in a number of malignancies. Despite this correlation, CIN can also lead to growth disadvantages. Here, we show that simultaneous overexpression of the mitotic checkpoint protein Mad2 with Kras(G12D) or Her2 in mammary glands of adult mice results in mitotic checkpoint overactivation and a delay in tumor onset. Time-lapse imaging of organotypic cultures and pathologic analysis prior to tumor establishment reveals error-prone mitosis, mitotic arrest, and cell death. Nonetheless, Mad2 expression persists and increases karyotype complexity in Kras tumors. Faced with the selective pressure of oncogene withdrawal, Mad2-positive tumors have a higher frequency of developing persistent subclones that avoid remission and continue to grow.
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http://dx.doi.org/10.1016/j.celrep.2016.05.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920917PMC
June 2016

Is aneuploidy deciding your fate?

Cell Cycle 2014 3;13(9):1370. Epub 2014 Apr 3.

Mouse Biology Unit; European Molecular Biology Laboratory (EMBL); Monterotondo, Italy.

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http://dx.doi.org/10.4161/cc.28753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050132PMC
April 2016

Mad2 is a critical mediator of the chromosome instability observed upon Rb and p53 pathway inhibition.

Cancer Cell 2011 Jun;19(6):701-14

Department of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, USA.

Multiple mechanisms have been proposed to explain how Rb and p53 tumor suppressor loss lead to chromosome instability (CIN). It was recently shown that Rb pathway inhibition causes overexpression of the mitotic checkpoint gene Mad2, but whether Mad2 overexpression is required to generate CIN in this context is unknown. Here, we show that CIN in cultured cells lacking Rb family proteins requires Mad2 upregulation and that this upregulation is also necessary for CIN and tumor progression in vivo. Mad2 is also repressed by p53 and its upregulation is required for CIN in a p53 mutant tumor model. These results demonstrate that Mad2 overexpression is a critical mediator of the CIN observed upon inactivation of two major tumor suppressor pathways.
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http://dx.doi.org/10.1016/j.ccr.2011.04.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3120099PMC
June 2011

Mad2-induced chromosome instability leads to lung tumour relapse after oncogene withdrawal.

Nature 2010 Mar 21;464(7287):436-40. Epub 2010 Feb 21.

Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

Inhibition of an initiating oncogene often leads to extensive tumour cell death, a phenomenon known as oncogene addiction. This has led to the search for compounds that specifically target and inhibit oncogenes as anticancer agents. However, there has been no systematic exploration of whether chromosomal instability generated as a result of deregulation of the mitotic checkpoint pathway, a frequent characteristic of solid tumours, has any effect on oncogene addiction. Here we show that induction of chromosome instability by overexpression of the mitotic checkpoint gene Mad2 in mice does not affect the regression of Kras-driven lung tumours when Kras is inhibited. However, tumours that experience transient Mad2 overexpression and consequent chromosome instability recur at markedly elevated rates. The recurrent tumours are highly aneuploid and have varied activation of pro-proliferative pathways. Thus, early chromosomal instability may be responsible for tumour relapse after seemingly effective anticancer treatments.
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http://dx.doi.org/10.1038/nature08803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2841716PMC
March 2010

Mitotic chromosomal instability and cancer: mouse modelling of the human disease.

Nat Rev Cancer 2010 Feb;10(2):102-15

Program in Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

The stepwise progression from an early dysplastic lesion to full-blown metastatic malignancy is associated with increases in genomic instability. Mitotic chromosomal instability - the inability to faithfully segregate equal chromosome complements to two daughter cells during mitosis - is a widespread phenomenon in solid tumours that is thought to serve as the fuel for tumorigenic progression. How chromosome instability (CIN) arises in tumours and what consequences it has are still, however, hotly debated issues. Here we review the recent literature with an emphasis on models that recapitulate observations from human disease.
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http://dx.doi.org/10.1038/nrc2781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526619PMC
February 2010

Very CIN-ful: whole chromosome instability promotes tumor suppressor loss of heterozygosity.

Cancer Cell 2009 Dec;16(6):451-2

Program in Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Mechanisms by which whole chromosome instability lead to tumorigenesis have eluded the cancer research field. In this issue of Cancer Cell, Baker et al. show that CIN induced by a defective mitotic checkpoint, under certain genetic and tissue contexts, leads to accelerated loss of heterozygosity of a tumor suppressor gene.
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http://dx.doi.org/10.1016/j.ccr.2009.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513159PMC
December 2009

Hec1 overexpression hyperactivates the mitotic checkpoint and induces tumor formation in vivo.

Proc Natl Acad Sci U S A 2008 Oct 21;105(43):16719-24. Epub 2008 Oct 21.

Program in Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

Hec1 (Highly Expressed in Cancer 1) is one of four proteins of the outer kinetochore Ndc80 complex involved in the dynamic interface between centromeres and spindle microtubules. Its overexpression is seen in a variety of human tumors and correlates with tumor grade and prognosis. We show here that the overexpression of Hec1 in an inducible mouse model results in mitotic checkpoint hyperactivation. As previously observed with overexpression of the Mad2 gene, hyperactivation of the mitotic checkpoint leads to aneuploidy in vitro and is sufficient to generate tumors in vivo that harbor significant levels of aneuploidy. These results underscore the role of chromosomal instability as a result of mitotic checkpoint hyperactivation in the initiation of tumorigenesis.
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http://dx.doi.org/10.1073/pnas.0803504105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2570608PMC
October 2008

Changing Mad2 levels affects chromosome segregation and spindle assembly checkpoint control in female mouse meiosis I.

PLoS One 2007 Nov 28;2(11):e1165. Epub 2007 Nov 28.

CNRS UMR7622 Biologie du Développement, Paris, France.

The spindle assembly checkpoint (SAC) ensures correct separation of sister chromatids in somatic cells and provokes a cell cycle arrest in metaphase if one chromatid is not correctly attached to the bipolar spindle. Prolonged metaphase arrest due to overexpression of Mad2 has been shown to be deleterious to the ensuing anaphase, leading to the generation of aneuploidies and tumorigenesis. Additionally, some SAC components are essential for correct timing of prometaphase. In meiosis, we and others have shown previously that the Mad2-dependent SAC is functional during the first meiotic division in mouse oocytes. Expression of a dominant-negative form of Mad2 interferes with the SAC in metaphase I, and a knock-down approach using RNA interference accelerates anaphase onset in meiosis I. To prove unambigiously the importance of SAC control for mammalian female meiosis I we analyzed oocyte maturation in Mad2 heterozygote mice, and in oocytes overexpressing a GFP-tagged version of Mad2. In this study we show for the first time that loss of one Mad2 allele, as well as overexpression of Mad2 lead to chromosome missegregation events in meiosis I, and therefore the generation of aneuploid metaphase II oocytes. Furthermore, SAC control is impaired in mad2+/- oocytes, also leading to the generation of aneuploidies in meiosis I.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001165PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2082075PMC
November 2007

Mad2 overexpression promotes aneuploidy and tumorigenesis in mice.

Cancer Cell 2007 Jan 28;11(1):9-23. Epub 2006 Dec 28.

Cancer Biology and Genetics Program, Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

Mad2 is an essential component of the spindle checkpoint that blocks activation of Separase and dissolution of sister chromatids until microtubule attachment to kinetochores is complete. We show here that overexpression of Mad2 in transgenic mice leads to a wide variety of neoplasias, appearance of broken chromosomes, anaphase bridges, and whole-chromosome gains and losses, as well as acceleration of myc-induced lymphomagenesis. Moreover, continued overexpression of Mad2 is not required for tumor maintenance, unlike the majority of oncogenes studied to date. These results demonstrate that transient Mad2 overexpression and chromosome instability can be an important stimulus in the initiation and progression of different cancer subtypes.
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http://dx.doi.org/10.1016/j.ccr.2006.10.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1850996PMC
January 2007

Cooperation between Cdk4 and p27kip1 in tumor development: a preclinical model to evaluate cell cycle inhibitors with therapeutic activity.

Cancer Res 2005 May;65(9):3846-52

Molecular Oncology, Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain.

Deregulation of the G1-S transition of the cell cycle is a common feature of human cancer. Tumor-associated alterations in this process frequently affect cyclin-dependent kinases (Cdk), their regulators (cyclins, INK4 inhibitors, or p27Kip1), and their substrates (retinoblastoma protein). Although these proteins are generally thought to act in a linear pathway, mutations in different components frequently cooperate in tumor development. Using gene-targeted mouse models, we report in this article that Cdk4 resistance to INK4 inhibitors, due to the Cdk4 R24C mutation, strongly cooperates with p27(Kip1) deficiency in tumor development. No such cooperation is observed between Cdk4 R24C and p18(INK4c) absence, suggesting that the only function of p18INK4c is inhibiting Cdk4 in this model. Cdk4(R/R) knock in mice, which express the Cdk4 R24C mutant protein, develop pituitary tumors with complete penetrance and short latency in a p27Kip1-/- or p27Kip1+/- background. We have investigated whether this tumor model could be useful to assess the therapeutic activity of cell cycle inhibitors. We show here that exposure to flavopiridol, a wide-spectrum Cdk inhibitor, significantly delays tumor progression and leads to tumor-free survival in a significant percentage of treated mice. These data suggest that genetically engineered tumor models involving key cell cycle regulators are a valuable tool to evaluate drugs with potential therapeutic benefit in human cancer.
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http://dx.doi.org/10.1158/0008-5472.CAN-04-4195DOI Listing
May 2005

Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6.

Cell 2004 Aug;118(4):493-504

Molecular Oncology Program, Centro Nacional de Investigaciones Oncológicas (CNIO), E-28029 Madrid, Spain.

Cdk4 and Cdk6 are thought to be essential for initiation of the cell cycle in response to mitogenic stimuli. Previous studies have shown that Cdk4 is dispensable for proliferation in most cell types, an observation attributed to a putative compensatory role by Cdk6. Cdk6-null mice are viable and develop normally although hematopoiesis is slightly impaired. Embryos defective for Cdk4 and Cdk6 die during the late stages of embryonic development due to severe anemia. However, these embryos display normal organogenesis and most cell types proliferate normally. In vitro, embryonic fibroblasts lacking Cdk4 and Cdk6 proliferate and become immortal upon serial passage. Moreover, quiescent Cdk4/Cdk6-null cells respond to serum stimulation and enter S phase with normal kinetics although with lower efficiency. These results indicate that D-type cyclin-dependent kinases are not essential for cell cycle entry and suggest the existence of alternative mechanisms to initiate cell proliferation upon mitogenic stimulation.
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http://dx.doi.org/10.1016/j.cell.2004.08.002DOI Listing
August 2004

Cyclin-dependent kinase 2 is essential for meiosis but not for mitotic cell division in mice.

Nat Genet 2003 Sep 17;35(1):25-31. Epub 2003 Aug 17.

Molecular Oncology Program, Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro 3, Madrid E-28029, Spain.

We targeted the locus encoding the cyclin-dependent kinase 2 (CDK2) by homologous recombination in mouse embryonic stem (ES) cells. Embryonic fibroblasts lacking CDK2 proliferate normally and become immortal after continuous passage in culture. Elimination of a conditional Cdk2 allele in immortal cells does not have a significant effect on proliferation. Cdk2-/- mice are viable and survive for up to two years, indicating that CDK2 is also dispensable for proliferation and survival of most cell types. But CDK2 is essential for completion of prophase I during meiotic cell division in male and female germ cells, an unforeseen role for this cell cycle kinase.
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http://dx.doi.org/10.1038/ng1232DOI Listing
September 2003

Genetic rescue of Cdk4 null mice restores pancreatic beta-cell proliferation but not homeostatic cell number.

Oncogene 2003 Aug;22(34):5261-9

Molecular Oncology Program, Centro Nacional de Investigaciones Oncológicas, Melchor Fernández Almagro 3, 28029 Madrid, Spain.

Lack of Cdk4 expression in mice leads to insulin-deficient diabetes and female infertility owing to a reduced number of pancreatic beta cells and prolactin-producing pituitary lactotrophs, respectively. Cdk4 null mice display also reduced body and organ size. Here, we show that Cdk4 is essential for the postnatal proliferation of pancreatic beta cells but not for embryonic neogenesis from ductal epithelial cells. Re-expression of endogenous Cdk4 in beta cells and in the pituitary gland of Cdk4 null mice restores cell proliferation and results in fertile and normoglycemic animals, thus, demonstrating that the proliferation defects in these cellular populations are cell autonomous because of the lack of Cdk4 expression. However, these mice remain small in size, indicating that this phenotype is not because of pancreatic- or pituitary-mediated endocrine defects. This phenotype is a consequence of reduced cell numbers rather than reduced cell size. Thus, mammalian Cdk4 is not only involved in controlling proliferation of specific cell types but may play a wider role in establishing homeostatic cell numbers.
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http://dx.doi.org/10.1038/sj.onc.1206506DOI Listing
August 2003

Driving the cell cycle to cancer.

Adv Exp Med Biol 2003 ;532:1-11

Molecular Oncology Programme, Centro Nacional de Investigaciones Oncológicas, Madrid, Spain.

Cell cycle progression requires the co-ordinated activation of several kinases, some of which are activated upon the binding of a cyclin subunit. At least four of these so-called cyclin-dependent kinases, namely Cdk4, Cdk6, Cdk2 and Cdk1, have specific roles at particular stages of the cell cycle, including passage through the various cell cycle transitions and the response to specific checkpoints. Not surprisingly, most human tumors carry mutations that deregulate at least one of these kinases. To analyze their specific role in vivo, we are generating strains of gene-targeted mice carrying either activated or defective alleles of these Cdks. As an example, Cdk4 expression appears to be expendable in most cell types since mice lacking Cdk4 are viable. Yet, Cdk4 mutant mice are smaller in size and infertile (only partial infertility in males). In addition, Cdk4 defective mice develop insulin dependent diabetes early in life. However, the importance of these Cdks in tumor cell cycles is underscored by the phenotype of knock in mice where the normal Cdk4 gene has been replaced by a Cdk4 R24C (insensitive to INK inhibitors) mutant. These animals develop a wide spectrum of spontaneous tumors and are highly susceptible to specific carcinogenic treatments. These models are being used now to understand how deregulation of these Cdks leads to cancer development and will be a valuable tool to design and validate new therapeutic strategies against tumour development.
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http://dx.doi.org/10.1007/978-1-4615-0081-0_1DOI Listing
February 2004