Experimental Oncology Group

Madrid, Spain

Experimental Oncology Group

Madrid, Spain

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PubMed | Experimental Oncology Group, French Institute of Health and Medical Research and Institute Paoli Calmettes IPC
Type: Journal Article | Journal: Oncotarget | Year: 2016

CDK4/CDK6 and RB proteins drive the progression through the G1 phase of the cell cycle. In acute myeloid leukemia (AML), the activity of the CDK/Cyclin D complex is increased. The mechanism involved is unknown, as are the respective roles played by CDK4 or CDK6 in this process. Here, we report that AML cells carrying FLT3-ITD mutations are dependent on CDK6 for cell proliferation while CDK4 is not essential. We showed that FLT3-ITD signaling is responsible for CDK6 overexpression, through a pathway involving the SRC-family kinase HCK. Accordingly, FLT3-ITD failed to transform primary hematopoietic progenitor cells from Cdk6-/- mice. Our results demonstrate that CDK6 is the primary target of CDK4/CDK6 inhibitors in FLT3-ITD positive AML. Furthermore, we delineate an essential protein kinase pathway -FLT3/HCK/CDK6- in the context of AML with FLT3-ITD mutations.

Hermann P.C.,Stem Cells and Cancer Group | Sancho P.,Stem Cells and Cancer Group | Canamero M.,Comparative Pathology Core Unit | Martinelli P.,Epithelial Carcinogenesis Group | And 14 more authors.
Gastroenterology | Year: 2014

Background & Aims Although smoking is a leading risk factor for pancreatic ductal adenocarcinoma (PDAC), little is known about the mechanisms by which smoking promotes initiation or progression of PDAC.Methods We studied the effects of nicotine administration on pancreatic cancer development in Kras+/LSLG12Vgeo;Elas-tTA/tetO-Cre (Ela-KRAS) mice, Kras+/LSLG12D;Trp53+/LSLR172H;Pdx-1-Cre (KPC) mice (which express constitutively active forms of KRAS), and C57/B6 mice. Mice were given nicotine for up to 86 weeks to produce blood levels comparable with those of intermediate smokers. Pancreatic tissues were collected and analyzed by immunohistochemistry and reverse transcriptase polymerase chain reaction; cells were isolated and assayed for colony and sphere formation and gene expression. The effects of nicotine were also evaluated in primary pancreatic acinar cells isolated from wild-type, nAChR7a-/-, Trp53-/-, and Gata6-/-;Trp53-/- mice. We also analyzed primary PDAC cells that overexpressed GATA6 from lentiviral expression vectors.Results Administration of nicotine accelerated transformation of pancreatic cells and tumor formation in Ela-KRAS and KPC mice. Nicotine induced dedifferentiation of acinar cells by activating AKT-ERK-MYC signaling; this led to inhibition of Gata6 promoter activity, loss of GATA6 protein, and subsequent loss of acinar differentiation and hyperactivation of oncogenic KRAS. Nicotine also promoted aggressiveness of established tumors as well as the epithelial-mesenchymal transition, increasing numbers of circulating cancer cells and their dissemination to the liver, compared with mice not exposed to nicotine. Nicotine induced pancreatic cells to acquire gene expression patterns and functional characteristics of cancer stem cells. These effects were markedly attenuated in K-Ras+/LSL-G12D;Trp53+/LSLR172H;Pdx-1-Cre mice given metformin. Metformin prevented nicotine-induced pancr.eatic carcinogenesis and tumor growth by up-regulating GATA6 and promoting differentiation toward an acinar cell program.Conclusions In mice, nicotine promotes pancreatic carcinogenesis and tumor development via down-regulation of Gata6 to induce acinar cell dedifferentiation. © 2014 by the AGA Institute.

Martinez-Bosch N.,Hospital Del Mar Medical Research Institute IMIM | Iglesias M.,Hospital Del Mar | Munne-Collado J.,Hospital Del Mar | Martinez-Caceres C.,Fundacion Formacion Investigacion Sanitaria Region Murcia FFIS | And 7 more authors.
Journal of Pathology | Year: 2014

Pancreatic cancer has a dismal prognosis and is currently the fourth leading cause of cancer-related death in developed countries. The inhibition of poly(ADP-ribose) polymerase-1 (Parp-1), the major protein responsible for poly(ADP-ribosy)lation in response to DNA damage, has emerged as a promising treatment for several tumour types. Here we aimed to elucidate the involvement of Parp-1 in pancreatic tumour progression. We assessed Parp-1 protein expression in normal, preneoplastic and pancreatic tumour samples from humans and from K-Ras- and c-myc-driven mouse models of pancreatic cancer. Parp-1 was highly expressed in acinar cells in normal and cancer tissues. In contrast, ductal cells expressed very low or undetectable levels of this protein, both in a normal and in a tumour context. The Parp-1 expression pattern was similar in human and mouse samples, thereby validating the use of animal models for further studies. To determine the in vivo effects of Parp-1 depletion on pancreatic cancer progression, Ela-myc-driven pancreatic tumour development was analysed in a Parp-1 knock-out background. Loss of Parp-1 resulted in increased tumour necrosis and decreased proliferation, apoptosis and angiogenesis. Interestingly, Ela-myc:Parp-1-/- mice displayed fewer ductal tumours than their Ela-myc:Parp-1+/+ counterparts, suggesting that Parp-1 participates in promoting acinar-to-ductal metaplasia, a key event in pancreatic cancer initiation. Moreover, impaired macrophage recruitment can be responsible for the ADM blockade found in the Ela-myc:Parp-1-/- mice. Finally, molecular analysis revealed that Parp-1 modulates ADM downstream of the Stat3-MMP7 axis and is also involved in transcriptional up-regulation of the MDM2, VEGFR1 and MMP28 cancer-related genes. In conclusion, the expression pattern of Parp-1 in normal and cancer tissue and the in vivo functional effects of Parp-1 depletion point to a novel role for this protein in pancreatic carcinogenesis and shed light into the clinical use of Parp-1 inhibitors. Copyright © 2014 Pathological Society of Great Britain and Ireland.

Murga M.,Genomic Instability Group | Campaner S.,Italian National Cancer Institute | Lopez-Contreras A.J.,Genomic Instability Group | Toledo L.I.,Genomic Instability Group | And 11 more authors.
Nature Structural and Molecular Biology | Year: 2011

Oncogene-induced replicative stress activates an Atr-and Chk1-dependent response, which has been proposed to be widespread in tumors. We explored whether the presence of replicative stress could be exploited for the selective elimination of cancer cells. To this end, we evaluated the impact of targeting the replicative stress-response on cancer development. In mice (Mus musculus), the reduced levels of Atr found on a mouse model of the Atr-Seckel syndrome completely prevented the development of Myc-induced lymphomas or pancreatic tumors, both of which showed abundant levels of replicative stress. Moreover, Chk1 inhibitors were highly effective in killing Myc-driven lymphomas. By contrast, pancreatic adenocarcinomas initiated by K-Ras G12V showed no detectable evidence of replicative stress and were nonresponsive to this therapy. Besides its impact on cancer, Myc overexpression aggravated the phenotypes of Atr-Seckel mice, revealing that oncogenes can modulate the severity of replicative stress-associated diseases. © 2011 Nature America, Inc. All rights reserved.

Lopez-Contreras A.J.,Genomic Instability Group | Lopez-Contreras A.J.,Copenhagen University | Specks J.,Genomic Instability Group | Barlow J.H.,U.S. National Institutes of Health | And 9 more authors.
Genes and Development | Year: 2015

In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2TG) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity. © 2015 Lopez-Contreras et al.

Trakala M.,Cell Division and Cancer Group | Rodriguez-Acebes S.,DNA Replication Group | Maroto M.,Cell Division and Cancer Group | Symonds C.E.,Experimental Oncology Group | And 5 more authors.
Developmental Cell | Year: 2015

Polyploidization is a natural process that frequently accompanies differentiation; its deregulation is linked to genomic instability and cancer. Despite its relevance, why cells select different polyploidization mechanisms is unknown. Here we report a systematic genetic analysis of endomitosis, a process inwhich megakaryocytes become polyploid by entering mitosis but aborting anaphase. Whereas ablation of the APC/C cofactor Cdc20 results in mitotic arrest and severe thrombocytopenia, lack of the kinases Aurora-B, Cdk1, or Cdk2 does not affect megakaryocyte polyploidization or platelet levels. Ablation of Cdk1 forces a switch to endocycles without mitosis, whereas polyploidization in the absence of Cdk1 and Cdk2 occurs in the presence of aberrant re-replication events. Importantly, ablation of these kinases rescues the defects in Cdc20 null megakaryocytes. These findings suggest that endomitosis can be functionally replaced by alternative polyploidization mechanisms invivo and provide the cellular basis for therapeutic approaches aimed to discriminate mitotic and polyploid cells. © 2015 Elsevier Inc.

Martinez-Bosch N.,Cancer Research Program | Fernandez-Barrena M.G.,Schulze Center for Novel Therapeutics | Moreno M.,Cancer Research Program | Ortiz-Zapater E.,Cancer Research Program | And 12 more authors.
Cancer Research | Year: 2014

Despite some advances, pancreatic ductal adenocarcinoma (PDAC) remains generally refractory to current treatments. Desmoplastic stroma, a consistent hallmark of PDAC, has emerged as a major source of therapeutic resistance and thus potentially promising targets for improved treatment. The glycan-binding protein galectin-1 (Gal1) is highly expressed in PDAC stroma, but its roles there have not been studied. Here we report functions and molecular pathways of Gal1 that mediate its oncogenic properties in this setting. Genetic ablation of Gal1 in a mouse model of PDAC (EIa-myc mice) dampened tumor progression by inhibiting proliferation, angiogenesis, desmoplasic reaction and by stimulating a tumor-associated immune response, yielding a 20% increase in relative lifesplan. Cellular analyses in vitro and in vivo suggested these effects were mediated through the tumor microenvironment. Importantly, acinar-to-ductal metaplasia, a crucial step for initiation of PDAC, was found to be regulated by Gal1. Mechanistic investigations revealed that Gal1 promoted Hedgehog pathway signaling in PDAC cells and stromal fibroblasts as well as in Ela-myc tumors. Taken together, our findings establish a function for Gal1 in tumor-stroma crosstalk in PDAC and provide a preclinical rationale for Gal1 targeting as a microenvironment-based therapeutic strategy. © 2014 AACR.

Monasor A.,Genomic Instability Group | Murga M.,Genomic Instability Group | Lopez-Contreras A.J.,Genomic Instability Group | Navas C.,Experimental Oncology Group | And 3 more authors.
Cell Cycle | Year: 2013

Replication stress (RS) is a source of DNA damage that has been linked to cancer and aging, which is suppressed by the ATR kinase. In mice, reduced ATR levels in a model of the ATR-Seckel syndrome lead to RS and accelerated aging. Similarly, ATR-Seckel embryonic fibroblasts (MEF) accumulate RS and undergo cellular senescence. We previously showed that senescence of ATR-Seckel MEF cannot be rescued by p53-deletion. Here, we show that the genetic ablation of the INK4a/ Arf locus fully rescues senescence on ATR mutant MEF, but also that induced by other conditions that generate RS such as low doses of hydroxyurea or ATR inhibitors. In addition, we show that a persistent exposure to RS leads to increased levels of INK4a/Arf products, revealing that INK4a/ARF behaves as a bona fide RS checkpoint. Our data reveal an unknown role for INK4a/ARF in limiting the expansion of cells suffering from persistent replication stress, linking this well-known tumor suppressor to the maintenance of genomic integrity. © 2013 Landes Bioscience.

Martinelli P.,Epithelial Carcinogenesis Group | Madriles F.,Epithelial Carcinogenesis Group | Canamero M.,Comparative Pathology Unit | Carrillo-De Santa Pau E.,Epithelial Carcinogenesis Group | And 4 more authors.
Gut | Year: 2016

Background and aims: Gata6 is required to complete and maintain acinar differentiation in the mouse pancreas. Pancreas-specific Gata6 ablation during development causes extensive and persistent acinarductal metaplasia, which is considered an initial step of mutant KRas-driven carcinogenesis. Therefore, the Gata6-null pancreas might represent a tumour-prone environment. We investigated whether Gata6 plays a role during pancreatic tumorigenesis. Design: We analysed genetically engineered mouse models and human pancreatic ductal adenocarcinoma (PDAC) cell lines, using a combination of histopathological studies, genome-wide expression and chromatin immunoprecipitation experiments to understand the role of Gata6 in the initiation and progression of KRasG12V-driven tumours Results We show that Gata6 maintains the acinar differentiation programme, both directly and indirectly, and it concomitantly suppresses ectopic programmes in the pancreas. Gata6 ablation renders acinar cells more sensitive to KRasG12V, thereby accelerating tumour development. Gata6 expression is spontaneously lost in a mouse model of KRasG12V-driven PDAC, in association with altered cell differentiation. Using a combination of ChIP-Seq and RNA-Seq, we show that Gata6 exerts its tumour-suppressive effect through the promotion of cell differentiation, the suppression of inflammatory pathways, and the direct repression of cancer-related pathways. Among them is the epidermal growth factor receptor (EGFR) pathway, the activity of which is upregulated in the normal and preneoplastic Gata6-null pancreas. Accordingly, GATA6-silencing in human PDAC cells leads to an upregulation of EGFR. Conclusions: We propose that, in the pancreas, Gata6 acts as a tumour suppressor by enforcing acinar cell differentiation, by directly and indirectly repressing ectopic differentiation programmes, and by regulating crucial cancer-related gene expression pathways.

PubMed | Experimental Oncology Group and Epithelial Cell Biology Group
Type: | Journal: Journal of cell science | Year: 2017

Epidermal homeostasis is tightly controlled by a balancing act of self-renewal or terminal differentiation of proliferating basal keratinocytes. An increase in DNA content as a consequence of a mitotic block is a recognized mechanism underlying keratinocyte differentiation, but the molecular mechanisms involved in this process are not yet fully understood. Using cultured primary keratinocytes, here we report that the expression of the mammalian Microtubule and Kinetochore-associated protein Clasp2 is intimately associated to the basal proliferative makeup of keratinocytes, and its deficiency leads to premature differentiation. Clasp2 deficient keratinocytes exhibit increased centrosomal numbers, and numerous mitotic alterations, including multipolar spindles and chromosomal misalignments that overall result in mitotic stress and a high DNA content. Such mitotic block prompts premature keratinocyte differentiation in a p53-dependent manner in the absence of cell death. Our findings reveal a novel role for Clasp2 in governing keratinocyte undifferentiated features and highlight the presence of surveillance mechanisms that avoid cell cycle entry in cells with alterations in the DNA content.

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