Analysis of Paired Primary-Metastatic Hormone-Receptor Positive Breast Tumors (HRPBC) Uncovers Potential Novel Drivers of Hormonal Resistance

We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in KMT2C and/or the T666I mutation in the kinase domain of EPHA7 conferred hormonal resistance in vitro. Novel inactivating mutations in KMT2C and EPHA7, which confer hormonal resistance, are linked to adverse clinical course in HRPBC

Estudios in vitro de la capacidad mutagénica y potencialidad carcinogénica de sales metálicas e hidrocarburos aromáticos policíclicos por análisis a nivel citológico y molecular

Los agentes carcinogénicos presentes en el ambiente son los principales agentes causales del desarrollo de la enfermedad neoplásica ya sea por la acción genotóxica directa (iniciador tumoral) o bien por la acción epigenética (promotor tumoral) que estimula la división celular y favorece la expresión del gen mutado antes, debido a la acción del iniciador. Por tanto, el estudio de la capacidad mutagénica y potencialidad carcinogénica de los contaminantes ambientales, podría brindar información para ayudar a dilucidar los mecanismos por los cuales estos compuestos ejercen su poder carcinogénico. Numerosos estudios han asociado la exposición de las sales de cadmio y de arsénico y de los hidrocarburos aromáticos policíclicos con el desarrollo de cáncer de pulmón. Sin embargo, los mecanismos mediante los cuales estas sustancias ejercen su poder carcinogénico no han sido elucidados cabalmente. A tal fin se empleó la línea de fibroblastos de pulmón humano MRC-5, y se evaluaron los efectos genotóxicos de las sales de cadmio (cloruro y sulfato de cadmio), las sales de arsénico (arsenito de sodio y ácido dimetilarsínico) y los hidrocarburos aromáticos policíclos (benzo[a]pireno y dibenzo[a,i]pireno). Se evaluó la capacidad de estos compuestos de inducir intercambios de cromátidas hermanas así como de rupturas de cadena simple de la molécula de ADN y /o la formación de aductos ADN-ADN o ADN-proteína e inducción de apoptosis mediante el empleo del ensayo cometa. Por otra parte, se evaluó la capacidad de inducción de mutaciones puntuales en el exón 1 del protooncogén K-ras y los exones 5 a 8 del gen supresor de tumores p53 mediante el empleo de la técnica de PCR-SSCP. Dado que las mutaciones puntuales en el codón 12 del protooncogén K-ras son las de mayor prevalencia e implicancia funcional del gen se evaluó su presencia mediante la técnica de PCR con enriquecimiento alélico.Facultad de Ciencias Naturales y Muse

Estudios in vitro de la capacidad mutagénica y potencialidad carcinogénica de sales metálicas e hidrocarburos aromáticos policíclicos por análisis a nivel citológico y molecular

Los agentes carcinogénicos presentes en el ambiente son los principales agentes causales del desarrollo de la enfermedad neoplásica ya sea por la acción genotóxica directa (iniciador tumoral) o bien por la acción epigenética (promotor tumoral) que estimula la división celular y favorece la expresión del gen mutado antes, debido a la acción del iniciador. Por tanto, el estudio de la capacidad mutagénica y potencialidad carcinogénica de los contaminantes ambientales, podría brindar información para ayudar a dilucidar los mecanismos por los cuales estos compuestos ejercen su poder carcinogénico. Numerosos estudios han asociado la exposición de las sales de cadmio y de arsénico y de los hidrocarburos aromáticos policíclicos con el desarrollo de cáncer de pulmón. Sin embargo, los mecanismos mediante los cuales estas sustancias ejercen su poder carcinogénico no han sido elucidados cabalmente. A tal fin se empleó la línea de fibroblastos de pulmón humano MRC-5, y se evaluaron los efectos genotóxicos de las sales de cadmio (cloruro y sulfato de cadmio), las sales de arsénico (arsenito de sodio y ácido dimetilarsínico) y los hidrocarburos aromáticos policíclos (benzo[a]pireno y dibenzo[a,i]pireno). Se evaluó la capacidad de estos compuestos de inducir intercambios de cromátidas hermanas así como de rupturas de cadena simple de la molécula de ADN y /o la formación de aductos ADN-ADN o ADN-proteína e inducción de apoptosis mediante el empleo del ensayo cometa. Por otra parte, se evaluó la capacidad de inducción de mutaciones puntuales en el exón 1 del protooncogén K-ras y los exones 5 a 8 del gen supresor de tumores p53 mediante el empleo de la técnica de PCR-SSCP. Dado que las mutaciones puntuales en el codón 12 del protooncogén K-ras son las de mayor prevalencia e implicancia funcional del gen se evaluó su presencia mediante la técnica de PCR con enriquecimiento alélico.Facultad de Ciencias Naturales y Muse

Mutational Analysis and Characterization of WFYY Motif in human PrimPol

Trabajo presentado en el XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Granada (España) del 09 al 12 de septiembre de 2014

PrimPol, a new human DNA primase/polymerase, is involved in translesion synthesis and recovery of stalled forks during nuclear DNA replication

Trabajo presentado en DNA Replication and Recombination, celebrado en Canadá el 03 de marzo de 2013

Repriming of DNA synthesis at stalled replication forks by human PrimPol

DNA replication forks that collapse during the process of genomic duplication lead to double-strand breaks and constitute a threat to genomic stability. The risk of fork collapse is higher in the presence of replication inhibitors or after UV irradiation, which introduces specific modifications in the structure of DNA. In these cases, fork progression may be facilitated by error-prone translesion synthesis (TLS) DNA polymerases. Alternatively, the replisome may skip the damaged DNA, leaving an unreplicated gap to be repaired after replication. This mechanism strictly requires a priming event downstream of the lesion. Here we show that PrimPol, a new human primase and TLS polymerase, uses its primase activity to mediate uninterrupted fork progression after UV irradiation and to reinitiate DNA synthesis after dNTP depletion. As an enzyme involved in tolerance to DNA damage, PrimPol might become a target for cancer therapy. © 2013 Nature America, Inc. All rights reserved.Comunidad de Madrid (S2011/BMD-236); Spanish Ministry of Economy and Competitiveness (BFU2010-21467, BFU2012-37969,Consolider CSD2007-00015)Peer Reviewe

Lafora bodies and neurological defects in malin-deficient mice correlate with impaired autophagy

Criado-García, Olga et al.Lafora disease (LD), a fatal neurodegenerative disorder characterized by the presence of intracellular inclusions called Lafora bodies (LBs), is caused by loss-of-function mutations in laforin or malin. Previous studies suggested a role of these proteins in the regulation of glycogen biosynthesis, in glycogen dephosphorylation and in the modulation of the intracellular proteolytic systems. However, the contribution of each of these processes to LD pathogenesis is unclear. We have generated a malin-deficient (Epm2b-/-) mouse with a phenotype similar to that of LD patients. By 3-6 months of age, Epm2b-/- mice present neurological and behavioral abnormalities that correlate with a massive presence of LBs in the cortex, hippocampus and cerebellum. Sixteen-day-old Epm2b-/- mice, without detectable LBs, show an impairment of macroautophagy (hereafter called autophagy), which remains compromised in adult animals. These data demonstrate similarities between the Epm2a-/- and Epm2b-/- mice that provide further insights into LD pathogenesis. They illustrate that the dysfunction of autophagy is a consequence of the lack of laforin-malin complexes and a common feature of both mouse models of LD. Because this dysfunction precedes other pathological manifestations, we propose that decreased autophagy plays a primary role in the formation of LBs and it is critical in LD pathogenesis. © The Author 2011. Published by Oxford University Press. All rights reserved.This work was funded by the Spanish Ministerio de Ciencia eInnovacio ́ n (SAF2008-00226 to S.R.C.; BFU2007-61774 andBFU2010-16031toP.B.;BFU2008-00186toE.K.;SAF2008-01907 to P.S.; and SAF2010-18586 to J.M.S.), theRed de Biobancos del FIS (RD09/0076/00011 to C.N.), theFondo de Investigacio ́ n Sanitaria (PI10/02628 to C.N.;PI070023 to M.S.), the Fundacio ́ n Marato TV3 (Ref. 100130to P.S., E.K. and S.R.C.) and the Centro de Investigacio ́ n Bio-me ́dica en Red de Enfermedades Raras (CIBERER).A.M.G.-C. holds a fellowship from the Fundacio ́ n ConchitaRa ́bagoPeer Reviewe

Targeting Tumor Mitochondrial Metabolism Overcomes Resistance to Antiangiogenics

Epithelial malignancies are effectively treated by antiangiogenics; however, acquired resistance is a major problem in cancer therapeutics. Epithelial tumors commonly have mutations in the MAPK/Pi3K-AKT pathways, which leads to high-rate aerobic glycolysis. Here, we show how multikinase inhibitor antiangiogenics (TKIs) induce hypoxia correction in spontaneous breast and lung tumor models. When this happens, the tumors downregulate glycolysis and switch to long-term reliance on mitochondrial respiration. A transcriptomic, metabolomic, and phosphoproteomic study revealed that this metabolic switch is mediated by downregulation of HIF1α and AKT and upregulation of AMPK, allowing uptake and degradation of fatty acids and ketone bodies. The switch renders mitochondrial respiration necessary for tumor survival. Agents like phenformin or ME344 induce synergistic tumor control when combined with TKIs, leading to metabolic synthetic lethality. Our study uncovers mechanistic insights in the process of tumor resistance to TKIs and may have clinical applicability

Malin knockout mice support a primary role of autophagy in the pathogenesis of Lafora disease

Knecht, Erwin et al.Lafora disease (LD), a fatal neurodegenerative disorder characterized by intracellular inclusions called Lafora bodies (LBs), is caused by recessive loss-of-function mutations in the genes encoding either laforin or malin. Previous studies suggested a role of these proteins in regulating glycogen biosynthesis, in glycogen dephosphorylation and in the modulation of intracellular proteolytic systems. However, the contribution of each of these processes to LD pathogenesis is unclear. Here we review our recent finding that dysfunction of autophagy is a common feature of both laforin- and malin-deficient mice, preceding other pathological manifestations. We propose that autophagy plays a primary role in LD pathogenesis and is a potential target for its treatment. © 2012 Landes Bioscience.Spanish Ministry of Science and Innovation (SAF2008-00226 to SRdeC, BFU2010-16031 to PB; BFU2008-00186, SAF2008-01907, SAF2010-18586); the Red de Biobancos del FIS (RD09/0076/00011); the Fondo de Investigación Sanitaria (PI10/02628, PI070023); the Fundación Marato TV3 (Ref.100130); the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER); Fundación Conchita RábagoPeer Reviewe