Gonzalo Gómez

Telomerase expression confers cardioprotection in the adult mouse heart after acute myocardial infarction

Coronary heart disease is one of the main causes of death in the developed world, and treatment success remains modest, with high mortality rates within 1 year after myocardial infarction (MI). Thus, new therapeutic targets and effective treatments are necessary. Short telomeres are risk factors for age-associated diseases, including heart disease. Here we address the potential of telomerase (Tert) activation in prevention of heart failure after MI in adult mice. We use adeno-associated viruses for cardiac-specific Tert expression. We find that upon MI, hearts expressing Tert show attenuated cardiac dilation, improved ventricular function and smaller infarct scars concomitant with increased mouse survival by 17% compared with controls. Furthermore, Tert treatment results in elongated telomeres, increased numbers of Ki67 and pH3-positive cardiomyocytes and a gene expression switch towards a regeneration signature of neonatal mice. Our work suggests telomerase activation could be a therapeutic strategy to prevent heart failure after MI.

Genetic inactivation of Cdk7 leads to cell cycle arrest and induces premature aging due to adult stem cell exhaustion

Cyclin‐dependent kinase (Cdk)7, the catalytic subunit of the Cdk‐activating kinase (CAK) complex has been implicated in the control of cell cycle progression and of RNA polymerase II (RNA pol II)‐mediated transcription. Genetic inactivation of the Cdk7 locus revealed that whereas Cdk7 is completely dispensable for global transcription, is essential for the cell cycle via phosphorylation of Cdk1 and Cdk2. In vivo, Cdk7 is also indispensable for cell proliferation except during the initial stages of embryonic development. Interestingly, widespread elimination of Cdk7 in adult tissues with low proliferative indexes had no phenotypic consequences. However, ablation of conditional Cdk7 alleles in tissues with elevated cellular turnover led to the efficient repopulation of these tissues with Cdk7‐expressing cells most likely derived from adult stem cells that may have escaped the inactivation of their targeted Cdk7 alleles. This process, a physiological attempt to maintain tissue homeostasis, led to the attrition of adult stem cell pools and to the appearance of age‐related phenotypes, including telomere shortening and early death.

ARID1A alterations are associated with FGFR3-wild type, poor-prognosis, urothelial bladder tumors.

Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological, genetic, and epigenetic levels. Exome sequencing has identified ARID1A as a novel tumor suppressor gene coding for a chromatin remodeling protein that is mutated in UBC. Here, we assess ARID1A alterations in two series of patients with UBC. In the first tumor series, we analyze exons 2–20 in 52 primary UBC and find that all mutant tumors belong to the aggressive UBC phenotype (high grade non-muscle invasive and muscle invasive tumors) (P = 0.05). In a second series (n = 84), we assess ARID1A expression using immunohistochemistry, a surrogate for mutation analysis, and find that loss of expression increases with higher stage/grade, it is inversely associated with FGFR3 overexpression (P = 0.03) but it is not correlated with p53 overexpression (P = 0.30). We also analyzed the expression of cytokeratins in the same set of tumor and find, using unsupervised clustering, that tumors with ARID1A loss of expression are generally KRT5/6-low. In this patient series, loss of ARID1A expression is also associated with worse prognosis, likely reflecting the higher prevalence of losses found in tumors of higher stage and grade. The independent findings in these two sets of patients strongly support the notion that ARID1A inactivation is a key player in bladder carcinogenesis occurring predominantly in FGFR3 wild type tumors.

Temperature effects on gametophyte life-history traits and geographic distribution of two cryptic kelp species.

A major determinant of the geographic distribution of a species is expected to be its physiological response to changing abiotic variables over its range. The range of a species often corresponds to the geographic extent of temperature regimes the organism can physiologically tolerate. Many species have very distinct life history stages that may exhibit different responses to environmental factors. In this study we emphasized the critical role of the haploid microscopic stage (gametophyte) of the life cycle to explain the difference of edge distribution of two related kelp species. Lessonia nigrescens was recently identified as two cryptic species occurring in parapatry along the Chilean coast: one located north and the other south of a biogeographic boundary at latitude 29–30°S. Six life history traits from microscopic stages were identified and estimated under five treatments of temperature in eight locations distributed along the Chilean coast in order to (1) estimate the role of temperature in the present distribution of the two cryptic L. nigrescens species, (2) compare marginal populations to central populations of the two cryptic species. In addition, we created a periodic matrix model to estimate the population growth rate (λ) at the five temperature treatments. Differential tolerance to temperature was demonstrated between the two species, with the gametophytes of the Northern species being more tolerant to higher temperatures than gametophytes from the south. Second, the two species exhibited different life history strategies with a shorter haploid phase in the Northern species contrasted with considerable vegetative growth in the Southern species haploid stage. These results provide strong ecological evidence for the differentiation process of the two cryptic species and show local adaptation of the life cycle at the range limits of the distribution. Ecological and evolutionary implications of these findings are discussed.

The UBC-40 Urothelial Bladder Cancer cell line index: a genomic resource for functional studies.

BackgroundUrothelial bladder cancer is a highly heterogeneous disease. Cancer cell lines are useful tools for its study. This is a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines including information on origin, mutation status of genes implicated in bladder cancer (FGFR3, PIK3CA, TP53, and RAS), copy number alterations assessed using high density SNP arrays, uniparental disomy (UPD) events, and gene expression.ResultsBased on gene mutation patterns and genomic changes we identify lines representative of the FGFR3-driven tumor pathway and of the TP53/RB tumor suppressor-driven pathway. High-density array copy number analysis identified significant focal gains (1q32, 5p13.1-12, 7q11, and 7q33) and losses (i.e. 6p22.1) in regions altered in tumors but not previously described as affected in bladder cell lines. We also identify new evidence for frequent regions of UPD, often coinciding with regions reported to be lost in tumors. Previously undescribed chromosome X losses found in UBC lines also point to potential tumor suppressor genes. Cell lines representative of the FGFR3-driven pathway showed a lower number of UPD events.ConclusionsOverall, there is a predominance of more aggressive tumor subtypes among the cell lines. We provide a cell line classification that establishes their relatedness to the major molecularly-defined bladder tumor subtypes. The compiled information should serve as a useful reference to the bladder cancer research community and should help to select cell lines appropriate for the functional analysis of bladder cancer genes, for example those being identified through massive parallel sequencing.

INK4a/ARF limits the expansion of cells suffering from replication stress

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.

Macrobenthic communities in a shallow normoxia to hypoxia gradient in the Humboldt upwelling ecosystem

Hypoxia is one of the most important stressors affecting the health conditions of coastal ecosystems. In highly productive ecosystems such as the Humboldt Current ecosystem, the oxygen minimum zone is an important abiotic factor modulating the structure of benthic communities over the continental shelf. Herein, we study soft-bottom macrobenthic communities along a depth gradient–at 10, 20, 30 and 50 m–for two years to understand how hypoxia affects the structure of shallow communities at two sites in Mejillones Bay (23°S) in northern Chile. We test the hypothesis that, during months with shallow hypoxic zones, community structure will be much more dissimilar, thereby depicting a clear structural gradient with depth and correlated abiotic variables (e.g. organic matter, temperature and salinity). Likewise, during conditions of deeper hypoxic zones, communities will be similar among habitats as they could develop structure via succession in conditions with less stress. Throughout the sampling period (October 2015 to October 2017), the water column was hypoxic (from 2 to 0.5ml/l O2) most of the time, reaching shallow depths of 20 to 10 m. Only one episode of oxygenation was detected in June 2016, where normoxia (>2ml/l O2) reached down to 50 m. The structure of the communities depicted a clear pattern of increasing dissimilarity from shallow normoxic and deep hypoxic habitat. This pattern was persistent throughout time despite the occurrence of an oxygenation episode. Contrasting species abundance and biomass distribution explained the gradient in structure, arguably reflecting variable levels of hypoxia adaptation, i.e. few polychaetes such as Magelona physilia and Paraprionospio pinnata were only located in low oxygen habitats. The multivariable dispersion of community composition as a proxy of beta diversity decreased significantly with depth, suggesting loss of community structure and variability when transitioning from normoxic to hypoxic conditions. Our results show the presence of semi-permanent shallow hypoxia at Mejillones Bay, constraining diverse and more variable communities at a very shallow depth (10–20 m). These results must be considered in the context of the current decline of dissolved oxygen in most oceans and coastal regions and their impact on seabed biota.

TWEAK increases CD74 expression and sensitizes to DDT proinflammatory actions in tubular cells

CD74 is a multifunctional protein and a receptor for Macrophage Migration Inhibitory Factor (MIF) and MIF-2 / D-dopachrome tautomerase (DDT) cytokines, upregulated in diabetic kidney disease. However, the drivers of CD74 expression and DDT function in kidney cells are poorly characterized. TWEAK is a proinflammatory cytokine that promotes kidney injury. We have now identified CD74 gene expression as upregulated in the kidneys in response to systemic TWEAK administration in mice, and have characterized the in vivo CD74 expression and the functional consequences in cultured cells. TWEAK administration to mice resulted in a progressive time-dependent (up to 24h) upregulation of kidney CD74 mRNA (RT-PCR) and protein (Western blot). Furthermore, the CD74 ligands MIF and DDT were also upregulated at the protein level 24h after TWEAK administration. Immunohistochemistry localized the increased CD74, MIF and DDT expression to tubular cells. In cultured tubular cells, TWEAK increased CD74 mRNA and protein expression dose-dependently, with a temporal pattern similar to in vivo. TWEAK-induced CD74 localized to the cell membrane, where it can function as a cytokine receptor. For the first time, we explored the actions of DDT in tubular cells and found that DDT amplified the increase in MCP-1 and RANTES expression in response to TWEAK. By contrast, DDT did not significantly modify TWEAK-induced Klotho downregulation. In conclusion, TWEAK upregulates CD74 and its ligands MIF and DDT in renal tubular cells. This may have functional consequences for kidney injury since DDT amplified the inflammatory response to TWEAK.

Abstract 3931: Generation of tumorgraft models to characterize novel tumor suppressor genes implicated in lung carcinogenesis

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The extremely poor prognosis and lack of efficient treatments throw lung cancer (LC) to the top of the deadliest types of cancer worldwide. Throughout the years, the role played by host genetic variation has been constantly highlighted as the key in determining tumor onset and progression. In this regard, the discovery of LC-related genes paved the way to the development of targeted therapies that nowadays are benefiting patients harboring specific mutational patterns, such as EGFR mutations or ALK rearrangements. Here, aiming to characterize novel tumor suppressor genes in LC, 8 patient-derived tumors were subcutaneously or orthotopically engrafted in nude mice for growing and subsequently carry out exome and transcriptome sequencing. The strategy avoided the common admixture exhibited by the presence of normal cells in the sample, which were successfully swept away in the engrafting step, as determined by microsatellite analyses. Among the 4430 mutations detected by exome-seq, we focused on the 79 ones that would presumably lead to biallelic gene inactivation, a common feature exhibited by tumor suppressors: homozygous frameshift indels, nonsense and splicing site mutations. We then chose to characterize one particular candidate gene, which codifies for a globular protein that might be involved in the ability of tumors to escape the action of the immune system. The genetic screening performed resulted in a mutation rate of 5.1% in 79 tested LC cell lines and of 3.9% in a panel of 179 lung primary tumors. The complete coding region was cloned in constitutive and inducible systems in order to restore the expression of the protein in gene-depleted LC-cell lines, and preliminary results pinpoint that it may indeed trigger an anti-tumoral effect. This in vitro approach is currently ongoing and hopefully, will enlighten us about its tumor suppressive potential in the near future. Citation Format: Carolina Pereira, Pol Gimenez-Xavier, Gonzalo Gomez, Enric Condom, Alberto Villanueva, David Pisano, Montse Sanchez Cespedes. Generation of tumorgraft models to characterize novel tumor suppressor genes implicated in lung carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3931. doi:10.1158/1538-7445.AM2015-3931

Erratum to: The UBC-40 Urothelial Bladder Cancer Cell Line Index: a genomic resource for functional studies

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