RESUMEN

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers among all solid tumors. First-line treatment relies on gemcitabine (Gem) and despite treatment improvements, refractoriness remains a universal challenge. Attempts to decipher how feedback-loops control signaling pathways towards drug resistance have gained attention in recent years, particularly focused on the role of phosphatases. In this study, a CRISPR/Cas9-based phenotypic screen was performed to identify members from the dual-specificity phosphatases (DUSP) family potentially acting on Gem response in PDAC cells. The approach revealed the atypical RNA phosphatase DUSP11 as a potential target, whose inhibition creates vulnerability of PDAC cells to Gem. DUSP11 genetic inhibition impaired cell survival and promoted apoptosis, synergistically enhancing Gem cytotoxicity. In silico transcriptome analysis of RNA-seq data from PDAC human samples identified NF-ĸB signaling pathway highly correlated with DUSP11 upregulation. Consistently, Gem-induced NF-ĸB phosphorylation was blocked upon DUSP11 inhibition in vitro. Mechanistically, we found that DUSP11 directly impacts nc886 expression and modulates PKR-NF-ĸB signaling cascade after Gem exposure in PDAC cells resulting in resistance to Gem-induced cell death. In conclusion, this study provides new insights on DUSP11 role in RNA biology and Gem response in PDAC cells.

Asunto(s)

Desoxicitidina , Fosfatasas de Especificidad Dual , Gemcitabina , FN-kappa B , Neoplasias Pancreáticas , Humanos , Apoptosis/efectos de los fármacos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Fosfatasas de Especificidad Dual/genética , Fosfatasas de Especificidad Dual/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/metabolismo , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , FN-kappa B/metabolismo , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología

RESUMEN

Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S 'foot'. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation.

Asunto(s)

Proteínas Nucleares/fisiología , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Fosfatasas de Especificidad Dual/metabolismo , GTP Fosfohidrolasas/metabolismo , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Biogénesis de Organelos , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo

RESUMEN

MYC overexpression is a common phenomenon in gastric carcinogenesis. In this study, we identified genes differentially expressed with a downregulated profile in gastric cancer (GC) cell lines with silenced MYC. The TTLL12, CDKN3, CDC16, PTPRA, MZT2B, UBE2T genes were validated using qRT-PCR, western blot and immunohistochemistry in tissues of 213 patients with diffuse and intestinal GC. We identified high levels of TTLL12, MZT2B, CDC16, UBE2T, associated with early and advanced stages, lymph nodes, distant metastases and risk factors such as H. pylori. Our results show that in the diffuse GC the overexpression of CDC16 and UBE2T indicate markers of poor prognosis higher than TTLL12. That is, patients with overexpression of these two genes live less than patients with overexpression of TTLL12. In the intestinal GC, patients who overexpressed CDC16 had a significantly lower survival rate than patients who overexpressed MZT2B and UBE2T, indicating in our data a worse prognostic value of CDC16 compared to the other two genes. PTPRA and CDKN3 proved to be important for assessing tumor progression in the early and advanced stages. In summary, in this study, we identified diagnostic and prognostic biomarkers of GC under the control of MYC, related to the cell cycle and the neoplastic process.

Asunto(s)

Adenocarcinoma/genética , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias Gástricas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Subunidad Apc6 del Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc6 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Línea Celular Tumoral , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Fosfatasas de Especificidad Dual/genética , Fosfatasas de Especificidad Dual/metabolismo , Femenino , Silenciador del Gen , Humanos , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Pronóstico , ARN Interferente Pequeño , RNA-Seq , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidad , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo

RESUMEN

Leishmaniasis is caused by intracellular parasites transmitted to vertebrates by sandfly bites. Clinical manifestations include cutaneous, mucosal or visceral involvement depending upon the host immune response and the parasite species. To assure their survival inside macrophages, these parasites developed a plethora of highly successful strategies to manipulate various immune system pathways. Considering that inflammasome activation is critical for the establishment of a protective immune response in many parasite infections, in this study we determined the transcriptome of THP-1 cells after infection with L. infantum, with a particular focus on the inflammasome components. To this end, the human cell line THP-1, previously differentiated into macrophages by PMA treatment, was infected with L. infantum promastigotes. Differentiated THP-1 cells were also stimulated with LPS to be used as a comparative parameter. The gene expression signature was determined 8 hours after by RNA-seq technique. Infected or uninfected THP-1 cells were stimulated with nigericin (NIG) to measure active caspase-1 and TNF-α, IL-6 and IL-1ß levels in culture supernatants after 8, 24 and 48 hours. L. infantum triggered a gene expression pattern more similar to non-infected THP-1 cells and very distinct from LPS-stimulated cells. Some of the most up-regulated genes in L. infantum-infected cells were CDC20, CSF1, RPS6KA1, CD36, DUSP2, DUSP5, DUSP7 and TNFAIP3. Some up-regulated GO terms in infected cells included cell coagulation, regulation of MAPK cascade, response to peptide hormone stimulus, negative regulation of transcription from RNA polymerase II promoter and nerve growth factor receptor signaling pathway. Infection was not able to induce the expression of genes associated with the inflammasome signaling pathway. This finding was confirmed by the absence of caspase-1 activation and IL-1ß production after 8, 24 and 48 hours of infection. Our results indicate that L. infantum was unable to activate the inflammasomes during the initial interaction with THP-1 cells.

Asunto(s)

Inflamasomas/inmunología , Leishmania infantum/fisiología , Leishmaniasis/genética , Monocitos/inmunología , Monocitos/parasitología , Caspasa 1/genética , Caspasa 1/inmunología , Fosfatasas de Especificidad Dual/genética , Fosfatasas de Especificidad Dual/inmunología , Humanos , Inflamasomas/genética , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Leishmaniasis/inmunología , Leishmaniasis/parasitología , Células THP-1 , Transcriptoma , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

Dual Specificity Phosphatase 12 is a member of the Atypical DUSP Protein Tyrosine Phosphatase family, meaning that it does not contain typical MAP kinase targeting motifs, while being able to dephosphorylate tyrosine and serine/threonine residues. DUSP12 contains, apart from its catalytic domain, a zinc finger domain, making it one of the largest DUSPs, which displays strong nuclear expression in several tissues. In this work we identified nuclear targets of DUSP12 in two different cancer cell lines (A549 and MCF-7), challenging them with genotoxic stimuli to observe the effect on the networks and to link existing information about DUSP12 functions to the data obtained though mass spectrometry. We found network connections to the cytoskeleton (e.g. IQGAP1), to the chromatin (e.g. HP1BP3), to the splicing machinery and to the previously known pathway of ribosome maturation (e.g. TCOF1), which draw insight into many of the functions of this phosphatase, much likely connecting it to distinct, previously unknown genomic stability mechanisms.

Asunto(s)

Núcleo Celular/metabolismo , Daño del ADN , Fosfatasas de Especificidad Dual/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interacción de Proteínas , Células A549 , Núcleo Celular/patología , Humanos , Células MCF-7

RESUMEN

African American women are disproportionately affected by type 2 diabetes. Genetic factors may explain part of the excess risk. More than 100 genetic variants have been associated with risk of type 2 diabetes, but most studies have been conducted in white populations. Two genome-wide association studies (GWAS) in African Americans have identified three novel genetic variants only. We conducted admixture mapping using 2918 ancestral informative markers in 2632 cases of type 2 diabetes, and 2596 controls nested in the ongoing Black Women's Health Study cohort, with the goal of identifying genomic loci with local African ancestry associated with type 2 diabetes. In addition, we performed replication analysis of 71 previously identified index SNPs, and fine-mapped those genetic loci to identify better or new genetic variants associated with type 2 diabetes in African Americans. We found that individual African ancestry was associated with higher risk of type 2 diabetes. In addition, we identified two genomic regions, 3q26 and 12q23, with excess of African ancestry associated with higher risk of type 2 diabetes. Lastly, we replicated 8 out of 71 index SNPs from previous GWAS, including, for the first time in African Americans, the X-linked rs5945326 SNP near the DUSP9 gene. In addition, our fine-mapping efforts suggest independent signals at five loci. Our detailed analysis identified two genomic regions associated with risk of type 2 diabetes, and showed that many genetic risk variants are shared across ancestries.

Asunto(s)

Negro o Afroamericano/genética , Mapeo Cromosómico , Diabetes Mellitus Tipo 2/genética , Sitios Genéticos , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Adulto , Anciano , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 3/genética , Fosfatasas de Especificidad Dual/genética , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Persona de Mediana Edad , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética

RESUMEN

Mitogen-activated protein kinases (MAPK) are integration points for multiple biochemical signals. We evaluated 13 MAPK genes with breast cancer risk and determined if diet and lifestyle factors mediated risk. Data from 3 population-based case-control studies conducted in Southwestern United States, California, and Mexico included 4183 controls and 3592 cases. Percent Indigenous American (IA) ancestry was determined from 104 ancestry informative markers. The adaptive rank truncated product (ARTP) was used to determine the significance of each gene and the pathway with breast cancer risk, by menopausal status, genetic ancestry level, and estrogen receptor (ER)/progesterone receptor (PR) strata. MAP3K9 was associated with breast cancer overall (P(ARTP) = 0.02) with strongest association among women with the highest IA ancestry (P(ARTP) = 0.04). Several SNPs in MAP3K9 were associated with ER+/PR+ tumors and interacted with dietary oxidative balance score (DOBS), dietary folate, body mass index (BMI), alcohol consumption, cigarette smoking, and a history of diabetes. DUSP4 and MAPK8 interacted with calories to alter breast cancer risk; MAPK1 interacted with DOBS, dietary fiber, folate, and BMI; MAP3K2 interacted with dietary fat; and MAPK14 interacted with dietary folate and BMI. The patterns of association across diet and lifestyle factors with similar biological properties for the same SNPs within genes provide support for associations.

Asunto(s)

Neoplasias de la Mama/genética , Dieta/estadística & datos numéricos , Estilo de Vida , Proteínas Quinasas Activadas por Mitógenos/genética , Adulto , Anciano , Índice de Masa Corporal , Neoplasias de la Mama/etnología , Neoplasias de la Mama/metabolismo , Estudios de Casos y Controles , Grasas de la Dieta/metabolismo , Fibras de la Dieta/metabolismo , Fosfatasas de Especificidad Dual/genética , Ingestión de Energía/genética , Femenino , Ácido Fólico/metabolismo , Disparidades en el Estado de Salud , Humanos , MAP Quinasa Quinasa Quinasa 2 , Quinasas Quinasa Quinasa PAM/genética , Menopausia/genética , México/epidemiología , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 8 Activada por Mitógenos/genética , Fosfatasas de la Proteína Quinasa Activada por Mitógenos/genética , Polimorfismo de Nucleótido Simple/genética , Grupos de Población/genética , Receptores de Estrógenos/sangre , Receptores de Progesterona/sangre , Sistema de Registros , Factores de Riesgo , San Francisco , Sudoeste de Estados Unidos

RESUMEN

We present a phylogenetic hypothesis of the anuran clade Terrarana based on partial sequences of nuclear (Tyr and RAG1) and mitochondrial (12S, tRNA-Val, and 16S) genes, testing the monophyly of Ischnocnema and its species series. We performed maximum parsimony, maximum likelihood, and Bayesian inference analyses on 364 terminals: 11 outgroup terminals and 353 ingroup Terrarana terminals, including 139 Ischnocnema terminals (accounting for 29 of the 35 named Ischnocnema species) and 214 other Terrarana terminals within the families Brachycephalidae, Ceuthomantidae, Craugastoridae, and Eleutherodactylidae. Different optimality criteria produced similar results and mostly recovered the currently accepted families and genera. According to these topologies, Ischnocnema is not a monophyletic group. We propose new combinations for three species, relocating them to Pristimantis, and render Eleutherodactylus bilineatus Bokermann, 1975 incertae sedis status within Holoadeninae. The rearrangements in Ischnocnema place it outside the northernmost Brazilian Atlantic rainforest, where the fauna of Terrarana comprises typical Amazonian genera.

Asunto(s)

Anuros/clasificación , Filogenia , Animales , Anuros/genética , Proteínas de Arabidopsis , Teorema de Bayes , Brasil , Núcleo Celular/genética , ADN Mitocondrial/genética , Fosfatasas de Especificidad Dual , Evolución Molecular , Modelos Genéticos , Análisis de Secuencia de ADN