RESUMEN
Cellular ontogeny and MLL breakpoint site influence the capacity of MLL-edited CD34+ hematopoietic cells to initiate and recapitulate infant patients' features in pro-B-cell acute lymphoblastic leukemia (B-ALL). We provide key insights into the leukemogenic determinants of MLL-AF4+ infant B-ALL.
Asunto(s)
Edición Génica , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Lactante , Humanos , Proteína de la Leucemia Mieloide-Linfoide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Células Madre Hematopoyéticas , Proteínas de Fusión Oncogénica/genéticaRESUMEN
BACKGROUND: Recent evidence suggests a role for the microbiome in pancreatic ductal adenocarcinoma (PDAC) aetiology and progression. OBJECTIVE: To explore the faecal and salivary microbiota as potential diagnostic biomarkers. METHODS: We applied shotgun metagenomic and 16S rRNA amplicon sequencing to samples from a Spanish case-control study (n=136), including 57 cases, 50 controls, and 29 patients with chronic pancreatitis in the discovery phase, and from a German case-control study (n=76), in the validation phase. RESULTS: Faecal metagenomic classifiers performed much better than saliva-based classifiers and identified patients with PDAC with an accuracy of up to 0.84 area under the receiver operating characteristic curve (AUROC) based on a set of 27 microbial species, with consistent accuracy across early and late disease stages. Performance further improved to up to 0.94 AUROC when we combined our microbiome-based predictions with serum levels of carbohydrate antigen (CA) 19-9, the only current non-invasive, Food and Drug Administration approved, low specificity PDAC diagnostic biomarker. Furthermore, a microbiota-based classification model confined to PDAC-enriched species was highly disease-specific when validated against 25 publicly available metagenomic study populations for various health conditions (n=5792). Both microbiome-based models had a high prediction accuracy on a German validation population (n=76). Several faecal PDAC marker species were detectable in pancreatic tumour and non-tumour tissue using 16S rRNA sequencing and fluorescence in situ hybridisation. CONCLUSION: Taken together, our results indicate that non-invasive, robust and specific faecal microbiota-based screening for the early detection of PDAC is feasible.
Asunto(s)
Carcinoma Ductal Pancreático , Microbiota , Neoplasias Pancreáticas , Biomarcadores de Tumor , Antígeno CA-19-9 , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Estudios de Casos y Controles , Humanos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , ARN Ribosómico 16S/genética , Neoplasias PancreáticasRESUMEN
B-cell acute lymphoblastic leukemia (ALL; B-ALL) is the most common pediatric cancer, and high hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD being an initiating oncogenic event affiliated with childhood B-ALL, the mitotic and chromosomal defects associated with HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated with chromosome-alignment defects at the metaphase plate leading to robust chromosome-segregation defects and nonmodal karyotypes. Mechanistically, biochemical, functional, and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness, and mislocalization of the chromosome passenger complex proteins Aurora B kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and an impaired spindle assembly checkpoint (SAC), thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated with a defective condensin complex, AURKB, and SAC.
Asunto(s)
Adenosina Trifosfatasas , Aurora Quinasa B , Aberraciones Cromosómicas , Cromosomas Humanos , Proteínas de Unión al ADN , Metafase/genética , Complejos Multiproteicos , Proteínas de Neoplasias , Ploidias , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Aurora Quinasa B/genética , Aurora Quinasa B/metabolismo , Cromosomas Humanos/genética , Cromosomas Humanos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genéticaRESUMEN
FOXP2 is a gene involved in language development and function. Neanderthals and humans share the same coding region of the gene, although the formers are thought to have exhibited less sophisticated language abilities. In this paper, we report on several human-specific changes in two functional enhancers of FOXP2. Two of these variants are located within the binding sites for the transcription factors POLR2A and SMARCC1, respectively. Interestingly, SMARCC1 is involved in brain development and vitamin D metabolism. We hypothesize that the human specific change in this position might have resulted in a different regulation pattern of FOXP2 expression in our species compared to extinct hominins, with a potential impact on our language abilities.
Asunto(s)
Factores de Transcripción Forkhead , Humanos , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismoRESUMEN
Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , ADN Glicosilasas/genética , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica , Poli(ADP-Ribosa) Polimerasa-1/inmunología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/mortalidad , Daño del ADN , ADN Glicosilasas/antagonistas & inhibidores , ADN Glicosilasas/metabolismo , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , ADN de Neoplasias/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , Guanina/metabolismo , Células HCT116 , Humanos , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Estrés Oxidativo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Análisis de Supervivencia , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CRISPR/Cas is a prokaryotic self-defense system, widely known for its use as a gene-editing tool. Because of their high specificity to detect DNA and RNA sequences, different CRISPR systems have been adapted for nucleic acid detection. CRISPR detection technologies differ highly among them, since they are based on four of the six major subtypes of CRISPR systems. In just 5 years, the CRISPR diagnostic field has rapidly expanded, growing from a set of specific molecular biology discoveries to multiple FDA-authorized COVID-19 tests and the establishment of several companies. CRISPR-based detection methods are coupled with pre-existing preamplification and readout technologies, achieving sensitivity and reproducibility comparable to the current gold standard nucleic acid detection methods. Moreover, they are very versatile, can be easily implemented to detect emerging pathogens and new clinically relevant mutations, and offer multiplexing capability. The advantages of the CRISPR-based diagnostic approaches are a short sample-to-answer time and no requirement of laboratory settings; they are also much more affordable than current nucleic acid detection procedures. In this review, we summarize the applications and development trends of the CRISPR/Cas13 system in the identification of particular pathogens and mutations and discuss the challenges and future prospects of CRISPR-based diagnostic platforms in biomedicine.
Asunto(s)
Técnicas y Procedimientos Diagnósticos/tendencias , Enfermedad/genética , Edición Génica/métodos , COVID-19/genética , Sistemas CRISPR-Cas/genética , ADN/genética , Diagnóstico , Humanos , Reproducibilidad de los Resultados , SARS-CoV-2/genética , SARS-CoV-2/patogenicidadRESUMEN
BACKGROUND: Mutations in the coding region of FOXP2 are known to cause speech and language impairment. However, it is not clear how dysregulation of the gene contributes to language deficit. Interestingly, microdeletions of the region downstream the gene have been associated with cognitive deficits. METHODS: Here, we investigate changes in FOXP2 expression in the SK-N-MC neuroblastoma human cell line after deletion by CRISPR-Cas9 of two enhancers located downstream of the gene. RESULTS: Deletion of any of these two functional enhancers downregulates FOXP2, but also upregulates the closest 3' gene MDFIC. Because this effect is not statistically significant in a HEK 293 cell line, derived from the human kidney, both enhancers might confer a tissue specific regulation to both genes. We have also found that the deletion of any of these enhancers downregulates six well-known FOXP2 target genes in the SK-N-MC cell line. CONCLUSIONS: We expect these findings contribute to a deeper understanding of how FOXP2 and MDFIC are regulated to pace neuronal development supporting cognition, speech and language.
Asunto(s)
Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead/genética , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Células HEK293 , HumanosRESUMEN
Germline mutations in BUB1 and BUB3 have been reported to increase the risk of developing colorectal cancer (CRC) at young age, in presence of variegated aneuploidy and reminiscent dysmorphic traits of mosaic variegated aneuploidy syndrome. We performed a mutational analysis of BUB1 and BUB3 in 456 uncharacterized mismatch repair-proficient hereditary non-polyposis CRC families and 88 polyposis cases. Four novel or rare germline variants, one splice-site and three missense, were identified in four families. Neither variegated aneuploidy nor dysmorphic traits were observed in carriers. Evident functional effects in the heterozygous form were observed for c.1965-1G>A, but not for c.2296G>A (p.E766K), in spite of the positive co-segregation in the family. BUB1 c.2473C>T (p.P825S) and BUB3 c.77C>T (p.T26I) remained as variants of uncertain significance. As of today, the rarity of functionally relevant mutations identified in familial and/or early onset series does not support the inclusion of BUB1 and BUB3 testing in routine genetic diagnostics of familial CRC.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Proteínas de Ciclo Celular/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Mutación de Línea Germinal , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas Serina-Treonina Quinasas/genética , Huso Acromático/genética , Proteínas de Ciclo Celular/química , Humanos , Modelos Moleculares , Linaje , Proteínas de Unión a Poli-ADP-Ribosa/química , Conformación Proteica , Proteínas Serina-Treonina Quinasas/químicaRESUMEN
PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors. METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed. RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma. CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias de las Glándulas Suprarrenales/patología , Adulto , Sistemas CRISPR-Cas/genética , Metilación de ADN , ADN Metiltransferasa 3A , Femenino , Mutación con Ganancia de Función , Predisposición Genética a la Enfermedad , Genotipo , Mutación de Línea Germinal/genética , Humanos , Masculino , Paraganglioma/patología , Feocromocitoma/patología , Secuenciación del ExomaRESUMEN
The cancer-modelling field is now experiencing a conversion with the recent emergence of the RNA-programmable CRISPR-Cas9 system, a flexible methodology to produce essentially any desired modification in the genome. Cancer is a multistep process that involves many genetic mutations and other genome rearrangements. Despite their importance, it is difficult to recapitulate the degree of genetic complexity found in patient tumors. The CRISPR-Cas9 system for genome editing has been proven as a robust technology that makes it possible to generate cellular and animal models that recapitulate those cooperative alterations rapidly and at low cost. In this review, we will discuss the innovative applications of the CRISPR-Cas9 system to generate new models, providing a new way to interrogate the development and progression of cancers.
Asunto(s)
Sistemas CRISPR-Cas , Carcinogénesis/genética , Animales , Marcación de Gen/métodos , Humanos , Modelos GenéticosRESUMEN
Primary hyperoxaluria type 1 (PH1) is a rare inherited metabolic disorder characterized by oxalate overproduction in the liver, resulting in renal damage. It is caused by mutations in the AGXT gene. Combined liver and kidney transplantation is currently the only permanent curative treatment. We combined locus-specific gene correction and hepatic direct cell reprogramming to generate autologous healthy induced hepatocytes (iHeps) from PH1 patient-derived fibroblasts. First, site-specific AGXT corrected cells were obtained by homology directed repair (HDR) assisted by CRISPR-Cas9, following two different strategies: accurate point mutation (c.731T>C) correction or knockin of an enhanced version of AGXT cDNA. Then, iHeps were generated, by overexpression of hepatic transcription factors. Generated AGXT-corrected iHeps showed hepatic gene expression profile and exhibited in vitro reversion of oxalate accumulation compared to non-edited PH1-derived iHeps. This strategy set up a potential alternative cellular source for liver cell replacement therapy and a personalized PH1 in vitro disease model.
RESUMEN
MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes , Proteínas Proto-Oncogénicas c-myc , Animales , Ratones , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Transcripción Genética , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Femenino , MasculinoRESUMEN
Actin dynamics control early T-cell receptor (TCR) signalling during T-cell activation. However, the precise regulation of initial actin rearrangements is not completely understood. Here, we have investigated the regulatory role of the phosphatase Slingshot-1 (SSH1) in this process. Our data show that SSH1 rapidly polarises to nascent cognate synaptic contacts and later relocalises to peripheral F-actin networks organised at the mature immunological synapse. Knockdown of SSH1 expression by CRISPR/Cas9-mediated genome editing or small interfering RNA reveal a regulatory role for SSH1 in CD3ε conformational change, allowing Nck binding and proper downstream signalling and immunological synapse organisation. TCR triggering induces SSH1-mediated activation of actin dynamics through a mechanism mediated by Limk-1 inactivation. These data suggest that during early TCR activation, SSH1 is required for rapid F-actin rearrangements that mediate initial conformational changes of the TCR, integrin organisation and proximal signalling events for proper synapse organisation. Therefore, the SSH1 and Limk-1 axis is a key regulatory element for full T cell activation.
Asunto(s)
Quinasas Lim , Fosfoproteínas Fosfatasas , Receptores de Antígenos de Linfocitos T , Humanos , Quinasas Lim/metabolismo , Quinasas Lim/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/genética , Actinas/metabolismo , Actinas/genética , Activación de Linfocitos , Células Jurkat , Linfocitos T/metabolismo , Linfocitos T/inmunología , Transducción de Señal , Sinapsis Inmunológicas/metabolismoRESUMEN
Pheochromocytomas and paragangliomas are rare neuroendocrine tumours. Around 20-25 % of patients develop metastases, for which there is an urgent need of prognostic markers and therapeutic stratification strategies. The presence of a MAML3-fusion is associated with increased metastatic risk, but neither the processes underlying disease progression, nor targetable vulnerabilities have been addressed. We have compiled a cohort of 850 patients, which has shown a 3.65 % fusion prevalence and represents the largest MAML3-positive series reported to date. While MAML3-fusions mainly cause single pheochromocytomas, we also observed somatic post-zygotic events, resulting in multiple tumours in the same patient. MAML3-tumours show increased expression of neuroendocrine-to-mesenchymal transition markers, MYC-targets, and angiogenesis-related genes, leading to a distinct tumour microenvironment with unique vascular and immune profiles. Importantly, our findings have identified MAML3-tumours specific vulnerabilities beyond Wnt-pathway dysregulation, such as a rich vascular network, and overexpression of PD-L1 and CD40, suggesting potential therapeutic targets.
RESUMEN
The TINCR (Terminal differentiation-Induced Non-Coding RNA) gene is selectively expressed in epithelium tissues and is involved in the control of human epidermal differentiation and wound healing. Despite its initial report as a long non-coding RNA, the TINCR locus codes for a highly conserved ubiquitin-like microprotein associated with keratinocyte differentiation. Here we report the identification of TINCR as a tumor suppressor in squamous cell carcinoma (SCC). TINCR is upregulated by UV-induced DNA damage in a TP53-dependent manner in human keratinocytes. Decreased TINCR protein expression is prevalently found in skin and head and neck squamous cell tumors and TINCR expression suppresses the growth of SCC cells in vitro and in vivo. Consistently, Tincr knockout mice show accelerated tumor development following UVB skin carcinogenesis and increased penetrance of invasive SCCs. Finally, genetic analyses identify loss-of-function mutations and deletions encompassing the TINCR gene in SCC clinical samples supporting a tumor suppressor role in human cancer. Altogether, these results demonstrate a role for TINCR as protein coding tumor suppressor gene recurrently lost in squamous cell carcinomas.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , ARN Largo no Codificante , Animales , Ratones , Humanos , Ubiquitina/metabolismo , Carcinoma de Células Escamosas/genética , Genes Supresores de Tumor , Queratinocitos/metabolismo , Neoplasias de Cabeza y Cuello/genética , ARN Largo no Codificante/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , MicropéptidosRESUMEN
KRASG12C inhibitors have revolutionized the clinical management of patients with KRASG12C-mutant lung adenocarcinoma. However, patient exposure to these inhibitors leads to the rapid onset of resistance. In this study, we have used genetically engineered mice to compare the therapeutic efficacy and the emergence of tumor resistance between genetic ablation of mutant Kras expression and pharmacological inhibition of oncogenic KRAS activity. Whereas Kras ablation induces massive tumor regression and prevents the appearance of resistant cells in vivo, treatment of KrasG12C/Trp53-driven lung adenocarcinomas with sotorasib, a selective KRASG12C inhibitor, caused a limited antitumor response similar to that observed in the clinic, including the rapid onset of resistance. Unlike in human tumors, we did not observe mutations in components of the RAS-signaling pathways. Instead, sotorasib-resistant tumors displayed amplification of the mutant Kras allele and activation of xenobiotic metabolism pathways, suggesting that reduction of the on-target activity of KRASG12C inhibitors is the main mechanism responsible for the onset of resistance. In sum, our results suggest that resistance to KRAS inhibitors could be prevented by achieving a more robust inhibition of KRAS signaling mimicking the results obtained upon Kras ablation.
Asunto(s)
Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Animales , Ratones , Adenocarcinoma del Pulmón/patología , Neoplasias Pulmonares/patología , Mutación , Oncogenes , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de SeñalRESUMEN
Introduction: The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. Methods: We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. Results: Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. Conclusion: We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.
Asunto(s)
Colitis Ulcerosa , Enfermedades Inflamatorias del Intestino , Humanos , Colitis Ulcerosa/patología , Células CACO-2 , Tapsigargina , Estrés del Retículo Endoplásmico/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Células Epiteliales/metabolismo , Hidroximetilglutaril-CoA SintasaRESUMEN
Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS-/-) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS-/- mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS-/- livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease.