RESUMEN
BACKGROUND & AIMS: The ß-catenin signaling pathway is one of the most commonly deregulated pathways in cancer cells. Amino acid substitutions within armadillo repeats 5 and 6 (K335, W383, and N387) of ß-catenin are found in several tumor types, including liver tumors. We investigated the mechanisms by which these substitutions increase signaling and the effects on liver carcinogenesis in mice. METHODS: Plasmids encoding tagged full-length ß-catenin (CTNNB1) or ß-catenin with the K335I or N387K substitutions, along with MET, were injected into tails of FVB/N mice. Tumor growth was monitored, and livers were collected and analyzed by histology, immunohistochemistry, and quantitative reverse-transcription polymerase chain reaction. Tagged full-length and mutant forms of ß-catenin were expressed in HEK293, HCT116, and SNU449 cells, which were analyzed by immunoblots and immunoprecipitation. A panel of ß-catenin variants and cell lines with knock-in mutations were analyzed for differences in N-terminal phosphorylation, half-life, and association with other proteins in the signaling pathway. RESULTS: Mice injected with plasmids encoding K335I or N387K ß-catenin and MET developed larger, more advanced tumors than mice injected with plasmids encoding WT ß-catenin and MET. K335I and N387K ß-catenin bound APC with lower affinity than WT ß-catenin but still interacted with scaffold protein AXIN1 and in the nucleus with TCF7L2. This interaction resulted in increased transcription of genes regulated by ß-catenin. Studies of protein structures supported the observed changes in relative binding affinities. CONCLUSION: Expression of ß-catenin with mutations in armadillo repeats 5 and 6, along with MET, promotes formation of liver tumors in mice. In contrast to N-terminal mutations in ß-catenin that directly impair its phosphorylation by GSK3 or binding to BTRC, the K335I or N387K substitutions increase signaling via reduced binding to APC. However, these mutant forms of ß-catenin still interact with the TCF family of transcription factors in the nucleus. These findings show how these amino acid substitutions increase ß-catenin signaling in cancer cells.
Asunto(s)
Carcinogénesis/genética , Genes APC/fisiología , Neoplasias Hepáticas/genética , Vía de Señalización Wnt/genética , beta Catenina/genética , Animales , Células HCT116 , Células HEK293 , Humanos , Hígado/metabolismo , Ratones , Mutación , Plásmidos/farmacología , Proteínas Proto-Oncogénicas c-met , Transcripción GenéticaRESUMEN
BACKGROUND & AIMS: Mature transfer RNAs (tRNA) charged with amino acids decode mRNA to synthesize proteins. Dysregulation of translational machineries has a fundamental impact on cancer biology. This study aims to map the tRNAome landscape in liver cancer patients and to explore potential therapeutic targets at the interface of charging amino acid with tRNA. METHODS: Resected tumour and paired tumour-free (TFL) tissues from hepatocellular carcinoma (HCC) patients (n = 69), and healthy liver tissues from organ transplant donors (n = 21), HCC cell lines, and cholangiocarcinoma (CC) patient-derived tumour organoids were used. RESULTS: The expression levels of different mature tRNAs were highly correlated and closely clustered within individual tissues, suggesting that different members of the tRNAome function cooperatively in protein translation. Interestingly, high expression of tRNA-Lys-CUU in HCC tumours was associated with more tumour recurrence (HR 1.1; P = .022) and worse patient survival (HR 1.1; P = .0037). The expression of Lysyl-tRNA Synthetase (KARS), the enzyme catalysing the charge of lysine to tRNA-Lys-CUU, was significantly upregulated in HCC tumour tissues compared to tumour-free liver tissues. In HCC cell lines, lysine deprivation, KARS knockdown or treatment with the KARS inhibitor cladosporin effectively inhibited overall cell growth, single cell-based colony formation and cell migration. This was mechanistically mediated by cell cycling arrest and induction of apoptosis. Finally, these inhibitory effects were confirmed in 3D cultured patient-derived CC organoids. CONCLUSIONS: The biological process of charging tRNA-Lys-CUU with lysine sustains liver cancer cell growth and migration, and is clinically relevant in HCC patients. This process can be therapeutically targeted and represents an unexplored territory for developing novel treatment strategies against liver cancer.
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Fenómenos Biológicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Lisina , Recurrencia Local de Neoplasia , Aminoacilación de ARN de TransferenciaRESUMEN
The current understanding of cancer biology and development of effective treatments for cancer remain far from satisfactory. This in turn heavily relies on the availability of easy and robust model systems that resemble the architecture/physiology of the tumors in patients to facilitate research. Cancer research in vitro has mainly been based on the use of immortalized 2D cancer cell lines that deviate in many aspects from the original primary tumors. The recent development of the organoid technology allowing generation of organ-buds in 3D culture from adult stem cells has endowed the possibility of establishing stable culture from primary tumors. Although culturing organoids from liver tumors is thought to be difficult, we now convincingly demonstrate the establishment of organoids from mouse primary liver tumors. We have succeeded in culturing 91 lines from 129 liver tissue/tumors. These organoids can be grown in long-term cultures in vitro. About 20% of these organoids form tumors in immunodeficient mice upon (serial) transplantation, confirming their tumorigenic and self-renewal properties. Interestingly, single cells from the tumor organoids have high efficiency of organoid initiation, and a single organoid derived from a cancer cell is able to initiate a tumor in mice, indicating the enrichment of tumor-initiating cells in the tumor organoids. Furthermore, these organoids recapitulate, to some extent, the heterogeneity of liver cancer in patients, with respect to phenotype, cancer cell composition and treatment response. These model systems shall provide enormous opportunities to advance our research on liver cancer (stem cell) biology, drug development and personalized medicine.
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Ensayos de Selección de Medicamentos Antitumorales/métodos , Neoplasias Hepáticas/patología , Organoides/patología , Animales , Antineoplásicos/uso terapéutico , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Ratones , Cultivo Primario de Células , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The transformation of normal colonic epithelium to colorectal cancer (CRC) involves a relatively ordered progression, and understanding the molecular alterations involved may aid rational design of strategies aimed at preventing or counteracting disease. Homeobox A9 (HOXA9) is an oncogene in leukemia and has been implicated in CRC pathology, although its role in disease etiology remains obscure at best. We observe that HOXA9 expression is increased in colonic adenomas compared with location-matched healthy colon epithelium. Its forced expression results in dramatic genetic and signaling changes, with increased expression of growth factors IGF1 and FLT3, super-activity of the AKT survival pathway and a concomitant increase in compartment size. Furthermore, a reduced mRNA expression of the epithelial to mesenchymal transition marker N-cadherin as well as reduced activity of the actin cytoskeletal mediator PAK was seen, which is in apparent agreement with an observed reduced migratory response in HOXA9-overexpressing cells. Thus, HOXA9 appears closely linked with adenoma growth while impairing migration and metastasis and hence is both a marker and driver of premalignant polyp growth. Colonic polyps grow but remain premalignant for up to decades. Here, we show that HOXA9 drives growth in premalignant polyps, but simultaneously prevents further transformation.
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Transformación Celular Neoplásica/metabolismo , Neoplasias del Colon/patología , Transición Epitelial-Mesenquimal/fisiología , Proteínas de Homeodominio/metabolismo , Lesiones Precancerosas/patología , Anciano , Transformación Celular Neoplásica/patología , Neoplasias del Colon/metabolismo , Pólipos del Colon/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Lesiones Precancerosas/metabolismoRESUMEN
A remarkable feature of regenerative processes is their ability to halt proliferation once an organ's structure has been restored. The Wnt signalling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. However, the mechanisms that counterbalance Wnt-driven proliferation are poorly understood. Here we demonstrate in mice and humans that yes-associated protein 1 (YAP; also known as YAP1)--a protein known for its powerful growth-inducing and oncogenic properties--has an unexpected growth-suppressive function, restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signalling independently of the AXIN-APC-GSK-3ß complex partly by limiting the activity of dishevelled (DVL). DVL signals in the nucleus of intestinal stem cells, and its forced expression leads to enhanced Wnt signalling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas, and that its expression can restrict the growth of colorectal carcinoma xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proliferación Celular , Intestinos/citología , Fosfoproteínas/metabolismo , Regeneración/fisiología , Células Madre/citología , Células Madre/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Proteínas Dishevelled , Genes Supresores de Tumor , Humanos , Intestinos/fisiología , Ratones , Ratones Noqueados , Trasplante de Neoplasias , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Nicho de Células Madre , Trombospondinas/genética , Trombospondinas/metabolismo , Factores de Transcripción , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Proteínas Señalizadoras YAPRESUMEN
BACKGROUND & AIMS: Adult liver stem cells are usually maintained in a quiescent/slow-cycling state. However, a proliferative population, marked by leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), was recently identified as an important liver stem cell population. We aimed to investigate the dynamics and functions of proliferative and quiescent stem cells in healthy and injured livers. METHODS: We studied LGR5-positive stem cells using diphtheria toxin receptor and green fluorescent protein (GFP) knock-in mice. In these mice, LGR5-positive cells specifically coexpress diphtheria toxin receptor and the GFP reporter. Lineage-tracing experiments were performed in mice in which LGR5-positive stem cells and their daughter cells expressed a yellow fluorescent protein/mTmG reporter. Slow-cycling stem cells were investigated using GFP-based, Tet-on controlled transgenic mice. We studied the dynamics of both stem cell populations during liver homeostasis and injury induced by carbon tetrachloride. Stem cells were isolated from mouse liver and organoid formation assays were performed. We analyzed hepatocyte and cholangiocyte lineage differentiation in cultured organoids. RESULTS: We did not detect LGR5-expressing stem cells in livers of mice at any stage of a lifespan, but only following liver injury induced by carbon tetrachloride. In the liver stem cell niche, where the proliferating LGR5+ cells are located, we identified a quiescent/slow-cycling cell population, called label-retaining cells (LRCs). These cells were present in the homeostatic liver, capable of retaining the GFP label over 1 year, and expressed a panel of progenitor/stem cell markers. Isolated single LRCs were capable of forming organoids that could be carried in culture, expanded for months, and differentiated into hepatocyte and cholangiocyte lineages in vitro, demonstrating their bona fide stem cell properties. More interestingly, LRCs responded to liver injury and gave rise to LGR5-expressing stem cells, as well as other potential progenitor/stem cell populations, including SOX9- and CD44-positive cells. CONCLUSIONS: Proliferative LGR5 cells are an intermediate stem cell population in the liver that emerge only during tissue injury. In contrast, LRCs are quiescent stem cells that are present in homeostatic liver, respond to tissue injury, and can give rise to LGR5 stem cells, as well as SOX9- and CD44-positive cells.
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Proliferación Celular , Senescencia Celular , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Regeneración Hepática , Hígado/patología , Células Madre/patología , Animales , Conductos Biliares/metabolismo , Conductos Biliares/patología , Tetracloruro de Carbono , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Genotipo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/metabolismo , Ratones Transgénicos , Fenotipo , Regiones Promotoras Genéticas , ARN no Traducido/genética , Receptores Acoplados a Proteínas G/genética , Nicho de Células Madre , Células Madre/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: The Wnt/ß-catenin pathway plays a central role in the pathogenesis of most hepatoblastomas (HBs), that is, up to 60-80% carry activating CTNNB1 mutations. HBs can however also be the first manifestation of familial adenomatous polyposis (FAP). As this is a severe disease, it is important for the patient and related family members to firmly exclude FAP at an early stage. Current diagnosis largely depends on APC germline mutation detection on genomic DNA, which is associated with 10-20% false-negative results. Here, we establish and validate a tissue-based ß-catenin gene and immunohistochemical analysis, which complements germline mutation screening to exclude the diagnosis of FAP among HB patients. METHODS: Tumor tissues of 18 HB patients, including three FAP cases were subjected to CTNNB1 exon 3 mutational analysis and immunohistochemistry comparing staining patterns for total and exon 3 specific ß-catenin antibodies. RESULTS: Our novel tissue-based method reliably identified all three FAP patients. Their tumors were characterized by a wild-type exon 3 sequence and a comparable nuclear staining for both antibodies. In contrast, the non-FAP tumors carried missense CTNNB1 mutations combined with a clearly reduced staining for the exon 3 antibody, or complete loss of staining in case of lesions with exon 3 deletions. CONCLUSION: We have successfully established and validated a novel ß-catenin gene and immunohistochemical diagnostic method, which, when combined with routine germline DNA testing, allows the exclusion of the diagnosis of FAP among HB patients.
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Poliposis Adenomatosa del Colon/diagnóstico , Mutación de Línea Germinal , Hepatoblastoma/diagnóstico , Neoplasias Hepáticas/diagnóstico , Mutación Missense , beta Catenina/genética , beta Catenina/metabolismo , Poliposis Adenomatosa del Colon/complicaciones , Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/metabolismo , Adolescente , Niño , Preescolar , Análisis Mutacional de ADN , Diagnóstico Diferencial , Femenino , Estudios de Seguimiento , Hepatoblastoma/complicaciones , Hepatoblastoma/genética , Hepatoblastoma/metabolismo , Humanos , Inmunohistoquímica , Lactante , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , PronósticoRESUMEN
Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt's base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus-surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt-villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.
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Linaje de la Célula , Células Epiteliales/citología , Células Epiteliales/metabolismo , Hormonas Gastrointestinales/biosíntesis , Intestinos/citología , Péptidos Natriuréticos/biosíntesis , Animales , Hormonas Gastrointestinales/análisis , Hormonas Gastrointestinales/genética , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos , Péptidos Natriuréticos/análisis , Péptidos Natriuréticos/genética , Transducción de SeñalAsunto(s)
Carcinoma Ductal Pancreático/patología , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Transducción de Señal/efectos de los fármacos , beta Catenina/fisiología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Piridinas/farmacología , Pirimidinas/farmacología , Vorinostat/farmacología , Proteína Wnt3A/fisiología , beta Catenina/genéticaRESUMEN
Lung development is determined by the coordinated expression of several key genes. Previously, we and others have shown the importance of the sex determining region Y-box 2 (Sox2) gene in lung development. Transgenic expression of Sox2 during lung development resulted in cystic airways, and here we show that modulating the timing of ectopic Sox2 expression in the branching regions of the developing lung results in variable cystic lesions resembling the spectrum of the human congenital disorder congenital cystic adenomatoid malformation (CCAM). Sox2 dominantly differentiated naive epithelial cells into the proximal lineage irrespective of the presence of Fgf10. Sox2 directly induced the expression of Trp63, the master switch toward the basal cell lineage and induced the expression of Gata6, a factor involved in the emergence of bronchoalveolar stem cells. We showed that SOX2 and TRP63 are coexpressed in the lungs of human patients with type II CCAM. The combination of premature differentiation toward the proximal cell lineage and the induction of proliferation resulted in the cyst-like structures. Thus, we show that Sox2 is directly responsible for the emergence of two lung progenitor cells: basal cells by regulating the master gene Trp63 and bronchoalveolar stem cells by regulating Gata6.
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Malformación Adenomatoide Quística Congénita del Pulmón/metabolismo , Células Epiteliales/metabolismo , Pulmón/metabolismo , Fosfoproteínas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Células Madre/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Activación Transcripcional , Proteínas Supresoras de Tumor/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Linaje de la Célula , Proliferación Celular , Malformación Adenomatoide Quística Congénita del Pulmón/genética , Malformación Adenomatoide Quística Congénita del Pulmón/patología , Células Epiteliales/patología , Factor 10 de Crecimiento de Fibroblastos/metabolismo , Factor de Transcripción GATA6/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genotipo , Edad Gestacional , Células HEK293 , Humanos , Pulmón/patología , Ratones , Ratones Transgénicos , Fenotipo , Fosfoproteínas/genética , Factores de Transcripción SOXB1/genética , Células Madre/patología , Técnicas de Cultivo de Tejidos , Transactivadores/genética , Factores de Transcripción/genética , Transfección , Proteínas Supresoras de Tumor/genética , Regulación hacia ArribaRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is a highly aggressive liver cancer with significant morbidity and mortality rates. AXIN1 is one of the top-mutated genes in HCC, but the mechanism by which AXIN1 mutations contribute to HCC development remains unclear. METHODS: In this study, we utilized CRISPR/Cas9 genome editing to repair AXIN1-truncated mutations in five HCC cell lines. RESULTS: For each cell line we successfully obtained 2-4 correctly repaired clones, which all show reduced ß-catenin signaling accompanied with reduced cell viability and colony formation. Although exposure of repaired clones to Wnt3A-conditioned medium restored ß-catenin signaling, it did not or only partially recover their growth characteristics, indicating the involvement of additional mechanisms. Through RNA-sequencing analysis, we explored the gene expression patterns associated with repaired AXIN1 clones. Except for some highly-responsive ß-catenin target genes, no consistent alteration in gene/pathway expression was observed. This observation also applies to the Notch and YAP/TAZ-Hippo signaling pathways, which have been associated with AXIN1-mutant HCCs previously. The AXIN1-repaired clones also cannot confirm a recent observation that AXIN1 is directly linked to YAP/TAZ protein stability and signaling. CONCLUSIONS: Our study provides insights into the effects of repairing AXIN1 mutations on ß-catenin signaling, cell viability, and colony formation in HCC cell lines. However, further investigations are necessary to understand the complex mechanisms underlying HCC development associated with AXIN1 mutations.
Asunto(s)
Proteína Axina , Sistemas CRISPR-Cas , Carcinoma Hepatocelular , Neoplasias Hepáticas , Mutación , beta Catenina , Proteína Axina/genética , Proteína Axina/metabolismo , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Línea Celular Tumoral , beta Catenina/metabolismo , beta Catenina/genética , Regulación Neoplásica de la Expresión Génica , Edición Génica , Transducción de Señal/genéticaRESUMEN
AXIN1 is a major component of the ß-catenin destruction complex and is frequently mutated in various cancer types, particularly liver cancers. Truncating AXIN1 mutations are recognized to encode a defective protein that leads to ß-catenin stabilization, but the functional consequences of missense mutations are not well characterized. Here, we first identified the GSK3ß, ß-catenin, and RGS/APC interaction domains of AXIN1 that are the most critical for proper ß-catenin regulation. Analysis of 80 tumor-associated variants in these domains identified 18 that significantly affected ß-catenin signaling. Coimmunoprecipitation experiments revealed that most of them lost binding to the binding partner corresponding to the mutated domain. A comprehensive protein structure analysis predicted the consequences of these mutations, which largely overlapped with the observed effects on ß-catenin signaling in functional experiments. The structure analysis also predicted that loss-of-function mutations within the RGS/APC interaction domain either directly affected the interface for APC binding or were located within the hydrophobic core and destabilized the entire structure. In addition, truncated AXIN1 length inversely correlated with the ß-catenin regulatory function, with longer proteins retaining more functionality. These analyses suggest that all AXIN1-truncating mutations at least partially affect ß-catenin regulation, whereas this is only the case for a subset of missense mutations. Consistently, most colorectal and liver cancers carrying missense variants acquire mutations in other ß-catenin regulatory genes such as APC and CTNNB1. These results will aid the functional annotation of AXIN1 mutations identified in large-scale sequencing efforts or in individual patients. SIGNIFICANCE: Characterization of 80 tumor-associated missense variants of AXIN1 reveals a subset of 18 mutations that disrupt its ß-catenin regulatory function, whereas the majority are passenger mutations.
Asunto(s)
Proteína Axina , Mutación Missense , beta Catenina , Proteína Axina/genética , Proteína Axina/metabolismo , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Transducción de Señal/genética , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Células HEK293 , Línea Celular Tumoral , Unión ProteicaRESUMEN
Whereas aberrant activation of canonical Wnt/ß-catenin signaling underlies the majority of colorectal cancer cases, the contribution of non-canonical Wnt signaling is unclear. As enhanced expression of the most extensively studied non-canonical Wnt ligand WNT5A is observed in various diseases including colon cancer, WNT5A is gaining attention nowadays. Numerous in vitro studies suggest modulating capacities of WNT5A on proliferation, differentiation, migration and invasion, affecting tumor and non-mutant cells. However, a possible contribution of WNT5A to colorectal cancer remains to be elucidated. We have analyzed WNT5A expression in colorectal cancer profiling data sets, altered WNT5A expression in colon cancer cells and used our inducible Wnt5a transgenic mouse model to gain more insight into the role of WNT5A in intestinal cancer. We observed that increased WNT5A expression is associated with poor prognosis of colorectal cancer patients. WNT5A knockdown in human colon cancer cells caused reduced directional migration, deregulated focal adhesion site formation and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Despite these observed protumorigenic activities of WNT5A, the induction of Wnt5a expression in intestinal tumors of Apc1638N mice was not sufficient to augment malignancy or metastasis by itself. In conclusion, WNT5A promotes adhesion sites to form in a focal fashion and promotes the directional migration and invasion of colon cancer cells. Although these activities appear insufficient by themselves to augment malignancy or metastasis in Apc1638N mice, they might explain the poor colon cancer prognosis associated with enhanced WNT5A expression.
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Proteína de la Poliposis Adenomatosa del Colon/fisiología , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/patología , Neoplasias del Colon/patología , Intestinos/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Wnt/metabolismo , Animales , Apoptosis , Western Blotting , Adhesión Celular , Transformación Celular Neoplásica/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Adhesiones Focales , Humanos , Técnicas para Inmunoenzimas , Mucosa Intestinal/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Invasividad Neoplásica , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteínas Wnt/genética , Proteína Wnt-5aRESUMEN
Wnt5a is essential during embryonic development, as indicated by mouse Wnt5a knockout embryos displaying outgrowth defects of multiple structures including the gut. The dynamics of Wnt5a involvement in these processes is unclear, and perinatal lethality of Wnt5a knockout embryos has hampered investigation of Wnt5a during postnatal stages in vivo. Although in vitro studies have suggested a relevant role for Wnt5a postnatally, solid evidence for a significant impact of Wnt5a within the complexity of an adult organism is lacking. We generated a tightly-regulated inducible Wnt5a transgenic mouse model and investigated the effects of Wnt5a induction during different time-frames of embryonic development and in adult mice, focusing on the gastrointestinal tract. When induced in embryos from 10.5 dpc onwards, Wnt5a expression led to severe outgrowth defects affecting the gastrointestinal tracts, limbs, facial structures and tails, closely resembling the defects observed in Wnt5a knockout mice. However, Wnt5a induction from 13.5 dpc onwards did not cause this phenotype, indicating that the most critical period for Wnt5a in embryonic development is prior to 13.5 dpc. In adult mice, induced Wnt5a expression did not reveal abnormalities, providing the first in vivo evidence that Wnt5a has no major impact on mouse intestinal homeostasis postnatally. Protein expression of Wnt5a receptor Ror2 was strongly reduced in adult intestine compared to embryonic stages. Moreover, we uncovered a regulatory process where induction of Wnt5a causes downregulation of its receptor Ror2. Taken together, our results indicate a role for Wnt5a during a restricted time-frame of embryonic development, but suggest no impact during homeostatic postnatal stages.
Asunto(s)
Envejecimiento/genética , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genética , Intestinos/embriología , Proteínas Wnt/metabolismo , Envejecimiento/efectos de los fármacos , Animales , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestinos/citología , Intestinos/efectos de los fármacos , Ratones , Ratones Transgénicos , Modelos Animales , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Tetraciclina/farmacología , Proteínas Wnt/genética , Proteína Wnt-5aRESUMEN
The Wnt signal-transduction pathway induces the nuclear translocation of membrane-bound beta-catenin (Catnb) and has a key role in cell-fate determination. Tight somatic regulation of this signal is essential, as uncontrolled nuclear accumulation of beta-catenin can cause developmental defects and tumorigenesis in the adult organism. The adenomatous polyposis coli gene (APC) is a major controller of the Wnt pathway and is essential to prevent tumorigenesis in a variety of tissues and organs. Here, we have investigated the effect of different mutations in Apc on the differentiation potential of mouse embryonic stem (ES) cells. We provide genetic and molecular evidence that the ability and sensitivity of ES cells to differentiate into the three germ layers is inhibited by increased doses of beta-catenin by specific Apc mutations. These range from a severe differentiation blockade in Apc alleles completely deficient in beta-catenin regulation to more specific neuroectodermal, dorsal mesodermal and endodermal defects in more hypomorphic alleles. Accordingly, a targeted oncogenic mutation in Catnb also affects the differentiation potential of ES cells. Expression profiling of wildtype and Apc-mutated teratomas supports the differentiation defects at the molecular level and pinpoints a large number of downstream structural and regulating genes. Chimeric experiments showed that this effect is cell-autonomous. Our results imply that constitutive activation of the Apc/beta-catenin signaling pathway results in differentiation defects in tissue homeostasis, and possibly underlies tumorigenesis in the colon and other self-renewing tissues.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/fisiología , Proteínas del Citoesqueleto/genética , Embrión de Mamíferos/citología , Células Madre Pluripotentes/fisiología , Transducción de Señal/genética , Transactivadores/genética , Proteína de la Poliposis Adenomatosa del Colon/genética , Alelos , Animales , Diferenciación Celular , Línea Celular , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Dosificación de Gen , Perfilación de la Expresión Génica , Genes APC , Genotipo , Mutación de Línea Germinal , Heterocigoto , Homocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Teratoma/genética , Transactivadores/metabolismo , Trasplante Isogénico , beta CateninaRESUMEN
ß-Catenin (Ctnnb1) has been shown to play critical roles in the development and maintenance of epithelial cells, including the retinal pigment epithelium (RPE). Ctnnb1 is not only a component of intercellular junctions in the epithelium, it also functions as a transcriptional regulator in the Wnt signaling pathway. To identify which of its functional modalities is critically involved in mouse RPE development and maintenance, we varied Ctnnb1 gene content and activity in mouse RPE lineage cells and tested their impacts on mouse eye development. We found that a Ctnnb1 double mutant (Ctnnb1dm), which exhibits impaired transcriptional activity, could not replace Ctnnb1 in the RPE, whereas Ctnnb1Y654E, which has reduced affinity for the junctions, could do so. Expression of the constitutively active Ctnnb1∆ex3 mutant also suppressed the development of RPE, instead facilitating a ciliary cell fate. However, the post-mitotic or mature RPE was insensitive to the loss, inactivation, or constitutive activation of Ctnnb1. Collectively, our results suggest that Ctnnb1 should be maintained within an optimal range to specify RPE through transcriptional regulation of Wnt target genes in the optic neuroepithelium.
Asunto(s)
Epitelio Pigmentado de la Retina , Vía de Señalización Wnt , Ratones , Animales , Epitelio Pigmentado de la Retina/metabolismo , Vía de Señalización Wnt/genética , Diferenciación Celular , beta Catenina/genética , beta Catenina/metabolismo , Regulación de la Expresión Génica , Neuronas/metabolismoRESUMEN
RNF43 is an important negative regulator of ß-catenin signaling by removing Wnt-receptors from the membrane. It is often mutated in cancers, leading to aberrant Wnt-dependent nuclear translocation of ß-catenin. RNF43 has also been suggested to regulate ß-catenin signaling directly within the nucleus, among other proposed nuclear functions. Given the importance of RNF43 in regulating Wnt/ß-catenin signaling and its potential therapeutic relevance, a proper understanding of RNF43 biology is required. However, the presumed nuclear location is mainly based on available antibodies. These same antibodies have also been used extensively for immunoblotting or immunohistochemical purposes. However, a proper evaluation of their quality to reliably detect endogenous RNF43 has not been performed. Here, using genome editing we have generated a cell line that entirely misses RNF43 exons 8 and 9, encoding the epitopes of commonly used RNF43 antibodies. Using this clone in addition to various other cell line tools, we show that four RNF43 antibodies only yield non-specific signals when applied in immunoblotting, immunofluorescence and immunohistochemical experiments. In other words, they cannot reliably detect endogenous RNF43. Our results suggest that the nuclear staining patterns are an antibody artifact and that RNF43 is unlikely to localize within the nucleus. More generally, reports using RNF43 antibodies should be interpreted with caution, at least for the RNF43 protein aspects described in these papers.
Asunto(s)
Proteínas de Unión al ADN , beta Catenina , beta Catenina/genética , beta Catenina/metabolismo , Proteínas de Unión al ADN/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Oncogénicas/genética , Vía de Señalización Wnt/genéticaRESUMEN
In contrast to the majority of sporadic colorectal cancer which predominantly occur in the distal colon, most mismatch repair deficient tumours arise at the proximal side. At present, these regional preferences have not been explained properly. Recently, we have screened colorectal tumours for mutations in Wnt-related genes focusing specifically on colorectal location. Combining this analysis with published data, we propose a mechanism underlying the side-related preferences of colorectal cancers, based on the specific acquired genetic defects in ß-catenin signalling.
Asunto(s)
Neoplasias Colorrectales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Neoplasias Encefálicas , Genes APC , Humanos , Datos de Secuencia Molecular , Mutación , Síndromes Neoplásicos Hereditarios , Especificidad de Órganos , Vía de Señalización Wnt , beta Catenina/genética , beta Catenina/fisiologíaRESUMEN
Germline mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant hereditary predisposition to the development of multiple colorectal adenomas and of a broad spectrum of extra-intestinal tumors. Moreover, somatic APC mutations play a rate-limiting and initiating role in the majority of sporadic colorectal cancers. Notwithstanding its multifunctional nature, the main tumor suppressing activity of the APC gene resides in its ability to regulate Wnt/beta-catenin signaling. Notably, genotype-phenotype correlations have been established at the APC gene between the length and stability of the truncated proteins encoded by different mutant alleles, the corresponding levels of Wnt/beta-catenin signaling activity they encode for, and the incidence and distribution of intestinal and extra-intestinal tumors. Here, we report a novel mouse model, Apc1572T, obtained by targeting a truncated mutation at codon 1572 in the endogenous Apc gene. This hypomorphic mutant allele results in intermediate levels of Wnt/beta-catenin signaling activation when compared with other Apc mutations associated with multifocal intestinal tumors. Notwithstanding the constitutive nature of the mutation, Apc(+/1572T) mice have no predisposition to intestinal cancer but develop multifocal mammary adenocarcinomas and subsequent pulmonary metastases in both genders. The histology of the Apc1572T primary mammary tumours is highly heterogeneous with luminal, myoepithelial, and squamous lineages and is reminiscent of metaplastic carcinoma of the breast in humans. The striking phenotype of Apc(+/1572T) mice suggests that specific dosages of Wnt/beta-catenin signaling activity differentially affect tissue homeostasis and initiate tumorigenesis in an organ-specific fashion.