RESUMEN
A highly recurrent somatic L265P mutation in the TIR domain of the signaling adapter MYD88 constitutively activates NF-κB. It occurs in nearly all human patients with Waldenström's macroglobulinemia (WM), a B cell malignancy caused by IgM-expressing cells. Here, we introduced an inducible leucine to proline point mutation into the mouse Myd88 locus, at the orthologous position L252P. When the mutation was introduced early during B cell development, B cells developed normally. However, IgM-expressing plasma cells accumulated with age in spleen and bone, leading to more than 20-fold elevated serum IgM titers. When introduced into germinal center B cells in the context of an immunization, the Myd88L252P mutation caused prolonged persistence of antigen-specific serum IgM and elevated numbers of antigen-specific IgM plasma cells. Myd88L252P-expressing B cells switched normally, but plasma cells expressing other immunoglobulin isotypes did not increase in numbers, implying that IgM expression may be required for the observed cellular expansion. In order to test whether the Myd88L252P mutation can cause clonal expansions, we introduced it into a small fraction of CD19-positive B cells. In this scenario, five out of five mice developed monoclonal IgM serum paraproteins accompanied by an expansion of clonally related plasma cells that expressed mostly hypermutated VDJ regions. Taken together, our data suggest that the Myd88L252P mutation is sufficient to promote aberrant survival and expansion of IgM-expressing plasma cells which in turn can cause IgM monoclonal gammopathy of undetermined significance (MGUS), the premalignant condition that precedes WM.
Asunto(s)
Linfocitos B/metabolismo , Marcación de Gen , Inmunoglobulina M/sangre , Gammopatía Monoclonal de Relevancia Indeterminada/genética , Factor 88 de Diferenciación Mieloide/genética , Células Plasmáticas/metabolismo , Mutación Puntual , Animales , Linfocitos B/inmunología , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Predisposición Genética a la Enfermedad , Inmunoglobulina M/inmunología , Activación de Linfocitos , Ratones Endogámicos C57BL , Gammopatía Monoclonal de Relevancia Indeterminada/sangre , Gammopatía Monoclonal de Relevancia Indeterminada/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Paraproteínas/metabolismo , Fenotipo , Células Plasmáticas/inmunologíaRESUMEN
Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis.
Asunto(s)
Transformación Celular Viral , Infecciones por Virus de Epstein-Barr/patología , Herpesvirus Humano 4/patogenicidad , Linfoma de Células B/patología , Células Plasmáticas/patología , Animales , Diferenciación Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Fibroblastos , Herpesvirus Humano 4/metabolismo , Humanos , Linfoma de Células B/virología , Ratones , Ratones Noqueados , Células Plasmáticas/virología , Cultivo Primario de Células , Transactivadores/genética , Transactivadores/metabolismo , Proteínas de la Matriz Viral/metabolismo , Proteínas Virales/metabolismoRESUMEN
BACKGROUND: The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes. RESULTS: We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9. CONCLUSIONS: Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.
Asunto(s)
Sistemas CRISPR-Cas/genética , Técnicas de Sustitución del Gen/métodos , ARN no Traducido/genética , Animales , Clonación Molecular , Embrión de Mamíferos , Ratones , Ratones Endogámicos C57BL , MicroinyeccionesRESUMEN
Colorectal cancer is one of the most common cancers worldwide and one of the leading causes of cancer-related death in the Western world. Tumor progression towards metastasis affects a large number of patients with colorectal cancer and seriously affects their clinical outcome. Therefore, considerable effort has been made towards the development of therapeutic strategies that can decrease or prevent colorectal cancer metastasis. Standard treatment of metastatic colorectal cancer with chemotherapy has been improved in the last 10 years by the addition of new targeted agents. The currently used antibodies bevacizumab, cetuximab and panitumumab target the VEGF and EGFR signaling pathways, which are crucial for tumor progression and metastasis. These antibodies have shown relevant efficacy in both first- and second-line treatment of metastatic colorectal cancer. Additionally, other signaling pathways, including the Wnt and HGF/Met pathways, have a well-established role in colorectal cancer progression and metastasis and constitute, therefore, promising targets for new therapeutic approaches. Several new drugs targeting these pathways, including different antibodies and small-molecule tyrosine kinase inhibitors, are currently being developed and tested in clinical trials. In this review, we summarize the new developments in this field, focusing on the inhibitors that show more promising results for use in colorectal cancer patients.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Terapia Molecular Dirigida , Animales , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Diseño de Fármacos , Humanos , Metástasis de la Neoplasia/prevención & control , Transducción de Señal/efectos de los fármacosRESUMEN
During the initiation stage of eukaryotic mRNA translation, the eIF4G (eukaryotic initiation factor 4G) proteins act as an aggregation point for recruiting the small ribosomal subunit to an mRNA. We previously used RNAi (RNA interference) to reduce expression of endogenous eIF4GI proteins, resulting in reduced protein synthesis rates and alterations in the morphology of cells. Expression of EIF4G1 cDNAs, encoding different isoforms (f-a) which arise through selection of alternative initiation codons, rescued translation to different extents. Furthermore, overexpression of the eIF4GII paralogue in the eIF4GI-knockdown background was unable to restore translation to the same extent as eIF4GIf/e isoforms, suggesting that translation events governed by this protein are different. In the present study we show that multiple isoforms of eIF4GII exist in mammalian cells, arising from multiple promoters and alternative splicing events, and have identified a non-canonical CUG initiation codon which extends the eIF4GII N-terminus. We further show that the rescue of translation in eIF4GI/eIF4GII double-knockdown cells by our novel isoforms of eIF4GII is as robust as that observed with either eIF4GIf or eIF4GIe, and more than that observed with the original eIF4GII. As the novel eIF4GII sequence diverges from eIF4GI, these data suggest that the eIF4GII N-terminus plays an alternative role in initiation factor assembly.
Asunto(s)
Codón Iniciador/genética , Factor 4G Eucariótico de Iniciación/genética , Regiones Promotoras Genéticas/genética , Sitios de Empalme de ARN/genética , Secuencia de Aminoácidos , Línea Celular Tumoral , ADN Complementario/genética , Factor 4G Eucariótico de Iniciación/biosíntesis , Exones/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Interferencia de ARN , Estabilidad del ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The metastasis-inducing protein S100A4 was found to be a prognostic indicator for the development of metachronous metastases. S100A4 expression levels correlate with the formation of human colorectal cancer metastases and shorter patients' survival. Inhibition of S100A4 expression in patients might therefore result in decreased metastasis formation and prolonged survival. In the present study, we used shRNA expression plasmids to inhibit S100A4 expression in the colorectal cancer cell lines HCT116, SW620 and DLD-1. Cell lines with reduced S100A4 expression showed reduced cell migration and invasion in vitro. The knock-down of S100A4 expression also led to significantly diminished formation of liver metastases when intrasplenically transplanted in mice (P = 0.004). We then focused on the therapeutic potential of systemically applied shRNA expression plasmids acting on S100A4 via repeated hydrodynamics-based tail vein injection of plasmid DNA. Mice, intrasplenically transplanted with HCT116 cells and treated systemically with S100A4shRNA plasmids, showed a decrease of S100A4 and MMP9 expression levels, resulting in significantly reduced liver metastases (P = 0.005). In summary, we show for the first time the intratumoral knock down of S100A4 via systemic application of S100A4shRNA plasmid DNA, which restricts metastasis formation in a xenografted mouse model of colorectal cancer.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Metástasis de la Neoplasia , Interferencia de ARN , Proteínas S100/genética , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundario , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , ARN Interferente Pequeño , Proteína de Unión al Calcio S100A4 , Proteínas S100/metabolismo , Trasplante HeterólogoRESUMEN
We previously identified the gene metastasis-associated in colon cancer-1 (MACC1) and demonstrated its important role for metastasis prediction in colorectal cancer. MACC1 induces cell motility and proliferation in vitro as well as metastasis in several mouse models. Here we report non-invasive real time imaging of inhibition of colorectal tumor progression and metastasis in xenografted mice by MACC1 shRNA. First, we demonstrated reduction of tumors and liver metastases by endpoint imaging of mice transplanted with MACC1 endogenously high expressing colorectal cancer cells and treated with shRNAs acting on MACC1 or Met. Next, we generated a novel bicistronic IRES vector simultaneously expressing the reporter gene firefly luciferase and MACC1 to ensure a direct correlation of bioluminescence signal with MACC1 expression. We transfected MACC1 endogenously low expressing colorectal cancer cells with this luciferase-IRES-MACC1 construct, transplanted them intrasplenically, and monitored MACC1 induced tumor growth and metastasis by in vivo imaging over time. Transfection of an IRES construct harboring the firefly luciferase reporter gene together with MACC1 lacking the SH3-domain reduced tumor growth and metastasis. Finally, we counteracted the luciferase-IRES-MACC1 induced effects by shRNA targeting MACC1 and monitored reduced tumor growth and metastasis by in vivo imaging over weeks. In summary, the new bicistronic luciferase-IRES-MACC1 construct is suitable for in vivo imaging of tumor progression and metastasis, and moreover, for imaging of therapy response such as treatment with MACC1 shRNA. Thereby, we provide proof-of-concept for employment of this MACC1-based in vivo model for evaluating therapeutic intervention strategies aiming at inhibition of tumor growth and metastasis.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Genes Reporteros , Vectores Genéticos , Humanos , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , ARN Interferente Pequeño/metabolismo , TransactivadoresRESUMEN
The calcium-binding protein S100A4 is a central mediator of metastasis formation in colon cancer. S100A4 is a target gene of the Wnt/ß-catenin pathway, which is constitutively active in the majority of colon cancers. In this study a high-throughput screen was performed to identify small-molecule compounds targeting the S100A4-promoter activity. In this screen calcimycin was identified as a transcriptional inhibitor of S100A4. In colon cancer cells calcimycin treatment reduced S100A4 mRNA and protein expression in a dose- and time-dependent manner. S100A4-induced cellular processes associated with metastasis formation, such as cell migration and invasion, were inhibited by calcimycin in an S100A4-specific manner. Calcimycin reduced ß-catenin mRNA and protein levels despite the expression of Δ45-mutated ß-catenin. Consequently, calcimycin inhibited Wnt/ß-catenin pathway activity and the expression of prominent ß-catenin target genes such as S100A4, cyclin D1, c-myc, and dickkopf-1. Finally, calcimycin treatment of human colon cancer cells inhibited metastasis formation in xenografted immunodeficient mice. Our results demonstrate that targeting the expression of S100A4 with calcimycin provides a functional strategy to restrict cell motility in colon cancer cells. Therefore calcimycin may be useful for studying S100A4 biology, and these studies may serve as a lead for the development of treatments for colon cancer metastasis.
Asunto(s)
Antineoplásicos/farmacología , Calcimicina/farmacología , Movimiento Celular/efectos de los fármacos , Proteínas S100/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , Animales , Antineoplásicos/uso terapéutico , Calcimicina/uso terapéutico , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Expresión Génica/efectos de los fármacos , Genes Reporteros , Células HCT116 , Humanos , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Luciferasas/biosíntesis , Luciferasas/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Regiones Promotoras Genéticas , Proteína de Unión al Calcio S100A4 , Proteínas S100/antagonistas & inhibidores , Proteínas S100/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Metastasis formation in colon cancer severely reduces the survival rate in patients. S100A4, a calcium-binding protein, is implicated in promoting metastasis formation in colon cancer. METHODS: To identify a transcription inhibitor of S100A4, high-throughput screening of 1280 pharmacologically active compounds was performed using a human colon cancer cell line expressing a S100A4 promoter-driven luciferase (LUC) reporter gene construct (HCT116-S1004p-LUC). Niclosamide, an antihelminthic agent, was identified as a potential candidate. Colon cancer cell lines (HCT116, SW620, LS174T, SW480, and DLD-1) were treated with 1 µM niclosamide to analyze the effect on S100A4 mRNA and protein expression by quantitative reverse transcription-polymerase chain reaction and immunoblot assays, and effects on cell migration, invasion, proliferation, and colony formation were also assessed in vitro. The effect of niclosamide on liver metastasis was assessed in a xenograft mouse model of human colon cancer (n = 8 mice) by in vivo imaging. The long-term effect of niclosamide on metastasis formation after discontinued treatment was quantified by scoring, and overall survival (n = 12 mice) was analyzed by Kaplan-Meier method after discontinuation of treatment. All statistical tests were two-sided. RESULTS: Reduced S100A4 mRNA and protein expression, and inhibited cell migration, invasion, proliferation, and colony formation were observed in niclosamide-treated colon cancer cells in vitro. In vivo imaging of niclosamide-treated mice showed reduced liver metastasis compared with solvent-treated control mice (n = 4 mice per group). Compared with the control group, discontinuation of treatment for 26 days showed reduced liver metastasis formation in mice (n = 6 mice per group) (control vs discontinuous treatment, mean metastasis score = 100% vs 34.9%, mean difference = 65.1%; 95% confidence interval [CI] = 18.4% to 111.9%, P < .01) and increased overall survival (n = 6 mice per group; control vs discontinuous treatment, median survival = 24 vs 46.5 days, ratio = 0.52, 95% CI = 0.19 to 0.84, P = .001). CONCLUSION: Niclosamide inhibits S100A4-induced metastasis formation in a mouse model of colon cancer and has therapeutic potential.
Asunto(s)
Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias Hepáticas/prevención & control , Niclosamida/farmacología , Proteínas S100/metabolismo , Animales , Antihelmínticos/farmacología , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inmunoprecipitación de Cromatina , Neoplasias del Colon/patología , Progresión de la Enfermedad , Ensayo de Cambio de Movilidad Electroforética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Infusiones Parenterales , Estimación de Kaplan-Meier , Neoplasias Hepáticas/secundario , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Niclosamida/administración & dosificación , Niclosamida/química , Pronóstico , ARN Mensajero/metabolismo , ARN Neoplásico/metabolismo , Distribución Aleatoria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína de Unión al Calcio S100A4 , Proteínas S100/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Trasplante Heterólogo , Ensayo de Tumor de Célula Madre , Proteínas Wnt/efectos de los fármacos , Proteínas Wnt/metabolismo , beta Catenina/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
Colon cancer metastasis is often associated with activation of the Wnt/ß-catenin signaling pathway and high expression of the metastasis mediator S100A4. We previously demonstrated the transcriptional regulation of S100A4 by ß-catenin and the importance of the interconnection of these cellular programs for metastasis. Here we probe the hypothesis that the nonsteroidal anti-inflammatory drug sulindac sulfide can inhibit colon cancer metastasis by intervening in ß-catenin signaling and thereby interdicting S100A4. We treated colon cancer cell lines heterozygous for gain-of-function and wild-type ß-catenin with sulindac. We analyzed sulindac's effects on ß-catenin expression and subcellular localization, ß-catenin binding to the T-cell factor (TCF)/S100A4 promoter complex, S100A4 promoter activity, S100A4 expression, cell motility, and proliferation. Mice intrasplenically transplanted with S100A4-overexpressing colon cancer cells were treated with sulindac. Tumor growth and metastasis, and their ß-catenin and S100A4 expressions, were determined. We report the expression knockdown of ß-catenin by sulindac, leading to its reduced nuclear accumulation. The binding of ß-catenin to TCF was clearly lowered, resulting in reduced S100A4 promoter activity and expression. This correlated well with the inhibition of cell migration and invasion, which could be rescued by ectopic S100A4 expression. In mice, sulindac treatment resulted in reduced tumor growth in the spleen (P = .014) and decreased liver metastasis in a human colon cancer xenograft model (P = .025). Splenic tumors and liver metastases of sulindac-treated mice showed lowered ß-catenin and S100A4 levels. These results suggest that modulators of ß-catenin signaling such as sulindac offer potential as antimetastatic agents by interdicting S100A4 expression.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Proteínas S100/genética , Sulindac/uso terapéutico , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Hígado/patología , Ratones , Metástasis de la Neoplasia , Proteína de Unión al Calcio S100A4 , Proteínas S100/metabolismo , Transducción de Señal , Bazo/patología , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/metabolismo , Activación Transcripcional , Proteínas Wnt/genéticaRESUMEN
Histone deacetylases (HD) represent a novel target in cancer treatment, particularly for scattered small tumours such as the hepatocellular carcinoma (HCC). However, only few studies address the toxicological impact of HD Inhibitors (HDIs) on malignantly transformed cells versus primary hepatocytes. We examined whether and how different classes of HDIs sensitise the human HCC cell line HepG2, primary healthy murine and human liver cells towards the death receptor agonists TNFα and CD95L. Apicidin, M344 (N-hydroxy-7-(-4-dimethylaminobenzol)aminoheptanamide), CBHA (m-carboxycinnamic acid bis-hydroxamide) and VPA (valproic acid) sensitised liver cell cultures towards CD95-triggered apoptosis with the following potency: apicidin > M344 ≈ CBHA â« VPA. Apicidin sensitised towards CD95 also in the intact organ, i.e. in the isolated perfused mouse liver. No significant sensitisation towards TNFα was found in vitro. Western blot analysis showed that all HDIs studied downregulated the anti-apoptotic protein cFLIP, but only VPA additionally affected the expression level of XIAP. Furthermore, in models of the intrinsic apoptosis pathway, i.e. in HepG2 cells treated with Melphalan and in primary hepatocytes irradiated with UV light, only VPA exhibited significant sensitisation. These findings extend the biochemical, pharmacological and toxicological basis for HDI therapy and provide a caveat for clinical use in patients with an accompanying critical inflammatory state in which the CD95 system might be pre-activated.
Asunto(s)
Antineoplásicos/inmunología , Antineoplásicos/toxicidad , Hepatocitos/efectos de los fármacos , Inhibidores de Histona Desacetilasas/inmunología , Inhibidores de Histona Desacetilasas/toxicidad , Animales , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular/efectos de los fármacos , Proteína Ligando Fas/inmunología , Proteína Ligando Fas/metabolismo , Células Hep G2 , Hepatocitos/enzimología , Hepatocitos/inmunología , Histona Desacetilasas/metabolismo , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Perfusión , Transfección , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Colon cancer is still a burden mainly due to metastasis formation. The latter is often associated with a constitutive activation of the Wnt/beta-catenin signaling pathway and high expression of the metastasis-inducing gene S100A4. We previously identified S100A4 as a transcriptional target of beta-catenin. Intervention strategies targeting Wnt/beta-catenin signaling might therefore represent promising approaches to inhibit tumor growth and metastasis formation when induced by S100A4. Many inhibitors, various strategies, as well as different routes of application targeting key molecules of the Wnt signaling pathway have been reported within the last decade. Consequently, downregulation of beta-catenin target genes lead to altered tumorigenic and metastastic abilities of cancer cells. This review focuses on the potential of Wnt/beta-catenin signaling intervention to restrict colon cancer metastasis formation by interdicting S100A4 expression.