RESUMEN
Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf-/- mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.
Asunto(s)
Elementos Transponibles de ADN , Resistencia a Antineoplásicos/genética , Vectores Genéticos/genética , Mutagénesis Insercional , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/genética , Aloinjertos , Animales , Antineoplásicos/farmacología , Biomarcadores de Tumor , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Modelos Animales de Enfermedad , Flujo Genético , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Noqueados , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/mortalidad , Neoplasias/patología , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMEN
BACKGROUND & AIMS: High-throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinomas (HCCs). However, high intratumor and intertumor heterogeneity, combined with large numbers of passenger mutations, have made it difficult to identify driver mutations that contribute to the development of HCC. We combined transposon mutagenesis with a high-throughput screen of a small-hairpin RNA (shRNA) library to identify genes and pathways that contribute to HCC development. METHODS: Sleeping beauty transposons were mobilized in livers of transgenic mice predisposed to develop hepatocellular adenoma and HCC owing to expression of the hepatitis B virus surface antigen. This whole-genome mutagenesis technique was used to generate an unbiased catalogue of candidate cancer genes (CCGs). Pooled shRNA libraries targeting 250 selected CCGs then were introduced into immortalized mouse liver cells and the cells were monitored for their tumor-forming ability after injection into nude mice. RESULTS: Transposon-mediated mutagenesis identified 1917 high-confident CCGs and highlighted the importance of Ras signaling in the development of HCC. Subsequent pooled shRNA library screening of 250 selected CCGs validated 27 HCC tumor-suppressor genes. Individual shRNA knockdown of 4 of these genes (Acaa2, Hbs1l, Ralgapa2, and Ubr2) increased the proliferation of multiple human HCC cell lines in culture and accelerated the formation of xenograft tumors in nude mice. The ability of Ralgapa2 to promote HCC cell proliferation and tumor formation required its inhibition of Rala and Ralb. Dual inhibition of Ras signaling via Ral and Raf, using a combination of small-molecule inhibitor RBC8 and sorafenib, reduced the proliferation of HCC cells in culture and completely inhibited their growth as xenograft tumors in nude mice. CONCLUSIONS: In a 2-step forward genetic screen in mice, we identified members of the Ral guanosine triphosphatase-activating protein pathway and other proteins as suppressors of HCC cell proliferation and tumor growth. These proteins might serve as therapeutic targets for liver cancer.
Asunto(s)
Carcinoma Hepatocelular/genética , Proteínas Activadoras de GTPasa/fisiología , Genes Supresores de Tumor , Neoplasias Hepáticas Experimentales/genética , Proteínas de Unión al GTP ral/fisiología , Animales , Proliferación Celular/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Hígado/metabolismo , Ratones , Ratones Transgénicos , Transducción de Señal/genéticaRESUMEN
Ecotropic viral integration site-1 (EVI1) is an oncogenic zinc finger transcription factor whose expression is frequently up-regulated in myeloid leukemia and epithelial cancers. To better understand the mechanisms underlying EVI1-associated disease, we sought to define the EVI1 interactome in cancer cells. By using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics, we could confidently assign 78 proteins as EVI1-interacting partners for FLAG-tagged EVI1. Subsequently, we showed that 22 of 27 tested interacting proteins could coimmunoprecipitate with endogenous EVI1 protein, which represented an 81.5% validation rate. Additionally, by comparing the stable isotope labeling by amino acids in cell culture (SILAC) data with high-throughput yeast two hybrid results, we showed that five of these proteins interacted directly with EVI1. Functional classification of EVI1-interacting proteins revealed associations with cellular transcription machinery; modulators of transcription; components of WNT, TGF-ß, and RAS pathways; and proteins regulating DNA repair, recombination, and mitosis. We also identified EVI1 phosphorylation sites by MS analysis and showed that Ser538 and Ser858 can be phosphorylated and dephosphorylated by two EVI1 interactome proteins, casein kinase II and protein phosphatase-1α. Finally, mutations that impair EVI1 phosphorylation at these sites reduced EVI1 DNA binding through its C-terminal zinc finger domain and induced cancer cell proliferation. Collectively, these combinatorial proteomic approaches demonstrate that EVI1 interacts with large and complex networks of proteins, which integrate signals from various different signaling pathways important for oncogenesis. Comprehensive analysis of the EVI1 interactome has thus provided an important resource for dissecting the molecular mechanisms of EVI1-associated disease.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Mitosis , Neoplasias/metabolismo , Proteínas Oncogénicas/metabolismo , Reparación del ADN por Recombinación , Factores de Transcripción/metabolismo , Vía de Señalización Wnt , Proteínas de Unión al ADN/genética , Células HeLa , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Neoplasias/genética , Neoplasias/patología , Proteínas Oncogénicas/genética , Fosforilación/genética , Proto-Oncogenes/genética , Factores de Transcripción/genéticaRESUMEN
Invasiveness underlies cancer aggressiveness and is a hallmark of malignancy. Most malignant tumors have elevated levels of Tn, an O-GalNAc glycan. Mechanisms underlying Tn up-regulation and its effects remain unclear. Here we show that Golgi-to-endoplasmic reticulum relocation of polypeptide N-acetylgalactosamine-transferases (GalNAc-Ts) drives high Tn levels in cancer cell lines and in 70% of malignant breast tumors. This process stimulates cell adhesion to the extracellular matrix, as well as migration and invasiveness. The GalNAc-Ts lectin domain, mediating high-density glycosylation, is critical for these effects. Interfering with the lectin domain function inhibited carcinoma cell migration in vitro and metastatic potential in mice. We also show that stimulation of cell migration is dependent on Tn-bearing proteins present in lamellipodia of migrating cells. Our findings suggest that relocation of GalNAc-Ts to the endoplasmic reticulum frequently occurs upon cancerous transformation to enhance tumor cell migration and invasiveness through modification of cell surface proteins.
Asunto(s)
Acetilgalactosamina/metabolismo , Retículo Endoplásmico/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Glicosiltransferasas/metabolismo , Invasividad Neoplásica/fisiopatología , Neoplasias/fisiopatología , Animales , Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Western Blotting , Línea Celular , Movimiento Celular/fisiología , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Glicosilación , Aparato de Golgi/metabolismo , Humanos , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos BALB C , Neoplasias/metabolismoRESUMEN
Ecotropic viral integration site 1 (EVI1) is an oncogenic dual domain zinc finger transcription factor that plays an essential role in the regulation of hematopoietic stem cell renewal, and its overexpression in myeloid leukemia and epithelial cancers is associated with poor patient survival. Despite the discovery of EVI1 in 1988 and its emerging role as a dominant oncogene in various types of cancer, few EVI1 target genes are known. This lack of knowledge has precluded a clear understanding of exactly how EVI1 contributes to cancer. Using a combination of ChIP-Seq and microarray studies in human ovarian carcinoma cells, we show that the two zinc finger domains of EVI1 bind to DNA independently and regulate different sets of target genes. Strikingly, an enriched fraction of EVI1 target genes are cancer genes or genes associated with cancer. We also show that more than 25% of EVI1-occupied genes contain linked EVI1 and activator protein (AP)1 DNA binding sites, and this finding provides evidence for a synergistic cooperative interaction between EVI1 and the AP1 family member FOS in the regulation of cell adhesion, proliferation, and colony formation. An increased number of dual EVI1/AP1 target genes are also differentially regulated in late-stage ovarian carcinomas, further confirming the importance of the functional cooperation between EVI1 and FOS. Collectively, our data indicate that EVI1 is a multipurpose transcription factor that synergizes with FOS in invasive tumors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , Adhesión Celular , Inmunoprecipitación de Cromatina , ADN/genética , ADN/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica , Células HeLa , Humanos , Proteína del Locus del Complejo MDS1 y EV11 , Invasividad Neoplásica , Unión Proteica , Proto-OncogenesRESUMEN
The YAP-TEAD protein-protein interaction mediates YAP oncogenic functions downstream of the Hippo pathway. To date, available YAP-TEAD pharmacologic agents bind into the lipid pocket of TEAD, targeting the interaction indirectly via allosteric changes. However, the consequences of a direct pharmacological disruption of the interface between YAP and TEADs remain largely unexplored. Here, we present IAG933 and its analogs as potent first-in-class and selective disruptors of the YAP-TEAD protein-protein interaction with suitable properties to enter clinical trials. Pharmacologic abrogation of the interaction with all four TEAD paralogs resulted in YAP eviction from chromatin and reduced Hippo-mediated transcription and induction of cell death. In vivo, deep tumor regression was observed in Hippo-driven mesothelioma xenografts at tolerated doses in animal models as well as in Hippo-altered cancer models outside mesothelioma. Importantly this also extended to larger tumor indications, such as lung, pancreatic and colorectal cancer, in combination with RTK, KRAS-mutant selective and MAPK inhibitors, leading to more efficacious and durable responses. Clinical evaluation of IAG933 is underway.
Asunto(s)
Vía de Señalización Hippo , Proteínas Serina-Treonina Quinasas , Factores de Transcripción , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Factores de Transcripción/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ratones , Línea Celular Tumoral , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas de Unión al ADN/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Transcripción de Dominio TEA , Proteínas ras/metabolismo , Femenino , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
OBJECTIVE AND DESIGN: We investigated the role and regulation of zinc transporters in the activation of the inflammatory response in macrophages. Our exploratory computational study found that Zip14 (SLC39A14) was consistently up-regulated in activated macrophages; we therefore focused subsequently on that gene in the mechanistic study. MATERIAL: The expression and function of Zip14 was assessed in primary macrophages obtained by in-vitro differentiation of monocytes from human blood. METHODS: Primary macrophages were subjected to treatments with lipopolysaccharides, cytokines, chemicals, and pharmacological agents. SLC39A14 and inflammatory cytokine gene expressions were assessed by RT-qPCR. Zip14 siRNA knockdown was performed to explore the gene function. RESULTS: Lipopolysaccharide's inflammatory stimulus was a strong inducer of SLC39A14 mRNA expression in macrophages. This induction was dependent on calcium signaling, GC-rich DNA-binding, and NF-κB down-regulation. Impregnation of lipopolysaccharide-stimulated macrophages with the glucocorticoid dexamethasone further enhanced Zip14 expression while reducing interleukin-6 and tumor necrosis factor-α production. Zip14 knockdown in macrophages attenuated the expression and secretion of cytokines, indicating a buffering function for this zinc transporter. CONCLUSIONS: Collectively, our results identified the zinc transporter Zip14 as expressed downstream of lipopolysaccharide signals in macrophages. Zip14 induction had a regulatory function in cytokine production.
Asunto(s)
Proteínas de Transporte de Catión/inmunología , Macrófagos/inmunología , Animales , Calcio/metabolismo , Proteínas de Transporte de Catión/genética , Diferenciación Celular , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunosupresores/farmacología , Inflamación/inmunología , Interferón gamma/farmacología , Lipopolisacáridos , Pulmón/inmunología , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Monocitos/inmunología , ARN Mensajero/metabolismo , Bazo/inmunologíaRESUMEN
Insulin receptor substrate-1 (IRS-1) and IRS-2 are known to transduce and amplify signals emanating from the insulin receptor. Here we show that Grb2-associated binder 1 (Gab1), despite its structural similarity to IRS proteins, is a negative modulator of hepatic insulin action. Liver-specific Gab1 knockout (LGKO) mice showed enhanced hepatic insulin sensitivity with reduced glycemia and improved glucose tolerance. In LGKO liver, basal and insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2 was elevated, accompanied by enhanced Akt/PKB activation. Conversely, Erk activation by insulin was suppressed in LGKO liver, leading to defective IRS-1 Ser612 phosphorylation. Thus, Gab1 acts to attenuate, through promotion of the Erk pathway, insulin-elicited signals flowing through IRS and Akt proteins, which represents a novel balancing mechanism for control of insulin signal strength in the liver.
Asunto(s)
Insulina/metabolismo , Hígado/metabolismo , Fosfoproteínas/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Análisis Químico de la Sangre , Glucemia , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ingeniería Genética , Prueba de Tolerancia a la Glucosa , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Transgénicos , Fosfoproteínas/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Tirosina/metabolismoRESUMEN
Liver regeneration is a rapid and concerted response to injury, in which growth factor-generated intracellular signals result in activation of transcription factors, DNA synthesis, and hepatocyte proliferation. However, the link between cytoplasmic signals resulting in proliferative response to liver injury remains to be elucidated. We show here that association of Gab1 adaptor protein and Shp2 tyrosine phosphatase is a critical event at the early phase of liver regeneration. Partial hepatectomy (PH) rapidly and transiently induced assembly of a complex comprising Shp2 and tyrosine-phosphorylated Gab1 in wild-type hepatocytes. Consistently, liver-specific Shp2 knockout (LSKO) and liver-specific Gab1 knockout (LGKO) mice displayed very similar phenotypes of defective liver regeneration triggered by PH, including blunted extracellular signal-regulated kinase 1/2 (Erk1/2) activation, decreased expression of immediate-early genes, and reduced levels of cyclins A, E, and B1, as well as suppression of hepatocyte proliferation. In contrast, the Akt and interleukin-6/Stat3 pathways were up-regulated posthepatectomy in LSKO and LGKO mice, accompanied by improved hepatoprotection. Collectively, this study establishes the physiological significance of the Gab1/Shp2 link in promoting mitogenic signaling through the Erk pathway in mammalian liver regeneration.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/fisiología , Regeneración Hepática/fisiología , Fosfoproteínas/fisiología , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Secuencia de Bases , Proliferación Celular , Citocinas/genética , ADN/genética , Regulación hacia Abajo , Genes Inmediatos-Precoces , Sustancias de Crecimiento/genética , Hepatectomía , Hepatocitos/citología , Hepatocitos/metabolismo , Interleucina-6/metabolismo , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Hígado/lesiones , Hígado/metabolismo , Regeneración Hepática/genética , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/deficiencia , Proteínas Tirosina Fosfatasas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Aberrant activation of the JAK/STAT pathway is thought to be the critical event in the pathogenesis of the chronic myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia and primary myelofibrosis. The most frequent genetic alteration in these pathologies is the activating JAK2V617F mutation, and expression of the mutant gene in mouse models was shown to cause a phenotype resembling the human diseases. Given the body of genetic evidence, it has come as a sobering finding that JAK inhibitor therapy only modestly suppresses the JAK2V617F allele burden, despite showing clear benefits in terms of reducing splenomegaly and constitutional symptoms in patients. To gain a better understanding if JAK2V617F is required for maintenance of myeloproliferative disease once it has evolved, we generated a conditional inducible transgenic JAK2V617F mouse model using the SCL-tTA-2S tet-off system. Our model corroborates that expression of JAK2V617F in hematopoietic stem and progenitor cells recapitulates key hallmarks of human myeloproliferative neoplasms, and exhibits gender differences in disease manifestation. The disease was found to be transplantable, and importantly, reversible when transgenic JAK2V617F expression was switched off. Our results indicate that mutant JAK2V617F-specific inhibitors should result in profound disease modification by disabling the myeloproliferative clone bearing mutant JAK2.
Asunto(s)
Regulación de la Expresión Génica , Células Madre Hematopoyéticas , Janus Quinasa 2 , Trastornos Mieloproliferativos , Transgenes , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Humanos , Janus Quinasa 2/biosíntesis , Janus Quinasa 2/genética , Ratones , Ratones Transgénicos , Mutación Missense , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Trastornos Mieloproliferativos/patologíaRESUMEN
Activation of p53 by inhibitors of the p53-MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53-MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis. shRNA screens identified PUMA as a mediator of the p53 response specifically in the pulsed regimen. Consistent with this, the single high-dose HDM201 regimen resulted in rapid and marked induction of PUMA expression and apoptosis together with downregulation of Bcl-xL in vivo Knockdown of Bcl-xL was identified as the top sensitizer to HDM201 in vitro, and Bcl-xL was enriched in relapsing tumors from mice treated with intermittent high doses of HDM201. These findings define a regimen-dependent mechanism by which disruption of MDM2-p53 elicits therapeutic efficacy when given with infrequent dosing. In an ongoing HDM201 trial, the observed exposure-response relationship indicates that the molecular mechanism elicited by pulse dosing is likely reproducible in patients. These data support the clinical comparison of daily and intermittent regimens of p53-MDM2 inhibitors.Significance: Pulsed high doses versus sustained low doses of the p53-MDM2 inhibitor HDM201 elicit a proapoptotic response from wild-type p53 cancer cells, offering guidance to current clinical trials with this and other drugs that exploit the activity of p53. Cancer Res; 78(21); 6257-67. ©2018 AACR.
Asunto(s)
Antineoplásicos/administración & dosificación , Imidazoles/administración & dosificación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Pirimidinas/administración & dosificación , Pirroles/administración & dosificación , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Apoptosis , Área Bajo la Curva , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Imidazoles/farmacología , Estimación de Kaplan-Meier , Dosis Máxima Tolerada , Ratones , Trasplante de Neoplasias , Pirimidinas/farmacología , Pirroles/farmacología , ARN Interferente Pequeño/metabolismo , Factores de Tiempo , Proteína bcl-X/metabolismoRESUMEN
The ecotropic viral integration site 1 (Evi1) oncogenic transcription factor is one of a number of alternative transcripts encoded by the Mds1 and Evi1 complex locus (Mecom). Overexpression of Evi1 has been observed in a number of myeloid disorders and is associated with poor patient survival. It is also amplified and/or overexpressed in many epithelial cancers including nasopharyngeal carcinoma, ovarian carcinoma, ependymomas, and lung and colorectal cancers. Two murine knockout models have also demonstrated Evi1's critical role in the maintenance of hematopoietic stem cell renewal with its absence resulting in the death of mutant embryos due to hematopoietic failure. Here we characterize a novel mouse model (designated Evi1(fl3)) in which Evi1 exon 3, which carries the ATG start, is flanked by loxP sites. Unexpectedly, we found that germline deletion of exon3 produces a hypomorphic allele due to the use of an alternative ATG start site located in exon 4, resulting in a minor Evi1 N-terminal truncation and a block in expression of the Mds1-Evi1 fusion transcript. Evi1(δex3/δex3) mutant embryos showed only a mild non-lethal hematopoietic phenotype and bone marrow failure was only observed in adult Vav-iCre/+, Evi1(fl3/fl3) mice in which exon 3 was specifically deleted in the hematopoietic system. Evi1(δex3/δex3) knockout pups are born in normal numbers but die during the perinatal period from congenital heart defects. Database searches identified 143 genes with similar mutant heart phenotypes as those observed in Evi1(δex3/δex3) mutant pups. Interestingly, 42 of these congenital heart defect genes contain known Evi1-binding sites, and expression of 18 of these genes are also effected by Evi1 siRNA knockdown. These results show a potential functional involvement of Evi1 target genes in heart development and indicate that Evi1 is part of a transcriptional program that regulates cardiac development in addition to the development of blood.
Asunto(s)
Alelos , Proteínas de Unión al ADN/genética , Estudios de Asociación Genética , Cardiopatías Congénitas/genética , Proto-Oncogenes/genética , Factores de Transcripción/genética , Animales , Animales Recién Nacidos , Secuencia de Bases , Médula Ósea/patología , Proteínas de Unión al ADN/química , Modelos Animales de Enfermedad , Exones , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Genes Letales , Cardiopatías Congénitas/mortalidad , Cardiopatías Congénitas/patología , Cardiopatías Congénitas/fisiopatología , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Inmunofenotipificación , Proteína del Locus del Complejo MDS1 y EV11 , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Fenotipo , Alineación de Secuencia , Índice de Severidad de la Enfermedad , Factores de Transcripción/químicaRESUMEN
The most common risk factor for developing hepatocellular carcinoma (HCC) is chronic infection with hepatitis B virus (HBV). To better understand the evolutionary forces driving HCC, we performed a near-saturating transposon mutagenesis screen in a mouse HBV model of HCC. This screen identified 21 candidate early stage drivers and a very large number (2,860) of candidate later stage drivers that were enriched for genes that are mutated, deregulated or functioning in signaling pathways important for human HCC, with a striking 1,199 genes being linked to cellular metabolic processes. Our study provides a comprehensive overview of the genetic landscape of HCC.
Asunto(s)
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , Hepatitis B Crónica/complicaciones , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virología , Mutagénesis , Animales , Carcinoma Hepatocelular/metabolismo , Elementos Transponibles de ADN , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/metabolismo , Redes y Vías Metabólicas/genética , Metabolómica/métodos , Ratones , Ratones Transgénicos , Mutagénesis Insercional , Ácido Pirúvico/metabolismoRESUMEN
The human gene Ptpn11, which encodes the tyrosine phosphatase Shp2, may act as a proto-oncogene because dominantly activating mutations have been detected in several types of leukemia. Herein we report a tumor-suppressor function of Shp2. Hepatocyte-specific deletion of Shp2 promotes inflammatory signaling through the Stat3 pathway and hepatic inflammation/necrosis, resulting in regenerative hyperplasia and development of tumors in aged mice. Furthermore, Shp2 ablation dramatically enhanced diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) development, which was abolished by concurrent deletion of Shp2 and Stat3 in hepatocytes. Decreased Shp2 expression was detected in a subfraction of human HCC specimens. Thus, in contrast to the leukemogenic effect of dominant-active mutants, Ptpn11/Shp2 has a tumor-suppressor function in liver.
Asunto(s)
Adenoma de Células Hepáticas/enzimología , Carcinoma Hepatocelular/enzimología , Neoplasias Hepáticas/enzimología , Hígado/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/análisis , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adenoma de Células Hepáticas/genética , Adenoma de Células Hepáticas/patología , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/prevención & control , Citocinas/sangre , Citocinas/genética , Dietilnitrosamina , Regulación de la Expresión Génica , Hepatitis/enzimología , Hepatitis/genética , Hepatitis/patología , Humanos , Hiperplasia , Mediadores de Inflamación/sangre , Interleucina-6/administración & dosificación , Lipopolisacáridos/administración & dosificación , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Regeneración Hepática , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Necrosis , Proteína Tirosina Fosfatasa no Receptora Tipo 11/deficiencia , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proto-Oncogenes Mas , Factor de Transcripción STAT3/deficiencia , Factor de Transcripción STAT3/genética , Transducción de Señal , Factores de Tiempo , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
Stat5 and Stat3, two members of the Stat (signal transducer and activator of transcription) family, are known to play critical roles in mammopoiesis/lactogenesis and involution, respectively, in the mammary gland. Phosphotyrosine phosphatase Shp2 has been shown to dephosphorylate and thus inactivate both Stat5 and Stat3 in vitro. Paradoxically, cell culture studies also suggest a positive role of Shp2 in promoting prolactin-stimulated Stat5 activation. We have shown here that selective deletion of Shp2 in mouse mammary glands suppresses Stat5 activity during pregnancy and lactation, resulting in significant impairment of lobulo-alveolar outgrowth and lactation. In contrast, Stat3 activity was slightly up-regulated shortly before/at involution, leading to normal epithelial cell apoptosis/involution in Shp2-deficient mammary gland. Thus, Shp2 acts to promote Stat5 activation by the JAK2.prolactin receptor complex, while negatively modulating Stat3 activity before the onset of involution. This is the first demonstration that Shp2 manipulates Stat5 and Stat3 activities reciprocally in mammary epithelial cells, providing novel insight into the complex mechanisms for regulation of various Stat family members by a cytoplasmic tyrosine phosphatase.