RESUMEN
CIC (also known as Capicua) is a transcriptional repressor negatively regulated by RAS/MAPK signaling. Whereas the functions of Cic have been well characterized in Drosophila, little is known about its role in mammals. CIC is inactivated in a variety of human tumors and has been implicated recently in the promotion of lung metastases. Here, we describe a mouse model in which we inactivated Cic by selectively disabling its DNA-binding activity, a mutation that causes derepression of its target genes. Germline Cic inactivation causes perinatal lethality due to lung differentiation defects. However, its systemic inactivation in adult mice induces T-cell acute lymphoblastic lymphoma (T-ALL), a tumor type known to carry CIC mutations, albeit with low incidence. Cic inactivation in mice induces T-ALL by a mechanism involving derepression of its well-known target, Etv4 Importantly, human T-ALL also relies on ETV4 expression for maintaining its oncogenic phenotype. Moreover, Cic inactivation renders T-ALL insensitive to MEK inhibitors in both mouse and human cell lines. Finally, we show that Ras-induced mouse T-ALL as well as human T-ALL carrying mutations in the RAS/MAPK pathway display a genetic signature indicative of Cic inactivation. These observations illustrate that CIC inactivation plays a key role in this human malignancy.
Asunto(s)
Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Represoras/genética , Proteínas E1A de Adenovirus/metabolismo , Alelos , Animales , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Desarrollo Embrionario/genética , Fibroblastos/metabolismo , Genes ras , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Mutación , Oligodendroglioma/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , Transcripción GenéticaRESUMEN
The epigenetic regulator TET2 is frequently mutated in hematological diseases. Mutations have been shown to arise in hematopoietic stem cells early in disease development and lead to altered DNA methylation landscapes and an increased risk of hematopoietic malignancy. Here, we show by genome-wide mapping of TET2 binding sites in different cell types that TET2 localizes to regions of open chromatin and cell-type-specific enhancers. We find that deletion of Tet2 in native hematopoiesis as well as fully transformed acute myeloid leukemia (AML) results in changes in transcription factor (TF) activity within these regions, and we provide evidence that loss of TET2 leads to attenuation of chromatin binding of members of the basic helix-loop-helix (bHLH) TF family. Together, these findings demonstrate that TET2 activity shapes the local chromatin environment at enhancers to facilitate TF binding and provides an example of how epigenetic dysregulation can affect gene expression patterns and drive disease development.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/genética , Proteínas Proto-Oncogénicas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Células Cultivadas , Dioxigenasas , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Ratones , Unión ProteicaRESUMEN
HMG-box proteins, including Sox/SRY (Sox) and TCF/LEF1 (TCF) family members, bind DNA via their HMG-box. This binding, however, is relatively weak and both Sox and TCF factors employ distinct mechanisms for enhancing their affinity and specificity for DNA. Here we report that Capicua (CIC), an HMG-box transcriptional repressor involved in Ras/MAPK signaling and cancer progression, employs an additional distinct mode of DNA binding that enables selective recognition of its targets. We find that, contrary to previous assumptions, the HMG-box of CIC does not bind DNA alone but instead requires a distant motif (referred to as C1) present at the C-terminus of all CIC proteins. The HMG-box and C1 domains are both necessary for binding specific TGAATGAA-like sites, do not function via dimerization, and are active in the absence of cofactors, suggesting that they form a bipartite structure for sequence-specific binding to DNA. We demonstrate that this binding mechanism operates throughout Drosophila development and in human cells, ensuring specific regulation of multiple CIC targets. It thus appears that HMG-box proteins generally depend on auxiliary DNA binding mechanisms for regulating their appropriate genomic targets, but that each sub-family has evolved unique strategies for this purpose. Finally, the key role of C1 in DNA binding also explains the fact that this domain is a hotspot for inactivating mutations in oligodendroglioma and other tumors, while being preserved in oncogenic CIC-DUX4 fusion chimeras associated to Ewing-like sarcomas.
Asunto(s)
ADN/genética , Proteínas de Drosophila/genética , Proteínas HMGB/genética , Proteínas del Grupo de Alta Movilidad/genética , Mutación , Neoplasias/genética , Proteínas Represoras/genética , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Sitios de Unión/genética , ADN/metabolismo , Drosophila/embriología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Células HEK293 , Dominios HMG-Box/genética , Proteínas HMGB/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunohistoquímica , Microscopía Confocal , Modelos Genéticos , Neoplasias/metabolismo , Unión Proteica , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The Ras family of small GTPases constitutes a central node in the transmission of mitogenic stimuli to the cell cycle machinery. The ultimate receptor of these mitogenic signals is the retinoblastoma (Rb) family of pocket proteins, whose inactivation is a required step to license cell proliferation. However, little is known regarding the molecular events that connect Ras signaling with the cell cycle. Here, we provide genetic evidence to illustrate that the p53/p21 Cdk-interacting protein 1 (Cip1)/Rb axis is an essential component of the Ras signaling pathway. Indeed, knockdown of p53, p21Cip1, or Rb restores proliferative properties in cells arrested by ablation of the three Ras loci, H-, N- and K-Ras. Ras signaling selectively inactivates p53-mediated induction of p21Cip1 expression by inhibiting acetylation of specific lysine residues in the p53 DNA binding domain. Proliferation of cells lacking both Ras proteins and p53 can be prevented by reexpression of the human p53 ortholog, provided that it retains an active DNA binding domain and an intact lysine residue at position 164. These results unveil a previously unidentified role for p53 in preventing cell proliferation under unfavorable mitogenic conditions. Moreover, we provide evidence that cells lacking Ras and p53 proteins owe their proliferative properties to the unexpected retroactivation of the Raf/Mek/Erk cascade by a Ras-independent mechanism.
Asunto(s)
Proliferación Celular , Sistema de Señalización de MAP Quinasas , Proteína p53 Supresora de Tumor/metabolismo , Proteínas ras/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Ciclo Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Genes ras , Humanos , Lisina/química , Ratones , Microscopía Fluorescente , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Neoplasias/metabolismo , Homología de Secuencia de Aminoácido , Quinasas raf/metabolismoRESUMEN
The Fragile Histidine Triad Diadenosine Triphosphatase (FHIT) gene is located in the Common Fragile Site FRA3B and encodes an enzyme that hydrolyzes the dinucleotide Ap3A. Although FHIT loss is one of the most frequent copy number alterations in cancer, its relevance for cancer initiation and progression remains unclear. FHIT is frequently lost in cancers from the digestive tract, which is compatible with being a cancer driver event in these tissues. However, FHIT loss could also be a passenger event due to the inherent fragility of the FRA3B locus. Moreover, the physiological relevance of FHIT enzymatic activity and the levels of Ap3A is largely unclear. We have conducted here a systematic pan-cancer analysis of FHIT status in connection with other mutations and phenotypic alterations, and we have critically discussed our findings in connection with the literature to provide an overall view of FHIT implications in cancer.
Asunto(s)
Ácido Anhídrido Hidrolasas , Proteínas de Neoplasias , Neoplasias , Humanos , Ácido Anhídrido Hidrolasas/genética , Sitios Frágiles del Cromosoma , Proteínas de Neoplasias/genética , Neoplasias/genéticaRESUMEN
Plk1-interacting checkpoint helicase (PICH) is a DNA translocase involved in resolving ultrafine anaphase DNA bridges and, therefore, is important to safeguard chromosome segregation and stability. PICH is overexpressed in various human cancers, particularly in lymphomas such as Burkitt lymphoma, which is caused by MYC translocations. To investigate the relevance of PICH in cancer development and progression, we have combined novel PICH-deficient mouse models with the Eµ-Myc transgenic mouse model, which recapitulates B-cell lymphoma development. We have observed that PICH deficiency delays the onset of MYC-induced lymphomas in Pich heterozygous females. Moreover, using a Pich conditional knockout mouse model, we have found that Pich deletion in adult mice improves the survival of Eµ-Myc transgenic mice. Notably, we show that Pich deletion in healthy adult mice is well tolerated, supporting PICH as a suitable target for anticancer therapies. Finally, we have corroborated these findings in two human Burkitt lymphoma cell lines and we have found that the death of cancer cells was accompanied by chromosomal instability. Based on these findings, we propose PICH as a potential therapeutic target for Burkitt lymphoma and for other cancers where PICH is overexpressed.
Asunto(s)
Linfoma de Burkitt , Adulto , Femenino , Animales , Humanos , Ratones , Linfoma de Burkitt/genética , Línea Celular , Inestabilidad Cromosómica , Modelos Animales de Enfermedad , Ratones Noqueados , Ratones Transgénicos , ADNRESUMEN
The transcriptional repressor Capicua (CIC) has emerged as an important rheostat of cell growth regulated by RAS/MAPK signaling. Cic was originally discovered in Drosophila, where it was shown to be inactivated by MAPK signaling downstream of the RTKs Torso and EGFR, which results in signal-dependent responses that are required for normal cell fate specification, proliferation and survival of developing and adult tissues. CIC is highly conserved in mammals, where it is also negatively regulated by MAPK signaling. Here, we review the roles of CIC during mammalian development, tissue homeostasis, tumor formation and therapy resistance. Available data indicate that CIC is involved in multiple biological processes, including lung development, liver homeostasis, autoimmunity and neurobehavioral processes. Moreover, CIC has been shown to be involved in tumor development as a tumor suppressor, both in human as well as in mouse models. Finally, several lines of evidence implicate CIC as a determinant of sensitivity to EGFR and MAPK pathway inhibitors, suggesting that CIC may play a broader role in human cancer than originally anticipated.
Asunto(s)
Proteínas Represoras/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Animales , Drosophila/crecimiento & desarrollo , Drosophila/metabolismo , Resistencia a Antineoplásicos/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Represoras/genéticaRESUMEN
Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Raslox (H-Ras -/-, N-Ras -/-, K-Ras lox/lox, RERTert/ert) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Estudios de Asociación Genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Transducción de Señal , Proteínas ras/metabolismo , Animales , Línea Celular , Proliferación Celular , Quinasas MAP Reguladas por Señal Extracelular/genética , Expresión Génica , Técnicas de Inactivación de Genes , Vectores Genéticos/genética , Humanos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Proteínas Proto-Oncogénicas c-raf/genética , Reproducibilidad de los Resultados , Retroviridae/genética , Transducción Genética , Proteínas ras/genéticaRESUMEN
Genetic studies in mice have provided evidence that H-Ras and K-Ras proteins are bioequivalent. However, human tumors display marked differences in the association of RAS oncogenes with tumor type. Thus, to further assess the bioequivalence of oncogenic H-Ras and K-Ras, we replaced the coding region of the murine K-Ras locus with H-RasG12V oncogene sequences. Germline expression of H-RasG12V or K-RasG12V from the K-Ras locus resulted in embryonic lethality. However, expression of these genes in adult mice led to different tumor phenotypes. Whereas H-RasG12V elicited papillomas and hematopoietic tumors, K-RasG12V induced lung tumors and gastric lesions. Pulmonary expression of H-RasG12V created a senescence-like state caused by excessive MAPK signaling. Likewise, H-RasG12V but not K-RasG12V induced senescence in mouse embryonic fibroblasts. Label-free quantitative analysis revealed that minor differences in H-RasG12V expression levels led to drastically different biological outputs, suggesting that subtle differences in MAPK signaling confer nonequivalent functions that influence tumor spectra induced by RAS oncoproteins. Cancer Res; 77(3); 707-18. ©2016 AACR.