RESUMEN
Highly proliferative Lgr5+ stem cells maintain the intestinal epithelium and are thought to be largely homogeneous. Although quiescent intestinal stem cell (ISC) populations have been described, the identity and features of such a population remain controversial. Here we report unanticipated heterogeneity within the Lgr5+ ISC pool. We found that expression of the RNA-binding protein Mex3a labels a slowly cycling subpopulation of Lgr5+ ISCs that contribute to all intestinal lineages with distinct kinetics. Single-cell transcriptome profiling revealed that Lgr5+ cells adopt two discrete states, one of which is defined by a Mex3a expression program and relatively low levels of proliferation genes. During homeostasis, Mex3a+ cells continually shift into the rapidly dividing, self-renewing ISC pool. Chemotherapy and radiation preferentially target rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, helping to promote regeneration of the intestinal epithelium following toxic insults. Thus, Mex3a defines a reserve-like ISC population within the Lgr5+ compartment.
Asunto(s)
Proliferación Celular/fisiología , Mucosa Intestinal/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/metabolismo , Animales , Mucosa Intestinal/citología , Ratones , Ratones Transgénicos , Proteínas de Unión al ARN/genética , Receptores Acoplados a Proteínas G/genética , Células Madre/citologíaRESUMEN
Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with ß-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.
Asunto(s)
Adenoma , Receptores de Proteínas Morfogenéticas Óseas/genética , Neoplasias Colorrectales/fisiopatología , Factor de Transcripción GATA6/metabolismo , Regulación Neoplásica de la Expresión Génica , Células Madre/citología , Células Madre/metabolismo , Adenoma/patología , Animales , Antineoplásicos/farmacología , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Proliferación Celular , Femenino , Técnica del Anticuerpo Fluorescente , Factor de Transcripción GATA6/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Pirazoles/farmacología , Pirimidinas/farmacología , Células Madre/efectos de los fármacos , Proteínas Wnt/metabolismoRESUMEN
Stress-induced monoubiquitination of p53 is a crucial event for the nuclear-cytoplasm-mitochondria trafficking and transcription-independent pro-apoptotic functions of p53. Although an intact ubiquitination pathway and a functional nuclear export sequence are required for p53 nuclear export, the role of specific residues within this region in regulating both processes remains largely unknown. Here we characterize the mechanisms accounting for the nuclear accumulation of a new point mutation (Lys-351 to Asn) in the nuclear export sequence of p53 identified in a cisplatin-resistant ovarian carcinoma cell line (A2780 CIS). We found that K351N substitution abrogates the monoubiquitination of p53 induced by both Mdm2 and MSL2 E3-ligases. As a consequence, cells expressing p53 K351N mutant showed defects in cisplatin-induced translocation of p53 to mitochondria, Bax oligomerization, and mitochondrial membrane depolarization. These data identify K351N as a critical mutation of p53 that contributes to the development and maintenance of resistance to cisplatin.
Asunto(s)
Resistencia a Antineoplásicos , Mitocondrias/metabolismo , Mutación Missense , Neoplasias Ováricas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinación , Sustitución de Aminoácidos , Antineoplásicos/farmacología , Línea Celular Tumoral , Cisplatino/farmacología , Femenino , Humanos , Potencial de la Membrana Mitocondrial/genética , Mitocondrias/genética , Señales de Exportación Nuclear/genética , Neoplasias Ováricas/genética , Transporte de Proteínas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Inactivation of the tumor suppressor p53 is central to carcinogenesis and acquisition of resistance to drug-induced apoptosis. The majority of alterations are missense mutations and occur within the DNA-binding domain. However, little is known about the point mutations in the tetramerization domain (TD). Here we investigated the properties of a new p53 mutant (Lys 351 to Asn) in the TD identified in a cisplatin-resistant ovarian carcinoma cell line (A2780 CIS). We found that K351N substitution significantly reduces the thermodynamic stability of p53 tetramers without affecting the overall half-life of the protein. Moreover, p53 K351N has a reduced ability to bind DNA and to trans-activate its specific target gene promoters, such as bax. Data obtained from the analysis of p53 subcellular localization revealed that K351N mutation inhibits the nuclear export of p53 and accumulation in the cytoplasm induced by cisplatin treatment. These results identify p53 K351N as a new cancer associated mutant with reduced tumor suppressor activity and altered functions in response to apoptotic stimuli.
