RESUMEN
Murine epidermal stem cells undergo alternate cycles of dormancy and activation, fuelling tissue renewal. However, only a subset of stem cells becomes active during each round of morphogenesis, indicating that stem cells coexist in heterogeneous responsive states. Using a circadian-clock reporter-mouse model, here we show that the dormant hair-follicle stem cell niche contains coexisting populations of cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. The core clock protein Bmal1 modulates the expression of stem cell regulatory genes in an oscillatory manner, to create populations that are either predisposed, or less prone, to activation. Disrupting this clock equilibrium, through deletion of Bmal1 (also known as Arntl) or Per1/2, resulted in a progressive accumulation or depletion of dormant stem cells, respectively. Stem cell arrhythmia also led to premature epidermal ageing, and a reduction in the development of squamous tumours. Our results indicate that the circadian clock fine-tunes the temporal behaviour of epidermal stem cells, and that its perturbation affects homeostasis and the predisposition to tumorigenesis.
Asunto(s)
Relojes Circadianos/fisiología , Ritmo Circadiano/fisiología , Folículo Piloso/citología , Células Madre/citología , Factores de Transcripción ARNTL/deficiencia , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Adhesión Celular/genética , Ciclo Celular/genética , Células Cultivadas , Senescencia Celular , Relojes Circadianos/genética , Ritmo Circadiano/genética , Señales (Psicología) , Femenino , Regulación de la Expresión Génica/genética , Homeostasis/genética , Homeostasis/fisiología , Masculino , Ratones , Ratones Noqueados , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Nicho de Células Madre , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/genética , Vía de Señalización Wnt/genéticaRESUMEN
Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal co-activation of these pathways in the adult Drosophila midgut induces "tumor-like" overgrowths. Here, we show that, in these clones and in CRC cell lines, Dpp/TGF-ß acts as a tumor suppressor. Moreover, we discover that the Iroquois/IRX-family-protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. We also show that this genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF-ß. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF-ß. In addition, Irx5 expression confers a growth advantage in the presence of TGF-ß, but is selected against in its absence. Together, our results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF-ß activity. We propose that during the characteristic adenoma-to-carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF-ß, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF-ß pathway components.
Asunto(s)
Adenocarcinoma/metabolismo , Carcinogénesis/metabolismo , Neoplasias Colorrectales/metabolismo , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Células Cultivadas , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/genética , Humanos , Mucosa Intestinal/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Regulación hacia ArribaRESUMEN
Eph receptor tyrosine kinases and their ligands, the ephrins, play key roles in the regulation of migration and cell adhesion during development, thereby influencing cell fate, morphogenesis and organogenesis. Recent findings suggest that Eph signalling also controls the architecture and physiology of different tissues in the adult body under normal and pathological conditions such as cancer. A prime example is the intestinal epithelium where EphB-ephrinB interactions regulate both cell positioning and tumor progression. Here, we will review recent advances on the role of Eph-ephrin signalling in the intestine and other organs.
Asunto(s)
Efrinas/metabolismo , Neoplasias/enzimología , Receptores de la Familia Eph/metabolismo , Transducción de Señal , Humanos , Morfogénesis , Neoplasias/metabolismo , Especificidad de ÓrganosRESUMEN
Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here, we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occurs in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA polymerase I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5+ and LGR5- tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins.
Asunto(s)
Neoplasias Colorrectales , Células Madre Neoplásicas , Línea Celular Tumoral , Neoplasias Colorrectales/genética , ADN Ribosómico , Humanos , Receptores Acoplados a Proteínas GRESUMEN
The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patient-derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage-tracing cassettes knocked in the LGR5 locus. Analysis of LGR5-EGFP+ cells isolated from organoid-derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage-tracing experiments showed that LGR5+ CRC cells self-renew and generate progeny over long time periods that undergo differentiation toward mucosecreting- and absorptive-like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Neoplasias Colorrectales/fisiopatología , Células Madre Neoplásicas/fisiología , Organoides , Animales , Diferenciación Celular , Proliferación Celular , Femenino , Edición Génica/métodos , Técnicas de Sustitución del Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Xenoinjertos , Humanos , Ratones SCID , Receptores Acoplados a Proteínas G/genética , Coloración y Etiquetado/métodosRESUMEN
Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart.
Asunto(s)
Diferenciación Celular , Transformación Celular Neoplásica/patología , Metilación de ADN , Progresión de la Enfermedad , Intestinos/patología , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Cromosomas de los Mamíferos/genética , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Sitios Genéticos , Genoma , Neoplasias Intestinales/patología , Ratones Endogámicos C57BL , Análisis de Secuencia de ADN , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
Whereas the series of genetic events leading to colorectal cancer (CRC) have been well established, the precise functions that these alterations play in tumor progression and how they disrupt intestinal homeostasis remain poorly characterized. Activation of the Wnt/Wg signaling pathway by a mutation in the gene APC is the most common trigger for CRC, inducing benign lesions that progress to carcinomas due to the accumulation of other genetic alterations. Among those, Ras mutations drive tumour progression in CRC, as well as in most epithelial cancers. As mammalian and Drosophila's intestines share many similarities, we decided to explore the alterations induced in the Drosophila midgut by the combined activation of the Wnt signaling pathway with gain of function of Ras signaling in the intestinal stem cells. Here we show that compound Apc-Ras clones, but not clones bearing the individual mutations, expand as aggressive intestinal tumor-like outgrowths. These lesions reproduce many of the human CRC hallmarks such as increased proliferation, blockade of cell differentiation and cell polarity and disrupted organ architecture. This process is followed by expression of tumoral markers present in human lesions. Finally, a metabolic behavioral assay shows that these flies suffer a progressive deterioration in intestinal homeostasis, providing a simple readout that could be used in screens for tumor modifiers or therapeutic compounds. Taken together, our results illustrate the conservation of the mechanisms of CRC tumorigenesis in Drosophila, providing an excellent model system to unravel the events that, upon mutation in Apc and Ras, lead to CRC initiation and progression.
Asunto(s)
Carcinogénesis/patología , Drosophila melanogaster/fisiología , Tracto Gastrointestinal/patología , Mutación/genética , Envejecimiento/patología , Animales , Apoptosis , Células Clonales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Tracto Gastrointestinal/fisiopatología , Oncogenes , Proteínas ras/metabolismoRESUMEN
DNA methylation and chromatin remodeling are frequently implicated in the silencing of genes involved in carcinogenesis. Long Range Epigenetic Silencing (LRES) is a mechanism of gene inactivation that affects multiple contiguous CpG islands and has been described in different human cancer types. However, it is unknown whether there is a coordinated regulation of the genes embedded in these regions in normal cells and in early stages of tumor progression. To better characterize the molecular events associated with the regulation and remodeling of these regions we analyzed two regions undergoing LRES in human colon cancer in the mouse model. We demonstrate that LRES also occurs in murine cancer in vivo and mimics the molecular features of the human phenomenon, namely, downregulation of gene expression, acquisition of inactive histone marks, and DNA hypermethylation of specific CpG islands. The genes embedded in these regions showed a dynamic and autonomous regulation during mouse intestinal cell differentiation, indicating that, in the framework considered here, the coordinated regulation in LRES is restricted to cancer. Unexpectedly, benign adenomas in Apc(Min/+) mice showed overexpression of most of the genes affected by LRES in cancer, which suggests that the repressive remodeling of the region is a late event. Chromatin immunoprecipitation analysis of the transcriptional insulator CTCF in mouse colon cancer cells revealed disrupted chromatin domain boundaries as compared with normal cells. Malignant regression of cancer cells by in vitro differentiation resulted in partial reversion of LRES and gain of CTCF binding. We conclude that genes in LRES regions are plastically regulated in cell differentiation and hyperproliferation, but are constrained to a coordinated repression by abolishing boundaries and the autonomous regulation of chromatin domains in cancer cells.
Asunto(s)
Cromatina/metabolismo , Neoplasias del Colon/metabolismo , Islas de CpG , Metilación de ADN , ADN de Neoplasias/metabolismo , Silenciador del Gen , Animales , Células CACO-2 , Cromatina/genética , Cromatina/patología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , ADN de Neoplasias/genética , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.
Asunto(s)
Colon/citología , Mucosa Intestinal/citología , Células Madre Multipotentes/fisiología , Receptor EphB2/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular , Citometría de Flujo , Humanos , Inmunohistoquímica , Microscopía Confocal , Células Madre Multipotentes/citología , Reacción en Cadena en Tiempo Real de la Polimerasa , Telómero/metabolismoRESUMEN
A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse.
Asunto(s)
Células Madre Adultas/metabolismo , Neoplasias del Colon/diagnóstico , Intestinos/patología , Células Madre Neoplásicas/metabolismo , Receptor EphB3/metabolismo , Células Madre Adultas/patología , Animales , Diferenciación Celular , Separación Celular , Extensiones de la Superficie Celular/patología , Células Cultivadas , Neoplasias del Colon/patología , Neoplasias del Colon/fisiopatología , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Recurrencia Local de Neoplasia , Células Madre Neoplásicas/patología , Pronóstico , Receptor EphB3/genética , Receptores Acoplados a Proteínas G/metabolismo , Nicho de Células MadreRESUMEN
Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-alpha-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and alpha1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in M1 and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER.