RESUMO
Somatic copy number gains are pervasive across cancer types, yet their roles in oncogenesis are insufficiently evaluated. This inadequacy is partly due to copy gains spanning large chromosomal regions, obscuring causal loci. Here, we employed organoid modeling to evaluate candidate oncogenic loci identified via integrative computational analysis of extreme copy gains overlapping with extreme expression dysregulation in The Cancer Genome Atlas. Subsets of "outlier" candidates were contextually screened as tissue-specific cDNA lentiviral libraries within cognate esophagus, oral cavity, colon, stomach, pancreas, and lung organoids bearing initial oncogenic mutations. Iterative analysis nominated the kinase DYRK2 at 12q15 as an amplified head and neck squamous carcinoma oncogene in p53-/- oral mucosal organoids. Similarly, FGF3, amplified at 11q13 in 41% of esophageal squamous carcinomas, promoted p53-/- esophageal organoid growth reversible by small molecule and soluble receptor antagonism of FGFRs. Our studies establish organoid-based contextual screening of candidate genomic drivers, enabling functional evaluation during early tumorigenesis.
Assuntos
Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Oncogenes , Transformação Celular Neoplásica/genética , Neoplasias/genética , Carcinogênese/genética , Amplificação de GenesRESUMO
The earliest events during human tumour initiation, although poorly characterized, may hold clues to malignancy detection and prevention1. Here we model occult preneoplasia by biallelic inactivation of TP53, a common early event in gastric cancer, in human gastric organoids. Causal relationships between this initiating genetic lesion and resulting phenotypes were established using experimental evolution in multiple clonally derived cultures over 2 years. TP53 loss elicited progressive aneuploidy, including copy number alterations and structural variants prevalent in gastric cancers, with evident preferred orders. Longitudinal single-cell sequencing of TP53-deficient gastric organoids similarly indicates progression towards malignant transcriptional programmes. Moreover, high-throughput lineage tracing with expressed cellular barcodes demonstrates reproducible dynamics whereby initially rare subclones with shared transcriptional programmes repeatedly attain clonal dominance. This powerful platform for experimental evolution exposes stringent selection, clonal interference and a marked degree of phenotypic convergence in premalignant epithelial organoids. These data imply predictability in the earliest stages of tumorigenesis and show evolutionary constraints and barriers to malignant transformation, with implications for earlier detection and interception of aggressive, genome-instable tumours.
Assuntos
Transformação Celular Neoplásica , Evolução Clonal , Lesões Pré-Cancerosas , Seleção Genética , Neoplasias Gástricas , Humanos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Evolução Clonal/genética , Instabilidade Genômica , Mutação , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologia , Organoides/metabolismo , Organoides/patologia , Aneuploidia , Variações do Número de Cópias de DNA , Análise de Célula Única , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Progressão da Doença , Linhagem da CélulaRESUMO
Homeless individuals are disproportionately likely to experience mental health conditions, and typically face many systemic barriers to access mental health services. This study sought to determine the mental health needs of homeless and recently housed individuals in Canada. A meta-ethnography was conducted to synthesise existing qualitative data and translate themes across a broader context. Thirty-five studies on the experiences of 1511 individuals with a history of homelessness were included. Themes were interpreted by comparing and contrasting findings across multiple contexts. Distinct, yet highly interrelated, unmet mental health needs were revealed through personal narratives of trauma, stigmatisation, victimisation, and a lack of basic necessities. Six themes that characterised this population's mental health needs were ontological security, autonomy, hope and purpose, empowerment, social connection and belonging, and access to services. This study revealed homeless individuals' unmet mental health needs to inform social and policy change and improve psychological well-being.
Assuntos
Pessoas Mal Alojadas , Saúde Mental , Humanos , Habitação , Canadá , Antropologia CulturalRESUMO
Mutations in ARID1A rank among the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated ARID1A knockout (KO) in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity, and mucinous differentiation. Genetic WNT/ß-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of ARID1A KO-mediated transformation. ARID1A mutation induced transcriptional regulatory modules characteristic of microsatellite instability and Epstein-Barr virus-associated subtype human gastric cancer, including FOXM1-associated mitotic genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of ARID1A KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with nonessential WNT-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells. SIGNIFICANCE: We establish the first human forward genetic modeling of a commonly mutated tumor suppressor gene, ARID1A. Our study integrates diverse modalities including CRISPR/Cas9 genome editing, organoid culture, systems biology, and small-molecule screening to derive novel insights into early transformation mechanisms of ARID1A-deficient gastric cancers.See related commentary by Zafra and Dow, p. 1327.This article is highlighted in the In This Issue feature, p. 1307.
Assuntos
Sistemas CRISPR-Cas , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/genética , Neoplasias Gástricas/genética , Fatores de Transcrição/genética , Humanos , Modelos Biológicos , MutaçãoRESUMO
The estrogen receptor (ER) mechanisms by which 17ß-estradiol influences depressive-like behaviour have primarily been investigated acutely and not within an animal model of depression. Therefore, the current study aimed to dissect the contribution of ERα and ERß to the effects of 17ß-estradiol under non-stress and chronic stress conditions. Ovariectomized (OVX) or sham-operated mice were treated chronically (47 days) with 17ß-estradiol (E2), the ERß agonist diarylpropionitrile (DPN), the ERα agonist propylpyrazole-triol (PPT), or vehicle. On day 15 of treatment, mice from each group were assigned to chronic unpredictable stress (CUS; 28 days) or non-CUS conditions. Mice were assessed for anxiety- and depressive-like behaviour and hypothalamic-pituitary-adrenal (HPA) axis function. Cytokine and chemokine levels, and postsynaptic density protein 95 were measured in the hippocampus and frontal cortex, and adult hippocampal neurogenesis was assessed. Overall, the effects of CUS were more robust that those of estrogenic treatments, as seen by increased immobility in the tail suspension test (TST), reduced PSD-95 expression, reduced neurogenesis in the ventral hippocampus, and HPA axis negative feedback dysregulation. However, we also observe CUS-dependent and -independent effects of ovarian status and estrogenic treatments. The effects of CUS on PSD-95 expression, the cytokine milieu, and in TST were largely driven by PPT and DPN, indicating that these treatments were not protective. Independent of CUS, estradiol increased neurogenesis in the dorsal hippocampus, blunted the corticosterone response to an acute stressor, and increased anxiety-like behaviour. These findings provide insights into the complexities of estrogen signaling in modulating depressive-like phenotypes under non-stress and chronic stress conditions.
Assuntos
Depressão/metabolismo , Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Estresse Psicológico/metabolismo , Animais , Doença Crônica , Corticosterona/metabolismo , Depressão/etiologia , Depressão/psicologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Reação de Congelamento Cataléptica/efeitos dos fármacos , Reação de Congelamento Cataléptica/fisiologia , Hipocampo/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nitrilas/farmacologia , Ovariectomia , Fenóis/farmacologia , Fenótipo , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/metabolismo , Propionatos/farmacologia , Pirazóis/farmacologia , Estresse Psicológico/complicações , Estresse Psicológico/patologia , Estresse Psicológico/psicologiaRESUMO
Human enteroids-epithelial spheroids derived from primary gastrointestinal tissue-are a promising model to study pathogen-epithelial interactions. However, accessing the apical enteroid surface is challenging because it is enclosed within the spheroid. We developed a technique to reverse enteroid polarity such that the apical surface everts to face the media. Apical-out enteroids maintain proper polarity and barrier function, differentiate into the major intestinal epithelial cell (IEC) types, and exhibit polarized absorption of nutrients. We used this model to study host-pathogen interactions and identified distinct polarity-specific patterns of infection by invasive enteropathogens. Salmonella enterica serovar Typhimurium targets IEC apical surfaces for invasion via cytoskeletal rearrangements, and Listeria monocytogenes, which binds to basolateral receptors, invade apical surfaces at sites of cell extrusion. Despite different modes of entry, both pathogens exit the epithelium within apically extruding enteroid cells. This model will enable further examination of IECs in health and disease.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Mucosa Intestinal/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Polaridade Celular , Células Epiteliais/metabolismo , Ácidos Graxos/metabolismo , Humanos , Listeria monocytogenes/fisiologia , Modelos Biológicos , Salmonella typhimurium/fisiologia , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/microbiologiaRESUMO
The intestinal epithelium is a rapidly renewing cellular compartment. This constant regeneration is a hallmark of intestinal homeostasis and requires a tightly regulated balance between intestinal stem cell (ISC) proliferation and differentiation. Since intestinal epithelial cells directly contact pathogenic environmental factors that continuously challenge their integrity, ISCs must also actively divide to facilitate regeneration and repair. Understanding niche adaptations that maintain ISC activity during homeostatic renewal and injury-induced intestinal regeneration is therefore a major and ongoing focus for stem cell biology. Here, we review recent concepts and propose an active interconversion of the ISC niche between homeostasis and injury-adaptive states that is superimposed upon an equally dynamic equilibrium between active and reserve ISC populations.
Assuntos
Homeostase , Mucosa Intestinal/metabolismo , Nicho de Células-Tronco , Animais , Diferenciação Celular , Proliferação de Células , Humanos , Mucosa Intestinal/patologiaRESUMO
RNA-binding proteins (RBPs) are expressed broadly during both development and malignant transformation, yet their mechanistic roles in epithelial homeostasis or as drivers of tumor initiation and progression are incompletely understood. Here we describe a novel interplay between RBPs LIN28B and IMP1 in intestinal epithelial cells. Ribosome profiling and RNA sequencing identified IMP1 as a principle node for gene expression regulation downstream from LIN28B In vitro and in vivo data demonstrate that epithelial IMP1 loss increases expression of WNT target genes and enhances LIN28B-mediated intestinal tumorigenesis, which was reversed when we overexpressed IMP1 independently in vivo. Furthermore, IMP1 loss in wild-type or LIN28B-overexpressing mice enhances the regenerative response to irradiation. Together, our data provide new evidence for the opposing effects of the LIN28B-IMP1 axis on post-transcriptional regulation of canonical WNT signaling, with implications in intestinal homeostasis, regeneration and tumorigenesis.
Assuntos
Carcinogênese , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regulon , Via de Sinalização Wnt , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Humanos , Mucosa Intestinal/fisiologia , Camundongos , Camundongos Transgênicos , Oncogenes , Biossíntese de Proteínas , Proteínas de Ligação a RNA/fisiologia , Regeneração , Células-Tronco/metabolismoRESUMO
Intestinal epithelial stem cells (IESCs) are critical to maintain intestinal epithelial function and homeostasis. We tested the hypothesis that aging promotes IESC dysfunction using old (18-22 months) and young (2-4 month) Sox9-EGFP IESC reporter mice. Different levels of Sox9-EGFP permit analyses of active IESC (Sox9-EGFPLow), activatable reserve IESC and enteroendocrine cells (Sox9-EGFPHigh), Sox9-EGFPSublow progenitors, and Sox9-EGFPNegative differentiated lineages. Crypt-villus morphology, cellular composition and apoptosis were measured by histology. IESC function was assessed by crypt culture, and proliferation by flow cytometry and histology. Main findings were confirmed in Lgr5-EGFP and Lgr5-LacZ mice. Aging-associated gene expression changes were analyzed by Fluidigm mRNA profiling. Crypts culture from old mice yielded fewer and less complex enteroids. Histology revealed increased villus height and Paneth cells per crypt in old mice. Old mice showed increased numbers and hyperproliferation of Sox9-EGFPLow IESC and Sox9-EGFPHigh cells. Cleaved caspase-3 staining demonstrated increased apoptotic cells in crypts and villi of old mice. Gene expression profiling revealed aging-associated changes in mRNAs associated with cell cycle, oxidative stress and apoptosis specifically in IESC. These findings provide new, direct evidence for aging associated IESC dysfunction, and define potential biomarkers and targets for translational studies to assess and maintain IESC function during aging.
Assuntos
Envelhecimento/patologia , Proliferação de Células , Células Epiteliais/patologia , Mucosa Intestinal/patologia , Jejuno/patologia , Células-Tronco/patologia , Fatores Etários , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Apoptose , Ciclo Celular , Linhagem da Célula , Enterócitos/metabolismo , Enterócitos/patologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Mucosa Intestinal/metabolismo , Jejuno/metabolismo , Óperon Lac , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estresse Oxidativo , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Fenótipo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Esferoides Celulares , Células-Tronco/metabolismo , Fatores de Tempo , Técnicas de Cultura de TecidosRESUMO
The canonical Wnt/ß-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling ß-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/ß-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.
Assuntos
Autorrenovação Celular , Intestinos/citologia , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Animais , Linhagem da Célula , Proliferação de Células , Feminino , Humanos , Ligantes , Masculino , Camundongos , Organoides/citologia , Organoides/crescimento & desenvolvimento , Análise de Célula Única , Nicho de Células-Tronco , Transcriptoma , Ubiquitina-Proteína Ligases/metabolismo , beta Catenina/metabolismoRESUMO
Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-ß-catenin signaling. Constitutive activation of Wnt-ß-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.
Assuntos
Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Glioblastoma/genética , Infarto da Artéria Cerebral Média/genética , Hemorragias Intracranianas/genética , Receptores Acoplados a Proteínas G/genética , Junções Íntimas/metabolismo , Animais , Barreira Hematoencefálica/ultraestrutura , Modelos Animais de Doenças , Células Endoteliais/ultraestrutura , Matriz Extracelular/metabolismo , Citometria de Fluxo , Imunofluorescência , Glioblastoma/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Hemorragias Intracranianas/metabolismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Microvasos , Pericitos/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real , Junções Íntimas/ultraestrutura , Via de Sinalização WntRESUMO
Gut microbiota play an important role in regulating the development of the host immune system, metabolic rate, and at times, disease pathogenesis. The factors and mechanisms that mediate interactions between microbiota and the intestinal epithelium are not fully understood. We provide novel evidence that microbiota may control intestinal epithelial stem cell (IESC) proliferation in part through microRNAs (miRNAs). We demonstrate that miRNA profiles differ dramatically across functionally distinct cell types of the mouse jejunal intestinal epithelium and that miRNAs respond to microbiota in a highly cell type-specific manner. Importantly, we also show that miRNAs in IESCs are more prominently regulated by microbiota compared with miRNAs in any other intestinal epithelial cell subtype. We identify miR-375 as one miRNA that is significantly suppressed by the presence of microbiota in IESCs. Using a novel method to knockdown gene and miRNA expression ex vivo enteroids, we demonstrate that we can knock down gene expression in Lgr5+ IESCs. Furthermore, when we knock down miR-375 in IESCs, we observe significantly increased proliferative capacity. Understanding the mechanisms by which microbiota regulate miRNA expression in IESCs and other intestinal epithelial cell subtypes will elucidate a critical molecular network that controls intestinal homeostasis and, given the heightened interest in miRNA-based therapies, may offer novel therapeutic strategies in the treatment of gastrointestinal diseases associated with altered IESC function.
Assuntos
Mucosa Intestinal/metabolismo , Células-Tronco/microbiologia , Transcriptoma , Animais , Feminino , Vida Livre de Germes , Proteínas de Fluorescência Verde/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Fatores de Transcrição SOX9/genética , Células-Tronco/citologiaRESUMO
Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the ApcMin mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling-induced proliferation, particularly during regeneration and adenoma formation.
Assuntos
Polipose Adenomatosa do Colo/metabolismo , Proliferação de Células , Intestino Delgado/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Experimentais/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Técnicas de Silenciamento de Genes , Intestino Delgado/patologia , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Receptores Acoplados a Proteínas G/genéticaRESUMO
Wnt signalling is involved in multiple aspects of embryonic development and adult tissue homeostasis, notably via controlling cellular proliferation and differentiation. Wnt signalling is subject to stringent positive and negative regulation to promote proper development and homeostasis yet avoid aberrant growth. Such multi-layer regulation includes post-translational modification and processing of Wnt proteins themselves, R-spondin (Rspo) amplification of Wnt signalling, diverse receptor families, and intracellular and extracellular antagonists and destruction and transcription complexes. In the gastrointestinal tract, Wnt signalling is crucial for development and renewal of the intestinal epithelium. Intestinal stem cells (ISCs) undergo symmetric division and neutral drift dynamics to renew the intestinal epithelium. Sources of Wnts and Wnt amplifers such as R-spondins are beginning to be elucidated as well as their functional contribution to intestinal homeostasis. In this review we focus on regulation of ISCs and intestinal homeostasis by the Wnt/Rspo pathway, the potential cellular sources of Wnt signalling regulators and highlight potential future areas of study.
Assuntos
Intestinos/citologia , Células-Tronco/fisiologia , Via de Sinalização Wnt , Animais , Intestinos/fisiologia , RegeneraçãoRESUMO
Proliferation and differentiation of intestinal epithelial cells (IECs) occur in part through precise regulation of key transcription factors, such as SOX9. MicroRNAs (miRNAs) have emerged as prominent fine-tuners of transcription factor expression and activity. We hypothesized that miRNAs, in part through the regulation of SOX9, may mediate IEC homeostasis. Bioinformatic analyses of the SOX9 3'-UTR revealed highly conserved target sites for nine different miRNAs. Of these, only the miR-30 family members were both robustly and variably expressed across functionally distinct cell types of the murine jejunal epithelium. Inhibition of miR-30 using complementary locked nucleic acids (LNA30bcd) in both human IECs and human colorectal adenocarcinoma-derived Caco-2 cells resulted in significant up-regulation of SOX9 mRNA but, interestingly, significant down-regulation of SOX9 protein. To gain mechanistic insight into this non-intuitive finding, we performed RNA sequencing on LNA30bcd-treated human IECs and found 2440 significantly increased genes and 2651 significantly decreased genes across three time points. The up-regulated genes are highly enriched for both predicted miR-30 targets, as well as genes in the ubiquitin-proteasome pathway. Chemical suppression of the proteasome rescued the effect of LNA30bcd on SOX9 protein levels, indicating that the regulation of SOX9 protein by miR-30 is largely indirect through the proteasome pathway. Inhibition of the miR-30 family led to significantly reduced IEC proliferation and a dramatic increase in markers of enterocyte differentiation. This in-depth analysis of a complex miRNA regulatory program in intestinal epithelial cell models provides novel evidence that the miR-30 family likely plays an important role in IEC homeostasis.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Enterócitos/metabolismo , Regulação da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Fatores de Transcrição SOX9/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células CACO-2 , Enterócitos/citologia , Humanos , Masculino , Camundongos , Camundongos Mutantes , MicroRNAs/genética , Fatores de Transcrição SOX9/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Current views suggest that apoptosis eliminates genetically damaged cells that may otherwise form tumors. Prior human studies link elevated insulin and reduced apoptosis to risk of colorectal adenomas. We hypothesized that hyperinsulinemia associated with obesity would lead to reduced colon epithelial cell (CEC) apoptosis after radiation and that this effect would be altered by deletion of the insulin-like growth factor (IGF) 1 receptor (IGF1R) or the insulin receptor (IR). Mice with villin-Cre-mediated IGF1R or IR deletion in CECs and floxed littermates were fed a high-fat diet to induce obesity and hyperinsulinemia or control low-fat chow. Mice were exposed to 5-Gy abdominal radiation to induce DNA damage and euthanized 4 h later for evaluation of apoptosis by localization of cleaved caspase-3. Obese mice exhibited decreased apoptosis of genetically damaged CECs. IGF1R deletion did not affect CEC apoptosis in lean or obese animals. In contrast, IR loss increased CEC apoptosis in both diet groups but did not prevent antiapoptotic effects of obesity. Levels of p53 protein were significantly reduced in CECs of obese mice with intact IR but increased in both lean and obese mice without IR. Levels of mRNAs encoding proapoptotic Perp and the cell cycle inhibitor Cdkn1b/p27 were reduced in CECs of obese mice and increased in lean mice lacking IR. Together, our studies provide novel evidence for antiapoptotic roles of obesity and IR, but not IGF1R, in colonic epithelium after DNA damage. However, neither IR nor IGF1R deletion prevented a reduction in radiation-induced CEC apoptosis during obesity and hyperinsulinemia.
Assuntos
Apoptose/efeitos da radiação , Colo/patologia , Mucosa Intestinal/metabolismo , Obesidade/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animais , Western Blotting , Caspase 3 , Colo/metabolismo , Imuno-Histoquímica , Masculino , Camundongos , Lesões Experimentais por Radiação , Reação em Cadeia da Polimerase em Tempo Real , Receptor IGF Tipo 1/genética , Receptor de Insulina/genéticaRESUMO
Insulin-like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used Sox9-enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9-EGFP(Low)) and reserve/facultative ISCs (Sox9-EGFP(High)) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high-dose radiation-induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini-pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9-EGFP(Low) ISCs and percentage of these cells in M-phase. IGF1 increased percentages of Sox9-EGFP(High) ISCs in S-phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9-EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9-EGFP(High) facultative ISCs but not Sox9-EGFP(Low) actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation.
Assuntos
Células-Tronco Adultas/classificação , Fator de Crescimento Insulin-Like I/fisiologia , Intestino Delgado/citologia , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/fisiologia , Animais , Ciclo Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/fisiologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/fisiologia , Camundongos , Camundongos Transgênicos , Células-Tronco Multipotentes/classificação , Células-Tronco Multipotentes/efeitos dos fármacos , Células-Tronco Multipotentes/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Lesões Experimentais por Radiação/tratamento farmacológico , Lesões Experimentais por Radiação/patologia , Lesões Experimentais por Radiação/fisiopatologia , Receptor IGF Tipo 1/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Regeneração/efeitos dos fármacos , Regeneração/fisiologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismoRESUMO
Myeloid translocation genes (MTGs) are transcriptional corepressors implicated in development, malignancy, differentiation, and stem cell function. While MTG16 loss renders mice sensitive to chemical colitis, the role of MTG16 in the small intestine is unknown. Histological examination revealed that Mtg16(-/-) mice have increased enterocyte proliferation and goblet cell deficiency. After exposure to radiation, Mtg16(-/-) mice exhibited increased crypt viability and decreased apoptosis compared with wild-type (WT) mice. Flow cytometric and immunofluorescence analysis of intestinal epithelial cells for phospho-histone H2A.X also indicated decreased DNA damage and apoptosis in Mtg16(-/-) intestines. To determine if Mtg16 deletion affected epithelial cells in a cell-autonomous fashion, intestinal crypts were isolated from Mtg16(-/-) mice. Mtg16(-/-) and WT intestinal crypts showed similar enterosphere forming efficiencies when cultured in the presence of EGF, Noggin, and R-spondin. However, when Mtg16(-/-) crypts were cultured in the presence of Wnt3a, they demonstrated higher enterosphere forming efficiencies and delayed progression to mature enteroids. Mtg16(-/-) intestinal crypts isolated from irradiated mice exhibited increased survival compared with WT intestinal crypts. Interestingly, Mtg16 expression was reduced in a stem cell-enriched population at the time of crypt regeneration. This is consistent with MTG16 negatively regulating regeneration in vivo. Taken together, our data demonstrate that MTG16 loss promotes radioresistance and impacts intestinal stem cell function, possibly due to shifting cellular response away from DNA damage-induced apoptosis and towards DNA repair after injury.
Assuntos
Proliferação de Células , Raios gama , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Proteínas Nucleares/metabolismo , Lesões Experimentais por Radiação/metabolismo , Regeneração , Fatores de Transcrição/metabolismo , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Dano ao DNA , Feminino , Regulação da Expressão Gênica , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Histonas/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fenótipo , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/patologia , Tolerância a Radiação , Regeneração/efeitos dos fármacos , Proteínas Repressoras , Transdução de Sinais , Células-Tronco/metabolismo , Células-Tronco/patologia , Técnicas de Cultura de Tecidos , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteína Wnt3A/farmacologiaRESUMO
The insulin receptor (IR) regulates nutrient uptake and utilization in multiple organs, but its role in the intestinal epithelium is not defined. This study developed a mouse model with villin-Cre (VC) recombinase-mediated intestinal epithelial cell (IEC)-specific IR deletion (VC-IR(Δ/Δ)) and littermate controls with floxed, but intact, IR (IR(fl/fl)) to define in vivo roles of IEC-IR in mice fed chow or high-fat diet (HFD). We hypothesized that loss of IEC-IR would alter intestinal growth, biomarkers of intestinal epithelial stem cells (IESC) or other lineages, body weight, adiposity, and glucose or lipid handling. In lean, chow-fed mice, IEC-IR deletion did not affect body or fat mass, plasma glucose, or IEC proliferation. In chow-fed VC-IR(Δ/Δ) mice, mRNA levels of the Paneth cell marker lysozyme (Lyz) were decreased, but markers of other differentiated lineages were unchanged. During HFD-induced obesity, IR(fl/fl) and VC-IR(Δ/Δ) mice exhibited similar increases in body and fat mass, plasma insulin, mRNAs encoding several lipid-handling proteins, a decrease in Paneth cell number, and impaired glucose tolerance. In IR(fl/fl) mice, HFD-induced obesity increased circulating cholesterol; numbers of chromogranin A (CHGA)-positive enteroendocrine cells (EEC); and mRNAs encoding Chga, glucose-dependent insulinotrophic peptide (Gip), glucagon (Gcg), Lyz, IESC biomarkers, and the enterocyte cholesterol transporter Scarb1. All these effects were attenuated or lost in VC-IR(Δ/Δ) mice. These results demonstrate that IEC-IR is not required for normal growth of the intestinal epithelium in lean adult mice. However, our findings provide novel evidence that, during HFD-induced obesity, IEC-IR contributes to increases in EEC, plasma cholesterol, and increased expression of Scarb1 or IESC-, EEC-, and Paneth cell-derived mRNAs.