RESUMEN
Colorectal cancer (CRC) is among the most frequent forms of cancer, and new strategies for its prevention and therapy are urgently needed1. Here we identify a metabolite signalling pathway that provides actionable insights towards this goal. We perform a dietary screen in autochthonous animal models of CRC and find that ketogenic diets exhibit a strong tumour-inhibitory effect. These properties of ketogenic diets are recapitulated by the ketone body ß-hydroxybutyrate (BHB), which reduces the proliferation of colonic crypt cells and potently suppresses intestinal tumour growth. We find that BHB acts through the surface receptor Hcar2 and induces the transcriptional regulator Hopx, thereby altering gene expression and inhibiting cell proliferation. Cancer organoid assays and single-cell RNA sequencing of biopsies from patients with CRC provide evidence that elevated BHB levels and active HOPX are associated with reduced intestinal epithelial proliferation in humans. This study thus identifies a BHB-triggered pathway regulating intestinal tumorigenesis and indicates that oral or systemic interventions with a single metabolite may complement current prevention and treatment strategies for CRC.
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Neoplasias Colorrectales , Transducción de Señal , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacología , Animales , Proliferación Celular , Transformación Celular Neoplásica , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/prevención & control , HumanosRESUMEN
Phagocytosis is the necessary first step to sense foreign microbes or particles and enables activation of innate immune pathways such as inflammasomes. However, the molecular mechanisms underlying how phagosomes modulate inflammasome activity are not fully understood. We show that in murine dendritic cells (DCs), the lysosomal histidine/peptide solute carrier transporter SLC15A4, associated with human inflammatory disorders, is recruited to phagosomes and is required for optimal inflammasome activity after infectious or sterile stimuli. Dextran sodium sulfate-treated SLC15A4-deficient mice exhibit decreased colon inflammation, reduced IL-1ß production by intestinal DCs, and increased autophagy. Similarly, SLC15A4-deficient DCs infected with Salmonella typhimurium show reduced caspase-1 cleavage and IL-1ß production. This correlates with peripheral NLRC4 inflammasome assembly and increased autophagy. Overexpression of constitutively active mTORC1 rescues decreased IL-1ß levels and caspase1 cleavage, and restores perinuclear inflammasome positioning. Our findings support that SLC15A4 couples phagocytosis with inflammasome perinuclear assembly and inhibition of autophagy through phagosomal content sensing. Our data also reveal the previously unappreciated importance of mTORC1 signaling pathways to promote and sustain inflammasome activity.
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Células Dendríticas , Inflamasomas , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de Transporte de Membrana , Animales , Autofagia , Caspasa 1/metabolismo , Células Dendríticas/metabolismo , Dextranos/metabolismo , Histidina , Humanos , Interleucina-1beta/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Fagosomas/metabolismoRESUMEN
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.
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Carcinogénesis , Regulación de la Expresión Génica , Mucosa Intestinal/metabolismo , Proteínas de Unión al ARN/metabolismo , Regulón , Vía de Señalización Wnt , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Humanos , Mucosa Intestinal/fisiología , Ratones , Ratones Transgénicos , Oncogenes , Biosíntesis de Proteínas , Proteínas de Unión al ARN/fisiología , Regeneración , Células Madre/metabolismoRESUMEN
Here, we introduce a facile, scalable engineering approach to enable long-term development and maturation of organoids. We have redesigned the configuration of conventional organoid culture to develop a platform that converts single injections of stem cell suspensions to radial arrays of organoids that can be maintained for extended periods without the need for passaging. Using this system, we demonstrate accelerated production of intestinal organoids with significantly enhanced structural and functional maturity, and their continuous development for over 4 weeks. Furthermore, we present a patient-derived organoid model of inflammatory bowel disease (IBD) and its interrogation using single-cell RNA sequencing to demonstrate its ability to reproduce key pathological features of IBD. Finally, we describe the extension of our approach to engineer vascularized, perfusable human enteroids, which can be used to model innate immune responses in IBD. This work provides an immediately deployable platform technology toward engineering more realistic organ-like structures in a dish.
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Enfermedades Inflamatorias del Intestino , Organoides , Humanos , Organogénesis , Células Madre , Intestinos , Enfermedades Inflamatorias del Intestino/genéticaRESUMEN
The intestinal epithelium exhibits a rapid and efficient regenerative response to injury. Emerging evidence supports a model where plasticity of differentiated cells, particularly those in the secretory lineages, contributes to epithelial regeneration upon ablation of injury-sensitive stem cells. However, such facultative stem cell activity is rare within secretory populations. Here, we ask whether specific functional properties predict facultative stem cell activity. We utilize in vivo labeling combined with ex vivo organoid formation assays to evaluate how cell age and autophagic state contribute to facultative stem cell activity within secretory lineages. Strikingly, we find that cell age (time elapsed since cell cycle exit) does not correlate with secretory cell plasticity. Instead, high autophagic vesicle content predicts plasticity and resistance to DNA damaging injury independently of cell lineage. Our findings indicate that autophagic status prior to injury serves as a lineage-agnostic marker for the prospective identification of facultative stem cells.
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Mucosa Intestinal , Células Madre , Estudios Prospectivos , Células Madre/metabolismo , Linaje de la Célula , Diferenciación Celular/genéticaRESUMEN
The profound complexity of the intestinal mucosa demands a spatial approach to the study of gut transcriptomics. Although single-cell RNA sequencing has revolutionized our ability to survey the diverse cell types of the intestine, knowledge of cell type alone cannot fully describe the cells that make up the intestinal mucosa. During homeostasis and disease, dramatic gradients of oxygen, nutrients, extracellular matrix proteins, morphogens, and microbiota collectively dictate intestinal cell state, and only spatial techniques can articulate differences in cellular transcriptomes at this level. Spatial transcriptomic techniques assign transcriptomic data to precise regions in a tissue of interest. In recent years, these protocols have become increasingly accessible, and their application in the intestinal mucosa has exploded in popularity. In the gut, spatial transcriptomics typically involve the application of tissue sections to spatially barcoded RNA sequencing or laser capture microdissection followed by RNA sequencing. In combination with single-cell RNA sequencing, these spatial sequencing approaches allow for the construction of spatial transcriptional maps at pseudosingle-cell resolution. In this review, we describe the spatial transcriptomic technologies recently applied in the gut and the previously unattainable discoveries that they have produced.
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Microbiota , Transcriptoma , Perfilación de la Expresión Génica , Intestinos , Mucosa IntestinalRESUMEN
Calorie restriction can enhance the regenerative capacity of the injured intestinal epithelium. Among other metabolic changes, calorie restriction can activate the autophagy pathway. Although independent studies have attributed the regenerative benefit of calorie restriction to downregulation of mTORC1, it is not known whether autophagy itself is required for the regenerative benefit of calorie restriction. We used mouse and organoid models with autophagy gene deletion to evaluate the contribution of autophagy to intestinal epithelial regeneration following calorie restriction. In the absence of injury, mice with intestinal epithelial-specific deletion of autophagy gene Atg7 (Atg7ΔIEC) exhibit weight loss and histological changes similar to wild-type mice following calorie restriction. Conversely, calorie-restricted Atg7ΔIEC mice displayed a significant reduction in regenerative crypt foci after irradiation compared with calorie-restricted wild-type mice. Targeted analyses of tissue metabolites in calorie-restricted mice revealed an association between calorie restriction and reduced glycocholic acid (GCA) in wild-type mice but not in Atg7ΔIEC mice. To evaluate whether GCA can directly modulate epithelial stem cell self-renewal, we performed enteroid formation assays with or without GCA. Wild-type enteroids exhibited reduced enteroid formation efficiency in response to GCA treatment, suggesting that reduced availability of GCA during calorie restriction may be one mechanism by which calorie restriction favors epithelial regeneration in a manner dependent upon epithelial autophagy. Taken together, our data support the premise that intestinal epithelial Atg7 is required for the regenerative benefit of calorie restriction, due in part to its role in modulating luminal GCA with direct effects on epithelial stem cell self-renewal.NEW & NOTEWORTHY Calorie restriction is associated with enhanced intestinal regeneration after irradiation, but the requirement of autophagy for this process is not known. Our data support the premise that intestinal epithelial autophagy is required for the regenerative benefit of calorie restriction. We also report that luminal levels of primary bile acid glycocholic acid are modulated by epithelial cell autophagy during calorie restriction with direct effects on epithelial stem cell function.
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Restricción Calórica , Intestinos , Ratones , Animales , Intestinos/fisiología , Mucosa Intestinal/metabolismo , Células Epiteliales , Autofagia/genéticaRESUMEN
Colorectal cancer (CRC) is a leading cause of cancer-related death. There is an urgent need for new methods of early CRC detection and monitoring to improve patient outcomes. Extracellular vesicles (EVs) are secreted, lipid-bilayer bound, nanoparticles that carry biological cargo throughout the body and in turn exhibit cancer-related biomarker potential. RNA binding proteins (RBPs) are posttranscriptional regulators of gene expression that may provide a link between host cell gene expression and EV phenotypes. Insulin-like growth factor 2 RNA binding protein 1 (IGF2BP1/IMP1) is an RBP that is highly expressed in CRC with higher levels of expression correlating with poor prognosis. IMP1 binds and potently regulates tumor-associated transcripts that may impact CRC EV phenotypes. Our objective was to test whether IMP1 expression levels impact EV secretion and/or cargo. We used RNA sequencing, in vitro CRC cell lines, ex vivo colonoid models, and xenograft mice to test the hypothesis that IMP1 influences EV secretion and/or cargo in human CRC. Our data demonstrate that IMP1 modulates the RNA expression of transcripts associated with extracellular vesicle pathway regulation, but it has no effect on EV secretion levels in vitro or in vivo. Rather, IMP1 appears to affect EV regulation by directly entering EVs in a transformation-dependent manner. These findings suggest that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.NEW & NOTEWORTHY This work demonstrates that the RNA binding protein IGF2BP1/IMP1 alters the transcript profile of colorectal cancer cell (CRC) mRNAs from extracellular vesicle (EV) pathways. IMP1 does not alter EV production or secretion in vitro or in vivo, but rather enters CRC cells where it may further impact EV cargo. Our work shows that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.
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Neoplasias Colorrectales , Vesículas Extracelulares , Humanos , Ratones , Animales , Vesículas Extracelulares/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Mensajero/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/patologíaRESUMEN
RNA binding proteins, including IMP1/IGF2BP1, are essential regulators of intestinal development and cancer. Imp1 hypomorphic mice exhibit gastrointestinal growth defects, yet the specific role for IMP1 in colon epithelial repair is unclear. Our prior work revealed that intestinal epithelial cell-specific Imp1 deletion (Imp1ΔIEC ) was associated with better regeneration in mice after irradiation. Here, we report increased IMP1 expression in patients with Crohn's disease and ulcerative colitis. We demonstrate that Imp1ΔIEC mice exhibit enhanced recovery following dextran sodium sulfate (DSS)-mediated colonic injury. Imp1ΔIEC mice exhibit Paneth cell granule changes, increased autophagy flux, and upregulation of Atg5. In silico and biochemical analyses revealed direct binding of IMP1 to MAP1LC3B, ATG3, and ATG5 transcripts. Genetic deletion of essential autophagy gene Atg7 in Imp1ΔIEC mice revealed increased sensitivity of double-mutant mice to colonic injury compared to control or Atg7 single mutant mice, suggesting a compensatory relationship between Imp1 and the autophagy pathway. The present study defines a novel interplay between IMP1 and autophagy, where IMP1 may be transiently induced during damage to modulate colonic epithelial cell responses to damage.
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Mucosa Intestinal/metabolismo , Proteínas de Unión al ARN/genética , Cicatrización de Heridas/genética , Adulto , Anciano , Animales , Autofagia/genética , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Biomarcadores , Estudios de Casos y Controles , Línea Celular , Colitis Ulcerosa/genética , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Colon , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Enfermedad de Crohn/patología , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Mucosa Intestinal/patología , Masculino , Ratones , Persona de Mediana Edad , Células de Paneth/metabolismo , Células de Paneth/patología , Unión Proteica , Biosíntesis de Proteínas , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Adulto JovenRESUMEN
The RNA-binding protein insulin-like growth factor 2 mRNA binding protein 1 (IMP1) is overexpressed in colorectal cancer (CRC); however, evidence for a direct role for IMP1 in CRC metastasis is lacking. IMP1 is regulated by let-7 microRNA, which binds in the 3' untranslated region (UTR) of the transcript. The availability of binding sites is in part controlled by alternative polyadenylation, which determines 3' UTR length. Expression of the short 3' UTR transcript (lacking all microRNA sites) results in higher protein levels and is correlated with increased proliferation. We used in vitro and in vivo model systems to test the hypothesis that the short 3' UTR isoform of IMP1 promotes CRC metastasis. Herein we demonstrate that 3' UTR shortening increases IMP1 protein expression and that this in turn enhances the metastatic burden to the liver, whereas expression of the long isoform (full length 3' UTR) does not. Increased tumor burden results from elevated tumor surface area driven by cell proliferation and cell survival mechanisms. These processes are independent of classical apoptosis pathways. Moreover, we demonstrate the shifts toward the short isoform are associated with metastasis in patient populations where IMP1-long expression predominates. Overall, our work demonstrates that different IMP1 expression levels result in different functional outcomes in CRC metastasis and that targeting IMP1 may reduce tumor progression in some patients.
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Regiones no Traducidas 3'/genética , Proliferación Celular , Neoplasias Colorrectales/patología , Neoplasias Hepáticas/secundario , Proteínas de Unión al ARN/genética , Animales , Apoptosis , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Desnudos , Proteínas de Unión al ARN/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Intestinal epithelial cells are among the most rapidly proliferating cell types in the human body. There are several different subtypes of epithelial cells, each with unique functional roles in responding to the ever-changing environment. The epithelium's ability for rapid and customized responses to environmental changes requires multitiered levels of gene regulation. An emerging paradigm in gastrointestinal epithelial cells is the regulation of functionally related mRNA families, or regulons, via RNA-binding proteins (RBPs). RBPs represent a rapid and efficient mechanism to regulate gene expression and cell function. In this review, we will provide an overview of intestinal epithelial RBPs and how they contribute specifically to intestinal epithelial stem cell dynamics. In addition, we will highlight key gaps in knowledge in the global understanding of RBPs in gastrointestinal physiology as an opportunity for future studies.
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Regulación de la Expresión Génica/genética , Homeostasis/genética , Proteínas de Unión al ARN/genética , ARN/metabolismo , Humanos , Procesamiento Postranscripcional del ARN/genética , Regulón/fisiologíaRESUMEN
OBJECTIVE: The influence of eosinophilic oesophagitis (EoE)-associated inflammation upon oesophageal epithelial biology remains poorly understood. We investigated the functional role of autophagy in oesophageal epithelial cells (keratinocytes) exposed to the inflammatory EoE milieu. DESIGN: Functional consequences of genetic or pharmacological autophagy inhibition were assessed in endoscopic oesophageal biopsies, human oesophageal keratinocytes, single cell-derived ex vivo murine oesophageal organoids as well as a murine model recapitulating EoE-like inflammation and basal cell hyperplasia. Gene expression, morphological and functional characterisation of autophagy and oxidative stress were performed by transmission electron microscopy, immunostaining, immunoblotting, live cell imaging and flow cytometry. RESULTS: EoE-relevant inflammatory conditions promoted autophagy and basal cell hyperplasia in three independent murine EoE models and oesophageal organoids. Inhibition of autophagic flux via chloroquine treatment augmented basal cell hyperplasia in these model systems. Oesophageal keratinocytes stimulated with EoE-relevant cytokines, including tumour necrosis factor-α and interleukin-13 exhibited activation of autophagic flux in a reactive oxygen species-dependent manner. Autophagy inhibition via chloroquine treatment or depletion of Beclin-1 or ATG-7, augmented oxidative stress induced by EoE-relevant stimuli in murine EoE, oesophageal organoids and human oesophageal keratinocytes. Oesophageal epithelia of paediatric EoE patients with active inflammation displayed increased autophagic vesicle content compared with normal and EoE remission subjects. Functional flow cytometric analysis revealed autophagic flux in human oesophageal biopsies. CONCLUSIONS: Our findings reveal for the first time that autophagy may function as a cytoprotective mechanism to maintain epithelial redox balance and homeostasis under EoE inflammation-associated stress, providing mechanistic insights into the role of autophagy in EoE pathogenesis.
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Autofagia/fisiología , Esofagitis Eosinofílica/metabolismo , Animales , Autofagia/efectos de los fármacos , Cloroquina/farmacología , Citocinas/farmacología , Esofagitis Eosinofílica/patología , Eosinófilos/metabolismo , Epitelio/metabolismo , Esofagoscopía , Esófago/patología , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Ratones , Modelos Animales , Estrés OxidativoRESUMEN
Esophageal cells overexpressing epidermal growth factor receptor (EGFR) and TP53 mutation can invade into the extracellular matrix when grown in 3D-organotypic cultures (OTC) and mimic early invasion in esophageal squamous cell carcinoma (ESCC). We have performed laser capture microdissection with RNA microarray analysis on the invasive and non-invasive tumor cells of p53(R175H)-overexpressing OTC samples to determine candidate genes facilitating tumor invasion. WNT10A was found to be >4-fold upregulated in the invasive front. Since WNT10A is also prominently upregulated during placode promotion in hair follicle development, a process that requires epithelial cells to thicken and elongate, in order to allow downward growth, we hypothesized that WNT10A may be important in mediating a similar mechanism of tumor cell invasion in ESCC. We have found that WNT10A expression is significantly upregulated in human ESCC, when compared with normal adjacent tissue. Furthermore, high WNT10A expression levels correlate with poor survival. Interestingly, we observe that WNT10A is expressed early in embryogenesis, but is reduced dramatically postnatally. We demonstrate that overexpression of WNT10a promotes migration and invasion, and proliferation of transformed esophageal cells. Lastly, we show that WNT10A overexpression induces a greater CD44(High)/CD24(Low) population, which are putative markers of cancer stem cells, and increases self-renewal capability. Taken together, we propose that WNT10A acts as an oncofetal factor that is highly expressed and may promote proper development of the esophagus. During tumorigenesis, it is aberrantly overexpressed in order to promote ESCC migration and invasion, and may be linked to self-renewal of a subset of ESCC cells.
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Carcinoma de Células Escamosas/patología , Diferenciación Celular , Transformación Celular Neoplásica/patología , Neoplasias Esofágicas/patología , Esófago/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Wnt/metabolismo , Animales , Apoptosis , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/mortalidad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos C57BL , Fenotipo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteínas Wnt/genéticaRESUMEN
BACKGROUND & AIMS: The intestinal epithelium interfaces with a diverse milieu of luminal contents while maintaining robust digestive and barrier functions. Facultative intestinal stem cells are cells that survive tissue injury and divide to re-establish the epithelium. Prior studies have shown autophagic state as functional marker of facultative intestinal stem cells, but regulatory mechanisms are not known. The current study evaluated a post-transcriptional regulation of autophagy as an important factor for facultative stem cell state and tissue regeneration. METHODS: We evaluated stem cell composition, autophagic vesicle content, organoid formation, and in vivo regeneration in mice with intestinal epithelial deletion of the RNA binding protein IGF2 messenger RNA binding protein 1 (IMP1). The contribution of autophagy to resulting in vitro and in vivo phenotypes was evaluated via genetic inactivation of Atg7. Molecular analyses of IMP1 modulation of autophagy at the protein and transcript localization levels were performed using IMP1 mutant studies and single-molecule fluorescent in situ hybridization. RESULTS: Epithelial Imp1 deletion reduced leucine rich repeat containing G protein coupled receptor 5 cell frequency but enhanced both organoid formation efficiency and in vivo regeneration after irradiation. We confirmed prior studies showing increased autophagy with IMP1 deletion. Deletion of Atg7 reversed the enhanced regeneration observed with Imp1 deletion. IMP1 deletion or mutation of IMP1 phosphorylation sites enhanced expression of essential autophagy protein microtubule-associated protein 1 light chain 3ß. Furthermore, immunofluorescence imaging coupled with single-molecule fluorescent in situ hybridization showed IMP1 colocalization with MAP1LC3B transcripts at homeostasis. Stress induction led to decreased colocalization. CONCLUSIONS: Depletion of IMP1 enhances autophagy, which promotes intestinal regeneration via expansion of facultative intestinal stem cells.
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Mucosa Intestinal , Intestinos , Animales , Ratones , Hibridación Fluorescente in Situ , Mucosa Intestinal/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Células Madre/metabolismoRESUMEN
BACKGROUND & AIMS: Epithelial disruption in eosinophilic esophagitis (EoE) encompasses both impaired differentiation and diminished barrier integrity. We have shown that lysyl oxidase (LOX), a collagen cross-linking enzyme, is up-regulated in the esophageal epithelium in EoE. However, the functional roles of LOX in the esophageal epithelium remains unknown. METHODS: We investigated roles for LOX in the human esophageal epithelium using 3-dimensional organoid and air-liquid interface cultures stimulated with interleukin (IL)13 to recapitulate the EoE inflammatory milieu, followed by single-cell RNA sequencing, quantitative reverse-transcription polymerase chain reaction, Western blot, histology, and functional analyses of barrier integrity. RESULTS: Single-cell RNA sequencing analysis on patient-derived organoids revealed that LOX was induced by IL13 in differentiated cells. LOX-overexpressing organoids showed suppressed basal and up-regulated differentiation markers. In addition, LOX overexpression enhanced junctional protein genes and transepithelial electrical resistance. LOX overexpression restored the impaired differentiation and barrier function, including in the setting of IL13 stimulation. Transcriptome analyses on LOX-overexpressing organoids identified an enriched bone morphogenetic protein (BMP) signaling pathway compared with wild-type organoids. In particular, LOX overexpression increased BMP2 and decreased the BMP antagonist follistatin. Finally, we found that BMP2 treatment restored the balance of basal and differentiated cells. CONCLUSIONS: Our data support a model whereby LOX exhibits noncanonical roles as a signaling molecule important for epithelial homeostasis in the setting of inflammation via activation of the BMP pathway in the esophagus. The LOX/BMP axis may be integral in esophageal epithelial differentiation and a promising target for future therapies.
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Diferenciación Celular , Esofagitis Eosinofílica , Organoides , Proteína-Lisina 6-Oxidasa , Humanos , Esofagitis Eosinofílica/patología , Esofagitis Eosinofílica/metabolismo , Proteína-Lisina 6-Oxidasa/metabolismo , Proteína-Lisina 6-Oxidasa/genética , Organoides/metabolismo , Organoides/patología , Interleucina-13/metabolismo , Interleucina-13/farmacología , Mucosa Esofágica/patología , Mucosa Esofágica/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Esófago/patología , Transducción de Señal , Análisis de la Célula Individual , Proteínas Morfogenéticas Óseas/metabolismoRESUMEN
BACKGROUND & AIMS: Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis. METHODS: We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining. RESULTS: Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer. CONCLUSIONS: Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.
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Autofagia , Proliferación Celular , Homeostasis , Ratones Noqueados , Organoides , Animales , Ratones , Organoides/metabolismo , Esófago/patología , Esófago/citología , Esófago/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Proteína 7 Relacionada con la Autofagia/metabolismo , Proteína 7 Relacionada con la Autofagia/genética , 4-Nitroquinolina-1-Óxido , Autorrenovación de las Células , Mucosa Esofágica/patología , Mucosa Esofágica/metabolismo , Mucosa Esofágica/citología , Análisis de la Célula IndividualRESUMEN
Igf2 mRNA binding protein 1 (IMP1, CRD-BP, ZBP-1) is a messenger RNA binding protein that we have shown previously to regulate colorectal cancer (CRC) cell growth in vitro. Furthermore, increased IMP1 expression correlates with enhanced metastasis and poor prognosis in CRC patients. In the current study, we sought to elucidate IMP1-mediated functions in CRC pathogenesis in vivo. Using CRC cell xenografts, we demonstrate that IMP1 overexpression promotes xenograft tumor growth and dissemination into the blood. Furthermore, intestine-specific knockdown of Imp1 dramatically reduces tumor number in the Apc (Min/+) mouse model of intestinal tumorigenesis. In addition, IMP1 knockdown xenografts exhibit a reduced number of tumor cells entering the circulation, suggesting that IMP1 may directly modulate this early metastatic event. We further demonstrate that IMP1 overexpression decreases E-cadherin expression, promotes survival of single tumor cell-derived colonospheres and promotes enrichment and maintenance of a population of CD24+CD44+ cells, signifying that IMP1 overexpressing cells display evidence of loss of epithelial identity and enhancement of a tumor-initiating cell phenotype. Taken together, these findings implicate IMP1 as a modulator of tumor growth and provide evidence for a novel role of IMP1 in early events in CRC metastasis.
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Proteína de la Poliposis Adenomatosa del Colon/fisiología , Transformación Celular Neoplásica/patología , Neoplasias Colorrectales/patología , Intestinos/patología , Células Madre Neoplásicas/patología , Proteínas de Unión al ARN/fisiología , Animales , Apoptosis , Western Blotting , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Neoplasias Colorrectales/metabolismo , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Transición Epitelial-Mesenquimal , Fibroblastos/metabolismo , Fibroblastos/patología , Xenoinjertos , Humanos , Técnicas para Inmunoenzimas , Integrasas/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Células Madre Neoplásicas/metabolismo , Fenotipo , Células Tumorales CultivadasAsunto(s)
Distinciones y Premios , Investigación Biomédica/historia , Enfermedades del Sistema Digestivo/historia , Gastroenterología/historia , Enfermedades del Sistema Digestivo/diagnóstico , Enfermedades del Sistema Digestivo/fisiopatología , Enfermedades del Sistema Digestivo/terapia , Historia del Siglo XX , Historia del Siglo XXI , HumanosRESUMEN
Understanding how macroautophagy/autophagy contributes to tissue homeostasis is essential for understanding organismal health. The intestinal epithelium is an ideal model to define mechanisms that regulate tissue homeostasis because it houses well-defined populations of intestinal stem cells. Active intestinal stem cells (a-ISCs) are defined by their active cycling and self-renewal during homeostasis, which supports continual tissue turnover in vivo. In vitro, this is observed as long-term organoid formation capacity. A second population of stem cells, called "facultative intestinal stem cells" (f-ISCs), are defined by their ability to 1) survive tissue damage that depletes the injury-sensitive a-ISCs and 2) reenter the cell cycle to repopulate the a-ISC compartment and regenerate the epithelium. The prospective identification of f-ISCs has been challenging, as cells expressing markers of multiple differentiated lineages, particularly secretory lineages, appear to function as f-ISCs in diverse injury contexts. We evaluated cell age (defined as time elapsed after cell cycle exit) and autophagic state (marked by autophagic vesicle content) as molecular features that may be related to f-ISC capacity. We found that autophagic state, but not cell age, prospectively identifies f-ISCs within multiple lineages. As such, we describe autophagy as a lineage-agnostic marker of f-ISC capacity in the mammalian intestine.