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BACKGROUND & AIMS: Human sporadic colorectal cancer (CRC) results from a multistep pathway with sequential acquisition of specific genetic mutations in the colorectal epithelium. Modeling CRC in vivo is critical for understanding the tumor microenvironment. To accurately recapitulate human CRC pathogenesis, mouse models must include these multi-step genetic abnormalities. AIMS: Generate a sporadic CRC model that more closely mimics this multi-step process and use this model to study the role of a novel Let7 target PLAGL2 in CRC pathogenesis. METHODS: We generated a CRISPR/Cas9 somatic mutagenesis mouse model that is inducible and multiplexed for simultaneous inactivation of multiple genes involved in CRC pathogenesis. We used both a doxycycline-inducible transcriptional activator and a dox-inactivated transcriptional repressor to achieve tight, non-leaky expression of the Cas9 nickase. This mouse has transgenic expression of multiple guide RNAs to induce sporadic inactivation in the gut epithelium of four tumor suppressor genes commonly mutated in CRC, Apc, Pten, Smad4 and Trp53. These were crossed to Vil-LCL-PLAGL2 mice which have Cre-inducible overexpression of PLAGL2 in the gut epithelium. RESULTS: These mice exhibited random somatic mutations in all four targeted tumor suppressor genes, resulting in multiple adenomas and adenocarcinomas in the small bowel and colon. Crosses with Vil-LCL-PLAGL2 mice demonstrated that gut-specific PLAGL2 overexpression increased colon tumor growth. CONCLUSIONS: This conditional model represents a new CRISPR/Cas9-mediated mouse model of colorectal carcinogenesis. These mice can be used to investigate the role of novel, previously uncharacterized genes in CRC, in the context of multiple commonly mutated tumor suppressor genes and thus more closely mimic human CRC pathogenesis.
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Colorectal cancer (CRC) tumorigenesis and progression are linked to common oncogenic mutations, especially in the tumor suppressor APC, whose loss triggers the deregulation of TCF4/ß-Catenin activity. CRC tumorigenesis is also driven by multiple epimutational modifiers such as transcriptional regulators. We describe the common (and near-universal) activation of the zinc finger transcription factor and Let-7 target PLAGL2 in CRC and find that it is a key driver of intestinal epithelial transformation. PLAGL2 drives proliferation, cell cycle progression, and anchorage-independent growth in CRC cell lines and nontransformed intestinal cells. Investigating effects of PLAGL2 on downstream pathways revealed very modest effects on canonical Wnt signaling. Alternatively, we find pronounced effects on the direct PLAGL2 target genes IGF2, a fetal growth factor, and ASCL2, an intestinal stem cell-specific bHLH transcription factor. Inactivation of PLAGL2 in CRC cell lines has pronounced effects on ASCL2 reporter activity. Furthermore, ASCL2 expression can partially rescue deficits of proliferation and cell cycle progression caused by depletion of PLAGL2 in CRC cell lines. Thus, the oncogenic effects of PLAGL2 appear to be mediated via core stem cell and onco-fetal pathways, with minimal effects on downstream Wnt signaling.NEW & NOTEWORTHY A Let-7 target called PLAGL2 drives oncogenic transformation via Wnt-independent pathways. This work illustrates the robust effects of this zinc finger transcription factor in colorectal cancer (CRC) cell lines and nontransformed intestinal epithelium, with effects mediated, in part, via the direct target genes ASCL2 and IGF2. This has implications for the role of PLAGL2 in activation of onco-fetal and onco-stem cell pathways, contributing to immature and highly proliferative phenotypes in CRC.
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Neoplasias Colorrectales , Humanos , Neoplasias Colorrectales/metabolismo , Línea Celular Tumoral , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , beta Catenina/metabolismo , Transformación Celular Neoplásica/genética , Vía de Señalización Wnt , Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Proliferación Celular/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
The use of T cells from healthy donors for allogeneic chimeric antigen receptor T (CAR-T) cell cancer therapy is attractive because healthy donor T cells can produce versatile off-the-shelf CAR-T treatments. To maximize safety and durability of allogeneic products, the endogenous T cell receptor and major histocompatibility complex class I molecules are often removed via knockout of T cell receptor beta constant (TRBC) (or T cell receptor alpha constant [TRAC]) and B2M, respectively. However, gene editing tools (e.g., CRISPR-Cas9) can display poor fidelity, which may result in dangerous off-target mutations. Additionally, many gene editing technologies require T cell activation, resulting in a low percentage of desirable stem cell memory T cells (TSCM). We characterize an RNA-guided endonuclease, called Cas-CLOVER, consisting of the Clo051 nuclease domain fused with catalytically dead Cas9. In primary T cells from multiple donors, we find that Cas-CLOVER is a high-fidelity site-specific nuclease, with low off-target activity. Notably, Cas-CLOVER yields efficient multiplexed gene editing in resting T cells. In conjunction with the piggyBac transposon for delivery of a CAR transgene against the B cell maturation antigen (BCMA), we produce allogeneic CAR-T cells composed of high percentages of TSCM cells and possessing potent in vivo anti-tumor cytotoxicity.
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[This corrects the article DOI: 10.1016/j.omtn.2022.06.003.].
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The RNA-binding protein Apobec1 complementation factor (A1CF) regulates posttranscriptional ApoB mRNA editing, but the range of RNA targets and the long-term effect of altered A1CF expression on liver function are unknown. Here we studied hepatocyte-specific A1cf-transgenic (A1cf+/Tg), A1cf+/Tg Apobec1-/-, and A1cf-/- mice fed chow or high-fat/high-fructose diets using RNA-Seq, RNA CLIP-Seq, and tissue microarrays from human hepatocellular cancer (HCC). A1cf+/Tg mice exhibited increased hepatic proliferation and steatosis, with increased lipogenic gene expression (Mogat1, Mogat2, Cidea, Cd36) associated with shifts in polysomal RNA distribution. Aged A1cf+/Tg mice developed spontaneous fibrosis, dysplasia, and HCC, and this development was accelerated on a high-fat/high-fructose diet and was independent of Apobec1. RNA-Seq revealed increased expression of mRNAs involved in oxidative stress (Gstm3, Gpx3, Cbr3), inflammatory response (Il19, Cxcl14, Tnfα, Ly6c), extracellular matrix organization (Mmp2, Col1a1, Col4a1), and proliferation (Kif20a, Mcm2, Mcm4, Mcm6), and a subset of mRNAs (including Sox4, Sox9, Cdh1) were identified in RNA CLIP-Seq. Increased A1CF expression in human HCC correlated with advanced fibrosis and with reduced survival in a subset with nonalcoholic fatty liver disease. In conclusion, we show that hepatic A1CF overexpression selectively alters polysomal distribution and mRNA expression, promoting lipogenic, proliferative, and inflammatory pathways leading to HCC.
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Carcinoma Hepatocelular/metabolismo , Hígado Graso/metabolismo , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Hígado Graso/genética , Hígado Graso/patología , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Chronic inflammation of the gastric mucosa, often caused by autoimmune gastritis and/or infection with Helicobacter pylori, can lead to atrophy of acid-secreting parietal cells with metaplasia of remaining cells. The histological pattern marks a critical step in the progression from chronic gastritis to gastric cancer, yet underlying mechanism(s) of inflammation-induced cell death of gastric epithelial cells are poorly understood. We investigated direct effects of a type 1 cytokine associated with autoimmunity and infection, interferon-γ (IFN-γ), on gastric epithelial cells. IFN-γ was applied to three-dimensional organoid cultures of gastric epithelial cells derived from gastric corpus gland (gastroids) of control and IFN-γ receptor-deficient mice. Gastroids were also treated with supernatants from activated immune cells isolated from a mouse model of autoimmune-mediated atrophic gastritis (TxA23) with and without IFN-γ expression. Finally, histopathological analysis of atrophy and metaplasia severity was performed in TxA23 mice and compared to TxA23 × Ifng-/- mice. Gastric epithelial cells in gastroid cultures expressed IFN-γ receptor in the basolateral membrane, and gastroids died when treated with IFN-γ in an IFN-γ receptor-dependent manner. Supernatants from immune cells containing high levels of IFN-γ were highly toxic to gastroids, and toxicity was tempered when IFN-γ was either neutralized using a monoclonal antibody or when supernatants from Ifng-/- mouse immune cells were used. Finally, TxA23 × Ifng-/- mice showed near-complete abrogation of pre-cancerous histopathological atrophy and metaplasia versus IFN-γ-sufficient controls. We identify IFN-γ as a critical promoter of parietal cell atrophy with metaplasia during the progression of gastritis to gastric atrophy and metaplasia. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Mucosa Gástrica/patología , Interferón gamma/fisiología , Neoplasias Gástricas/patología , Animales , Atrofia/patología , Muerte Celular/fisiología , Transformación Celular Neoplásica/patología , Progresión de la Enfermedad , Células Epiteliales/patología , Gastritis , Interferón gamma/deficiencia , Interferón gamma/farmacología , Metaplasia/patología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células Parietales Gástricas/patología , Células Tumorales CultivadasRESUMEN
Mammalian C to U RNA is mediated by APOBEC1, the catalytic deaminase, together with RNA binding cofactors (including A1CF and RBM47) whose relative physiological requirements are unresolved. Although A1CF complements APOBEC1 for in vitro RNA editing, A1cf-/- mice exhibited no change in apolipoproteinB (apoB) RNA editing, while Rbm47 mutant mice exhibited impaired intestinal RNA editing of apoB as well as other targets. Here we examined the role of A1CF and RBM47 in adult mouse liver and intestine, following deletion of either one or both gene products and also following forced (liver or intestinal) transgenic A1CF expression. There were minimal changes in hepatic and intestinal apoB RNA editing in A1cf-/- mice and no changes in either liver- or intestine-specific A1CF transgenic mice. Rbm47 liver-specific knockout (Rbm47LKO ) mice demonstrated reduced editing in a subset (11 of 20) of RNA targets, including apoB. By contrast, apoB RNA editing was virtually eliminated (<6% activity) in intestine-specific (Rbm47IKO ) mice with only five of 53 targets exhibiting C-to-U RNA editing. Double knockout of A1cf and Rbm47 in liver (ARLKO ) eliminated apoB RNA editing and reduced editing in the majority of other targets, with no changes following adenoviral APOBEC1 administration. Intestinal double knockout mice (ARIKO ) demonstrated further reduced editing (<10% activity) in four of five of the residual APOBEC1 targets identified in ARIKO mice. These data suggest that A1CF and RBM47 each function independently, yet interact in a tissue-specific manner, to regulate the activity and site selection of APOBEC1 dependent C-to-U RNA editing.
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Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Edición de ARN , Proteínas de Unión al ARN/metabolismo , Desaminasas APOBEC-1/genética , Desaminasas APOBEC-1/metabolismo , Animales , Secuencia de Bases , Técnicas de Inactivación de Genes , Ribonucleoproteínas Nucleares Heterogéneas/deficiencia , Ribonucleoproteínas Nucleares Heterogéneas/genética , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Especificidad de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
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
Intestinal epithelial stem cell (IESC) fate is promoted by two major transcriptional regulators, the TCF4/ß-catenin complex and ASCL2, which drive expression of IESC-specific factors, including Lgr5, Ephb2, and Rnf43. Canonical Wnt signaling via TCF4/ß-catenin directly transactivates Ascl2, which in turn auto-regulates its own expression. Conversely, Let-7 microRNAs antagonize the IESC lineage by repressing specific mRNA targets. Here, we identify the zinc finger transcription factor PLAGL2 as a Let-7 target that regulates IESC fate. PLAGL2 drives an IESC expression signature, activates Wnt gene expression, and enhances a TCF/LEF reporter in intestinal organoids. In parallel, via cell-autonomous mechanisms, PLAGL2 is required for lineage clonal expansion and directly enhances expression of ASCL2. PLAGL2 also supports enteroid growth and survival in the context of Wnt ligand depletion. PLAGL2 expression is strongly associated with an IESC signature in colorectal cancer and may be responsible for contributing to the aberrant activation of an immature phenotype.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al ADN/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Madre/citología , Células Madre/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Biomarcadores , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Humanos , Ratones , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Transducción de Señal , Factores de Transcripción/genéticaRESUMEN
MicroRNAs (miRNAs) are small single-stranded RNAs that repress mRNA translation and trigger mRNA degradation. Of the â¼1900 miRNA-encoding genes present in the human genome, â¼250 miRNAs are reported to have changes in abundance or altered functions in colorectal cancer. Thousands of studies have documented aberrant miRNA levels in colorectal cancer, with some miRNAs reported to actively regulate tumorigenesis. A recurrent phenomenon with miRNAs is their frequent participation in feedback loops, which probably serve to reinforce or magnify biological outcomes to manifest a particular cellular phenotype. Here, we review the roles of oncogenic miRNAs (oncomiRs), tumor suppressive miRNAs (anti-oncomiRs) and miRNA regulators in colorectal cancer. Given their stability in patient-derived samples and ease of detection with standard and novel techniques, we also discuss the potential use of miRNAs as biomarkers in the diagnosis of colorectal cancer and as prognostic indicators of this disease. MiRNAs also represent attractive candidates for targeted therapies because their function can be manipulated through the use of synthetic antagonists and miRNA mimics.
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Neoplasias Colorrectales/genética , MicroARNs/metabolismo , Transducción de Señal/genética , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Neoplasias Colorrectales/patología , Pleiotropía Genética , Humanos , MicroARNs/genética , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patologíaRESUMEN
The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) after aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway or, alternatively, for enhancing homology-directed repair to facilitate the generation of a specific mutation (or "knock-in"). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA activity. Reliable methods for enriching and identifying desired mutants are also lacking. Here we describe a method using the Piggybac transposon for stable genomic integration of an H2B-GFP reporter or a hygromycin resistance gene for assaying Cas9 target cleavage and homology-directed repair. The H2B-GFP fusion protein provides increased stability and an obvious pattern of nuclear localization. This method, called SRIRACCHA (i.e. a stable, but reversible, integrated reporter for assaying CRISPR/Cas-stimulated HDR activity), enables the enrichment of mutants via selection of GFP-positive or hygromycin-resistant mammalian cells (immortalized or non-immortalized) as a surrogate for the modification of the endogenous target site. Currently available hyperactive Piggybac transposase mutants allow both delivery and removal of the surrogate reporters, with minimal risk of generating undesirable mutations. This assay permits rapid screening for efficient guide RNAs and the accelerated identification of mutant clones and is applicable to many cell types. We foresee the utility of this approach in contexts in which the maintenance of genomic integrity is essential, for example, when engineering cells for therapeutic purposes.
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Sistemas CRISPR-Cas , Eliminación de Gen , Marcación de Gen/métodos , Vectores Genéticos/genética , Animales , Línea Celular Tumoral , RatonesRESUMEN
We hypothesized that basic helix-loop-helix (bHLH) MIST1 (BHLHA15) is a "scaling factor" that universally establishes secretory morphology in cells that perform regulated secretion. Here, we show that targeted deletion of MIST1 caused dismantling of the secretory apparatus of diverse exocrine cells. Parietal cells (PCs), whose function is to pump acid into the stomach, normally lack MIST1 and do not perform regulated secretion. Forced expression of MIST1 in PCs caused them to expand their apical cytoplasm, rearrange mitochondrial/lysosome trafficking, and generate large secretory granules. Mist1 induced a cohort of genes regulated by MIST1 in multiple organs but did not affect PC function. MIST1 bound CATATG/CAGCTG E boxes in the first intron of genes that regulate autophagosome/lysosomal degradation, mitochondrial trafficking, and amino acid metabolism. Similar alterations in cell architecture and gene expression were also caused by ectopically inducing MIST1 in vivo in hepatocytes. Thus, MIST1 is a scaling factor necessary and sufficient by itself to induce and maintain secretory cell architecture. Our results indicate that, whereas mature cell types in each organ may have unique developmental origins, cells performing similar physiological functions throughout the body share similar transcription factor-mediated architectural "blueprints."
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Regulación de la Expresión Génica/genética , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Células Parietales Gástricas/citología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Vías Secretoras/genética , Células Acinares/citología , Células Acinares/efectos de los fármacos , Células Acinares/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Línea Celular , Expresión Génica Ectópica/efectos de los fármacos , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Ratones , Células Parietales Gástricas/efectos de los fármacos , Células Parietales Gástricas/metabolismo , Células Parietales Gástricas/ultraestructura , Tamoxifeno/farmacologíaRESUMEN
RNA-binding protein (RBP) is a key player in regulating gene expression at the posttranscriptional level. CLIP-Seq, with the ability to provide a genome-wide map of protein-RNA interactions, has been increasingly used to decipher RBP-mediated posttranscriptional regulation. Generating highly reliable binding sites from CLIP-Seq requires not only stringent library preparation but also considerable computational efforts. Here we presented a first systematic evaluation of major computational steps for identifying RBP binding sites from CLIP-Seq data, including preprocessing, the choice of control samples, peak normalization, and motif discovery. We found that avoiding PCR amplification artifacts, normalizing to input RNA or mRNAseq, and defining the background model from control samples can reduce the bias introduced by RNA abundance and improve the quality of detected binding sites. Our findings can serve as a general guideline for CLIP experiments design and the comprehensive analysis of CLIP-Seq data.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Sitios de Unión/genética , Células CACO-2 , Regulación de la Expresión Génica , Humanos , MicroARNs/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de ARNRESUMEN
A more complete understanding of aberrant oncogenic signaling in neuroblastoma, a malignancy of the developing sympathetic nervous system, is paramount to improving patient outcomes. Recently, we identified LIN28B as an oncogenic driver in high-risk neuroblastoma. Here, we identify the oncogene RAN as a LIN28B target and show regional gain of chromosome 12q24 as an additional somatic alteration resulting in increased RAN expression. We show that LIN28B influences RAN expression by promoting RAN Binding Protein 2 expression and by directly binding RAN mRNA. Further, we demonstrate a convergence of LIN28B and RAN signaling on Aurora kinase A activity. Collectively, these findings demonstrate that LIN28B-RAN-AURKA signaling drives neuroblastoma oncogenesis, suggesting that this pathway may be amenable to therapeutic targeting.
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Aurora Quinasa A/genética , Neuroblastoma/genética , Proteínas de Unión al ARN/genética , Transducción de Señal/genética , Proteína de Unión al GTP ran/genética , Aurora Quinasa A/metabolismo , Western Blotting , Carcinogénesis/genética , Línea Celular Tumoral , Niño , Cromosomas Humanos Par 12/genética , Variaciones en el Número de Copia de ADN , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , MicroARNs/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteína Proto-Oncogénica N-Myc , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína de Unión al GTP ran/metabolismoRESUMEN
Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.
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Adenocarcinoma/genética , Proteína HMGA2/metabolismo , Neoplasias Intestinales/genética , MicroARNs/fisiología , Células Madre Neoplásicas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Proteína HMGA2/genética , Humanos , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Células Tumorales CultivadasRESUMEN
The RNA-binding proteins LIN28A and LIN28B have diverse functions in embryonic stem cells, cellular reprogramming, growth, and oncogenesis. Many of these effects occur via direct inhibition of Let-7 microRNAs (miRNAs), although Let-7-independent effects have been surmised. We report that intestine targeted expression of LIN28B causes intestinal hypertrophy, crypt expansion, and Paneth cell loss. Furthermore, LIN28B fosters intestinal polyp and adenocarcinoma formation. To examine potential Let-7-independent functions of LIN28B, we pursued ribonucleoprotein cross-linking, immunoprecipitation, and high-throughput sequencing (CLIP-seq) to identify direct RNA targets. This revealed that LIN28B bound a substantial number of mRNAs and modestly augmented protein levels of these target mRNAs in vivo. Conversely, Let-7 had a profound effect; modulation of Let-7 levels via deletion of the mirLet7c2/mirLet7b genes recapitulated effects of Lin28b overexpression. Furthermore, intestine-specific Let-7 expression could reverse hypertrophy and Paneth cell depletion caused by Lin28b. This was independent of effects on insulin-PI3K-mTOR signaling. Our study reveals that Let-7 miRNAs are critical for repressing intestinal tissue growth and promoting Paneth cell differentiation. Let-7-dependent effects of LIN28B may supersede Let-7-independent effects on intestinal tissue growth. In summary, LIN28B can definitively act as an oncogene in the absence of canonical genetic alterations.
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Carcinogénesis/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Mucosa Intestinal/fisiopatología , MicroARNs/genética , Animales , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/crecimiento & desarrollo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos BALB C , MicroARNs/metabolismo , Células de Paneth/citología , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Transducción de SeñalRESUMEN
The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed.