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BACKGROUND & AIMS: RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been characterized sufficiently. In this study, we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 in exocrine cells. METHODS: We generated Ptf1a-Cre;LSL-KrasG12D;Rnf43flox/flox (KRC) and Ptf1a-Cre; LSL-KrasG12D (KC) mice and animal survival was assessed. KRC mice were sacrificed at 2 months, 4 months, and at moribund status followed by analysis of pancreata by single-cell RNA sequencing. Comparative analyses between moribund KRC and a moribund Kras/Tp53-driven PDAC GEMM (KPC) was performed. Cell lines were isolated from KRC and KC tumors and interrogated by cytokine array analyses, ATAC sequencing, and in vitro drug assays. KRC GEMMs were also treated with an anti-CTLA4 neutralizing antibody with treatment response measured by magnetic response imaging. RESULTS: We demonstrate that KRC mice display a marked increase in incidence of high-grade cystic lesions of the pancreas and PDAC compared with KC. Importantly, KRC mice have a significantly decreased survival compared with KC mice. Using single-cell RNA sequencing, we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes, with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the KPC PDAC GEMM. Furthermore, expression of the chemokine CXCL5 was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. CONCLUSIONS: The KRC GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC. This GEMM features a markedly different immune microenvironment compared with previously reported PDAC GEMMs and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.
Assuntos
Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ubiquitina-Proteína Ligases , Adenocarcinoma/genética , Animais , Carcinoma Ductal Pancreático/patologia , Modelos Animais de Doenças , Epigênese Genética , Humanos , Camundongos , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Microambiente Tumoral , Ubiquitina-Proteína Ligases/genética , Neoplasias PancreáticasRESUMO
Adeno-associated virus' (AAV) relatively simple structure makes it accommodating for engineering into controllable delivery platforms. Cancer, such as pancreatic ductal adenocarcinoma (PDAC), are often characterized by upregulation of membrane-bound proteins, such as MMP-14, that propagate survival integrin signaling. In order to target tumors, we have engineered an MMP-14 protease-activatable AAV vector that responds to both membrane-bound and extracellularly active MMPs. This "provector" was generated by inserting a tetra-aspartic acid inactivating motif flanked by the MMP-14 cleavage sequence IPESLRAG into the capsid subunits. The MMP-14 provector shows lower background transduction than previously developed provectors, leading to a 9.5-fold increase in transduction ability. In a murine model of PDAC, the MMP-14 provector shows increased delivery to an allograft tumor. This proof-of-concept study illustrates the possibilities of membrane-bound protease-activatable gene therapies to target tumors.
Assuntos
Vetores Genéticos , Neoplasias Pancreáticas , Animais , Dependovirus/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Metaloproteinase 14 da Matriz/genética , Metaloproteinases da Matriz/genética , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Peptídeo Hidrolases/genéticaRESUMO
Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single "workhorse" mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic Kras G12D allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic KrasG12D allele at the endogenous locus, and insertion deletion ("indel") and frameshift mutations of targeted tumor suppressor alleles. By using a single "workhorse" mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we present a facile autochthonous platform for interrogation of the PDAC genome.
Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Neoplasias Experimentais , Neoplasias Pancreáticas , Recombinação Genética/genética , Animais , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
BACKGROUND & AIMS: Mutations at hotspots in GNAS, which encodes stimulatory G-protein, α subunits, are detected in approximately 60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We generated mice with KRAS-induced IPMNs that also express a constitutively active form of GNAS in pancreas and studied tumor development. METHODS: We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice); pancreatic tissues of these mice express activated KRAS and also express a mutant form of GNAS (GNASR201C) upon doxycycline administration. Mice that were not given doxycycline were used as controls, and survival times were compared by Kaplan-Meier analysis. Pancreata were collected at different time points after doxycycline administration and analyzed by histology. Pancreatic ductal adenocarcinomas (PDACs) were isolated from mice and used to generate cell lines, which were analyzed by reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and colony formation and invasion assays. Full-length and mutant forms of yes-associated protein (YAP) were expressed in PDAC cells. IPMN specimens were obtained from 13 patients with IPMN undergoing surgery and analyzed by immunohistochemistry. RESULTS: All Kras;Gnas mice developed pancreatic cystic lesions that resemble human IPMNs; the grade of epithelial dysplasia increased with time. None of the control mice developed cystic lesions. Approximately one third of Kras;Gnas mice developed PDACs at a median of 30 weeks after doxycycline administration, whereas 33% of control mice developed PDACs. Expression of GNASR201C did not accelerate the development of PDACs compared with control mice. However, the neoplasms observed in Kras;Gnas mice were more differentiated, and expressed more genes associated with ductal phenotypes, than in control mice. PDACs isolated from Kras;Gnas mice had activation of the Hippo pathway; in cells from these tumors, phosphorylated YAP1 was sequestered in the cytoplasm, and this was also observed in human IPMNs with GNAS mutations. Sequestration of YAP1 was not observed in PDAC cells from control mice. CONCLUSIONS: In mice that express activated KRAS in the pancreas, we found expression of GNASR201C to cause development of more differentiated tumors, with gene expression pattern associated with the ductal phenotype. Expression of mutant GNAS caused phosphorylated YAP1 to be sequestered in the cytoplasm, altering tumor progression.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Carcinoma Ductal Pancreático/etiologia , Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Mutação , Neoplasias Císticas, Mucinosas e Serosas/etiologia , Neoplasias Pancreáticas/etiologia , Fosfoproteínas/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromograninas/fisiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Humanos , Camundongos , Fosfoproteínas/análise , Fosfoproteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Proteínas de Sinalização YAPRESUMO
Dicer is an essential ribonuclease involved in the biogenesis of miRNAs. Previous studies have reported downregulation of Dicer in multiple cancers including hepatocellular carcinoma. To identify signaling pathways that are altered upon Dicer depletion, we carried out quantitative phosphotyrosine profiling of liver tissue from Dicer knockout mice. We employed antibody-based enrichment of phosphotyrosine containing peptides coupled with SILAC spike-in approach for quantitation. High resolution mass spectrometry-based analysis identified 349 phosphotyrosine peptides corresponding to 306 unique phosphosites of which 75 were hyperphosphorylated and 78 were hypophosphorylated. Several receptor tyrosine kinases including MET, PDGF receptor alpha, Insulin-like growth factor 1 and Insulin receptor as well as non-receptor tyrosine kinases such as Src family kinases were found to be hyperphosphorylated upon depletion of Dicer. In addition, signaling molecules such as IRS-2 and STAT3 were hyperphosphorylated. Activation of these signaling pathways has been implicated previously in various types of cancers. Interestingly, we observed hypophosphorylation of molecules including focal adhesion kinase and paxillin. Our study profiles the perturbed signaling pathways in response to dysregulated miRNAs resulting from depletion of Dicer. Our findings warrant further studies to investigate oncogenic effects of downregulation of Dicer in cancers.
Assuntos
RNA Helicases DEAD-box/genética , Fosfotirosina/metabolismo , Ribonuclease III/genética , Transdução de Sinais , Sequência de Aminoácidos , Animais , Linhagem Celular , RNA Helicases DEAD-box/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fosfopeptídeos/análise , Fosfopeptídeos/metabolismo , Fosfotirosina/análise , Mapas de Interação de Proteínas , Receptores Proteína Tirosina Quinases/metabolismo , Ribonuclease III/metabolismoRESUMO
Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated >2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems.
Assuntos
RNA Helicases DEAD-box/genética , Metabolismo dos Lipídeos/genética , Proteômica/métodos , Ribonuclease III/genética , Animais , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Engenharia Genética/métodos , Histocitoquímica , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteoma/análise , Proteoma/genética , Proteoma/metabolismo , Reprodutibilidade dos Testes , Ribonuclease III/metabolismoRESUMO
Notch signaling regulates cell fate decisions in a wide variety of adult and embryonic tissues. Here we show that Notch pathway components and Notch target genes are upregulated in invasive pancreatic cancer, as well as in pancreatic cancer precursors from both mouse and human. In mouse pancreas, ectopic Notch activation results in accumulation of nestin-positive precursor cells and expansion of metaplastic ductal epithelium, previously identified as a precursor lesion for pancreatic cancer. Notch is also activated as a direct consequence of EGF receptor activation in exocrine pancreas and is required for TGF alpha-induced changes in epithelial differentiation. These findings suggest that Notch mediates the tumor-initiating effects of TG alpha by expanding a population of undifferentiated precursor cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/patologia , Proteínas de Filamentos Intermediários/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso , Neoplasias Pancreáticas/metabolismo , Fator de Crescimento Transformador alfa/farmacologia , Animais , Biomarcadores/análise , Carcinoma Ductal/metabolismo , Células Cultivadas , Progressão da Doença , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Nestina , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/patologia , Receptores Notch , Transdução de Sinais , Regulação para CimaRESUMO
Intrahepatic cholangiocarcinoma (ICC) is a primary biliary malignancy that harbors a dismal prognosis. Oncogenic mutations of KRAS and loss-of-function mutations of BRCA1-associated protein 1 (BAP1) have been identified as recurrent somatic alterations in ICC. However, an autochthonous genetically engineered mouse model of ICC that genocopies the co-occurrence of these mutations has never been developed. By crossing Albumin-Cre mice bearing conditional alleles of mutant Kras and/or floxed Bap1, Cre-mediated recombination within the liver was induced. Mice with hepatic expression of mutant KrasG12D alone (KA), bi-allelic loss of hepatic Bap1 (BhomoA), and heterozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhetKA) developed primary hepatocellular carcinoma (HCC), but no discernible ICC. In contrast, mice with homozygous loss of Bap1 in conjunction with mutant KrasG12D expression (BhomoKA) developed discrete foci of HCC and ICC. Further, the median survival of BhomoKA mice was significantly shorter at 24 weeks when compared to the median survival of ≥40 weeks in BhetKA mice and approximately 50 weeks in BhomoA and KA mice (p < 0.001). Microarray analysis performed on liver tissue from KA and BhomoKA mice identified differentially expressed genes in the setting of BAP1 loss and suggests that deregulation of ferroptosis might be one mechanism by which loss of BAP1 cooperates with oncogenic Ras in hepato-biliary carcinogenesis. Our autochthonous model provides an in vivo platform to further study this lethal class of neoplasm.
RESUMO
Background & Aims: ARID1A is postulated to be a tumor suppressor gene owing to loss-of-function mutations in human pancreatic ductal adenocarcinomas (PDAC). However, its role in pancreatic pathogenesis is not clear despite recent studies using genetically engineered mouse (GEM) models. We aimed at further understanding of its direct functional role in PDAC, using a combination of GEM model and PDAC cell lines. Methods: Pancreas-specific mutant Arid1a-driven GEM model (Ptf1a-Cre; KrasG12D; Arid1af/f or "KAC") was generated by crossing Ptf1a-Cre; KrasG12D ("KC") mice with Arid1af/f mice and characterized histologically with timed necropsies. Arid1a was also deleted using CRISPR-Cas9 system in established human and murine PDAC cell lines to study the immediate effects of Arid1a loss in isogenic models. Cell lines with or without Arid1a expression were developed from respective autochthonous PDAC GEM models, compared functionally using various culture assays, and subjected to RNA-sequencing for comparative gene expression analysis. DNA damage repair was analyzed in cultured cells using immunofluorescence and COMET assay. Results: Retention of Arid1a is critical for early progression of mutant Kras-driven pre-malignant lesions into PDAC, as evident by lower Ki-67 and higher apoptosis staining in "KAC" as compared to "KC" mice. Enforced deletion of Arid1a in established PDAC cell lines caused suppression of cellular growth and migration, accompanied by compromised DNA damage repair. Despite early development of relatively indolent cystic precursor lesions called intraductal papillary mucinous neoplasms (IPMNs), a subset of "KAC" mice developed aggressive PDAC in later ages. PDAC cells obtained from older autochthonous "KAC" mice revealed various compensatory ("escaper") mechanisms to overcome the growth suppressive effects of Arid1a loss. Conclusions: Arid1a is an essential survival gene whose loss impairs cellular growth, and thus, its expression is critical during early stages of pancreatic tumorigenesis in mouse models. In tumors that arise in the setting of ARID1A loss, a multitude of "escaper" mechanisms drive progression.
RESUMO
A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here, we explored the role of oncogenic KRAS in protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS*) drives cell-autonomous expression of type I cytokine receptor complexes (IL2rγ-IL4rα and IL2rγ-IL13rα1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS* and Th2 cells producing IL4 and IL13. Activated IL2rγ-IL4rα and IL2rγ-IL13rα1 receptors signal primarily via JAK1-STAT6. Integrated transcriptomic, chromatin occupancy, and metabolomic studies identified MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus, paracrine signaling in the tumor microenvironment plays a key role in the KRAS*-driven metabolic reprogramming of PDAC. SIGNIFICANCE: Type II cytokines, secreted by Th2 cells in the tumor microenvironment, can stimulate cancer cell-intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS*-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions, providing candidate therapeutic targets in the KRAS* pathway for this intractable disease.
Assuntos
Citocinas/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Reprogramação Celular/genética , Humanos , Camundongos , Oncogenes , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transfecção , Microambiente TumoralRESUMO
In the developing pancreas, the onset of exocrine differentiation is driven by the activity of the PTF1 (pancreas transcription factor 1) transcriptional complex, which is comprised of the class II bHLH (basic helix-loop-helix) protein, Ptf1-p48 [also known as Ptf1a (pancreas specific transcription factor 1a)], and a class I E-box binding partner. Activity of the PTF1 complex is normally inhibited by the Notch signalling pathway, a process mediated by Notch effector proteins in the HES (Hairy/Enhancer of Split) family of bHLH transcriptional repressors. In the present study, we show that this inhibitory effect occurs through direct interaction between HES family members and Ptf1-p48. The HES family members Hey1 (hairy/enhancer-of-split related with YRPW motif 1) and Hey2 co-immunoprecipitate with Ptf1-p48, and Ptf1-p48 binding by Hes1 is also evident in yeast two-hybrid and GST (glutathione S-transferase) pull-down assays. The ability of Hes1 to interact with Ptf1-p48 resides within a fragment comprised of the bHLH, Orange and C-terminal domains, and does not require the N-terminal or WRPW elements. The ability of truncated versions of Hes1 to bind Ptf1-p48 correlates with their ability to down-regulate the activity of the PTF1 transcriptional complex, defining Ptf1-p48 binding as the most likely mechanism by which Notch effector proteins delay exocrine pancreatic differentiation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Células COS , Chlorocebus aethiops , Regulação para Baixo , Complexos Multiproteicos/metabolismo , Ligação Proteica , Ratos , Fatores de Transcrição HES-1 , Técnicas do Sistema de Duplo-HíbridoRESUMO
The developmental plasticity of adult pancreas is evidenced by the ability to undergo conversion between different epithelial cell types. Specific examples of such conversions include acinar to ductal metaplasia, ductal to islet metaplasia, and generation of ductal structures within islets. Although 90% of human pancreatic cancers are classified as ductal adenocarcinoma, markers of all pancreatic epithelial cell types (acini, ductal, and endocrine) as well as markers of gastric and intestinal lineages can be detected in these tumors. In recent years considerable knowledge has been gained regarding regulation of cellular differentiation and various signaling pathways involved in normal and neoplastic pancreas through studies of pancreatic cancer and immortalized ductal cell lines. However, these studies provide little insight into the context of normal developmental cues, the disruption of which leads to organ pathology. Here we have described a detailed method for preparation, maintenance, and manipulation of adult and embryonic mouse pancreas. These methods may be utilized in studies involving normal epithelial differentiation, contributing to improved understanding of pancreatic development and disease.
Assuntos
Técnicas de Cultura de Órgãos/métodos , Animais , Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Imuno-Histoquímica , Masculino , Camundongos , Pâncreas/citologia , Pâncreas/embriologia , RatosRESUMO
The role of miRNA processing in the maintenance of adult pancreatic acinar cell identity and during the initiation and progression of pancreatic neoplasia has not been studied in detail. In this work, we deleted Dicer specifically in adult pancreatic acinar cells, with or without simultaneous activation of oncogenic Kras. We found that Dicer is essential for the maintenance of acinar cell identity. Acinar cells lacking Dicer showed increased plasticity, as evidenced by loss of polarity, initiation of epithelial-to-mesenchymal transition (EMT) and acinar-to-ductal metaplasia (ADM). In the context of oncogenic Kras activation, the initiation of ADM and pancreatic intraepithelial neoplasia (PanIN) were both highly sensitive to Dicer gene dosage. Homozygous Dicer deletion accelerated the formation of ADM but not PanIN. In contrast, heterozygous Dicer deletion accelerated PanIN initiation, revealing complex roles for Dicer in the regulation of both normal and neoplastic pancreatic epithelial identity.
Assuntos
Células Acinares/metabolismo , RNA Helicases DEAD-box/metabolismo , Pâncreas/citologia , Neoplasias Pancreáticas/enzimologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ribonuclease III/metabolismo , Animais , Polaridade Celular/fisiologia , RNA Helicases DEAD-box/deficiência , Transição Epitelial-Mesenquimal/fisiologia , Imunofluorescência , Imuno-Histoquímica , Camundongos , Ribonuclease III/deficiênciaRESUMO
Dicer is a crucial RNase III enzyme in miRNA biogenesis pathway. Although numerous studies have been carried out to investigate the role of miRNAs and Dicer in the regulation of biological processes, few studies have examined proteomic alterations upon knockout of Dicer. We employed a Cre-loxP-based inducible knockout mouse system to investigate the proteome regulated by Dicer-dependent miRNAs. We utilized spiked liver lysates from metabolically labeled mice to quantify the subtle changes in the liver proteome upon deletion of Dicer. We identified 2137 proteins using high resolution tandem mass spectrometry analysis. The upregulated proteins included several enzymes involved in peroxisomal ß-oxidation of fatty acids and a large majority of the upregulated proteins involved in lipid metabolism were known PPARα targets. MRM-based assays were carried out to confirm the upregulation of enzymes including peroxisomal bifunctional enzyme, phosphoenolpyruvate carboxykinase 1, cytochrome P450 3A13, cytochrome P450 3A41 and myristoylated alanine-rich protein kinase C substrate. Further, miRNA-124 which is predicted to regulate expression of peroxisomal bifunctional enzyme was confirmed to be downregulated in the Dicer knockout mice. Our study demonstrates the strength of coupling knockout mouse models and quantitative proteomic strategies to investigate functions of individual proteins in vivo. BIOLOGICAL SIGNIFICANCE: Dicer dependent miRNA biogenesis is the major pathway for generation of mature miRNAs. We developed SILAC mouse-based proteomics screen to identify protein targets of Dicer-dependent miRNAs in liver of Dicer knockout mice. We spiked liver lysates of induced and uninduced Dicer knockout mice with liver lysate of SILAC labeled mice for identification of dysregulated proteome. We quantitated 1217 proteins of which 257 were upregulated in induced Dicer knockout mice. We observed enrichment of PPAR-α targets and proteins involved in lipid metabolism among upregulated proteins. We further carried out MRM-based validation of peroxisomal bifunctional enzyme, phosphoenolpyruvate carboxykinase 1, Cyp3A13, Cyp3A41 and myristoylated alanine-rich protein kinase C substrate. We further validated upregulation of peroxisomal bifunctional enzyme using Western blot analysis and downregulation of its predicted upstream miRNA, miR-124 using qRT-PCR. Our study demonstrates that upon ablation of Dicer, certain Dicer-dependent miRNAs are dysregulated which result in dysregulation of their target proteins such as proteins associated with lipid metabolism. Our study illustrates the use of SILAC strategy for quantitative proteomic investigations of animal model systems.
Assuntos
RNA Helicases DEAD-box/metabolismo , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , PPAR alfa/metabolismo , Proteoma/metabolismo , Proteômica , Ribonuclease III/metabolismo , Animais , RNA Helicases DEAD-box/genética , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , PPAR alfa/genética , Peroxissomos/genética , Peroxissomos/metabolismo , Proteoma/genética , Ribonuclease III/genética , Espectrometria de Massas em TandemRESUMO
Mammalian studies have implicated important roles for the basic helix-loop-helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Pâncreas/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem da Célula/fisiologia , Primers do DNA , DNA Complementar/genética , Imunofluorescência , Sequências Hélice-Alça-Hélice/genética , Técnicas Histológicas , Hibridização In Situ , Insulina/metabolismo , Dados de Sequência Molecular , Morfogênese , Pâncreas/citologia , Pâncreas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Transativadores/metabolismo , Peixe-Zebra/metabolismoRESUMO
Mammalian studies have implicated important roles for the basic helix-loop-helix transcription factor Ptf1a-p48 in the development of both exocrine and endocrine pancreas. We have cloned the Ptf1a-p48 ortholog in Danio rerio. Early zebrafish ptf1a expression is observed in developing hindbrain and in endodermal pancreatic precursors. Analysis of ptf1a and insulin expression reveals a population of exocrine precursors that, throughout early development, are temporally and spatially segregated from endocrine elements. Morpholino-mediated knockdown of ptf1a confirms early divergence of these endocrine and exocrine lineages. Ptf1a morphants lack differentiated exocrine pancreas, but maintain normal differentiation and organization of the principal islet. In addition to the exocrine phenotype, ptf1a knockdown also reduces the prevalence of a small population of anterior endocrine cells normally found outside the principal islet. Together, these findings suggest the presence of distinct ptf1a-dependent and ptf1a-independent precursor populations in developing zebrafish pancreas.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Pâncreas/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem da Célula/fisiologia , Primers do DNA , DNA Complementar/genética , Imunofluorescência , Sequências Hélice-Alça-Hélice/genética , Hibridização In Situ , Insulina/metabolismo , Dados de Sequência Molecular , Morfogênese , Pâncreas/citologia , Pâncreas/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Transativadores/metabolismo , Peixe-Zebra/metabolismoRESUMO
Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.