RESUMO
The epithelial signaling pathways involved in damage and regeneration, and neoplastic transformation are known to be similar. We noted upregulation of argininosuccinate synthetase (ASS1) in hyperproliferative intestinal epithelium. Since ASS1 leads to de novo synthesis of arginine, an important amino acid for the growth of intestinal epithelial cells, its upregulation can contribute to epithelial proliferation necessary to be sustained during oncogenic transformation and regeneration. Here we investigated the function of ASS1 in the gut epithelium during tissue regeneration and tumorigenesis, using intestinal epithelial conditional Ass1 knockout mice and organoids, and tissue specimens from colorectal cancer patients. We demonstrate that ASS1 is strongly expressed in the regenerating and Apc-mutated intestinal epithelium. Furthermore, we observe an arrest in amino acid flux of the urea cycle, which leads to an accumulation of intracellular arginine. However, loss of epithelial Ass1 does not lead to a reduction in proliferation or increase in apoptosis in vivo, also in mice fed an arginine-free diet. Epithelial loss of Ass1 seems to be compensated by altered arginine metabolism in other cell types and the liver.
Assuntos
Argininossuccinato Sintase/metabolismo , Carcinogênese/patologia , Células Epiteliais/enzimologia , Intestinos/patologia , Regeneração , Adenoma/sangue , Adenoma/genética , Adenoma/patologia , Polipose Adenomatosa do Colo/sangue , Polipose Adenomatosa do Colo/genética , Aminoácidos/metabolismo , Animais , Arginina/metabolismo , Argininossuccinato Sintase/genética , Linhagem Celular Tumoral , Dieta , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/patologia , Fígado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Organoides/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genéticaRESUMO
Enforcing differentiation of cancer stem cells is considered as a potential strategy to sensitize colorectal cancer cells to irradiation and chemotherapy. Activation of the unfolded protein response, due to endoplasmic reticulum (ER) stress, causes rapid stem cell differentiation in normal intestinal and colon cancer cells. We previously found that stem cell differentiation was mediated by a Protein kinase R-like ER kinase (PERK) dependent arrest of mRNA translation, resulting in rapid protein depletion of WNT-dependent transcription factor c-MYC. We hypothesize that ER stress dependent stem cell differentiation may rely on the depletion of additional transcriptional regulators with a short protein half-life that are rapidly depleted due to a PERK-dependent translational pause. Using a novel screening method, we identify novel transcription factors that regulate the intestinal stem cell fate upon ER stress. ER stress was induced in LS174T cells with thapsigargin or subtilase cytotoxin (SubAB) and immediate alterations in nuclear transcription factor activity were assessed by the CatTFRE assay in which transcription factors present in nuclear lysate are bound to plasmid DNA, co-extracted and quantified using mass-spectrometry. The role of altered activity of transcription factor CtBP2 was further examined by modification of its expression levels using CAG-rtTA3-CtBP2 overexpression in small intestinal organoids, shCtBP2 knockdown in LS174T cells, and familial adenomatous polyposis patient-derived organoids. CtBP2 overexpression organoids were challenged by ER stress and ionizing irradiation. We identified a unique set of transcription factors with altered activation upon ER stress. Gene ontology analysis showed that transcription factors with diminished binding were involved in cellular differentiation processes. ER stress decreased CtBP2 protein expression in mouse small intestine. ER stress induced loss of CtBP2 expression which was rescued by inhibition of PERK signaling. CtBP2 was overexpressed in mouse and human colorectal adenomas. Inducible CtBP2 overexpression in organoids conferred higher clonogenic potential, resilience to irradiation-induced damage and a partial rescue of ER stress-induced loss of stemness. Using an unbiased proteomics approach, we identified a unique set of transcription factors for which DNA-binding activity is lost directly upon ER stress. We continued investigating the function of co-regulator CtBP2, and show that CtBP2 mediates ER stress-induced loss of stemness which supports the intestinal stem cell state in homeostatic stem cells and colorectal cancer cells.
Assuntos
Oxirredutases do Álcool/metabolismo , Diferenciação Celular/genética , Proteínas Correpressoras/metabolismo , Estresse do Retículo Endoplasmático/genética , Mucosa Intestinal/citologia , Células-Tronco/fisiologia , Oxirredutases do Álcool/genética , Linhagem Celular Tumoral , Proteínas Correpressoras/genética , Colo/citologia , Colo/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Mucosa Intestinal/metabolismo , Organoides , Tapsigargina/farmacologia , Resposta a Proteínas não Dobradas/genética , eIF-2 Quinase/metabolismoRESUMO
INTRODUCTION: Clinical trials are currently investigating whether an extended mesenteric resection for ileocecal resections could reduce postoperative recurrence in Crohn's disease. Resection of the mesorectum, which contains proinflammatory macrophages, during proct(ocol)ectomy, is associated with reduced recurrent inflammation and improved wound healing. We aimed to characterize the macrophages in the ileocecal mesentery, which were compared with those in the mesorectum, to provide a biological rationale for the ongoing trials. METHODS: In 13 patients with Crohn's disease and 4 control patients undergoing a proctectomy, tissue specimens were sampled at 3 locations from the mesorectum: distal (rectum), middle, and proximal (sigmoid). In 38 patients with Crohn's disease and 7 control patients undergoing ileocecal resections, tissue specimens also obtained from 3 locations: adjacent to the inflamed terminal ileum, adjacent to the noninflamed ileal resection margin, and centrally along the ileocolic artery. Immune cells from these tissue specimens were analyzed by flow cytometry for expression of CD206 to determine their inflammatory status. RESULTS: In the mesorectum, a gradient from proinflammatory to regulatory macrophages from distal to proximal was observed, corresponding to the adjacent inflammation of the intestine. By contrast, the ileocecal mesentery did not contain high amounts of proinflammatory macrophages adjacent to the inflamed tissue, and a gradient toward a more proinflammatory phenotype was seen in the central mesenteric area. DISCUSSION: Although the mesentery is a continuous structure, the mesorectum and the ileocecal mesentery show different immunological characteristics. Therefore, currently, there is no basis to perform an extended ileocecal resection in patients with Crohn's disease.
Assuntos
Colectomia/métodos , Doença de Crohn/cirurgia , Macrófagos/imunologia , Mesentério/citologia , Protectomia/métodos , Adulto , Idoso , Ceco/citologia , Ceco/imunologia , Ceco/patologia , Ceco/cirurgia , Estudos de Coortes , Colo Sigmoide/citologia , Colo Sigmoide/imunologia , Colo Sigmoide/patologia , Colo Sigmoide/cirurgia , Doença de Crohn/imunologia , Doença de Crohn/patologia , Feminino , Humanos , Íleo/citologia , Íleo/imunologia , Íleo/patologia , Íleo/cirurgia , Masculino , Mesentério/imunologia , Mesentério/patologia , Mesentério/cirurgia , Pessoa de Meia-Idade , Reto/citologia , Reto/imunologia , Reto/patologia , Reto/cirurgia , Recidiva , Prevenção Secundária/métodos , Adulto JovemRESUMO
BACKGROUND & AIMS: Activation factor-1 transcription factor family members activating transcription factors 2 and 7 (ATF2 and ATF7) have highly redundant functions owing to highly homologous DNA binding sites. Their role in intestinal epithelial homeostasis and repair is unknown. Here, we assessed the role of these proteins in these conditions in an intestine-specific mouse model. METHODS: We performed in vivo and ex vivo experiments using Villin-CreERT2Atf2fl/flAtf7ko/ko mice. We investigated the effects of intestinal epithelium-specific deletion of the Atf2 DNA binding region in Atf7-/- mice on cellular proliferation, differentiation, apoptosis, and epithelial barrier function under homeostatic conditions. Subsequently, we exposed mice to 2% dextran sulfate sodium (DSS) for 7 days and 12 Gy whole-body irradiation and assessed the response to epithelial damage. RESULTS: Activating phosphorylation of ATF2 and ATF7 was detected mainly in the crypts of the small intestine and the lower crypt region of the colonic epithelium. Under homeostatic conditions, no major phenotypic changes were detectable in the intestine of ATF mutant mice. However, on DSS exposure or whole-body irradiation, the intestinal epithelium showed a clearly impaired regenerative response. Mutant mice developed severe ulceration and inflammation associated with increased epithelial apoptosis on DSS exposure and were less able to regenerate colonic crypts on irradiation. In vitro, organoids derived from double-mutant epithelium had a growth disadvantage compared with wild-type organoids, impaired wound healing capacity in scratch assay, and increased sensitivity to tumor necrosis factor-α-induced damage. CONCLUSIONS: ATF2 and ATF7 are dispensable for epithelial homeostasis, but are required to maintain epithelial regenerative capacity and protect against cell death during intestinal epithelial damage and repair.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Fatores Ativadores da Transcrição/metabolismo , Colite Ulcerativa/patologia , Mucosa Intestinal/patologia , Regeneração , Fator 2 Ativador da Transcrição/genética , Fatores Ativadores da Transcrição/genética , Animais , Apoptose , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Colite Ulcerativa/induzido quimicamente , Colo/efeitos dos fármacos , Colo/patologia , Colo/efeitos da radiação , Sulfato de Dextrana/administração & dosagem , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos da radiação , Camundongos , Camundongos Transgênicos , Organoides , Cultura Primária de Células , Irradiação Corporal TotalRESUMO
BACKGROUND AND AIMS: Rectal resection in inflammatory bowel disease [IBD] is frequently complicated by disturbed perineal wound healing. Close rectal dissection, where the mesorectum remains in situ, is hypothesized to reduce complications by minimizing dead space, compared to total mesorectal excision. The aim of this study was to analyse post-operative outcomes of both techniques. In addition, immune activity in mesorectal tissue was assessed. METHODS: Perineal complications and healing were retrospectively assessed in a series of 74 IBD patients undergoing proctectomy using close rectal dissection or total mesorectal excision. The mesorectums of 15 patients were analysed by fluorescence-activated cell sorting, immunofluorescence and in situ hybridization. Based on the clinical and in vitro findings, a novel surgical approach for Crohn's disease patients with disturbed perineal healing after proctectomy was developed. RESULTS: In Crohn's disease, perineal complications were more frequent after close rectal dissection than after total mesorectal excision [59.5% vs 17.6%; p = 0.007] with lower healing rates [51.4% vs 88.2%; p = 0.014]. No differences were observed in ulcerative colitis. The mesorectal tissue in Crohn's disease contained enhanced numbers of tumour necrosis factor α-producing CD14+ macrophages, with less expression of the wound-healing marker CD206. Based on these findings, mesorectal excision with omentoplasty was performed in eight patients with perineal complications after close rectal dissection, resulting in complete perineal wound closure in six. Pro-inflammatory characteristics remained present in the mesorectum after close rectal dissection in these patients. CONCLUSIONS: In Crohn's disease, close rectal dissection resulted in more perineal complications, associated with a pro-inflammatory immune status of the mesorectal tissue. Excision of this pro-inflammatory mesenteric tissue resulted in improved perineal healing rates.
Assuntos
Doença de Crohn , Neoplasias Retais , Humanos , Mesentério , Recidiva Local de Neoplasia , Períneo , Protectomia , Reto , Estudos RetrospectivosRESUMO
Variations in the reporting of potentially confounding variables in studies investigating systemic treatments for unresectable pancreatic cancer pose challenges in drawing accurate comparisons between findings. In this Review, we establish the first international consensus on mandatory baseline and prognostic characteristics in future trials for the treatment of unresectable pancreatic cancer. We did a systematic literature search to find phase 3 trials investigating first-line systemic treatment for locally advanced or metastatic pancreatic cancer to identify baseline characteristics and prognostic variables. We created a structured overview showing the reporting frequencies of baseline characteristics and the prognostic relevance of identified variables. We used a modified Delphi panel of two rounds involving an international panel of 23 leading medical oncologists in the field of pancreatic cancer to develop a consensus on the various variables identified. In total, 39 randomised controlled trials that had data on 15â863 patients were included, of which 32 baseline characteristics and 26 prognostic characteristics were identified. After two consensus rounds, 23 baseline characteristics and 12 prognostic characteristics were designated as mandatory for future pancreatic cancer trials. The COnsensus statement on Mandatory Measurements in unresectable PAncreatic Cancer Trials (COMM-PACT) identifies a mandatory set of baseline and prognostic characteristics to allow adequate comparison of outcomes between pancreatic cancer studies.
Assuntos
Ensaios Clínicos Fase III como Assunto/normas , Confiabilidade dos Dados , Neoplasias Pancreáticas/terapia , Ensaios Clínicos Controlados Aleatórios como Assunto/normas , Projetos de Pesquisa/normas , Biomarcadores/sangue , Consenso , Técnica Delphi , Nível de Saúde , Humanos , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Resultado do TratamentoRESUMO
The Na+ -taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild-type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1-transgenic mice. Effects of fibroblast growth factor 19 (FGF19) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno-associated virus-mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7α-hydroxylase Cyp7a1 expression was strongly down-regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF19) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B-treated cholestatic mice, whereas plasma FGF19 was not induced in humans treated with myrcludex B. Fgf15/FGF19 expression was induced in polarized human enterocyte-models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids. CONCLUSION: NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/19, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631-1643).
Assuntos
Ácidos e Sais Biliares/metabolismo , Enterócitos/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Animais , Linhagem Celular , Colesterol 7-alfa-Hidroxilase/metabolismo , Regulação para Baixo , Feminino , Humanos , Íleo/metabolismo , Lipopeptídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Isoformas de Proteínas/metabolismo , RatosRESUMO
TAM receptors (Tyro3, Axl, and Mer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion, and migration. TAM receptors can be activated by the vitamin K-dependent proteins Gas6 and protein S. Protein S is more commonly known as an important cofactor for protein C as well as a direct inhibitor of multiple coagulation factors. To our knowledge, the functions of Gas6 are limited to TAM receptor activation. When activated, the TAM receptors have effects on primary hemostasis and coagulation and display an anti-inflammatory or a proinflammatory effect, depending on cell type. To comprehend the effects that the TAM receptors and their ligands have on hemostasis and inflammation, we compare studies that report the different phenotypes displayed by mice with deficiencies in the genes of this receptor family and its ligands (protein S(+/-), Gas6(-/-), TAM(-/-), and variations of these). In this manner, we aim to display which features are attributable to the different ligands. Because of the effects TAM receptors have on hemostasis, inflammation, and cancer growth, their modulation could make interesting therapeutic targets in thromboembolic disease, atherosclerosis, sepsis, autoimmune disease, and cancer.
Assuntos
Hemostasia/genética , Inflamação/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteína S/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Humanos , Inflamação/metabolismo , Camundongos , Proteínas Proto-Oncogênicas/fisiologia , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
The processing of pro-interleukin-1beta depends on activation of caspase-1. Controversy has arisen whether Toll-like receptor (TLR) ligands alone can activate caspase-1 for release of interleukin-1beta (IL-1beta). Here we demonstrate that human blood monocytes release processed IL-1beta after a one-time stimulation with either TLR2 or TLR4 ligands, resulting from constitutively activated caspase-1 and release of endogenous adenosine triphosphate. The constitutive activation of caspase-1 depends on the inflammasome components, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NALP3, but in monocytes caspase-1 activation is uncoupled from pathogen-associated molecular pattern recognition. In contrast, macrophages are unable to process and release IL-1beta solely by TLR ligands and require a second adenosine triphosphate stimulation. We conclude that IL-1beta production is differentially regulated in monocytes and macrophages, and this reflects their separate functions in host defense and inflammation.