RESUMO
Single-nucleotide variations in C13orf31 (LACC1) that encode p.C284R and p.I254V in a protein of unknown function (called 'FAMIN' here) are associated with increased risk for systemic juvenile idiopathic arthritis, leprosy and Crohn's disease. Here we set out to identify the biological mechanism affected by these coding variations. FAMIN formed a complex with fatty acid synthase (FASN) on peroxisomes and promoted flux through de novo lipogenesis to concomitantly drive high levels of fatty-acid oxidation (FAO) and glycolysis and, consequently, ATP regeneration. FAMIN-dependent FAO controlled inflammasome activation, mitochondrial and NADPH-oxidase-dependent production of reactive oxygen species (ROS), and the bactericidal activity of macrophages. As p.I254V and p.C284R resulted in diminished function and loss of function, respectively, FAMIN determined resilience to endotoxin shock. Thus, we have identified a central regulator of the metabolic function and bioenergetic state of macrophages that is under evolutionary selection and determines the risk of inflammatory and infectious disease.
Assuntos
Artrite Juvenil/genética , Doença de Crohn/genética , Infecções/genética , Hanseníase/genética , Macrófagos/imunologia , Proteínas/genética , Choque Séptico/genética , Trifosfato de Adenosina/metabolismo , Animais , Bacteriólise , Células Cultivadas , Metabolismo Energético , Ácido Graxo Sintase Tipo I/metabolismo , Predisposição Genética para Doença , Humanos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Metabolismo dos Lipídeos/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/metabolismo , Oxirredução , Polimorfismo de Nucleotídeo Único , RiscoRESUMO
As neuroendocrine tumors (NETs) often present as metastatic lesions, immunohistochemical assignment to a site of origin is one of the most important tasks in their pathologic assessment. Because a fraction of NETs eludes the typical expression profiles of their primary localization, additional sensitive and specific markers are required to improve diagnostic certainty. We investigated the expression of the transcription factor Pituitary Homeobox 2 (PITX2) in a large-scale cohort of 909 NET and 248 neuroendocrine carcinomas (NEC) according to the immunoreactive score (IRS) and correlated PITX2 expression groups with general tumor groups and primary localization. PITX2 expression (all expression groups) was highly sensitive (98.1%) for midgut-derived NET, but not perfectly specific, as non-midgut NET (especially pulmonary/duodenal) were quite frequently weak or moderately positive. The specificity rose to 99.5% for a midgut origin of NET if only a strong PITX2 expression was considered, which was found in only 0.5% (one pancreatic/one pulmonary) of non-midgut NET. In metastases of midgut-derived NET, PITX2 was expressed in all cases (87.5% strong, 12.5% moderate), whereas CDX2 was negative or only weakly expressed in 31.3% of the metastases. In NEC, a fraction of cases (14%) showed a weak or moderate PITX2 expression, which was not associated with a specific tumor localization. Our study independently validates PITX2 as a very sensitive and specific immunohistochemical marker of midgut-derived NET in a very large collective of neuroendocrine neoplasms. Therefore, our data argue toward implementation into diagnostic panels applied for NET as a firstline midgut marker.
Assuntos
Carcinoma Neuroendócrino , Neoplasias Intestinais , Tumores Neuroendócrinos , Neoplasias Pancreáticas , Neoplasias Gástricas , Humanos , Tumores Neuroendócrinos/patologia , Biomarcadores Tumorais/metabolismo , Carcinoma Neuroendócrino/patologia , Fatores de Transcrição , Neoplasias Pancreáticas/patologiaRESUMO
BACKGROUND & AIMS: Throughout life, the intestinal epithelium undergoes constant self-renewal from intestinal stem cells. Together with genotoxic stressors and failing DNA repair, this self-renewal causes susceptibility toward malignant transformation. X-box binding protein 1 (XBP1) is a stress sensor involved in the unfolded protein response (UPR). We hypothesized that XBP1 acts as a signaling hub to regulate epithelial DNA damage responses. METHODS: Data from The Cancer Genome Atlas were analyzed for association of XBP1 with colorectal cancer (CRC) survival and molecular interactions between XBP1 and p53 pathway activity. The role of XBP1 in orchestrating p53-driven DNA damage response was tested in vitro in mouse models of chronic intestinal epithelial cell (IEC) DNA damage (Xbp1/H2bfl/fl, Xbp1ΔIEC, H2bΔIEC, H2b/Xbp1ΔIEC) and via orthotopic tumor organoid transplantation. Transcriptome analysis of intestinal organoids was performed to identify molecular targets of Xbp1-mediated DNA damage response. RESULTS: In The Cancer Genome Atlas data set of CRC, low XBP1 expression was significantly associated with poor overall survival and reduced p53 pathway activity. In vivo, H2b/Xbp1ΔIEC mice developed spontaneous intestinal carcinomas. Orthotopic tumor organoid transplantation revealed a metastatic potential of H2b/Xbp1ΔIEC-derived tumors. RNA sequencing of intestinal organoids (H2b/Xbp1fl/fl, H2bΔIEC, H2b/Xbp1ΔIEC, and H2b/p53ΔIEC) identified a transcriptional program downstream of p53, in which XBP1 directs DNA-damage-inducible transcript 4-like (Ddit4l) expression. DDIT4L inhibits mechanistic target of rapamycin-mediated phosphorylation of 4E-binding protein 1. Pharmacologic mechanistic target of rapamycin inhibition suppressed epithelial hyperproliferation via 4E-binding protein 1. CONCLUSIONS: Our data suggest a crucial role for XBP1 in coordinating epithelial DNA damage responses and stem cell function via a p53-DDIT4L-dependent feedback mechanism.
Assuntos
Adenocarcinoma/metabolismo , Adenoma/metabolismo , Transformação Celular Neoplásica/metabolismo , Dano ao DNA , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenoma/tratamento farmacológico , Adenoma/genética , Adenoma/patologia , Animais , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Bases de Dados Genéticas , Estresse do Retículo Endoplasmático , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Neoplasias Intestinais/tratamento farmacológico , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Inibidores de MTOR/farmacologia , Camundongos Knockout , Transdução de Sinais , Sirolimo/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação a X-Box/genéticaRESUMO
BACKGROUND & AIMS: Crohn's disease (CD) globally emerges with Westernization of lifestyle and nutritional habits. However, a specific dietary constituent that comprehensively evokes gut inflammation in human inflammatory bowel diseases remains elusive. We aimed to delineate how increased intake of polyunsaturated fatty acids (PUFAs) in a Western diet, known to impart risk for developing CD, affects gut inflammation and disease course. We hypothesized that the unfolded protein response and antioxidative activity of glutathione peroxidase 4 (GPX4), which are compromised in human CD epithelium, compensates for metabolic perturbation evoked by dietary PUFAs. METHODS: We phenotyped and mechanistically dissected enteritis evoked by a PUFA-enriched Western diet in 2 mouse models exhibiting endoplasmic reticulum (ER) stress consequent to intestinal epithelial cell (IEC)-specific deletion of X-box binding protein 1 (Xbp1) or Gpx4. We translated the findings to human CD epithelial organoids and correlated PUFA intake, as estimated by a dietary questionnaire or stool metabolomics, with clinical disease course in 2 independent CD cohorts. RESULTS: PUFA excess in a Western diet potently induced ER stress, driving enteritis in Xbp1-/-IEC and Gpx4+/-IEC mice. ω-3 and ω-6 PUFAs activated the epithelial endoplasmic reticulum sensor inositol-requiring enzyme 1α (IRE1α) by toll-like receptor 2 (TLR2) sensing of oxidation-specific epitopes. TLR2-controlled IRE1α activity governed PUFA-induced chemokine production and enteritis. In active human CD, ω-3 and ω-6 PUFAs instigated epithelial chemokine expression, and patients displayed a compatible inflammatory stress signature in the serum. Estimated PUFA intake correlated with clinical and biochemical disease activity in a cohort of 160 CD patients, which was similarly demonstrable in an independent metabolomic stool analysis from 199 CD patients. CONCLUSIONS: We provide evidence for the concept of PUFA-induced metabolic gut inflammation which may worsen the course of human CD. Our findings provide a basis for targeted nutritional therapy.
Assuntos
Doença de Crohn , Enterite , Ácidos Graxos Ômega-3 , Animais , Doença de Crohn/tratamento farmacológico , Endorribonucleases , Enterite/induzido quimicamente , Enterite/tratamento farmacológico , Ácidos Graxos Insaturados , Humanos , Inflamação/tratamento farmacológico , Camundongos , Proteínas Serina-Treonina Quinases , Receptor 2 Toll-LikeRESUMO
BACKGROUND: Pathological TNM staging (pTNM) is the strongest prognosticator in colorectal carcinoma (CRC) and the foundation of its post-operative clinical management. Tumours that invade pericolic/perirectal adipose tissue generally fall into the pT3 category without further subdivision. METHODS: The histological depth of invasion into the pericolic/perirectal fat was digitally and conventionally measured in a training cohort of 950 CRCs (Munich). We biostatistically calculated the optimal cut-off to stratify pT3 CRCs into novel pT3a (≤3 mm)/pT3b (>3 mm) subgroups, which were then validated in two independent cohorts (447 CRCs, Bayreuth/542 CRCs, Mainz). RESULTS: Compared to pT3a tumours, pT3b CRCs showed significantly worse disease-specific survival, including in pN0 vs pN+ and colonic vs. rectal cancers (DSS: P < 0.001, respectively, pooled analysis of all cohorts). Furthermore, the pT3a/pT3b subclassification remained an independent predictor of survival in multivariate analyses (e.g. DSS: P < 0.001, hazard ratio: 4.41 for pT3b, pooled analysis of all cohorts). While pT2/pT3a CRCs showed similar survival characteristics, pT3b cancers remained a distinct subgroup with dismal survival. DISCUSSION: The delineation of pT3a/pT3b subcategories of CRC based on the histological depth of adipose tissue invasion adds valuable prognostic information to the current pT3 classification and implementation into current staging practices of CRC should be considered.
Assuntos
Carcinoma , Neoplasias Retais , Humanos , Carcinoma/patologia , Invasividade Neoplásica/patologia , Estadiamento de Neoplasias , Prognóstico , Neoplasias Retais/patologia , Estudos RetrospectivosRESUMO
BACKGROUND: Immunohistochemical loss of CDX2 has been proposed as a biomarker of dismal survival in colorectal carcinoma (CRC), especially in UICC Stage II/III. However, it remains unclear, how CDX2 expression is related to central hematoxylin-eosin (HE)-based morphologic parameters defined by 2019 WHO classification and how its prognostic relevance is compared to these parameters. METHODS: We evaluated CDX2 expression in 1003 CRCs and explored its prognostic relevance compared to CRC subtypes, tumour budding and WHO grade in the overall cohort and in specific subgroups. RESULTS: CDX2-low/absent CRCs were enriched in specific morphologic subtypes, right-sided and microsatellite-instable (MSI-H) CRCs (P < 0.001) and showed worse survival characteristics in the overall cohort/UICC Stage II/III (e.g. DFS: P = 0.005) and in microsatellite stable and left-sided CRCs, but not in MSI-H or right-sided CRCs. Compared with CDX2, all HE-based markers showed a significantly better prognostic discrimination in all scenarios. In multivariate analyses including all morphologic parameters, CDX2 was not an independent prognostic factor. CONCLUSION: CDX2 loss has some prognostic impact in univariate analyses, but its prognostic relevance is considerably lower compared to central HE-based morphologic parameters defined by the WHO classification and vanishes in multivariate analyses incorporating these factors.
Assuntos
Fator de Transcrição CDX2/metabolismo , Neoplasias Colorretais/genética , Amarelo de Eosina-(YS)/metabolismo , Hematoxilina/metabolismo , Feminino , Humanos , Masculino , Instabilidade de Microssatélites , Prognóstico , Organização Mundial da SaúdeRESUMO
The recognition of autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn's disease. Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity, causes Paneth cell dysfunction. As Atg16l1(HM) mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells, the human orthologue of which harbours rare inflammatory bowel disease risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis. Unresolved ER stress is a common feature of inflammatory bowel disease epithelium, and several genetic risk factors of Crohn's disease affect Paneth cells. Here we show that impairment in either UPR (Xbp1(ΔIEC)) or autophagy function (Atg16l1(ΔIEC) or Atg7(ΔIEC)) in intestinal epithelial cells results in each other's compensatory engagement, and severe spontaneous Crohn's-disease-like transmural ileitis if both mechanisms are compromised. Xbp1(ΔIEC) mice show autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased intestinal epithelial cell death, inositol requiring enzyme 1α (IRE1α)-regulated NF-κB activation and tumour-necrosis factor signalling, which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity, and augmentation of autophagy in intestinal epithelial cells ameliorates ER stress-induced intestinal inflammation and eases NF-κB overactivation and intestinal epithelial cell death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth-cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal Crohn's disease as a specific disorder of Paneth cells.
Assuntos
Enteropatias/fisiopatologia , Mucosa Intestinal/patologia , Celulas de Paneth/patologia , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático/genética , Inflamação , Enteropatias/genética , Mucosa Intestinal/citologia , Camundongos , Fatores de Transcrição de Fator Regulador X , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Proteína 1 de Ligação a X-Box , eIF-2 Quinase/metabolismoRESUMO
The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.
Assuntos
Estresse do Retículo Endoplasmático , Enterocolite Necrosante/metabolismo , Mucosa Intestinal/metabolismo , Células-Tronco/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Apoptose/genética , Enterocolite Necrosante/genética , Enterocolite Necrosante/patologia , Células HEK293 , Humanos , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Células-Tronco/patologia , Receptor 4 Toll-Like/genética , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
OBJECTIVE: Intestinal epithelial cells (IECs) at the internal/external interface orchestrate the mucosal immune response. Paneth cells secrete antimicrobial peptides and inflammatory mediators, protect from pathogens and shape the commensal microbiota. Prompted by the genetic association of the locus harbouring the type I interferon (IFN) receptor (IFNAR1) with Crohn's disease, and a transcriptional signature for type I IFN signalling in Paneth cells, we studied the function of IFNAR1 in IECs. DESIGN: Type I IFN signalling was studied in mice with conditional deletion of Ifnar1 in IECs. Phenotype was characterised at baseline, and gut microbiota composition was assessed by 16S rDNA ribotyping. The role of IFNAR1 was also investigated in experimental colitis induced by dextran sodium sulfate (DSS) and colitis-associated cancer induced by DSS in conjunction with azoxymethane (AOM). RESULTS: Ifnar1(-/-(IEC)) mice displayed expansion of Paneth cell numbers and epithelial hyperproliferation compared with Ifnar1-sufficient littermates. While Ifnar1(-/-(IEC)) mice did not exhibit spontaneous inflammation or increased severity in DSS colitis compared with Ifnar1(+/+(IEC)) mice, they exhibited an increased tumour burden in the AOM/DSS model. Both hyperproliferation and tumour promotion were dependent on the microbial flora, as the differences between genotypes were marked upon separately housing mice, but disappeared when Ifnar1(-/-(IEC)) and Ifnar1(+/+(IEC)) mice were co-housed. Accordingly, ribotyping revealed marked differences between Ifnar1(-/-(IEC)) and Ifnar1(+/+(IEC)) mice that where diminished upon co-housing. CONCLUSIONS: IFNAR1 in IECs, and Paneth cells in particular, contributes to the regulation of the host-microbiota relationship, with consequences for intestinal regeneration and colitis-associated tumour formation.
Assuntos
Colite , Doença de Crohn , Células Caliciformes , Celulas de Paneth , Receptor de Interferon alfa e beta/genética , Animais , Peptídeos Catiônicos Antimicrobianos , Proliferação de Células/genética , Colite/etiologia , Colite/imunologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Doença de Crohn/patologia , Sulfato de Dextrana/farmacologia , Predisposição Genética para Doença , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Imunidade nas Mucosas/genética , Mediadores da Inflamação , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL , Microbiota , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Transcrição GênicaRESUMO
PURPOSE: Preoperative (neoadjuvant) radiation therapy (RT) is an essential part of multimodal rectal cancer therapy. Recently, total neoadjuvant therapy (TNT), which combines simultaneous radiochemotherapy with additional courses of chemotherapy, has emerged as an effective approach. TNT achieves a pathologic complete remission in approximately 30% of resected patients, opening avenues for treatment strategies that avoid radical organ resection. Furthermore, recent studies have demonstrated that anti-programmed cell death protein 1 immunotherapy can induce clinical complete responses in patients with specific genetic alterations. There is significant potential to enhance outcomes through intensifying, personalizing, and de-escalating treatment approaches. However, the heterogeneous response rates to RT or TNT and strategies to sensitize patients without specific genetic changes to immunotherapy remain poorly understood. METHODS AND MATERIALS: We developed a novel orthotopic mouse model of rectal cancer based on precisely defined endoscopic injections of tumor organoids that reflect tumor heterogeneity. Subsequently, we employed endoscopic- and computed tomography-guided RT and validated rectal tumor growth and response rates to therapy using small-animal magnetic resonance imaging and endoscopic follow-up. RESULTS: Rectal tumor formation was successfully induced in all mice after 2 organoid injections. Clinically relevant RT regimens with 5 × 5 Gy significantly delayed clinical signs of tumor progression and significantly improved survival. Consistent with human disease, rectal tumor progression correlated with the development of liver and lung metastases. Notably, long-term survivors after RT showed no evidence of tumor recurrence, as demonstrated by in vivo radiologic tumor staging and histopathologic examination. CONCLUSIONS: Our novel mouse model combines orthotopic tumor growth via noninvasive and precise rectal organoid injection and small-animal RT. This model holds significant promise for investigating the effect of tumor cell-intrinsic aspects, genetic alterations of the host, and exogenous factors (eg, nutrition or microbiota) on RT outcomes. Furthermore, it allows for the exploration of combination therapies involving chemotherapy, immunotherapy, or novel targeted therapies.
Assuntos
Radioterapia Guiada por Imagem , Neoplasias Retais , Humanos , Animais , Camundongos , Recidiva Local de Neoplasia/patologia , Neoplasias Retais/patologia , Terapia Combinada , Quimiorradioterapia , Terapia Neoadjuvante , Estadiamento de NeoplasiasRESUMO
The gut microbiota influences human health and the development of chronic diseases. However, our understanding of potentially protective or harmful microbe-host interactions at the molecular level is still in its infancy. To gain further insights into the hidden gut metabolome and its impact, we identified a cryptic non-ribosomal peptide BGC in the genome of Bacillus cereus DSM 28590 from the mouse intestine ( www.dsmz.de/miBC ), which was predicted to encode a thiazol(in)e substructure. Cloning and heterologous expression of this BGC revealed that it produces bacillamide D. In-depth functional evaluation showed potent cytotoxicity and inhibition of cell migration using the human cell lines HCT116 and HEK293, which was validated using primary mouse organoids. This work establishes the bacillamides as selective cytotoxins from a bacterial gut isolate that affect mammalian cells. Our targeted structure-function-predictive approach is demonstrated to be a streamlined method to discover deleterious gut microbial metabolites with potential effects on human health.
Assuntos
Bacillus cereus , Microbioma Gastrointestinal , Bacillus cereus/metabolismo , Bacillus cereus/genética , Animais , Camundongos , Humanos , Células HEK293 , Citotoxinas/metabolismo , Citotoxinas/genética , Células HCT116 , Intestinos/microbiologia , Movimento Celular , Organoides/metabolismoRESUMO
BACKGROUND: Esophageal cancer is one of the 10 most common cancers worldwide and its incidence is dramatically increasing. Despite some improvements, the current surveillance protocol with white light endoscopy and random untargeted biopsies collection (Seattle protocol) fails to diagnose dysplastic and cancerous lesions in up to 50% of patients. Therefore, new endoscopic imaging technologies in combination with tumor-specific molecular probes are needed to improve early detection. Herein, we investigated the use of the fluorescent Poly (ADP-ribose) Polymerase 1 (PARP1)-inhibitor PARPi-FL for early detection of dysplastic lesions in patient-derived organoids and transgenic mouse models, which closely mimic the transformation from non-malignant Barrett's Esophagus (BE) to invasive esophageal adenocarcinoma (EAC). METHODS: We determined PARP1 expression via immunohistochemistry (IHC) in human biospecimens and mouse tissues. We also assessed PARPi-FL uptake in patient- and mouse-derived organoids. Following intravenous injection of 75 nmol PARPi-FL/mouse in L2-IL1B (n = 4) and L2-IL1B/IL8Tg mice (n = 12), we conducted fluorescence molecular endoscopy (FME) and/or imaged whole excised stomachs to assess PARPi-FL accumulation in dysplastic lesions. L2-IL1B/IL8Tg mice (n = 3) and wild-type (WT) mice (n = 2) without PARPi-FL injection served as controls. The imaging results were validated by confocal microscopy and IHC of excised tissues. RESULTS: IHC on patient and murine tissue revealed similar patterns of increasing PARP1 expression in presence of dysplasia and cancer. In human and murine organoids, PARPi-FL localized to PARP1-expressing epithelial cell nuclei after 10 min of incubation. Injection of PARPi-FL in transgenic mouse models of BE resulted in the successful detection of lesions via FME, with a mean target-to-background ratio > 2 independently from the disease stage. The localization of PARPi-FL in the lesions was confirmed by imaging of the excised stomachs and confocal microscopy. Without PARPi-FL injection, identification of lesions via FME in transgenic mice was not possible. CONCLUSION: PARPi-FL imaging is a promising approach for clinically needed improved detection of dysplastic and malignant EAC lesions in patients with BE. Since PARPi-FL is currently evaluated in a phase 2 clinical trial for oral cancer detection after topical application, clinical translation for early detection of dysplasia and EAC in BE patients via FME screening appears feasible.
Assuntos
Adenocarcinoma , Esôfago de Barrett , Neoplasias Esofágicas , Humanos , Camundongos , Animais , Detecção Precoce de Câncer , Neoplasias Esofágicas/diagnóstico por imagem , Neoplasias Esofágicas/genética , Esôfago de Barrett/diagnóstico , Esôfago de Barrett/genética , Esôfago de Barrett/patologia , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/genética , Camundongos Transgênicos , Endoscopia , Poli(ADP-Ribose) Polimerase-1/genéticaRESUMO
SNAIL is a key transcriptional regulator in embryonic development and cancer. Its effects in physiology and disease are believed to be linked to its role as a master regulator of epithelial-to-mesenchymal transition (EMT). Here, we report EMT-independent oncogenic SNAIL functions in cancer. Using genetic models, we systematically interrogated SNAIL effects in various oncogenic backgrounds and tissue types. SNAIL-related phenotypes displayed remarkable tissue- and genetic context-dependencies, ranging from protective effects as observed in KRAS- or WNT-driven intestinal cancers, to dramatic acceleration of tumorigenesis, as shown in KRAS-induced pancreatic cancer. Unexpectedly, SNAIL-driven oncogenesis was not associated with E-cadherin downregulation or induction of an overt EMT program. Instead, we show that SNAIL induces bypass of senescence and cell cycle progression through p16INK4A-independent inactivation of the Retinoblastoma (RB)-restriction checkpoint. Collectively, our work identifies non-canonical EMT-independent functions of SNAIL and unravel its complex context-dependent role in cancer.
Assuntos
Neoplasias Pancreáticas , Fatores de Transcrição da Família Snail , Carcinogênese , Transformação Celular Neoplásica , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas p21(ras) , Animais , Fatores de Transcrição da Família Snail/genéticaRESUMO
The intestinal mucosa is a highly absorptive organ and simultaneously constitutes the physical barrier between the host and a complex outer ecosystem. Intestinal epithelial cells (IECs) represent a special node that receives signals from the host and the environment and translates them into corresponding responses. Specific molecular communication systems such as metabolites are known to transmit information across the intestinal boundary. The gut microbiota or food-derived metabolites are extrinsic factors that influence the homeostasis of the intestinal epithelium, while mitochondrial and host-derived cellular metabolites determine the identity, fitness, and regenerative capacity of IECs. Little is known, however, about the role of intrinsic and extrinsic metabolites of IECs in the initiation and progression of pathological processes such as inflammatory bowel disease and colorectal cancer as well as about their impact on intestinal immunity. In this review, we will highlight the most recent contributions on the modulatory effects of intestinal metabolites in gut pathophysiology, with a particular focus on metabolites in promoting intestinal inflammation or colorectal tumorigenesis. In addition, we will provide a perspective on the role of newly identified oncometabolites from the commensal and opportunistic microbiota in shaping response and resistance to antitumor therapy.
RESUMO
Growth and metastasis of colorectal cancer is closely connected to the biosynthetic capacity of tumor cells, and colorectal cancer stem cells that reside at the top of the intratumoral hierarchy are especially dependent on this feature. By performing disease modeling on patient-derived tumor organoids, we found that elevated expression of the ribosome biogenesis factor NLE1 occurs upon SMAD4 loss in TGFß1-exposed colorectal cancer organoids. TGFß signaling-mediated downregulation of NLE1 was prevented by ectopic expression of c-MYC, which occupied an E-box-containing region within the NLE1 promoter. Elevated levels of NLE1 were found in colorectal cancer cohorts compared with normal tissues and in colorectal cancer subtypes characterized by Wnt/MYC and intestinal stem cell gene expression. In colorectal cancer cells and organoids, NLE1 was limiting for de novo protein biosynthesis. Upon NLE1 ablation, colorectal cancer cell lines activated p38/MAPK signaling, accumulated p62- and LC3-positive structures indicative of impaired autophagy, and displayed more reactive oxygen species. Phenotypically, knockout of NLE1 inhibit.ed proliferation, migration and invasion, clonogenicity, and anchorage-independent growth. NLE1 loss also increased the fraction of apoptotic tumor cells, and deletion of TP53 further sensitized NLE1-deficient colorectal cancer cells to apoptosis. In an endoscopy-guided orthotopic mouse transplantation model, ablation of NLE1 impaired tumor growth in the colon and reduced primary tumor-derived liver metastasis. In patients with colorectal cancer, NLE1 mRNA levels predicted overall and relapse-free survival. Taken together, these data reveal a critical role of NLE1 in colorectal cancer growth and progression and suggest that NLE1 represents a potential therapeutic target in colorectal cancer patients. SIGNIFICANCE: NLE1 limits de novo protein biosynthesis and the tumorigenic potential of advanced colorectal cancer cells, suggesting NLE1 could be targeted to improve the treatment of metastatic colorectal cancer.
Assuntos
Neoplasias Colorretais , Genes myc , Proteínas dos Microfilamentos , Proteína Smad4 , Animais , Camundongos , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Camundongos Nus , Proteínas dos Microfilamentos/genética , Biossíntese de Proteínas , Proteína Smad4/genética , Regulação para Cima , HumanosRESUMO
Mast cells (MCs) are crucial players in the relationship between the tumor microenvironment (TME) and cancer cells and have been shown to influence angiogenesis and progression of human colorectal cancer (CRC). However, the role of MCs in the TME is controversially discussed as either pro- or anti-tumorigenic. Genetically engineered mouse models (GEMMs) are the most frequently used in vivo models for human CRC research. In the murine intestine there are at least three different MC subtypes: interepithelial mucosal mast cells (ieMMCs), lamina proprial mucosal mast cells (lpMMCs) and connective tissue mast cells (CTMCs). Interepithelial mucosal mast cells (ieMMCs) in (pre-)neoplastic intestinal formalin-fixed paraffin-embedded (FFPE) specimens of mouse models (total lesions n = 274) and human patients (n = 104) were immunohistochemically identified and semiquantitatively scored. Scores were analyzed along the adenoma-carcinoma sequence in humans and 12 GEMMs of small and large intestinal cancer. The presence of ieMMCs was a common finding in intestinal adenomas and carcinomas in mice and humans. The number of ieMMCs decreased in the course of colonic adenoma-carcinoma sequence in both species (p < 0.001). However, this dynamic cellular state was not observed for small intestinal murine tumors. Furthermore, ieMMC scores were higher in GEMMs with altered Wnt signaling (active ß-catenin) than in GEMMs with altered MAPK signaling and wildtypes (WT). In conclusion, we hypothesize that, besides stromal MCs (lpMMCs/CTMCs), particularly the ieMMC subset is important for onset and progression of intestinal neoplasia and may interact with the adjacent neoplastic epithelial cells in dependence on the molecular environment. Moreover, our study indicates the need for adequate GEMMs for the investigation of the intestinal immunologic TME.
RESUMO
Colorectal cancer (CRC) is a complex condition with heterogeneous aetiology, caused by a combination of various environmental, genetic, and epigenetic factors. The presence of a homeostatic gut microbiota is critical to maintaining host homeostasis and determines the delicate boundary between health and disease. The gut microbiota has been identified as a key environmental player in the pathogenesis of CRC. Perturbations of the gut microbiota structure (loss of equilibrium and homeostasis) are associated with several intestinal diseases including cancer. Such dysbiosis encompasses the loss of beneficial microorganisms, outgrowth of pathogens and pathobionts and a general loss of local microbiota diversity and richness. Notably, several mechanisms have recently been identified how bacteria induce cellular transformation and promote tumour progression. In particular, the formation of biofilms, the production of toxic metabolites or the secretion of genotoxins that lead to DNA damage in intestinal epithelial cells are newly discovered processes by which the microbiota can initiate tumour formation. The gut microbiota has also been implicated in the metabolism of therapeutic drugs (conventional chemotherapy) as well as in the modulation of radiotherapy responses and targeted immunotherapy. These new findings suggest that the efficacy of a given therapy depends on the composition of the host's gut microbiota and may therefore vary from patient to patient. In this review we discuss the role of host-microbiota interactions in cancer with a focus on CRC pathogenesis. Additionally, we show how gut bacteria can be exploited in current therapies and how mechanisms directed by microbiota, such as immune cell boost, probiotics and oncolytic bacteria, can be applied in the development of novel therapies.
RESUMO
BACKGROUND: Special AT-rich sequence-binding protein 2 (SATB2) has emerged as an alternative immunohistochemical marker to CDX2 for colorectal differentiation. However, the distribution and prognostic relevance of SATB2 expression in colorectal carcinoma (CRC) have to be further elucidated. METHODS: SATB2 expression was analysed in 1039 CRCs and correlated with clinicopathological and morphological factors, CDX2 expression as well as survival parameters within the overall cohort and in clinicopathological subgroups. RESULTS: SATB2 loss was a strong prognosticator in univariate analyses of the overall cohort (p < 0.001 for all survival comparisons) and in numerous subcohorts including high-risk scenarios (UICC stage III/high tumour budding). SATB2 retained its prognostic relevance in multivariate analyses of these high-risk scenarios (e.g., UICC stage III: DSS: p = 0.007, HR: 1.95), but not in the overall cohort (DSS: p = 0.1, HR: 1.25). SATB2 loss was more frequent than CDX2 loss (22.2% vs. 10.2%, p < 0.001) and of higher prognostic relevance with only moderate overlap between SATB2/CDX2 expression groups. CONCLUSIONS: SATB2 loss is able to identify especially aggressive CRCs in high-risk subgroups. While SATB2 is the prognostically superior immunohistochemical parameter compared to CDX2 in univariate analyses, it appears to be the less sensitive marker for colorectal differentiation as it is lost more frequently.
RESUMO
The 2019 World Health Organization (WHO) classification of colorectal carcinoma (CRC) profoundly reclassified CRC subtypes and introduces tumor budding as a second major grading criterion, while condensing conventional grade into a 2-tiered system. So far it remains largely unexplored how these parameters interact with each other and whether they truly have an independent impact on patient prognosis. We reclassified a large single-center cohort of 1004 CRCs spanning 2 decades for adjusted WHO grade (low vs. high), tumor budding (Bd1/Bd2/Bd3), and CRC subtype (adenocarcinoma not otherwise specified, micropapillary, mucinous, serrated, medullary, adenoma-like, signet-ring cell, mixed adenoneuroendocrine carcinoma/neuroendocrine carcinoma, undifferentiated) according to the criteria of the 2019 WHO classification. We investigated the interaction of these parameters, their connection to stage/microsatellite status, and their significance for patient survival in the different subgroups. Specific subtypes other than adenocarcinoma not otherwise specified represented one third of all CRCs and were unevenly distributed throughout stage and microsatellite subgroups. Subtypes, WHO grade and tumor budding profoundly impacted all survival parameters (P<0.001 for all analyses), with CRC subtypes and tumor budding-but not WHO grade-being stage-independent prognosticators for all survival comparisons. WHO grade had very limited prognostic value in CRC subtypes, while tumor budding retained its strong prognostic impact in most scenarios. Accurate delineation of CRC subtypes introduced in the 2019 WHO classification provides strong stage-independent prognostic information, arguing that they should be considered in pathology reports and in clinical trials. Of the morphology-based grading schemes included in the 2019 WHO, tumor budding outperforms WHO grade.
Assuntos
Carcinoma/patologia , Movimento Celular , Neoplasias Colorretais/patologia , Idoso , Biópsia , Carcinoma/classificação , Carcinoma/mortalidade , Carcinoma/cirurgia , Colectomia , Neoplasias Colorretais/classificação , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/cirurgia , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Instabilidade de Microssatélites , Gradação de Tumores , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Organização Mundial da SaúdeRESUMO
Biliary tract cancer ranks among the most lethal human malignancies, representing an unmet clinical need. Its abysmal prognosis is tied to an increasing incidence and a fundamental lack of mechanistic knowledge regarding the molecular basis of the disease. Here, we show that the Pdx1-positive extrahepatic biliary epithelium is highly susceptible toward transformation by activated PIK3CAH1047R but refractory to oncogenic KrasG12D. Using genome-wide transposon screens and genetic loss-of-function experiments, we discover context-dependent genetic interactions that drive extrahepatic cholangiocarcinoma (ECC) and show that PI3K signaling output strength and repression of the tumor suppressor p27Kip1 are critical context-specific determinants of tumor formation. This contrasts with the pancreas, where oncogenic Kras in concert with p53 loss is a key cancer driver. Notably, inactivation of p27Kip1 permits KrasG12D-driven ECC development. These studies provide a mechanistic link between PI3K signaling, tissue-specific tumor suppressor barriers, and ECC pathogenesis, and present a novel genetic model of autochthonous ECC and genes driving this highly lethal tumor subtype. SIGNIFICANCE: We used the first genetically engineered mouse model for extrahepatic bile duct carcinoma to identify cancer genes by genome-wide transposon-based mutagenesis screening. Thereby, we show that PI3K signaling output strength and p27Kip1 function are critical determinants for context-specific ECC formation. This article is highlighted in the In This Issue feature, p. 2945.