ABSTRACT
INTRODUCTION: Minimally invasive liver resection (MILR) is performed for other gastrointestinal applications. At our centre, all liver resections are systematically performed using a minimally invasive approach. This study aimed to describe our experience in minimising open surgery and emphasised the importance of minimally invasive surgery. PATIENTS AND METHODS: We retrospectively reviewed 260 patients who underwent liver surgery and compared the surgical outcomes between the open and MILR groups. RESULTS: A total of 154 patients (68%) underwent MILR. The proportion of patients who underwent prior abdominal surgery and resection was higher in the open surgery group. However, the proportion of patients with liver cirrhosis was similar between the two groups. The MILR group was superior in terms of operative time, blood loss, Pringle manoeuvre rate and mean hospital stay. In addition, major complication and bile leak rates were lower in the MILR group. No significant differences in the tumour size, number of lesions or underlying liver pathology were observed between the two groups. CONCLUSION: Acceptable outcomes can be achieved even when the minimally invasive approach is considered the primary option for all patients who require liver resection. Minimally invasive tools are necessary for the modern practice of liver surgery; therefore, laparoscopic or robotic surgery should be included in the armamentarium of liver surgeons.
ABSTRACT
PURPOSE: Intrahepatic cholangiocarcinoma (IHC) is a heterogeneous tumor. The hidden-genome classifier, a supervised machine learning-based algorithm, was used to quantify tumor heterogeneity and improve classification. EXPERIMENTAL DESIGN: A retrospective review of 1,370 patients with IHC, extrahepatic cholangiocarcinoma (EHC), gallbladder cancer (GBC), hepatocellular carcinoma (HCC), or biphenotypic tumors was conducted. A hidden-genome model classified 527 IHC based on genetic similarity to EHC/GBC or HCC. Genetic, histologic, and clinical data were correlated. RESULTS: In this study, 410 IHC (78%) had >50% genetic homology with EHC/GBC; 122 (23%) had >90% homology ("biliary class"), characterized by alterations of KRAS, SMAD4, and CDKN2A loss; 117 IHC (22%) had >50% genetic homology with HCC; and 30 (5.7%) had >90% homology ("HCC class"), characterized by TERT alterations. Patients with biliary- versus non-biliary-class IHC had median overall survival (OS) of 1 year (95% CI, 0.77, 1.5) versus 1.8 years (95% CI, 1.6, 2.0) for unresectable disease and 2.4 years (95% CI, 2.1, NR) versus 5.1 years (95% CI, 4.8, 6.9) for resectable disease. Large-duct IHC (n = 28) was more common in the biliary class (n = 27); the HCC class was composed mostly of small-duct IHC (64%, P = 0.02). The hidden genomic classifier predicted OS independent of FGFR2 and IDH1 alterations. By contrast, the histology subtype did not predict OS. CONCLUSIONS: IHC genetics form a spectrum with worse OS for tumors genetically aligned with EHC/GBC. The classifier proved superior to histologic subtypes for predicting OS independent of FGFR2 and IDH1 alterations. These results may explain the differential treatment responses seen in IHC and may direct therapy by helping stratify patients in future clinical trials.
Subject(s)
Bile Duct Neoplasms , Cholangiocarcinoma , Humans , Cholangiocarcinoma/genetics , Cholangiocarcinoma/pathology , Cholangiocarcinoma/mortality , Male , Female , Bile Duct Neoplasms/genetics , Bile Duct Neoplasms/pathology , Bile Duct Neoplasms/mortality , Middle Aged , Aged , Retrospective Studies , Receptor, Fibroblast Growth Factor, Type 2/genetics , Isocitrate Dehydrogenase/genetics , Biomarkers, Tumor/genetics , Adult , Mutation , Prognosis , Genetic Heterogeneity , Algorithms , Aged, 80 and over , Machine Learning , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/mortality , Gallbladder Neoplasms/genetics , Gallbladder Neoplasms/pathology , Gallbladder Neoplasms/mortality , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Liver Neoplasms/mortalityABSTRACT
KRAS mutations, mainly G12D and G12V, are found in more than 90% of pancreatic ductal adenocarcinoma (PDAC) cases. The success of drugs targeting KRASG12C suggests the potential for drugs specifically targeting these alternative PDAC-associated KRAS mutations. Here, we report a high-throughput drug-screening platform using a series of isogenic murine pancreatic organoids that are wild type (WT) or contain common PDAC driver mutations, representing both classical and basal PDAC phenotypes. We screened over 6,000 compounds and identified perhexiline maleate, which can inhibit the growth and induce cell death of pancreatic organoids carrying the KrasG12D mutation both in vitro and in vivo and primary human PDAC organoids. scRNA-seq analysis suggests that the cholesterol synthesis pathway is upregulated specifically in the KRAS mutant organoids, including the key cholesterol synthesis regulator SREBP2. Perhexiline maleate decreases SREBP2 expression levels and reverses the KRAS mutant-induced upregulation of the cholesterol synthesis pathway.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Animals , Mice , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Mutation/genetics , Organoids/metabolism , CholesterolABSTRACT
Inflammation is essential to the disruption of tissue homeostasis and can destabilize the identity of lineage-committed epithelial cells. Here, we employ lineage-traced mouse models, single-cell transcriptomic and chromatin analyses, and CUT&TAG to identify an epigenetic memory of inflammatory injury in the pancreatic acinar cell compartment. Despite resolution of pancreatitis, our data show that acinar cells fail to return to their molecular baseline, with retention of elevated chromatin accessibility and H3K4me1 at metaplasia genes, such that memory represents an incomplete cell fate decision. In vivo, we find this epigenetic memory controls lineage plasticity, with diminished metaplasia in response to a second insult but increased tumorigenesis with an oncogenic Kras mutation. The lowered threshold for oncogenic transformation, in turn, can be restored by blockade of MAPK signaling. Together, we define the chromatin dynamics, molecular encoding, and recall of a prolonged epigenetic memory of inflammatory injury that impacts future responses but remains reversible.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Mice , Animals , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Epigenetic Memory , Cell Transformation, Neoplastic/pathology , Acinar Cells/pathology , Pancreas/pathology , Chromatin/genetics , Metaplasia/pathology , Carcinoma, Pancreatic Ductal/geneticsABSTRACT
Introduction: Transcriptional activation depends on the interplay of chromatin modifiers to establish a permissive epigenetic landscape. While histone 3 lysine 9 (H3K9) methylation has long been associated with gene repression, there is limited evidence to support a role for H3K9 demethylases in gene activation. Methods: We leveraged knockdown and overexpression of JMJD2d / Kdm4d in mouse embryonic fibroblasts, coupled with extensive epigenomic analysesm to decipher the role of histone 3 lysine 9 demethylases in the innate immune response. Results: Here we describe the H3K9 demethylase Kdm4d/JMJD2d as a positive regulator of type I interferon responses. In mouse embryonic fibroblasts (MEFs), depletion of JMJD2d attenuates the transcriptional response, conferring increased viral susceptibility, while overexpression of the demethylase results in more robust IFN activation. We find that the underlying mechanism of JMJD2d in type I interferon responses consists of an effect both on the transcription of enhancer RNAs (eRNAs) and on dynamic H3K9me2 at associated promoters. In support of these findings, we establish that JMJD2d is associated with enhancer regions throughout the genome prior to stimulation but is redistributed to inducible promoters in conjunction with transcriptional activation. Discussion: Taken together, our data reveal JMJD2d as a chromatin modifier that connects enhancer transcription with promoter demethylation to modulate transcriptional responses.
Subject(s)
Histone Demethylases , Interferon Type I , Animals , Mice , Histone Demethylases/genetics , Histone Demethylases/metabolism , Histones/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Interferon Type I/genetics , Lysine/genetics , Fibroblasts/metabolism , Chromatin/geneticsABSTRACT
OBJECTIVE: To determine whether genomic risk groups identified by somatic mutation testing of colorectal liver metastasis (CRLM) can be used for "molecularly-guided" selection for adjuvant systemic chemotherapy and hepatic artery infusion of FUDR (SYS+HAI-FUDR). BACKGROUND: Several genomic biomarkers have been associated with clinical phenotype and survival for patients with resectable CRLM. It is unknown whether prognostication afforded by genomic stratification translates into enhanced patient selection for adjuvant hepatic artery infusion therapy. METHODS: Consecutive patients with resected CRLM and available mutational characterization via Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets were reviewed from a prospective institutional database. Patients were stratified into three genomic risk groups based on previously defined alterations in SMAD4, EGFR and the RAS/RAF pathway. The association between SYS+HAI-FUDR and overall survival, relative to adjuvant chemotherapy alone (SYS), was evaluated in each genomic risk group by Cox proportional hazard regression and propensity score matched analyses. RESULTS: A total of 334 patients (SYS+HAI-FUDR 204; SYS 130) were identified; the rates of RAS/RAF alterations and SMAD4 inactivation were 47.4% and 11.7%, respectively. After a median follow-up of 58âmonths, adjuvant SYS+HAI-FUDR was independently associated with a reduced risk of death (HR 0.50, 95%CI 0.26-0.98, P = 0.045) in the low-risk genomic group, but not in the moderate-risk (HR 1.07, 95%CI 0.5-2.07, P = 0.749) or high-risk (HR 1.62, 95%CI 0.29-9.12, P = 0.537) cohorts. Following propensity score matching, adjuvant SYS+HAI-FUDR remained associated with significant improvements in long-term survival selectively in the low-risk genomic cohort (5-year actuarial survival: 89% vs. 68%, P = 0.019). CONCLUSIONS: Genomic alterations in RAS/RAF, SMAD4, and EGFR may be useful to guide treatment selection in resectable CRLM patients and warrant external validation and integration in future clinical trial design.
Subject(s)
Colorectal Neoplasms/pathology , Liver Neoplasms/genetics , Liver Neoplasms/secondary , Aged , Chemotherapy, Adjuvant , Female , Genome , Humans , Liver Neoplasms/mortality , Liver Neoplasms/therapy , Male , Middle Aged , Mutation , Risk Assessment , Survival RateABSTRACT
The pancreas consists of several specialized cell types that display a remarkable ability to alter cellular identity in injury, regeneration, and repair. The abundant cellular plasticity within the pancreas appears to be exploited in tumorigenesis, with metaplastic, dedifferentiation, and transdifferentiation processes central to the development of pancreatic intraepithelial neoplasia and intraductal papillary neoplasms, precursor lesions to pancreatic ductal adenocarcinoma. In the face of shifting cellular identity, the cell of origin of pancreatic cancer has been difficult to elucidate. However, with the extensive utilization of in vivo lineage-traced mouse models coupled with insights from human samples, it has emerged that the acinar cell is most efficiently able to give rise to both intraductal papillary neoplasms and pancreatic intraepithelial neoplasia but that acinar and ductal cells can undergo malignant transformation to pancreatic ductal adenocarcinoma. In this review, we discuss the cellular reprogramming that takes place in both the normal and malignant pancreas and evaluate the current state of evidence that implicate both the acinar and ductal cell as context-dependent origins of this deadly disease.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Acinar Cells/pathology , Animals , Carcinoma, Pancreatic Ductal/pathology , Cell Plasticity , Mice , Pancreas/pathology , Pancreatic Neoplasms/pathologyABSTRACT
SARS-CoV-2, the etiological agent of COVID-19, is characterized by a delay in type I interferon (IFN-I)-mediated antiviral defenses alongside robust cytokine production. Here, we investigate the underlying molecular basis for this imbalance and implicate virus-mediated activation of NF-κB in the absence of other canonical IFN-I-related transcription factors. Epigenetic and single-cell transcriptomic analyses show a selective NF-κB signature that was most prominent in infected cells. Disruption of NF-κB signaling through the silencing of the NF-κB transcription factor p65 or p50 resulted in loss of virus replication that was rescued upon reconstitution. These findings could be further corroborated with the use of NF-κB inhibitors, which reduced SARS-CoV-2 replication in vitro. These data suggest that the robust cytokine production in response to SARS-CoV-2, despite a diminished IFN-I response, is the product of a dependency on NF-κB for viral replication. IMPORTANCE The COVID-19 pandemic has caused significant mortality and morbidity around the world. Although effective vaccines have been developed, large parts of the world remain unvaccinated while new SARS-CoV-2 variants keep emerging. Furthermore, despite extensive efforts and large-scale drug screenings, no fully effective antiviral treatment options have been discovered yet. Therefore, it is of the utmost importance to gain a better understanding of essential factors driving SARS-CoV-2 replication to be able to develop novel approaches to target SARS-CoV-2 biology.
Subject(s)
COVID-19/metabolism , Cytokines/metabolism , Interferon Type I/metabolism , SARS-CoV-2 , Transcription Factor RelA/metabolism , Transcriptome , Virus Replication , A549 Cells , Animals , COVID-19/virology , Chlorocebus aethiops , Epigenomics , Gene Expression Regulation , HEK293 Cells , HeLa Cells , Host Microbial Interactions , Humans , Signal Transduction , Single-Cell Analysis , Transcription Factor RelA/antagonists & inhibitors , Transcription Factor RelA/genetics , Transcription Factors/metabolism , Vero CellsABSTRACT
PURPOSE: The clinical behavior of ampullary adenocarcinoma varies widely. Targeted tumor sequencing may better define biologically distinct subtypes to improve diagnosis and management. EXPERIMENTAL DESIGN: The hidden-genome algorithm, a multilevel meta-feature regression model, was trained on a prospectively sequenced cohort of 3,411 patients (1,001 pancreatic adenocarcinoma, 165 distal bile-duct adenocarcinoma, 2,245 colorectal adenocarcinoma) and subsequently applied to targeted panel DNA-sequencing data from ampullary adenocarcinomas. Genomic classification (i.e., colorectal vs. pancreatic) was correlated with standard histologic classification [i.e., intestinal (INT) vs. pancreatobiliary (PB)] and clinical outcome. RESULTS: Colorectal genomic subtype prediction was primarily influenced by mutations in APC and PIK3CA, tumor mutational burden, and DNA mismatch repair (MMR)-deficiency signature. Pancreatic genomic-subtype prediction was dictated by KRAS gene alterations, particularly KRAS G12D, KRAS G12R, and KRAS G12V. Distal bile-duct adenocarcinoma genomic subtype was most influenced by copy-number gains in the MDM2 gene. Despite high (73%) concordance between immunomorphologic subtype and genomic category, there was significant genomic heterogeneity within both histologic subtypes. Genomic scores with higher colorectal probability were associated with greater survival compared with those with a higher pancreatic probability. CONCLUSIONS: The genomic classifier provides insight into the heterogeneity of ampullary adenocarcinoma and improves stratification, which is dictated by the proportion of colorectal and pancreatic genomic alterations. This approach is reproducible with available molecular testing and obviates subjective histologic interpretation.
Subject(s)
Adenocarcinoma/classification , Adenocarcinoma/genetics , Ampulla of Vater , Colorectal Neoplasms/classification , Colorectal Neoplasms/genetics , Common Bile Duct Neoplasms/classification , Common Bile Duct Neoplasms/genetics , Duodenal Neoplasms/classification , Duodenal Neoplasms/genetics , Genome , Aged , Correlation of Data , Female , Humans , Male , Middle AgedABSTRACT
BACKGROUND AND AIM: Genetic alterations in intrahepatic cholangiocarcinoma (iCCA) are increasingly well characterized, but their impact on outcome and prognosis remains unknown. APPROACH AND RESULTS: This bi-institutional study of patients with confirmed iCCA (n = 412) used targeted next-generation sequencing of primary tumors to define associations among genetic alterations, clinicopathological variables, and outcome. The most common oncogenic alterations were isocitrate dehydrogenase 1 (IDH1; 20%), AT-rich interactive domain-containing protein 1A (20%), tumor protein P53 (TP53; 17%), cyclin-dependent kinase inhibitor 2A (CDKN2A; 15%), breast cancer 1-associated protein 1 (15%), FGFR2 (15%), polybromo 1 (12%), and KRAS (10%). IDH1/2 mutations (mut) were mutually exclusive with FGFR2 fusions, but neither was associated with outcome. For all patients, TP53 (P < 0.0001), KRAS (P = 0.0001), and CDKN2A (P < 0.0001) alterations predicted worse overall survival (OS). These high-risk alterations were enriched in advanced disease but adversely impacted survival across all stages, even when controlling for known correlates of outcome (multifocal disease, lymph node involvement, bile duct type, periductal infiltration). In resected patients (n = 209), TP53mut (HR, 1.82; 95% CI, 1.08-3.06; P = 0.03) and CDKN2A deletions (del; HR, 3.40; 95% CI, 1.95-5.94; P < 0.001) independently predicted shorter OS, as did high-risk clinical variables (multifocal liver disease [P < 0.001]; regional lymph node metastases [P < 0.001]), whereas KRASmut (HR, 1.69; 95% CI, 0.97-2.93; P = 0.06) trended toward statistical significance. The presence of both or neither high-risk clinical or genetic factors represented outcome extremes (median OS, 18.3 vs. 74.2 months; P < 0.001), with high-risk genetic alterations alone (median OS, 38.6 months; 95% CI, 28.8-73.5) or high-risk clinical variables alone (median OS, 37.0 months; 95% CI, 27.6-not available) associated with intermediate outcome. TP53mut, KRASmut, and CDKN2Adel similarly predicted worse outcome in patients with unresectable iCCA. CDKN2Adel tumors with high-risk clinical features were notable for limited survival and no benefit of resection over chemotherapy. CONCLUSIONS: TP53, KRAS, and CDKN2A alterations were independent prognostic factors in iCCA when controlling for clinical and pathologic variables, disease stage, and treatment. Because genetic profiling can be integrated into pretreatment therapeutic decision-making, combining clinical variables with targeted tumor sequencing may identify patient subgroups with poor outcome irrespective of treatment strategy.
Subject(s)
Bile Duct Neoplasms/genetics , Bile Ducts, Intrahepatic , Cholangiocarcinoma/genetics , Adult , Aged , Aged, 80 and over , Bile Duct Neoplasms/therapy , Biliary Tract Surgical Procedures , Chemotherapy, Adjuvant , Cholangiocarcinoma/therapy , Cyclin-Dependent Kinase Inhibitor p16/genetics , DNA-Binding Proteins/genetics , Female , Humans , Isocitrate Dehydrogenase/genetics , Male , Middle Aged , Mutation , Neoadjuvant Therapy , Prognosis , Proto-Oncogene Proteins p21(ras)/genetics , Receptor, Fibroblast Growth Factor, Type 2/genetics , Transcription Factors/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/genetics , Young AdultABSTRACT
Tissue damage increases the risk of cancer through poorly understood mechanisms1. In mouse models of pancreatic cancer, pancreatitis associated with tissue injury collaborates with activating mutations in the Kras oncogene to markedly accelerate the formation of early neoplastic lesions and, ultimately, adenocarcinoma2,3. Here, by integrating genomics, single-cell chromatin assays and spatiotemporally controlled functional perturbations in autochthonous mouse models, we show that the combination of Kras mutation and tissue damage promotes a unique chromatin state in the pancreatic epithelium that distinguishes neoplastic transformation from normal regeneration and is selected for throughout malignant evolution. This cancer-associated epigenetic state emerges within 48 hours of pancreatic injury, and involves an 'acinar-to-neoplasia' chromatin switch that contributes to the early dysregulation of genes that define human pancreatic cancer. Among the factors that are most rapidly activated after tissue damage in the pre-malignant pancreatic epithelium is the alarmin cytokine interleukin 33, which recapitulates the effects of injury in cooperating with mutant Kras to unleash the epigenetic remodelling program of early neoplasia and neoplastic transformation. Collectively, our study demonstrates how gene-environment interactions can rapidly produce gene-regulatory programs that dictate early neoplastic commitment, and provides a molecular framework for understanding the interplay between genetic and environmental cues in the initiation of cancer.
Subject(s)
Cell Transformation, Neoplastic/genetics , Epigenesis, Genetic , Gene-Environment Interaction , Pancreas/metabolism , Pancreas/pathology , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Transformation, Neoplastic/pathology , Chromatin/genetics , Chromatin/metabolism , Chromatin/pathology , Disease Models, Animal , Female , Genomics , Humans , Interleukin-33/metabolism , Male , Mice , Mice, Inbred C57BL , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at an advanced stage, which limits surgical options and portends a dismal prognosis. Current oncologic PDAC therapies confer marginal benefit and, thus, a significant unmet clinical need exists for new therapeutic strategies. To identify effective PDAC therapies, we leveraged a syngeneic orthotopic PDAC transplant mouse model to perform a large-scale, in vivo screen of 16 single-agent and 41 two-drug targeted therapy combinations in mice. Among 57 drug conditions screened, combined inhibition of heat shock protein (Hsp)-90 and MEK was found to produce robust suppression of tumor growth, leading to an 80% increase in the survival of PDAC-bearing mice with no significant toxicity. Mechanistically, we observed that single-agent MEK inhibition led to compensatory activation of resistance pathways, including components of the PI3K/AKT/mTOR signaling axis, which was overcome with the addition of HSP90 inhibition. The combination of HSP90(i) + MEK(i) was also active in vitro in established human PDAC cell lines and in vivo in patient-derived organoid PDAC transplant models. These findings encourage the clinical development of HSP90(i) + MEK(i) combination therapy and highlight the power of clinically relevant in vivo model systems for identifying cancer therapies.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Evaluation, Preclinical , Drug Screening Assays, Antitumor , Adenocarcinoma/metabolism , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Animals , Antineoplastic Agents/therapeutic use , Benzodioxoles/pharmacology , Biomarkers, Tumor , Cell Line, Tumor , Disease Models, Animal , Drug Evaluation, Preclinical/methods , Drug Screening Assays, Antitumor/methods , Drug Synergism , Gene Expression , Humans , Immunohistochemistry , MAP Kinase Signaling System/drug effects , Mice , Molecular Targeted Therapy , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/mortality , Pancreatic Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Purines/pharmacology , Pyridones/pharmacology , Pyrimidinones/pharmacology , Signal Transduction/drug effects , Survival Rate , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
The tumour suppressor TP53 is mutated in the majority of human cancers, and in over 70% of pancreatic ductal adenocarcinoma (PDAC)1,2. Wild-type p53 accumulates in response to cellular stress, and regulates gene expression to alter cell fate and prevent tumour development2. Wild-type p53 is also known to modulate cellular metabolic pathways3, although p53-dependent metabolic alterations that constrain cancer progression remain poorly understood. Here we find that p53 remodels cancer-cell metabolism to enforce changes in chromatin and gene expression that favour a premalignant cell fate. Restoring p53 function in cancer cells derived from KRAS-mutant mouse models of PDAC leads to the accumulation of α-ketoglutarate (αKG, also known as 2-oxoglutarate), a metabolite that also serves as an obligate substrate for a subset of chromatin-modifying enzymes. p53 induces transcriptional programs that are characteristic of premalignant differentiation, and this effect can be partially recapitulated by the addition of cell-permeable αKG. Increased levels of the αKG-dependent chromatin modification 5-hydroxymethylcytosine (5hmC) accompany the tumour-cell differentiation that is triggered by p53, whereas decreased 5hmC characterizes the transition from premalignant to de-differentiated malignant lesions that is associated with mutations in Trp53. Enforcing the accumulation of αKG in p53-deficient PDAC cells through the inhibition of oxoglutarate dehydrogenase-an enzyme of the tricarboxylic acid cycle-specifically results in increased 5hmC, tumour-cell differentiation and decreased tumour-cell fitness. Conversely, increasing the intracellular levels of succinate (a competitive inhibitor of αKG-dependent dioxygenases) blunts p53-driven tumour suppression. These data suggest that αKG is an effector of p53-mediated tumour suppression, and that the accumulation of αKG in p53-deficient tumours can drive tumour-cell differentiation and antagonize malignant progression.
Subject(s)
Carcinoma, Pancreatic Ductal , Cell Differentiation/genetics , Ketoglutaric Acids/metabolism , Pancreatic Neoplasms , Tumor Suppressor Protein p53/metabolism , Animals , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/physiopathology , Cell Line, Tumor , Chromatin Assembly and Disassembly/drug effects , Chromatin Assembly and Disassembly/genetics , Disease Models, Animal , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Ketoglutaric Acids/pharmacology , Mice , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/physiopathology , Protein Binding , Succinic Acid/metabolism , Transcriptional ActivationABSTRACT
Synovial sarcoma is an aggressive cancer invariably associated with a chromosomal translocation involving genes encoding the SWI-SNF complex component SS18 and an SSX (SSX1 or SSX2) transcriptional repressor. Using functional genomics, we identify KDM2B, a histone demethylase and component of a non-canonical polycomb repressive complex 1 (PRC1.1), as selectively required for sustaining synovial sarcoma cell transformation. SS18-SSX1 physically interacts with PRC1.1 and co-associates with SWI/SNF and KDM2B complexes on unmethylated CpG islands. Via KDM2B, SS18-SSX1 binds and aberrantly activates expression of developmentally regulated genes otherwise targets of polycomb-mediated repression, which is restored upon KDM2B depletion, leading to irreversible mesenchymal differentiation. Thus, SS18-SSX1 deregulates developmental programs to drive transformation by hijacking a transcriptional repressive complex to aberrantly activate gene expression.
Subject(s)
F-Box Proteins/metabolism , Jumonji Domain-Containing Histone Demethylases/metabolism , Neoplasm Proteins/metabolism , Polycomb Repressive Complex 1/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , Sarcoma, Synovial/metabolism , Cell Nucleus/metabolism , Cell Transformation, Neoplastic/genetics , Gene Expression/physiology , Humans , Neoplasm Proteins/genetics , Oncogene Proteins, Fusion/genetics , Repressor Proteins/genetics , Sarcoma, Synovial/geneticsABSTRACT
Cystic neoplasms of the pancreas are being identified at an increasing frequency largely due to the increased use of abdominal cross-sectional imaging. These neoplasms represent a heterogeneous group of tumors with various genetic alterations, molecular features, and risks of malignancy. Despite the use of high-resolution radiographic studies, endoscopic evaluation, cyst fluid analysis, and novel molecular diagnostics, many of these lesions remain difficult to classify without operative resection. These diagnostic challenges are coupled with an improving but limited understanding of the natural history of these neoplasms. Treatment of pancreatic cystic neoplasms therefore remains controversial but consists largely of a selective tumor-specific approach to surgical resection. Future research remains necessary to better discriminate the biological behavior of these tumors in order to more appropriately select patients for operative intervention.
Subject(s)
Neoplasms, Cystic, Mucinous, and Serous/diagnosis , Pancreatic Neoplasms/diagnosis , Cyst Fluid/chemistry , Cystadenoma, Serous/diagnosis , Cystadenoma, Serous/therapy , Endosonography , Humans , Neoplasms, Cystic, Mucinous, and Serous/epidemiology , Neoplasms, Cystic, Mucinous, and Serous/genetics , Neoplasms, Cystic, Mucinous, and Serous/therapy , Pancreatic Ducts , Pancreatic Neoplasms/epidemiology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/therapyABSTRACT
Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3' untranslated region (3'UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3'UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3'UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3'UTR targeting as to harness the activity of several Ago proteins.
Subject(s)
Argonaute Proteins/metabolism , Eukaryotic Initiation Factors/metabolism , Fibroblasts/cytology , Liver/metabolism , RNA, Messenger/genetics , Animals , Argonaute Proteins/genetics , Cells, Cultured , Embryo, Mammalian/cytology , Female , Fibroblasts/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , NIH 3T3 Cells , RNA InterferenceABSTRACT
Effective antiviral immunity depends on the ability of infected cells or cells triggered with virus-derived nucleic acids to produce type I interferon (IFN), which activates transcription of numerous antiviral genes. However, disproportionately strong or chronic IFN expression is a common cause of inflammatory and autoimmune diseases. We describe an epigenetic mechanism that determines cell type-specific differences in IFN and IFN-stimulated gene (ISG) expression in response to exogenous signals. We identify di-methylation of histone H3 at lysine 9 (H3K9me2) as a suppressor of IFN and IFN-inducible antiviral gene expression. We show that levels of H3K9me2 at IFN and ISG correlate inversely with the scope and amplitude of IFN and ISG expression in fibroblasts and dendritic cells. Accordingly, genetic ablation or pharmacological inactivation of lysine methyltransferase G9a, which is essential for the generation of H3K9me2, resulted in phenotypic conversion of fibroblasts into highly potent IFN-producing cells and rendered these cells resistant to pathogenic RNA viruses. In summary, our studies implicate H3K9me2 and enzymes controlling its abundance as key regulators of innate antiviral immunity.
Subject(s)
Epigenesis, Genetic , Histones/metabolism , Interferons/biosynthesis , Virus Diseases/immunology , Animals , Histone-Lysine N-Methyltransferase/physiology , Immunity, Innate , Methylation , Mice , Mice, Inbred C57BLABSTRACT
Interaction of pathogens with cells of the immune system results in activation of inflammatory gene expression. This response, although vital for immune defence, is frequently deleterious to the host due to the exaggerated production of inflammatory proteins. The scope of inflammatory responses reflects the activation state of signalling proteins upstream of inflammatory genes as well as signal-induced assembly of nuclear chromatin complexes that support mRNA expression. Recognition of post-translationally modified histones by nuclear proteins that initiate mRNA transcription and support mRNA elongation is a critical step in the regulation of gene expression. Here we present a novel pharmacological approach that targets inflammatory gene expression by interfering with the recognition of acetylated histones by the bromodomain and extra terminal domain (BET) family of proteins. We describe a synthetic compound (I-BET) that by 'mimicking' acetylated histones disrupts chromatin complexes responsible for the expression of key inflammatory genes in activated macrophages, and confers protection against lipopolysaccharide-induced endotoxic shock and bacteria-induced sepsis. Our findings suggest that synthetic compounds specifically targeting proteins that recognize post-translationally modified histones can serve as a new generation of immunomodulatory drugs.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Gene Expression Regulation/drug effects , Heterocyclic Compounds, 4 or More Rings/pharmacology , Inflammation , Macrophages/drug effects , Acetylation/drug effects , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/therapeutic use , Benzodiazepines , Cells, Cultured , Epigenomics , Genome-Wide Association Study , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/therapeutic use , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Inflammation/drug therapy , Inflammation/prevention & control , Kaplan-Meier Estimate , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Models, Molecular , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Salmonella Infections/drug therapy , Salmonella Infections/immunology , Salmonella Infections/physiopathology , Salmonella Infections/prevention & control , Salmonella typhimurium , Sepsis/drug therapy , Sepsis/prevention & control , Shock, Septic/drug therapy , Shock, Septic/prevention & controlABSTRACT
Canonical animal microRNAs (miRNAs) are generated by sequential cleavage of precursor substrates by the Drosha and Dicer RNase III enzymes. Several variant pathways exploit other RNA metabolic activities to generate functional miRNAs. However, all of these pathways culminate in Dicer cleavage, suggesting that this is a unifying feature of miRNA biogenesis. Here, we show that maturation of miR-451, a functional miRNA that is perfectly conserved among vertebrates, is independent of Dicer. Instead, structure-function and knockdown studies indicate that Drosha generates a short pre-mir-451 hairpin that is directly cleaved by Ago2 and followed by resection of its 3' terminus. We provide stringent evidence for this model by showing that Dicer knockout cells can generate mature miR-451 but not other miRNAs, whereas Ago2 knockout cells reconstituted with wild-type Ago2, but not Slicer-deficient Ago2, can process miR-451. Finally, we show that the mir-451 backbone is amenable to reprogramming, permitting vector-driven expression of diverse functional miRNAs in the absence of Dicer. Beyond the demonstration of an alternative strategy to direct gene silencing, these observations open the way for transgenic rescue of Dicer conditional knockouts.