RESUMEN
Acute myeloid leukemia (AML) is a heterogeneous disease characterized by clonal expansion of myeloid blasts in the bone marrow (BM). Despite advances in therapy, the prognosis for AML patients remains poor, and there is a need to identify novel molecular pathways regulating tumor cell survival and proliferation. F-box ubiquitin E3 ligase, FBXO21, has low expression in AML, but expression correlates with survival in AML patients and patients with higher expression have poorer outcomes. Silencing FBXO21 in human-derived AML cell lines and primary patient samples leads to differentiation, inhibition of tumor progression, and sensitization to chemotherapy agents. Additionally, knockdown of FBXO21 leads to up-regulation of cytokine signaling pathways. Through a mass spectrometry-based proteomic analysis of FBXO21 in AML, we identified that FBXO21 ubiquitylates p85α, a regulatory subunit of the phosphoinositide 3-kinase (PI3K) pathway, for degradation resulting in decreased PI3K signaling, dimerization of free p85α and ERK activation. These findings reveal the ubiquitin E3 ligase, FBXO21, plays a critical role in regulating AML pathogenesis, specifically through alterations in PI3K via regulation of p85α protein stability.
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Proteínas F-Box , Leucemia Mieloide Aguda , Humanos , Proliferación Celular/fisiología , Proteínas F-Box/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Proteómica , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismoRESUMEN
Progesterone is an essential steroid hormone that is required to initiate and maintain pregnancy in mammals and serves as a metabolic intermediate in the synthesis of endogenously produced steroids, including sex hormones and corticosteroids. Steroidogenic luteal cells of the corpus luteum have the tremendous capacity to synthesize progesterone. These specialized cells are highly enriched with lipid droplets that store lipid substrate, which can be used for the synthesis of steroids. We recently reported that hormone-stimulated progesterone synthesis by luteal cells requires protein kinase A-dependent mobilization of cholesterol substrate from lipid droplets to mitochondria. We hypothesize that luteal lipid droplets are enriched with steroidogenic enzymes and facilitate the synthesis of steroids in the corpus luteum. In the present study, we analyzed the lipid droplet proteome, conducted the first proteomic analysis of lipid droplets under acute cyclic adenosine monophosphate (cAMP)-stimulated conditions, and determined how specific lipid droplet proteins affect steroidogenesis. Steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 and 3 beta-hydroxysteroid dehydrogenase (HSD3B), were highly abundant on lipid droplets of the bovine corpus luteum. High-resolution confocal microscopy confirmed the presence of active HSD3B on the surface of luteal lipid droplets. We report that luteal lipid droplets have the capacity to synthesize progesterone from pregnenolone. Lastly, we analyzed the lipid droplet proteome following acute stimulation with cAMP analog, 8-Br-cAMP, and report increased association of HSD3B with luteal lipid droplets following stimulation. These findings provide novel insights into the role of luteal lipid droplets in steroid synthesis.
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Gotas Lipídicas , Progesterona , Embarazo , Femenino , Bovinos , Animales , Progesterona/metabolismo , Gotas Lipídicas/metabolismo , Proteoma/metabolismo , Proteómica , Cuerpo Lúteo/metabolismo , Esteroides , Hormonas/metabolismo , Mamíferos/metabolismoRESUMEN
Immunoprecipitation-mass spectrometry (IP-MS) is a versatile tool to probe for global protein-protein interactions (PPIs) in biological samples. Such interactions coordinate complex biological processes, such as the DNA damage response (DDR). Induction of DNA damage activates signaling networks where posttranslational modifications cause PPI that facilitate DNA repair and cell cycle coordination. Protein interactome profiling of DDR sensors, transducers, and effectors has the potential to identify novel DDR mechanisms that could advance our understanding and treatment of diseases associated with DDR defects, such as cancer. The protocol described here is a routine PPI analysis procedure that can be performed on samples stimulated with DNA damage. All processes and reagents are optimized for maximum sensitivity on the interactome and minimal contamination for the mass spectrometer.
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Daño del ADN , Reparación del ADN , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Espectrometría de MasasRESUMEN
In this study, we identify USP1 as a transcriptional target of EWS::FLI1 and demonstrate the requisite function of USP1 in Ewing sarcoma (EWS) cell survival in response to endogenous replication stress. EWS::FLI1 oncogenic transcription factor drives most EWS, a pediatric bone cancer. EWS cells display elevated levels of R-loops and replication stress. The mechanism by which EWS cells override activation of apoptosis or cellular senescence in response to increased replication stress is not known. We show that USP1 is overexpressed in EWS and EWS::FLI1 regulates USP1 transcript levels. USP1 knockdown or inhibition arrests EWS cell growth and induces cell death by apoptosis. Mechanistically, USP1 regulates Survivin (BIRC5/API4) protein stability and the activation of caspase-9 and caspase-3/7 in response to endogenous replication stress. Notably, USP1 inhibition sensitizes cells to doxorubicin and etoposide treatment. Together, our study demonstrates that USP1 is regulated by EWS::FLI1, the USP1-Survivin axis promotes EWS cell survival, and USP1 inhibition sensitizes cells to standard of care chemotherapy. IMPLICATIONS: High USP1 and replication stress levels driven by EWS::FLI1 transcription factor in EWS are vulnerabilities that can be exploited to improve existing treatment avenues and overcome drug resistance.
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Sarcoma de Ewing , Humanos , Niño , Sarcoma de Ewing/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Survivin/genética , Survivin/metabolismo , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Línea Celular Tumoral , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
BACKGROUND & AIMS: Epidemiological studies have established alcohol and smoking as independent risk factors for recurrent acute pancreatitis and chronic pancreatitis. However, the molecular players responsible for the progressive loss of pancreatic parenchyma and fibroinflammatory response are poorly characterized. METHODS: Tandem mass tag-based proteomic and bioinformatics analyses were performed on the pancreata of mice exposed to alcohol, cigarette smoke, or a combination of alcohol and cigarette smoke. Biochemical, immunohistochemistry, and transcriptome analyses were performed on the pancreatic tissues and primary acinar cells treated with cerulein in combination with ethanol (50 mmol/L) and cigarette smoke extract (40 µg/mL) for the mechanistic studies. RESULTS: A unique alteration in the pancreatic proteome was observed in mice exposed chronically to the combination of alcohol and cigarette smoke (56.5%) compared with cigarette smoke (21%) or alcohol (17%) alone. The formation of toxic metabolites (P < .001) and attenuated unfolded protein response (P < .04) were the significantly altered pathways on combined exposure. The extracellular matrix (ECM) proteins showed stable malondialdehyde-acetaldehyde (MAA) adducts in the pancreata of the combination group and chronic pancreatitis patients with a history of smoking and alcohol consumption. Interestingly, MAA-ECM adducts significantly suppressed expression of X-box-binding protein-1, leading to acinar cell death in the presence of alcohol and smoking. The stable MAA-ECM adducts persist even after alcohol and smoking cessation, and significantly delay pancreatic regeneration by abrogating the expression of cyclin-dependent kinases (CDK7 and CDK5) and regeneration markers. CONCLUSIONS: The combined alcohol and smoking generate stable MAA-ECM adducts that increase endoplasmic reticulum stress and acinar cell death due to attenuated unfolded protein response and suppress expression of cell cycle regulators. Targeting aldehyde adducts might provide a novel therapeutic strategy for the management of recurrent acute pancreatitis and chronic pancreatitis.
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Acetaldehído , Pancreatitis Crónica , Acetaldehído/metabolismo , Enfermedad Aguda , Aldehídos , Animales , Ceruletida , Quinasas Ciclina-Dependientes/metabolismo , Etanol/toxicidad , Proteínas de la Matriz Extracelular/metabolismo , Malondialdehído/metabolismo , Ratones , Proteoma/metabolismo , Proteómica , Fumar/efectos adversos , Respuesta de Proteína DesplegadaRESUMEN
Acute anemia induces rapid expansion of erythroid precursors and accelerated differentiation to replenish erythrocytes. Paracrine signals-involving cooperation between stem cell factor (SCF)/Kit signaling and other signaling inputs-are required for the increased erythroid precursor activity in anemia. Our prior work revealed that the sterile alpha motif (SAM) domain 14 (Samd14) gene increases the regenerative capacity of the erythroid system in a mouse genetic model and promotes stress-dependent Kit signaling. However, the mechanism underlying Samd14's role in stress erythropoiesis is unknown. We identified a protein-protein interaction between Samd14 and the α- and ß-heterodimers of the F-actin capping protein (CP) complex. Knockdown of the CP ß subunit increased erythroid maturation in murine ex vivo cultures and decreased colony forming potential of stress erythroid precursors. In a genetic complementation assay for Samd14 activity, our results revealed that the Samd14-CP interaction is a determinant of erythroid precursor cell levels and function. Samd14-CP promotes SCF/Kit signaling in CD71med spleen erythroid precursors. Given the roles of Kit signaling in hematopoiesis and Samd14 in Kit pathway activation, this mechanism may have pathological implications in acute/chronic anemia.
Anemia is a condition in which the body has a shortage of healthy red blood cells to carry enough oxygen to support its organs. A range of factors are known to cause anemia, including traumatic blood loss, toxins or nutritional deficiency. An estimated one-third of all women of reproductive age are anemic, which can cause tiredness, weakness and shortness of breath. Severe anemia drives the release of hormones and growth factors, leading to a rapid regeneration of precursor red blood cells to replenish the supply in the blood. To understand how red blood cell regeneration is controlled, Ray et al. studied proteins involved in regenerating blood using mice in which anemia had been induced with chemicals. Previous research had shown that the protein Samd14 is produced at higher quantities in individuals with anemia, and is involved with the recovery of lost red blood cells. However, it is not known how the Samd14 protein plays a role in regenerating blood cells, or whether Samd14 interacts with other proteins required for red blood cell production. To shed light on these questions, mouse cells exposed to anemia conditions were used to see what proteins Samd14 binds to. Purifying Samd14 revealed that it interacts with the actin capping protein. This interaction relies on a specific region of Samd14 that is similar to regions in other proteins that bind capping proteins. Ray et al. found that the interaction between Samd14 and the actin capping protein increased the signals needed for the development and survival of new red blood cells. These results identify a signaling mechanism that, if disrupted, could cause anemia to develop. They lead to a better understanding of how our bodies recover from anemia, and potential avenues to treat this condition.
Asunto(s)
Anemia , Eritropoyesis , Animales , Diferenciación Celular , Eritrocitos , Células Precursoras Eritroides/metabolismo , Eritropoyesis/fisiología , Ratones , Proteínas/metabolismoRESUMEN
The unfolded protein response (UPR) is an adaptation mechanism activated to resolve transient accumulation of unfolded/misfolded proteins in the endoplasmic reticulum. Failure to resolve the transient accumulation of such proteins results in UPR-mediated programmed cell death. Loss of tumor suppressor gene or oncogene addiction in cancer cells can result in sustained higher basal UPR levels; however, it is not clear if these higher basal UPR levels in cancer cells can be exploited as a therapeutic strategy. We hypothesized that covalent modification of surface-exposed cysteine (SEC) residues could simulate unfolded/misfolded proteins to activate the UPR, and that higher basal UPR levels in cancer cells would provide the necessary therapeutic window. To test this hypothesis, here we synthesized analogs that can covalently modify multiple SEC residues and evaluated them as UPR activators. We identified a spirocyclic dimer, SpiD7, and evaluated its effects on UPR activation signals, that is, XBP1 splicing, phosphorylation of eIF2α, and a decrease in ATF 6 levels, in normal and cancer cells, which were further confirmed by RNA-Seq analyses. We found that SpiD7 selectively induced caspase-mediated apoptosis in cancer cells, whereas normal cells exhibited robust XBP1 splicing, indicating adaptation to stress. Furthermore, SpiD7 inhibited the growth of high-grade serous carcinoma cell lines ~3-15-fold more potently than immortalized fallopian tube epithelial (paired normal control) cells and reduced clonogenic growth of high-grade serous carcinoma cell lines. Our results suggest that induction of the UPR by covalent modification of SEC residues represents a cancer cell vulnerability and can be exploited to discover novel therapeutics.
Asunto(s)
Apoptosis , Carcinoma , Respuesta de Proteína Desplegada , Carcinoma/tratamiento farmacológico , Carcinoma/metabolismo , Línea Celular Tumoral , Descubrimiento de Drogas , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/metabolismo , HumanosRESUMEN
Pancreatic Ductal adenocarcinoma (PDAC) is an aggressive cancer commonly exhibiting KRAS-activating mutations. Alcohol contributes to the risk of developing PDAC in humans, and murine models have shown alcohol consumption in the context of KRAS mutation in the pancreas promotes the development of PDAC. The molecular signatures in pancreas cells altered by alcohol exposure in the context of mutant KRAS could identify pathways related to the etiology of PDAC. In this study, we evaluated the combined effects of alcohol exposure and KRAS mutation status on the transcriptome and proteome of pancreatic HPNE cell models. These analyses identified alterations in transcription and translational processes in mutant KRAS cells exposed to alcohol. In addition, multi-omics analysis suggests an increase in the correlation between mRNA transcript and protein abundance in cells exposed to alcohol with an underlying KRAS mutation. Through differential co-expression, SERPINE1 was found to be influential for PDAC development in the context of mutant KRAS and ethanol. In terms of PDAC subtypes, alcohol conditioning of HPNE cells expressing mutant KRAS decreases the Inflammatory subtype signature and increases the Proliferative and Metabolic signatures, as we previously observed in patient samples. The alterations in molecular subtypes were associated with an increased sensitivity to chemotherapeutic agents gemcitabine, irinotecan, and oxaliplatin. These results provide a framework for distinguishing the molecular dysregulation associated with combined alcohol and mutant KRAS in a pancreatic cell line model.
RESUMEN
This protocol represents an optimized proteomics-based protocol for the endogenous protein enrichment and protein-protein interaction analysis. This 2-step protocol consists of: 1) co-immunoprecipitation of the bait protein; 2) the bait-protein interactions analysis using LC-MS/MS. Here, we used Dynabeads® for the enrichment of the target protein (the bait) and its interactors. We have tested the protocol using several different cell lines. Our conclusion is that the protocol is applicable to different cell lines and species. For complete details on the use and execution of this protocol, please refer to Lagundzin et al. (2019).
Asunto(s)
Proteómica , Espectrometría de Masas en Tándem , Línea Celular , Cromatografía Liquida , Inmunoprecipitación , Proteínas/química , Proteómica/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Tumor necrosis factor (TNF) α-induced nuclear translocation of the NF-κB subunit RELA has been implicated in several pathological conditions. Here we report the discovery of a spirocyclic dimer (SpiD7) that covalently modifies RELA to inhibit TNFα-induced nuclear translocation. This is a previously unexplored strategy to inhibit TNFα-induced NF-κB activation.
RESUMEN
Genome wide-association studies (GWAS) have established over 400 breast cancer risk loci defined by common single nucleotide polymorphisms (SNPs), including several associated with estrogen-receptor (ER)-negative disease. Most of these loci have not been studied systematically and the mechanistic underpinnings of risk are largely unknown. Here we explored the landscape of genomic features at an ER-negative breast cancer susceptibility locus at chromosome 2p23.2 and assessed the functionality of 81 SNPs with strong evidence of association from previous fine mapping. Five candidate regulatory regions containing risk-associated SNPs were identified. Regulatory Region 1 in the first intron of WDR43 contains SNP rs4407214, which showed allele-specific interaction with the transcription factor USF1 in in vitro assays. CRISPR-mediated disruption of Regulatory Region 1 led to expression changes in the neighboring PLB1 gene, suggesting that the region acts as a distal enhancer. Regulatory Regions 2, 4, and 5 did not provide sufficient evidence for functionality in in silico and experimental analyses. Two SNPs (rs11680458 and rs1131880) in Regulatory Region 3, mapping to the seed region for miRNA-recognition sites in the 3' untranslated region of WDR43, showed allele-specific effects of ectopic expression of miR-376 on WDR43 expression levels. Taken together, our data suggest that risk of ER-negative breast cancer associated with the 2p23.2 locus is likely driven by a combinatorial effect on the regulation of WDR43 and PLB1.
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Neoplasias de la Mama , Neoplasias de la Mama Triple Negativas , Neoplasias de la Mama/genética , Estrógenos , Femenino , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
Cyclin-dependent kinase 9 (CDK9) is a member of the cyclin-dependent kinase (CDK) family which is involved in transcriptional regulation of several genes, including the oncogene Myc, and is a validated target for pancreatic cancer. Here we report the development of an aminopyrazole based proteolysis targeting chimera (PROTAC 2) that selectively degrades CDK9 (DC50 = 158 ± 6 nM). Mass spectrometry-based kinome profiling shows PROTAC 2 selectively degrades CDK9 in MiaPaCa2 cells and sensitizes them to Venetoclax mediated growth inhibition.
Asunto(s)
Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Proliferación Celular/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/química , Proteolisis/efectos de los fármacos , Pirazoles/química , Relación Estructura-Actividad , Sulfonamidas/farmacologíaRESUMEN
Lipid droplets (LDs) are composed of neutral lipids enclosed in a phospholipid monolayer, which harbors membrane-associated proteins that regulate LD functions. Despite the crucial role of LDs in lipid metabolism, remodeling of LD protein composition in disease contexts, such as steatosis, remains poorly understood. We hypothesized that chronic ethanol consumption, subsequent abstinence from ethanol, or fasting differentially affects the LD membrane proteome content and that these changes influence how LDs interact with other intracellular organelles. Here, male Wistar rats were pair-fed liquid control or ethanol diets for 6 weeks, and then, randomly chosen animals from both groups were either refed a control diet for 7 days or fasted for 48 h before euthanizing. From all groups, LD membrane proteins from purified liver LDs were analyzed immunochemically and by MS proteomics. Liver LD numbers and sizes were greater in ethanol-fed rats than in pair-fed control, 7-day refed, or fasted rats. Compared with control rats, ethanol feeding markedly altered the LD membrane proteome, enriching LD structural perilipins and proteins involved in lipid biosynthesis, while lowering LD lipase levels. Ethanol feeding also lowered LD-associated mitochondrial and lysosomal proteins. In 7-day refed (i.e., ethanol-abstained) or fasted-ethanol-fed rats, we detected distinct remodeling of the LD proteome, as judged by lower levels of lipid biosynthetic proteins, and enhanced LD interaction with mitochondria and lysosomes. Our study reveals evidence of significant remodeling of the LD membrane proteome that regulates ethanol-induced steatosis, its resolution after withdrawal and abstinence, and changes in LD interactions with other intracellular organelles.
Asunto(s)
Gotas LipídicasRESUMEN
RNA polymerase II subunit A Carboxy-Terminal Domain Phosphatase 1 (CTDP1), a member of the haloacid dehalogenase superfamily phosphatases, has a defined role in transcriptional regulation, but emerging evidence suggests an expanded functional repertoire in the cell cycle and DNA damage response. In humans, a splice site mutation in CTDP1 gives rise to the rare Congenital Cataracts Facial Dysmorphism and Neuropathy syndrome, and recent evidence from our lab indicates CTDP1 is required for breast cancer growth and proliferation. To explore the physiological function of CTDP1 in a mammalian system, we generated a conditional Ctdp1 knockout mouse model by insertion of loxP sites upstream of exon 3 and downstream of exon 4. Biallelic deletion of Ctdp1 results in lethality before embryonic day 7.5, with morphological features indicating embryo cell death and resorption. However, Ctdp1+/- mice are haplosufficient for phenotypic traits including body weight, hematological parameters, exploratory and locomotive functions. To investigate the potential mechanisms of the embryonic death caused by biallelic Ctdp1 knockout, mouse embryonic fibroblasts (MEFs) were established from Ctdp1+/+ and Ctdp1flox/flox mice. Lentivirus delivered Cre-mediated biallelic deletion of Ctdp1 in MEFs results in cell death preceded by impaired proliferation characterized by an increase in G1- and G2-phase populations and a reduction in the S-phase population. These cell cycle alterations caused by deletion of Ctdp1 are associated with an increase in p27 protein expression and a decrease in phosphorylated RB, phosphorylated Histone H3, and Cyclin B expression. Together, these results reveal that Ctdp1 plays an essential role in early mouse embryo development and cell growth and survival in part by regulating the cell cycle.
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Ciclo Celular/genética , Desarrollo Embrionario/genética , Fibroblastos/metabolismo , Genes Letales , Fosfoproteínas Fosfatasas/deficiencia , Animales , Puntos de Control del Ciclo Celular/genética , Muerte Celular/genética , Línea Celular , Eliminación de Gen , Marcación de Gen , Vectores Genéticos/genética , Inmunohistoquímica , Ratones , Ratones Noqueados , FenotipoRESUMEN
Coordination of a number of molecular mechanisms including transcription, alternative splicing, and class switch recombination are required to facilitate development, activation, and survival of B cells. Disruption of these pathways can result in malignant transformation. Recently, next-generation sequencing has identified a number of novel mutations in mantle cell lymphoma (MCL) patients including mutations in the ubiquitin E3 ligase UBR5. Approximately 18% of MCL patients were found to have mutations in UBR5, with the majority of mutations within the HECT domain of the protein that can accept and transfer ubiquitin molecules to the substrate. Determining if UBR5 controls the maturation of B cells is important to fully understand malignant transformation to MCL. To elucidate the role of UBR5 in B-cell maturation and activation, we generated a conditional mutant disrupting UBR5's C-terminal HECT domain. Loss of the UBR5 HECT domain leads to a block in maturation of B cells in the spleen and upregulation of proteins associated with messenger RNA splicing via the spliceosome. Our studies reveal a novel role of UBR5 in B-cell maturation by stabilization of spliceosome components during B-cell development and suggests UBR5 mutations play a role in MCL transformation.
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Linfocitos B/enzimología , Linfoma de Células del Manto/enzimología , Mutación , Proteínas de Neoplasias/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Humanos , Linfoma de Células del Manto/genética , Ratones , Ratones Mutantes , Proteínas de Neoplasias/genética , Dominios Proteicos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease that can be separated into distinct subtypes based on molecular signatures. Identifying PDAC subtype-specific therapeutic vulnerabilities is necessary to develop precision medicine approaches to treat PDAC. EXPERIMENTAL DESIGN: A total of 56 PDAC liver metastases were obtained from the UNMC Rapid Autopsy Program and analyzed with quantitative proteomics. PDAC subtypes were identified by principal component analysis based on protein expression profiling. Proteomic subtypes were further characterized by the associated clinical information, including but not limited to survival analysis, drug treatment response, and smoking and drinking status. RESULTS: Over 3,960 proteins were identified and used to delineate four distinct PDAC microenvironment subtypes: (i) metabolic; (ii) progenitor-like; (iii) proliferative; and (iv) inflammatory. PDAC risk factors of alcohol and tobacco consumption correlate with subtype classifications. Enhanced survival is observed in FOLFIRINOX treated metabolic and progenitor-like subtypes compared with the proliferative and inflammatory subtypes. In addition, TYMP, PDCD6IP, ERAP1, and STMN showed significant association with patient survival in a subtype-specific manner. Gemcitabine-induced alterations in the proteome identify proteins, such as serine hydroxymethyltransferase 1, associated with drug resistance. CONCLUSIONS: These data demonstrate that proteomic analysis of clinical PDAC liver metastases can identify molecular signatures unique to disease subtypes and point to opportunities for therapeutic development to improve the treatment of PDAC.
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Adenocarcinoma/patología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/patología , Neoplasias Hepáticas/secundario , Neoplasias Pancreáticas/patología , Proteoma/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Femenino , Fluorouracilo/administración & dosificación , Regulación Neoplásica de la Expresión Génica , Humanos , Irinotecán/administración & dosificación , Leucovorina/administración & dosificación , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Masculino , Tipificación Molecular/métodos , Oxaliplatino/administración & dosificación , Neoplasias Pancreáticas/clasificación , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteoma/análisis , Proteómica/métodos , Tasa de Supervivencia , Resultado del Tratamiento , GemcitabinaRESUMEN
Deleterious mutations in Breast Cancer 1 (BRCA1) are associated with an increased risk of breast and ovarian cancer. Mutations in the tandem BRCA1 C-terminal (tBRCT) protein domain disrupt critical protein interactions required for the faithful repair of DNA through homologous recombination, which contributes to oncogenesis. Our studies have identified RICTOR, PRR5, and SIN1 subunits of the mammalian target of rapamycin complex 2 (mTORC2) as interacting partners with the tBRCT domain of BRCA1 leading to the disruption of the mTORC2 complex. However, the interplay between mTORC2 signaling and BRCA1 function in the DNA damage response (DDR) remains to be determined. In this study, we used protein interaction assays to determine the binary interactions between the tBRCT domain and mTORC2 subunits, evaluated the impact of mTOR inhibition on the transcriptional function of the tBRCT, evaluated the impact of mTOR signaling on BRCA1 recruitment to DNA damage-induced foci and determined the breast cancer cell line response to mTOR inhibition dependent upon BRCA1 expression and mutation. This study determined that PRR5, RICTOR, and SIN1 could each independently interact with the BRCA1 tBRCT. Inhibition of mTORC1, but not mTORC1/2, increases BRCA1 transcriptional activation activity. Treatment with pan-mTOR inhibitor PP242 diminishes DNA damage-induced γH2AX and BRCA1 foci formation. Breast cancer cells lacking expression of functional BRCA1 are more sensitive to mTOR inhibitors. These data suggest that mTOR signaling is required for BRCA1 response to DNA damage and breast cancer cells lacking BRCA1 are more sensitive to pan-mTOR inhibition. This work suggests chemotherapeutic strategies using mTOR inhibitors could be tailored for patients that lack functional BRCA1.
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Proteína BRCA1/metabolismo , Neoplasias de la Mama/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Proteína BRCA1/genética , Neoplasias de la Mama/genética , Línea Celular Tumoral , Supervivencia Celular , Daño del ADN/genética , Daño del ADN/fisiología , Técnica del Anticuerpo Fluorescente , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Fosforilación/genética , Fosforilación/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Técnicas del Sistema de Dos HíbridosRESUMEN
Hyperinsulinemia affects 72% of Fanconi anemia (FA) patients and an additional 25% experience lowered glucose tolerance or frank diabetes. The underlying molecular mechanisms contributing to the dysfunction of FA pancreas ß cells is unknown. Therefore, we sought to evaluate the functional role of FANCA, the most commonly mutated gene in FA, in glucose-stimulated insulin secretion (GSIS). This study reveals that FANCA or FANCB knockdown impairs GSIS in human pancreas ß cell line EndoC-ßH3. To identify potential pathways by which FANCA might regulate GSIS, we employed a proteomics approach to identify FANCA protein interactions in EndoC-ßH3 differentially regulated in response to elevated glucose levels. Glucose-dependent changes in the FANCA interaction network were observed, including increased association with other FA family proteins, suggesting an activation of the DNA damage response in response to elevated glucose levels. Reactive oxygen species increase in response to glucose stimulation and are necessary for GSIS in EndoC-ßH3 cells. Glucose-induced activation of the DNA damage response was also observed as an increase in the DNA damage foci marker γ-H2AX and dependent upon the presence of reactive oxygen species. These results illuminate the role of FANCA in GSIS and its protein interactions regulated by glucose stimulation that may explain the prevalence of ß cell-specific endocrinopathies in FA patients.
Asunto(s)
Proteína del Grupo de Complementación A de la Anemia de Fanconi/metabolismo , Glucosa/farmacología , Secreción de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Línea Celular , Daño del ADN , Humanos , Secreción de Insulina/fisiología , Células Secretoras de Insulina/efectos de los fármacosRESUMEN
Many intracellular bacteria, including the obligate intracellular pathogen Chlamydia trachomatis, grow within a membrane-bound bacterium-containing vacuole (BCV). Secreted cytosolic effectors modulate host activity, but an understanding of the host-pathogen interactions that occur at the BCV membrane is limited by the difficulty in purifying membrane fractions from infected host cells. We used the ascorbate peroxidase (APEX2) proximity labeling system, which labels proximal proteins with biotin in vivo, to study the protein-protein interactions that occur at the chlamydial vacuolar, or inclusion, membrane. An in vivo understanding of the secreted chlamydial inclusion membrane protein (Inc) interactions (e.g., Inc-Inc and Inc-eukaryotic protein) and how these contribute to overall host-chlamydia interactions at this unique membrane is lacking. We hypothesize some Incs organize the inclusion membrane, whereas other Incs bind eukaryotic proteins to promote chlamydia-host interactions. To study this, Incs fused to APEX2 were expressed in C. trachomatis L2. Affinity purification-mass spectrometry (AP-MS) identified biotinylated proteins, which were analyzed for statistical significance using significance analysis of the interactome (SAINT). Broadly supporting both Inc-Inc and Inc-host interactions, our Inc-APEX2 constructs labeled Incs as well as known and previously unreported eukaryotic proteins localizing to the inclusion. We demonstrate, using bacterial two-hybrid and coimmunoprecipitation assays, that endogenous LRRFIP1 (LRRF1) is recruited to the inclusion by the Inc CT226. We further demonstrate interactions between CT226 and the Incs used in our study to reveal a model for inclusion membrane organization. Combined, our data highlight the utility of APEX2 to capture the complex in vivo protein-protein interactions at the chlamydial inclusion.