RESUMO
Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.
Assuntos
Neurônios/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Biossíntese de Proteínas , Serina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Animais , Axônios/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Códon/genética , Feminino , Glicina/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Tecido Nervoso/patologia , Consumo de Oxigênio , Neoplasias Pancreáticas/patologia , Pirazóis , Pirimidinas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RatosRESUMO
Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Movimento Celular , Proteína Duplacortina , Quinases Semelhantes a Duplacortina , Ensaios de Seleção de Medicamentos Antitumorais , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteínas Quinases/farmacocinética , Proteômica , Ratos , Relação Estrutura-Atividade , Peixe-Zebra , Neoplasias PancreáticasRESUMO
Ubiquitination is an essential post-translational modification that regulates protein stability or function. Its substrate specificity is dictated by various E3 ligases. The human C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit really interesting new gene (RING) E3 ligase with only a few known ubiquitination targets. Here, we used mass spectrometry-based proteomic techniques to gain insight into CTLH complex function and ubiquitination substrates in HeLa cells. First, global proteomics determined proteins that were significantly increased, and thus may be substrates targeted for degradation, in cells depleted of CTLH complex member RanBPM. RanBPM-dependent ubiquitination determined using diGLY-enriched proteomics and the endogenous RanBPM interactome further revealed candidate ubiquitination targets. Three glycolysis enzymes alpha-enolase, L-lactate dehydrogenase A chain (LDHA), and pyruvate kinase M1/2 (PKM) had decreased ubiquitin sites in shRanBPM cells and were found associated with RanBPM in the interactome. Reduced polyubiquitination was validated for PKM2 and LDHA in cells depleted of RanBPM and CTLH complex RING domain subunit RMND5A. PKM2 and LDHA protein levels were unchanged, yet their activity was increased in extracts of cells with downregulated RanBPM. Finally, RanBPM deficient cells displayed enhanced glycolysis and deregulated central carbon metabolism. Overall, this study identifies potential CTLH complex ubiquitination substrates and uncovers that the CTLH complex inhibits glycolysis via non-degradative ubiquitination of PKM2 and LDHA.
Assuntos
Glicólise/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , L-Lactato Desidrogenase/metabolismo , Camundongos , Proteômica/métodos , Especificidade por Substrato , Ubiquitina/metabolismoRESUMO
Multipotent/mesenchymal stromal cells (MSCs) exist within a variety of postnatal tissues; however, global proteomic analyses comparing tissue-specific MSC are limited. Using human bone marrow (BM)-derived MSCs as a gold standard, we used label-free mass spectrometry and functional assays to characterize the proteome, secretome, and corresponding function of human pancreas-derived MSCs (Panc-MSCs) with a classical phenotype (CD90+/CD73+/CD105+/CD45-/CD31-). Both MSC subtypes expressed mesenchymal markers vimentin, α-SMA, and STRO-1; however, expression of nestin was increased in Panc-MSCs. Accordingly, these Vimentinhigh /Nestinhigh cells were isolated from fresh human pancreatic islet and non-islet tissues. Next, we identified expression of >60 CD markers shared between Panc-MSCs and BM-MSCs, including validated expression of CD14. An additional 19 CD markers were differentially expressed, including reduced pericyte-marker CD146 expression on Panc-MSCs. Panc-MSCs also showed reduced expression of proteins involved in lipid and retinoid metabolism. Accordingly, Panc-MSCs showed restricted responses to adipogenic stimuli in vitro, although both MSC types demonstrated trilineage differentiation. In contrast, Panc-MSCs demonstrated accelerated growth kinetics and competency to pro-neurogenic stimuli in vitro. The secretome of Panc-MSCs was highly enriched for proteins associated with vascular development, wound healing and chemotaxis. Similar to BM-MSCs, Panc-MSCs conditioned media augmented endothelial cell survival, proliferation, and tubule formation in vitro. Importantly, the secretome of both MSC types was capable of stimulating chemotactic infiltration of murine endothelial cells in vivo and reduced hyperglycemia in STZ-treated mice following intrapancreatic injection. Overall, this study provides foundational knowledge to develop Panc-MSCs as a unique MSC subtype with functional properties beneficial in regenerative medicine for diabetes and vascular disease.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Regeneração Nervosa/genética , Nestina/metabolismo , Pâncreas/metabolismo , Proteoma/metabolismo , Medicina Regenerativa/métodos , Vimentina/metabolismo , Animais , Diferenciação Celular , Humanos , Camundongos , Camundongos Endogâmicos NODRESUMO
BACKGROUND: Wolfram syndrome (WFS) is a rare autosomal recessive syndrome in which diabetes mellitus and neurodegenerative disorders occur as a result of Wolframin deficiency and increased ER stress. In addition, WFS1 deficiency leads to calcium homeostasis disturbances and can change mitochondrial dynamics. The aim of this study was to evaluate protein levels and changes in gene transcription on human WFS cell model under experimental ER stress. METHODS: We performed transcriptomic and proteomic analysis on WFS human cell model-skin fibroblasts reprogrammed into induced pluripotent stem (iPS) cells and then into neural stem cells (NSC) with subsequent ER stress induction using tunicamycin (TM). Results were cross-referenced with publicly available RNA sequencing data in hippocampi and hypothalami of mice with WFS1 deficiency. RESULTS: Proteomic analysis identified specific signal pathways that differ in NSC WFS cells from healthy ones. Next, detailed analysis of the proteins involved in the mitochondrial function showed the down-regulation of subunits of the respiratory chain complexes in NSC WFS cells, as well as the up-regulation of proteins involved in Krebs cycle and glycolysis when compared to the control cells. Based on pathway enrichment analysis we concluded that in samples from mice hippocampi the mitochondrial protein import machinery and OXPHOS were significantly down-regulated. CONCLUSIONS: Our results show the functional and morphological secondary mitochondrial damage in patients with WFS. Video Abstract.
Assuntos
Síndrome de WolframRESUMO
Human multipotent stromal cells (hMSC) can induce islet regeneration after transplantation via the secretion of proteins that establish an islet regenerative niche. However, the identity of hMSC-secreted signals and the mechanisms by which pancreatic islet regeneration is induced remain unknown. Recently, mammalian pancreatic α-cells have been shown to possess considerable plasticity, and differentiate into ß-like cells after near complete ß-cell loss or overexpression of key transcriptional regulators. These studies have generated new excitement that islet regeneration during diabetes may be possible if we can identify clinically applicable stimuli to modulate these key regulatory pathways. Herein, we demonstrate that intrapancreatic-injection of concentrated hMSC-conditioned media (CM) stimulated islet regeneration without requiring cell transfer. hMSC CM-injection significantly reduced hyperglycemia, increased circulating serum insulin concentration, and improved glucose tolerance in streptozotocin-treated mice. The rate and extent of endogenous ß-cell mass recovery was dependent on total protein dose administered and was further augmented by the activation of Wnt-signaling using GSK3-inhibition during CM generation. Intrapancreatic hMSC CM-injection immediately set in motion a cascade of regenerative events that included the emergence of proliferating insulin+ clusters adjacent to ducts, NKX6.1 expression in glucagon+ cells at days 1-4 suggesting the acquisition of ß-cell phenotype by α-cells, and accelerated ß-cell maturation with increased MAFA-expression for >1 month postinjection. Discovery and validation of islet regenerative hMSC-secreted protein may lead to the development of cell-free regenerative therapies able to tip the balance in favor of ß-cell regeneration versus destruction during diabetes. Stem Cells 2019;37:516-528.
Assuntos
Ilhotas Pancreáticas/metabolismo , Células-Tronco Multipotentes/metabolismo , Regeneração/genética , Animais , Diferenciação Celular , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Mice lacking equilibrative nucleoside transporter 1 (ENT1 -/- ) demonstrate progressive calcification of spinal tissues including the annulus fibrosus (AF) of the intervertebral disc (IVD). We previously established ENT1 as the primary nucleoside transporter in the AF and demonstrated dysregulation of biomineralization pathways. To identify cellular pathways altered by loss of ENT1, we conducted microarray analysis of AF tissue from wild-type (WT) and ENT1 -/- mice before calcification (2 months of age) and associated with calcification (6 months of age). Bioinformatic analyses identified cell cycle dysregulation in ENT1 -/- AF tissues and implicated the E2f family of transcription factors as potential effectors. Quantitative polymerase chain reaction analysis confirmed increased expression of multiple E2f transcription factors and E2f interacting proteins ( Rb1 and Cdk2) in ENT1 -/- AF cells compared with WT at 6 months of age. At this time point, ENT1 -/- AF tissues showed increased JNK MAPK pathway activation, CDK1, minichromosome maintenance complex component 5 (Mcm5), and proliferating cell nuclear antigen (PCNA) protein expression, and PCNA-positive proliferating cells compared with WT controls. The current study demonstrates that loss of ENT1-mediated adenosine transport leads to increased cell proliferation in the AF of the IVD.
Assuntos
Anel Fibroso/metabolismo , Anel Fibroso/patologia , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Adenosina/metabolismo , Animais , Calcinose/metabolismo , Proliferação de Células/fisiologia , Camundongos , Camundongos KnockoutRESUMO
Blood-derived progenitor cell transplantation holds potential for the treatment of severe vascular diseases. Human umbilical cord blood (UCB)-derived hematopoietic progenitor cells purified using high aldehyde dehydrogenase (ALDHhi ) activity demonstrate pro-angiogenic functions following intramuscular (i.m.) transplantation into immunodeficient mice with hind-limb ischemia. Unfortunately, UCB ALDHhi cells are rare and prolonged ex vivo expansion leads to loss of high ALDH-activity and diminished vascular regenerative function. ALDH-activity generates retinoic acid, a potent driver of hematopoietic differentiation, creating a paradoxical challenge to expand UCB ALDHhi cells while limiting differentiation and retaining pro-angiogenic functions. We investigated whether inhibition of ALDH-activity during ex vivo expansion of UCB ALDHhi cells would prevent differentiation and expand progeny that retained pro-angiogenic functions after transplantation into non-obese diabetic/severe combined immunodeficient mice with femoral artery ligation-induced unilateral hind-limb ischemia. Human UCB ALDHhi cells were cultured under serum-free conditions for 9 days, with or without the reversible ALDH-inhibitor, diethylaminobenzaldehyde (DEAB). Although total cell numbers were increased >70-fold, the frequency of cells that retained ALDHhi /CD34+ phenotype was significantly diminished under basal conditions. In contrast, DEAB-inhibition increased total ALDHhi /CD34+ cell number by ≥10-fold, reduced differentiation marker (CD38) expression, and enhanced the retention of multipotent colony-forming cells in vitro. Proteomic analysis revealed that DEAB-treated cells upregulated anti-apoptotic protein expression and diminished production of proteins implicated with megakaryocyte differentiation. The i.m. transplantation of DEAB-treated cells into mice with hind-limb ischemia stimulated endothelial cell proliferation and augmented recovery of hind-limb perfusion. DEAB-inhibition of ALDH-activity delayed hematopoietic differentiation and expanded multipotent myeloid cells that accelerated vascular regeneration following i.m. transplantation in vivo. Stem Cells 2018;36:723-736.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Multipotentes/citologia , Regeneração/fisiologia , Animais , Proliferação de Células/fisiologia , Hematopoese/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Multipotentes/transplante , Neovascularização Fisiológica/fisiologiaRESUMO
During culture expansion, multipotent mesenchymal stromal cells (MSCs) differentially express aldehyde dehydrogenase (ALDH), an intracellular detoxification enzyme that protects long-lived cells against oxidative stress. Thus, MSC selection based on ALDH-activity may be used to reduce heterogeneity and distinguish MSC subsets with improved regenerative potency. After expansion of human bone marrow-derived MSCs, cell progeny was purified based on low versus high ALDH-activity (ALDHhi ) by fluorescence-activated cell sorting, and each subset was compared for multipotent stromal and provascular regenerative functions. Both ALDHl ° and ALDHhi MSC subsets demonstrated similar expression of stromal cell (>95% CD73+ , CD90+ , CD105+ ) and pericyte (>95% CD146+ ) surface markers and showed multipotent differentiation into bone, cartilage, and adipose cells in vitro. Conditioned media (CDM) generated by ALDHhi MSCs demonstrated a potent proliferative and prosurvival effect on human microvascular endothelial cells (HMVECs) under serum-free conditions and augmented HMVEC tube-forming capacity in growth factor-reduced matrices. After subcutaneous transplantation within directed in vivo angiogenesis assay implants into immunodeficient mice, ALDHhi MSC or CDM produced by ALDHhi MSC significantly augmented murine vascular cell recruitment and perfused vessel infiltration compared with ALDHl ° MSC. Although both subsets demonstrated strikingly similar mRNA expression patterns, quantitative proteomic analyses performed on subset-specific CDM revealed the ALDHhi MSC subset uniquely secreted multiple proangiogenic cytokines (vascular endothelial growth factor beta, platelet derived growth factor alpha, and angiogenin) and actively produced multiple factors with chemoattractant (transforming growth factor-ß, C-X-C motif chemokine ligand 1, 2, and 3 (GRO), C-C motif chemokine ligand 5 (RANTES), monocyte chemotactic protein 1 (MCP-1), interleukin [IL]-6, IL-8) and matrix-modifying functions (tissue inhibitor of metalloprotinase 1 & 2 (TIMP1/2)). Collectively, MSCs selected for ALDHhi demonstrated enhanced proangiogenic secretory functions and represent a purified MSC subset amenable for vascular regenerative applications. Stem Cells 2017;35:1542-1553.
Assuntos
Aldeído Desidrogenase/metabolismo , Vasos Sanguíneos/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/enzimologia , Regeneração , Biomarcadores/metabolismo , Prótese Vascular , Vasos Sanguíneos/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Microvasos/citologia , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Pericitos/citologia , Pericitos/efeitos dos fármacos , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacosRESUMO
Numerous workflows exist for large-scale bottom-up proteomics, many of which achieve exceptional proteome depth. Herein, we evaluated the performance of several commonly used sample preparation techniques for proteomic characterization of HeLa lysates [unfractionated in-solution digests, SDS-PAGE coupled with in-gel digestion, gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) technology, SCX StageTips and high-/low-pH reversed phase fractionation (HpH)]. HpH fractionation was found to be superior in terms of proteome depth (>8400 proteins detected) and fractionation efficiency compared to other techniques. SCX StageTip fractionation required minimal sample handling and was also a substantial improvement over SDS-PAGE separation and GELFrEE technology. Sequence coverage of the HeLa proteome increased to 38% when combining all workflows, however, total proteins detected improved only slightly to 8710. In summary, HpH fractionation and SCX StageTips are robust techniques and highly suited for complex proteome analysis.
Assuntos
Proteoma/análise , Proteômica/métodos , Eletroforese em Gel de Poliacrilamida , Células HeLa , HumanosRESUMO
AIMS/HYPOTHESIS: Novel strategies to stimulate the expansion of beta cell mass in situ are warranted for diabetes therapy. The aim of this study was to elucidate the secretome of human bone marrow (BM)-derived multipotent stromal cells (MSCs) with documented islet regenerative paracrine function. We hypothesised that regenerative MSCs will secrete a unique combination of protein factors that augment islet regeneration. METHODS: Human BM-derived MSCs were examined for glucose-lowering capacity after transplantation into streptozotocin-treated NOD/severe combined immunodeficiency (SCID) mice and segregated into samples with regenerative (MSCR) vs nonregenerative (MSCNR) capacity. Secreted proteins associated with islet regenerative function were identified using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics. To functionally validate the importance of active Wnt signalling, we stimulated the Wnt-signalling pathway in MSCNR samples during ex vivo expansion using glycogen synthase kinase 3 (GSK3) inhibition (CHIR99201), and the conditioned culture media (CM) generated was tested for the capacity to support cultured human islet cell survival and proliferation in vitro. RESULTS: MSCR showed increased secretion of proteins associated with cell growth, matrix remodelling, immunosuppressive and proangiogenic properties. In contrast, MSCNR uniquely secreted proteins known to promote inflammation and negatively regulate angiogenesis. Most notably, MSCR maintained Wnt signalling via Wnt5A/B (~2.5-fold increase) autocrine activity during ex vivo culture, while MSCNR repressed Wnt signalling via Dickkopf-related protein (DKK)1 (~2.5-fold increase) and DKK3 secretion. Inhibition of GSK3 activity in MSCNR samples increased the accumulation of nuclear ß-catenin and generated CM that augmented beta cell survival (13% increases) and proliferation when exposed to cultured human islets. CONCLUSIONS/INTERPRETATION: Maintenance of active Wnt signalling within human MSCs promotes the secretion of matricellular and proangiogenic proteins that formulate a niche for islet regeneration.
Assuntos
Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Células Secretoras de Insulina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Via de Sinalização Wnt/fisiologia , Animais , Diabetes Mellitus Experimental/metabolismo , Humanos , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , ProteômicaRESUMO
Defining the cellular response to pharmacological agents is critical for understanding the mechanism of action of small molecule perturbagens. Here, we developed a 96-well-plate-based high-throughput screening infrastructure for quantitative proteomics and profiled 875 compounds in a human cancer cell line with near-comprehensive proteome coverage. Examining the 24-h proteome changes revealed ligand-induced changes in protein expression and uncovered rules by which compounds regulate their protein targets while identifying putative dihydrofolate reductase and tankyrase inhibitors. We used protein-protein and compound-compound correlation networks to uncover mechanisms of action for several compounds, including the adrenergic receptor antagonist JP1302, which we show disrupts the FACT complex and degrades histone H1. By profiling many compounds with overlapping targets covering a broad chemical space, we linked compound structure to mechanisms of action and highlighted off-target polypharmacology for molecules within the library.
Assuntos
Neoplasias , Proteoma , Humanos , Proteoma/metabolismo , Proteômica , Ensaios de Triagem em Larga Escala , Linhagem CelularRESUMO
Alström syndrome (ALMS) and Bardet-Biedl syndrome (BBS) are rare genetic diseases with a number of common clinical features ranging from early-childhood obesity and retinal degeneration. ALMS and BBS belong to the ciliopathies, which are known to have the expression products of genes, encoding them as cilia-localized proteins in multiple target organs. The aim of this study was to perform transcriptomic and proteomic analysis on cellular models of ALMS and BBS syndromes to identify common and distinct pathological mechanisms present in both syndromes. For this purpose, epithelial cells were isolated from the urine of patients and healthy subjects, which were then cultured and reprogrammed into induced pluripotent stem (iPS) cells. The pathways of genes associated with the metabolism of lipids and glycosaminoglycan and the transport of small molecules were found to be concomitantly downregulated in both diseases, while transcripts related to signal transduction, the immune system, cell cycle control and DNA replication and repair were upregulated. Furthermore, protein pathways associated with autophagy, apoptosis, cilium assembly and Gli1 protein were upregulated in both ciliopathies. These results provide new insights into the common and divergent pathogenic pathways between two similar genetic syndromes, particularly in relation to primary cilium function and abnormalities in cell differentiation.
Assuntos
Síndrome de Alstrom , Síndrome de Bardet-Biedl , Ciliopatias , Obesidade Infantil , Criança , Humanos , Síndrome de Bardet-Biedl/genética , Transcriptoma/genética , Proteômica , Obesidade Infantil/complicações , Síndrome de Alstrom/genética , Proteínas/genéticaRESUMO
Microbial dysbiosis is a colorectal cancer (CRC) hallmark and contributes to inflammation, tumor growth, and therapy response. Gut microbes signal via metabolites, but how the metabolites impact CRC is largely unknown. We interrogated fecal metabolites associated with mouse models of colon tumorigenesis with varying mutational load. We find that microbial metabolites from healthy mice or humans are growth-repressive, and this response is attenuated in mice and patients with CRC. Microbial profiling reveals that Lactobacillus reuteri and its metabolite, reuterin, are downregulated in mouse and human CRC. Reuterin alters redox balance, and reduces proliferation and survival in colon cancer cells. Reuterin induces selective protein oxidation and inhibits ribosomal biogenesis and protein translation. Exogenous Lactobacillus reuteri restricts colon tumor growth, increases tumor reactive oxygen species, and decreases protein translation in vivo. Our findings indicate that a healthy microbiome and specifically, Lactobacillus reuteri, is protective against CRC through microbial metabolite exchange.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Microbioma Gastrointestinal , Gliceraldeído/análogos & derivados , Oxirredução , Propano/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Metabolismo Energético , Glutationa/metabolismo , Gliceraldeído/metabolismo , Gliceraldeído/farmacologia , Interações entre Hospedeiro e Microrganismos , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Metabolômica/métodos , Metagenômica/métodos , Camundongos , Modelos Biológicos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo , Propano/farmacologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic ductal adenocarcinomas (PDAC) depend on autophagy for survival; however, the metabolic substrates that autophagy provides to drive PDAC progression are unclear. Ferritin, the cellular iron storage complex, is targeted for lysosomal degradation (ferritinophagy) by the selective autophagy adaptor NCOA4, resulting in release of iron for cellular utilization. Using patient-derived and murine models of PDAC, we demonstrate that ferritinophagy is upregulated in PDAC to sustain iron availability, thereby promoting tumor progression. Quantitative proteomics reveals that ferritinophagy fuels iron-sulfur cluster protein synthesis to support mitochondrial homeostasis. Targeting NCOA4 leads to tumor growth delay and prolonged survival but with the development of compensatory iron acquisition pathways. Finally, enhanced ferritinophagy accelerates PDAC tumorigenesis, and an elevated ferritinophagy expression signature predicts for poor prognosis in patients with PDAC. Together, our data reveal that the maintenance of iron homeostasis is a critical function of PDAC autophagy, and we define NCOA4-mediated ferritinophagy as a therapeutic target in PDAC. SIGNIFICANCE: Autophagy and iron metabolism are metabolic dependencies in PDAC. However, targeted therapies for these pathways are lacking. We identify NCOA4-mediated selective autophagy of ferritin ("ferritinophagy") as upregulated in PDAC. Ferritinophagy supports PDAC iron metabolism and thereby tumor progression and represents a new therapeutic target in PDAC. See related commentary by Jain and Amaravadi, p. 2023. See related article by Ravichandran et al., p. 2198. This article is highlighted in the In This Issue feature, p. 2007.
Assuntos
Carcinoma Ductal Pancreático , Proteínas Ferro-Enxofre , Neoplasias Pancreáticas , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Disponibilidade Biológica , Carcinoma Ductal Pancreático/genética , Ferritinas/genética , Ferritinas/metabolismo , Humanos , Ferro/metabolismo , Ferro/farmacologia , Proteínas Ferro-Enxofre/metabolismo , Camundongos , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Neoplasias Pancreáticas/genética , Enxofre/metabolismo , Fatores de Transcrição/metabolismo , Neoplasias PancreáticasRESUMO
The mechanisms underlying metabolic adaptation of pancreatic ductal adenocarcinoma (PDA) cells to pharmacologic inhibition of RAS-MAPK signaling are largely unknown. Using transcriptome and chromatin immunoprecipitation profiling of PDA cells treated with the MEK inhibitor (MEKi) trametinib, we identify transcriptional antagonism between c-MYC and the master transcription factors for lysosome gene expression, the MiT/TFE proteins. Under baseline conditions, c-MYC and MiT/TFE factors compete for binding to lysosome gene promoters to fine-tune gene expression. Treatment of PDA cells or patient organoids with MEKi leads to c-MYC downregulation and increased MiT/TFE-dependent lysosome biogenesis. Quantitative proteomics of immunopurified lysosomes uncovered reliance on ferritinophagy, the selective degradation of the iron storage complex ferritin, in MEKi-treated cells. Ferritinophagy promotes mitochondrial iron-sulfur cluster protein synthesis and enhanced mitochondrial respiration. Accordingly, suppressing iron utilization sensitizes PDA cells to MEKi, highlighting a critical and targetable reliance on lysosome-dependent iron supply during adaptation to KRAS-MAPK inhibition. SIGNIFICANCE: Reduced c-MYC levels following MAPK pathway suppression facilitate the upregulation of autophagy and lysosome biogenesis. Increased autophagy-lysosome activity is required for increased ferritinophagy-mediated iron supply, which supports mitochondrial respiration under therapy stress. Disruption of ferritinophagy synergizes with KRAS-MAPK inhibition and blocks PDA growth, thus highlighting a key targetable metabolic dependency. See related commentary by Jain and Amaravadi, p. 2023. See related article by Santana-Codina et al., p. 2180. This article is highlighted in the In This Issue feature, p. 2007.
Assuntos
Carcinoma Ductal Pancreático , Proteínas Ferro-Enxofre , Neoplasias Pancreáticas , Humanos , Disponibilidade Biológica , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Ferro/metabolismo , Ferro/uso terapêutico , Proteínas Ferro-Enxofre/metabolismo , Proteínas Ferro-Enxofre/uso terapêutico , Coativadores de Receptor Nuclear/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Enxofre/metabolismo , Enxofre/uso terapêutico , Fatores de Transcrição/metabolismo , Neoplasias PancreáticasRESUMO
CRISPR-Cas9-based genetic screens have successfully identified cell type-dependent liabilities in cancer, including acute myeloid leukemia (AML), a devastating hematologic malignancy with poor overall survival. Because most of these screens have been performed in vitro using established cell lines, evaluating the physiologic relevance of these targets is critical. We have established a CRISPR screening approach using orthotopic xenograft models to validate and prioritize AML-enriched dependencies in vivo, including in CRISPR-competent AML patient-derived xenograft (PDX) models tractable for genome editing. Our integrated pipeline has revealed several targets with translational value, including SLC5A3 as a metabolic vulnerability for AML addicted to exogenous myo-inositol and MARCH5 as a critical guardian to prevent apoptosis in AML. MARCH5 repression enhanced the efficacy of BCL2 inhibitors such as venetoclax, further highlighting the clinical potential of targeting MARCH5 in AML. Our study provides a valuable strategy for discovery and prioritization of new candidate AML therapeutic targets. SIGNIFICANCE: There is an unmet need to improve the clinical outcome of AML. We developed an integrated in vivo screening approach to prioritize and validate AML dependencies with high translational potential. We identified SLC5A3 as a metabolic vulnerability and MARCH5 as a critical apoptosis regulator in AML, both of which represent novel therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 275.
Assuntos
Antineoplásicos/uso terapêutico , Sistemas CRISPR-Cas , Leucemia Mieloide Aguda/tratamento farmacológico , Medicina de Precisão , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Humanos , Leucemia Mieloide Aguda/genéticaRESUMO
The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03-69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03-69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03-69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03-69 led to an in vivo compatible compound MYF-03-176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration.
Assuntos
Cisteína , Via de Sinalização Hippo , Humanos , Animais , Camundongos , Projetos de Pesquisa , Ativação Transcricional , Transplante HeterólogoRESUMO
The tumor microenvironment (TME) is an important mediator of breast cancer progression. Cancer-associated fibroblasts constitute a major component of the TME and may originate from tissue-associated fibroblasts or infiltrating mesenchymal stromal cells (MSCs). The mechanisms by which cancer cells activate fibroblasts and recruit MSCs to the TME are largely unknown, but likely include deposition of a pro-tumorigenic secretome. The secreted embryonic protein NODAL is clinically associated with breast cancer stage and promotes tumor growth, metastasis, and vascularization. Herein, we show that NODAL expression correlates with the presence of activated fibroblasts in human triple-negative breast cancers and that it directly induces Cancer-associated fibroblasts phenotypes. We further show that NODAL reprograms cancer cell secretomes by simultaneously altering levels of chemokines (e.g., CXCL1), cytokines (e.g., IL-6) and growth factors (e.g., PDGFRA), leading to alterations in MSC chemotaxis. We therefore demonstrate a hitherto unappreciated mechanism underlying the dynamic regulation of the TME.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteína Nodal/genética , Proteína Nodal/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/fisiologia , Actinas/metabolismo , Linhagem Celular Tumoral , Quimiocina CXCL1/metabolismo , Quimiotaxia/fisiologia , Feminino , Humanos , Interleucina-6/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Neoplasias de Mama Triplo Negativas/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Current methods used for measuring amino acid side-chain reactivity lack the throughput needed to screen large chemical libraries for interactions across the proteome. Here we redesigned the workflow for activity-based protein profiling of reactive cysteine residues by using a smaller desthiobiotin-based probe, sample multiplexing, reduced protein starting amounts and software to boost data acquisition in real time on the mass spectrometer. Our method, streamlined cysteine activity-based protein profiling (SLC-ABPP), achieved a 42-fold improvement in sample throughput, corresponding to profiling library members at a depth of >8,000 reactive cysteine sites at 18 min per compound. We applied it to identify proteome-wide targets of covalent inhibitors to mutant Kirsten rat sarcoma (KRAS)G12C and Bruton's tyrosine kinase (BTK). In addition, we created a resource of cysteine reactivity to 285 electrophiles in three human cell lines, which includes >20,000 cysteines from >6,000 proteins per line. The goal of proteome-wide profiling of cysteine reactivity across thousand-member libraries under several cellular contexts is now within reach.