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Distinguishing quiescent from rupture-prone atherosclerotic lesions has significant translational and clinical implications. Electrochemical impedance spectroscopy (EIS) characterizes biological tissues by assessing impedance and phase delay responses to alternating current at multiple frequencies. We evaluated invasive 6-point stretchable EIS sensors over a spectrum of experimental atherosclerosis and compared results with intravascular ultrasound (IVUS), molecular positron emission tomography (PET) imaging, and histology. Male New Zealand White rabbits (n = 16) were placed on a high-fat diet, with or without endothelial denudation via balloon injury of the infrarenal abdominal aorta. Rabbits underwent in vivo micro-PET imaging of the abdominal aorta with 68Ga-DOTATATE, 18F-NaF, and 18F-FDG, followed by invasive interrogation via IVUS and EIS. Background signal-corrected values of impedance and phase delay were determined. Abdominal aortic samples were collected for histology. Analyses were performed blindly. EIS impedance was associated with markers of plaque activity including macrophage infiltration (r = .813, p = .008) and macrophage/smooth muscle cell (SMC) ratio (r = .813, p = .026). Moreover, EIS phase delay correlated with anatomic markers of plaque burden, namely intima/media ratio (r = .883, p = .004) and %stenosis (r = .901, p = .002), similar to IVUS. 68Ga-DOTATATE correlated with intimal macrophage infiltration (r = .861, p = .003) and macrophage/SMC ratio (r = .831, p = .021), 18F-NaF with SMC infiltration (r = -.842, p = .018), and 18F-FDG correlated with macrophage/SMC ratio (r = .787, p = .036). EIS with phase delay integrates key atherosclerosis features that otherwise require multiple complementary invasive and non-invasive imaging approaches to capture. These findings indicate the potential of invasive EIS to comprehensively evaluate human coronary artery disease.
Assuntos
Aterosclerose , Espectroscopia Dielétrica , Animais , Coelhos , Espectroscopia Dielétrica/métodos , Masculino , Aterosclerose/patologia , Aterosclerose/diagnóstico por imagem , Aorta Abdominal/patologia , Aorta Abdominal/diagnóstico por imagem , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/patologia , Tomografia por Emissão de Pósitrons/métodos , Fenótipo , Modelos Animais de Doenças , Macrófagos/patologia , Macrófagos/metabolismoRESUMO
Adenocarcinomas from multiple tissues can converge to treatment-resistant small cell neuroendocrine (SCN) cancers comprised of ASCL1, POU2F3, NEUROD1, and YAP1 subtypes. We investigated how mitochondrial metabolism influences SCN cancer (SCNC) progression. Extensive bioinformatics analyses encompassing thousands of patient tumors and human cancer cell lines uncovered enhanced expression of PGC-1α, a potent regulator of mitochondrial oxidative phosphorylation (OXPHOS), across several SCNC types. PGC-1α correlated tightly with increased expression of the lineage marker ASCL1 through a positive feedback mechanism. Analyses using a human prostate tissue-based SCN transformation system showed that the ASCL1 subtype has heightened PGC-1α expression and OXPHOS activity. PGC-1α inhibition diminished OXPHOS, reduced SCNC cell proliferation, and blocked SCN prostate tumor formation. PGC-1α overexpression enhanced OXPHOS, tripled the SCN prostate tumor formation rate, and promoted commitment to the ASCL1 lineage. These findings reveal the metabolic heterogeneity among SCNC subtypes and identify PGC-1α-induced OXPHOS as a regulator of SCNC lineage plasticity.
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Epithelial membrane protein-2 (EMP2) is upregulated in a number of tumors and therefore remains a promising target for mAb-based therapy. In the current study, image-guided therapy for an anti-EMP2 mAb was evaluated by PET in both syngeneic and immunodeficient cancer models expressing different levels of EMP2 to enable a better understanding of its tumor uptake and off target accumulation and clearance. The therapeutic efficacy of the anti-EMP2 mAb was initially evaluated in high- and low-expressing tumors, and the mAb reduced tumor load for the high EMP2-expressing 4T1 and HEC-1-A tumors. To create an imaging agent, the anti-EMP2 mAb was conjugated to p-SCN-Bn-deferoxamine (DFO) and radiolabeled with 89Zr. Tumor targeting and tissue biodistribution were evaluated in syngeneic tumor models (4T1, CT26, and Panc02) and human tumor xenograft models (Ramos, HEC-1-A, and U87MG/EMP2). PET imaging revealed radioactive accumulation in EMP2-positive tumors within 24 hours after injection, and the signal was retained for 5 days. High specific uptake was observed in tumors with high EMP2 expression (4T1, CT26, HEC-1-A, and U87MG/EMP2), with less accumulation in tumors with low EMP2 expression (Panc02 and Ramos). Biodistribution at 5 days after injection revealed that the tumor uptake ranged from 2 to approximately 16%ID/cc. The results show that anti-EMP2 mAbs exhibit EMP2-dependent tumor uptake with low off-target accumulation in preclinical cancer models. The development of improved anti-EMP2 Ab fragments may be useful to track EMP2-positive tumors for subsequent therapeutic interventions.
Assuntos
Glicoproteínas de Membrana , Radioisótopos , Zircônio , Animais , Humanos , Camundongos , Glicoproteínas de Membrana/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Linhagem Celular Tumoral , Feminino , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Distribuição Tecidual , Anticorpos Monoclonais , Modelos Animais de DoençasRESUMO
Significant evidence suggests that the failure of clinically tested epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (e.g. erlotinib, lapatinib, gefitinib) in glioblastoma (GBM) patients is primarily attributed to insufficient brain penetration, resulting in inadequate exposure to the targeted cells. Molecular imaging tools can facilitate GBM drug development by visualizing drug biodistribution and confirming target expression and localization. To assess brain exposure via PET molecular imaging, we synthesized fluorine-18 isotopologues of two brain-penetrant EGFR tyrosine kinase inhibitors developed specifically for GBM. Adapting our recently reported radiofluorination of N-arylsydnones, we constructed an ortho-disubstituted [18F]fluoroarene as the key intermediate. The radiotracers were produced on an automated synthesis module in 7-8% activity yield with high molar activity. In vivo PET imaging revealed rapid brain uptake in rodents and tumor accumulation in an EGFR-driven orthotopic GBM xenograft model.
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Lactate is a mitochondrial substrate for many tissues including neuron, muscle, skeletal and cardiac, as well as many cancer cells, however little is known about the processes that regulate its utilization in mitochondria. Based on the close association of Hexokinases (HK) with mitochondria, and the known cardio-protective role of HK in cardiac muscle, we have investigated the regulation of lactate and pyruvate metabolism by hexokinases (HKs), utilizing wild-type HEK293 cells and HEK293 cells in which the endogenous HKI and/or HKII have been knocked down to enable overexpression of wild type and mutant HKs. To assess the real-time changes in intracellular lactate levels the cells were transfected with a lactate specific FRET probe. In the HKI/HKII double knockdown cells, addition of extracellular pyruvate caused a large and sustained decrease in lactate. This decrease was rapidly reversed upon inhibition of the malate aspartate shuttle by aminooxyacetate, or inhibition of mitochondrial oxidative respiration by NaCN. These results suggest that in the absence of HKs, pyruvate-dependent activation of the TCA cycle together with the malate aspartate shuttle facilitates lactate transformation into pyruvate and its utilization by mitochondria. With replacement by overexpression of HKI or HKII the cellular response to pyruvate and NaCN was modified. With either hexokinase present, both the decrease in lactate due to the addition of pyruvate and the increase following addition of NaCN were either transient or suppressed altogether. Blockage of the pentose phosphate pathway with the inhibitor 6-aminonicotinamide (6-AN), abolished the effects of HK replacement. These results suggest that blocking of the malate aspartate shuttle by HK may involve activation of the pentose phosphate pathway and increased NADPH production.
Assuntos
Ácido Láctico , Ácido Pirúvico , Humanos , Hexoquinase/metabolismo , Malatos/metabolismo , Ácido Aspártico/metabolismo , Células HEK293RESUMO
BACKGROUND: Resistance to existing therapies is a significant challenge in improving outcomes for glioblastoma (GBM) patients. Metabolic plasticity has emerged as an important contributor to therapy resistance, including radiation therapy (RT). Here, we investigated how GBM cells reprogram their glucose metabolism in response to RT to promote radiation resistance. METHODS: Effects of radiation on glucose metabolism of human GBM specimens were examined in vitro and in vivo with the use of metabolic and enzymatic assays, targeted metabolomics, and FDG-PET. Radiosensitization potential of interfering with M2 isoform of pyruvate kinase (PKM2) activity was tested via gliomasphere formation assays and in vivo human GBM models. RESULTS: Here, we show that RT induces increased glucose utilization by GBM cells, and this is accompanied with translocation of GLUT3 transporters to the cell membrane. Irradiated GBM cells route glucose carbons through the pentose phosphate pathway (PPP) to harness the antioxidant power of the PPP and support survival after radiation. This response is regulated in part by the PKM2. Activators of PKM2 can antagonize the radiation-induced rewiring of glucose metabolism and radiosensitize GBM cells in vitro and in vivo. CONCLUSIONS: These findings open the possibility that interventions designed to target cancer-specific regulators of metabolic plasticity, such as PKM2, rather than specific metabolic pathways, have the potential to improve the radiotherapeutic outcomes in GBM patients.
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Glioblastoma , Piruvato Quinase , Humanos , Piruvato Quinase/metabolismo , Glioblastoma/metabolismo , Antioxidantes , Isoformas de Proteínas , Glucose/metabolismo , Linhagem Celular TumoralRESUMO
Mitochondria are critical to the governance of metabolism and bioenergetics in cancer cells1. The mitochondria form highly organized networks, in which their outer and inner membrane structures define their bioenergetic capacity2,3. However, in vivo studies delineating the relationship between the structural organization of mitochondrial networks and their bioenergetic activity have been limited. Here we present an in vivo structural and functional analysis of mitochondrial networks and bioenergetic phenotypes in non-small cell lung cancer (NSCLC) using an integrated platform consisting of positron emission tomography imaging, respirometry and three-dimensional scanning block-face electron microscopy. The diverse bioenergetic phenotypes and metabolic dependencies we identified in NSCLC tumours align with distinct structural organization of mitochondrial networks present. Further, we discovered that mitochondrial networks are organized into distinct compartments within tumour cells. In tumours with high rates of oxidative phosphorylation (OXPHOSHI) and fatty acid oxidation, we identified peri-droplet mitochondrial networks wherein mitochondria contact and surround lipid droplets. By contrast, we discovered that in tumours with low rates of OXPHOS (OXPHOSLO), high glucose flux regulated perinuclear localization of mitochondria, structural remodelling of cristae and mitochondrial respiratory capacity. Our findings suggest that in NSCLC, mitochondrial networks are compartmentalized into distinct subpopulations that govern the bioenergetic capacity of tumours.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Metabolismo Energético , Neoplasias Pulmonares , Mitocôndrias , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/ultraestrutura , Ácidos Graxos/metabolismo , Glucose/metabolismo , Gotículas Lipídicas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/ultraestrutura , Microscopia Eletrônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Fosforilação Oxidativa , Fenótipo , Tomografia por Emissão de PósitronsRESUMO
The low-dose mixture hypothesis of carcinogenesis proposes that exposure to an environmental chemical that is not individually oncogenic may nonetheless be capable of enabling carcinogenesis when it acts in concert with other factors. A class of ubiquitous environmental chemicals that are hypothesized to potentially function in this low-dose capacity are synthesized polybrominated diphenyl ethers (PBDEs). PBDEs can affect correlates of carcinogenesis that include genomic instability and inflammation. However, the effect of low-dose PBDE exposure on such correlates in mammary tissue has not been examined. In the present study, low-dose long-term (16 weeks) administration of PBDE to mice modulated transcriptomic indicators of genomic integrity and innate immunity in normal mammary tissue. PBDE increased transcriptome signatures for the Nuclear Factor Erythroid 2 Like 2 (NFE2L2) response to oxidative stress and decreased signatures for non-homologous end joining DNA repair (NHEJ). PBDE also decreased transcriptome signatures for the cyclic GMP-AMP Synthase - Stimulator of Interferon Genes (cGAS-STING) response, decreased indication of Interferon Stimulated Gene Factor 3 (ISGF3) and Nuclear Factor Kappa B (NF-κB) transcription factor activity, and increased digital cytometry estimates of immature dendritic cells (DCs) in mammary tissue. Replication of the PBDE exposure protocol in mice susceptible to mammary carcinogenesis resulted in greater tumor development. The results support the notion that ongoing exposure to low levels of PBDE can disrupt facets of genomic integrity and innate immunity in mammary tissue. Such effects affirm that synthesized PBDEs are a class of environmental chemicals that reasonably fit the low-dose mixture hypothesis.
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Chemically synthesized, small peptides that bind with high affinity and specificity to CD8-expressing (CD8+) tumor-infiltrating T cells, yet retain the desirable characteristics of small molecules, hold valuable potential for diagnostic molecular imaging of immune response. Here, we report the development of 18F-labeled peptides targeting human CD8α with nanomolar affinity via the strain-promoted sydnone-alkyne cycloaddition with 4-[18F]fluorophenyl sydnone. The 18F-sydnone is produced in one step, in high radiochemical yield, and the peptide labeling proceeds rapidly. A hydrophilic chemical linker results in a tracer with favorable pharmacokinetic properties and improved image contrast, as demonstrated by in vivo PET imaging studies.
Assuntos
Alcinos , Tomografia por Emissão de Pósitrons , Animais , Reação de Cicloadição , Radioisótopos de FlúorRESUMO
Unlike other epithelial cancer types, circulating tumor cells (CTCs) are less frequently detected in the peripheral blood of non-small cell lung cancer (NSCLC) patients using epithelial marker-based detection approaches despite the aggressive nature of NSCLC. Here, we demonstrate hexokinase-2 (HK2) as a metabolic function-associated marker for the detection of CTCs. In 59 NSCLC patients bearing cytokeratin-positive (CKpos) primary tumors, HK2 enables resolving cytokeratin-negative (HK2high/CKneg) CTCs as a prevalent population in about half of the peripheral blood samples with positive CTC counts. However, HK2high/CKneg tumor cells are a minority population in pleural effusions and cerebrospinal fluids. Single-cell analysis shows that HK2high/CKneg CTCs exhibit smaller sizes but consistent copy number variation profiles compared with CKpos counterparts. Single-cell transcriptome profiling reveals that CK expression levels of CTCs are independent of their epithelial-to-mesenchymal transition (EMT) status, challenging the long-standing association between CK expression and EMT. HK2high/CKneg CTCs display metastasis and EGFR inhibitor resistance-related molecular signatures and are selectively enriched in patients with EGFRL858R driver oncogene mutation as opposed to EGFR19Del , which is more frequently found in patients with prevalent CKpos CTCs in the blood. Consistently, treatment-naïve patients with a larger number or proportion of HK2high/CKneg CTCs in the blood exhibit poor therapy response and shorter progression-free survival. Collectively, our approach resolves a more complete spectrum of CTCs in NSCLC that can potentially be exploited to identify patient prognosis before therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Hexoquinase/sangue , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/patologia , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Transição Epitelial-Mesenquimal , Receptores ErbB/genética , Genótipo , Humanos , Queratinas/sangue , Biópsia Líquida , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/enzimologia , PrognósticoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Positron emission tomography (PET) reporter genes (PRGs), when coupled with positron-emitting PET reporter probes (PRPs), are useful for tracking specific cell populations in cell-based therapies, in transgenic animal models, and in xenograft tumor progression experiments. The activities of incorporated PRGs in targeted cells can be monitored noninvasively by PET imaging in preclinical in vivo studies and clinical applications following systemic administration of the appropriate PRG. Here we describe a method that minimizes both design and variability of vector delivery vehicles for alternative PRGs and biological variability of the in vivo target when comparing the efficacy, sensitivity, and specificity of alternative PRG/PRP combinations for in vivo PRG imaging. The principles described for comparing alternative PRG/PRP reporter gene systems can be applied to comparisons of alternative fluorescence, bioluminescence, single-photon emission computerized tomography (SPECT), and magnetic resonance imaging (MRI) reporter genes.
Assuntos
Adenoviridae/genética , Genes Reporter , Imagem Molecular/métodos , Tomografia por Emissão de Pósitrons/métodos , Timidina Quinase/análise , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Arginine (Arg) deprivation is a promising therapeutic approach for tumors with low argininosuccinate synthetase 1 (ASS1) expression. However, its efficacy as a single agent therapy needs to be improved as resistance is frequently observed. Methods: A tissue microarray was performed to assess ASS1 expression in surgical specimens of pancreatic ductal adenocarcinoma (PDAC) and its correlation with disease prognosis. An RNA-Seq analysis examined the role of ASS1 in regulating the global gene transcriptome. A high throughput screen of FDA-approved oncology drugs identified synthetic lethality between histone deacetylase (HDAC) inhibitors and Arg deprivation in PDAC cells with low ASS1 expression. We examined HDAC inhibitor panobinostat (PAN) and Arg deprivation in a panel of human PDAC cell lines, in ASS1-high and -knockdown/knockout isogenic models, in both anchorage-dependent and -independent cultures, and in multicellular complex cultures that model the PDAC tumor microenvironment. We examined the effects of combined Arg deprivation and PAN on DNA damage and the protein levels of key DNA repair enzymes. We also evaluated the efficacy of PAN and ADI-PEG20 (an Arg-degrading agent currently in Phase 2 clinical trials) in xenograft models with ASS1-low and -high PDAC tumors. Results: Low ASS1 protein level is a negative prognostic indicator in PDAC. Arg deprivation in ASS1-deficient PDAC cells upregulated asparagine synthetase (ASNS) which redirected aspartate (Asp) from being used for de novo nucleotide biosynthesis, thus causing nucleotide insufficiency and impairing cell cycle S-phase progression. Comprehensively validated, HDAC inhibitors and Arg deprivation showed synthetic lethality in ASS1-low PDAC cells. Mechanistically, combined Arg deprivation and HDAC inhibition triggered degradation of a key DNA repair enzyme C-terminal-binding protein interacting protein (CtIP), resulting in DNA damage and apoptosis. In addition, S-phase-retained ASS1-low PDAC cells (due to Arg deprivation) were also sensitized to DNA damage, thus yielding effective cell death. Compared to single agents, the combination of PAN and ADI-PEG20 showed better efficacy in suppressing ASS1-low PDAC tumor growth in mouse xenograft models. Conclusion: The combination of PAN and ADI-PEG20 is a rational translational therapeutic strategy for treating ASS1-low PDAC tumors through synergistic induction of DNA damage.
Assuntos
Arginina/deficiência , Argininossuccinato Sintase/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Histona Desacetilases/química , Hidrolases/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Panobinostat/farmacologia , Polietilenoglicóis/farmacologia , Animais , Antineoplásicos/farmacologia , Argininossuccinato Sintase/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Feminino , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Prognóstico , Mutações Sintéticas LetaisRESUMO
Chronic inflammation facilitates tumor progression. We discovered that a subset of non-small cell lung cancer cells underwent a gradually progressing epithelial-to-mesenchymal (EMT) phenotype following a 21-day exposure to IL-1ß, an abundant proinflammatory cytokine in the at-risk for lung cancer pulmonary and the lung tumor microenvironments. Pathway analysis of the gene expression profile and in vitro functional studies revealed that the EMT and EMT-associated phenotypes, including enhanced cell invasion, PD-L1 upregulation, and chemoresistance, were sustained in the absence of continuous IL-1ß exposure. We referred to this phenomenon as EMT memory. Utilizing a doxycycline-controlled SLUG expression system, we found that high expression of the transcription factor SLUG was indispensable for the establishment of EMT memory. High SLUG expression in tumors of lung cancer patients was associated with poor survival. Chemical or genetic inhibition of SLUG upregulation prevented EMT following the acute IL-1ß exposure but did not reverse EMT memory. Chromatin immunoprecipitation and methylation-specific PCR further revealed a SLUG-mediated temporal regulation of epigenetic modifications, including accumulation of H3K27, H3K9, and DNA methylation, in the CDH1 (E-cadherin) promoter following the chronic IL-1ß exposure. Chemical inhibition of DNA methylation not only restored E-cadherin expression in EMT memory, but also primed cells for chemotherapy-induced apoptosis.
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Carcinoma Pulmonar de Células não Pequenas/patologia , Epigênese Genética , Transição Epitelial-Mesenquimal , Memória Imunológica/imunologia , Inflamação/imunologia , Interleucina-1beta/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/imunologia , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Memória Imunológica/genética , Inflamação/genética , Interleucina-1beta/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Fenótipo , Células Tumorais CultivadasRESUMO
Biosynthesis of the pyrimidine nucleotide uridine monophosphate (UMP) is essential for cell proliferation and is achieved by the activity of convergent de novo and salvage metabolic pathways. Here we report the development and application of a cell-based metabolic modifier screening platform that leverages the redundancy in pyrimidine metabolism for the discovery of selective UMP biosynthesis modulators. In evaluating a library of protein kinase inhibitors, we identified multiple compounds that possess nucleotide metabolism modifying activity. The JNK inhibitor JNK-IN-8 was found to potently inhibit nucleoside transport and engage ENT1. The PDK1 inhibitor OSU-03012 (also known as AR-12) and the RAF inhibitor TAK-632 were shown to inhibit the therapeutically relevant de novo pathway enzyme DHODH and their affinities were unambiguously confirmed through in vitro assays and co-crystallization with human DHODH.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/antagonistas & inibidores , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Nucleosídeos de Pirimidina/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Di-Hidro-Orotato Desidrogenase , Desenho de Fármacos , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Humanos , Simulação de Dinâmica Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Since Warburg's observation that most cancers exhibit elevated glycolysis, decades of research have attempted to reduce tumor glucose utilization as a therapeutic approach. Hexokinase (HK) activity is the first glycolytic enzymatic step; despite many attempts to inhibit HK activity, none has reached clinical application. Identification of HK isoforms, and recognition that most tissues express only HK1 while most tumors express HK1 and HK2, stimulated reducing HK2 activity as a therapeutic option. However, studies using HK2 shRNA and isogenic HK1+HK2- and HK1+HK2+ tumor cell pairs demonstrated that tumors expressing only HK1, while exhibiting reduced glucose consumption, progressed in vivo as well as tumors expressing both HK1 and HK2. However, HK1-HK2+ tumor subpopulations exist among many cancers. shRNA HK2 suppression in HK1-HK2+ liver cancer cells reduced xenograft tumor progression, in contrast to HK1+HK2+ cells. HK2 inhibition, and partial inhibition of both oxidative phosphorylation and fatty acid oxidation using HK2 shRNA and small-molecule drugs, prevented human liver HK1-HK2+ cancer xenograft progression. Using human multiple myeloma xenografts and mouse allogeneic models to identify potential clinical translational agents, triple therapies that include antisense HK2 oligonucleotides, metformin, and perhexiline prevent progression. These results suggest an agnostic approach for HK1-HK2+ cancers, regardless of tissue origin.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Glicólise/efeitos dos fármacos , Hexoquinase/antagonistas & inibidores , Hexoquinase/genética , Neoplasias/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Glicólise/genética , Hexoquinase/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Neoplasias/genética , Neoplasias/patologia , Oniocompostos/farmacologia , Oniocompostos/uso terapêutico , Fosforilação Oxidativa/efeitos dos fármacos , Perexilina/farmacologia , Perexilina/uso terapêutico , RNA Interferente Pequeno/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aberrant overexpression of endoplasmic reticulum (ER)-resident oxidoreductase protein disulfide isomerase (PDI) plays an important role in cancer progression. In this study, we demonstrate that PDI promotes glioblastoma (GBM) cell growth and describe a class of allosteric PDI inhibitors that are selective for PDI over other PDI family members. Methods: We performed a phenotypic screening triage campaign of over 20,000 diverse compounds to identify PDI inhibitors cytotoxic to cancer cells. From this screen, BAP2 emerged as a lead compound, and we assessed BAP2-PDI interactions with gel filtration, thiol-competition assays, and site-directed mutagenesis studies. To assess selectivity, we compared BAP2 activity across several PDI family members in the PDI reductase assay. Finally, we performed in vivo studies with a mouse xenograft model of GBM combining BAP2 and the standard of care (temozolomide and radiation), and identified affected gene pathways with nascent RNA sequencing (Bru-seq). Results: BAP2 and related analogs are novel PDI inhibitors that selectively inhibit PDIA1 and PDIp. Though BAP2 contains a weak Michael acceptor, interaction with PDI relies on Histidine 256 in the b' domain of PDI, suggesting allosteric binding. Furthermore, both in vitro and in vivo, BAP2 reduces cell and tumor growth. BAP2 alters the transcription of genes involved in the unfolded protein response, ER stress, apoptosis and DNA repair response. Conclusion: These results indicate that BAP2 has anti-tumor activity and the suppressive effect on DNA repair gene expression warrants combination with DNA damaging agents to treat GBM.
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Antineoplásicos/farmacologia , Reparo do DNA/efeitos dos fármacos , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Glioblastoma/tratamento farmacológico , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/isolamento & purificação , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Transplante de Neoplasias , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Transplante Heterólogo , Resultado do TratamentoRESUMO
Colony-stimulating factor 1 receptor is a type III receptor protein tyrosine kinase belonging to PDGFR family. CSF1R signaling is essential for differentiation, proliferation and survival of macrophages. Aberrant expression of CSF1R appears to be an attractive target in several cancer types. Higher expression of CSF1R ligands correlates to tumor progression. CSF1R inhibitors have been shown to suppress cancers. We have attempted an in silico fragment derived drug discovery approach by screening Ë25,000 in-house compounds as potential CSF1R inhibitors. Using FBDD approach we have identified six diverse fragments that exhibit affinity towards hinge region of CSF1R. Some of the fragments 5-nitroindole and 7-azaindole and their derivatives were synthesized for further evaluation. The in silico and in vitro enzyme activity studies reveal moderate inhibition of CSF1R kinase activity by 5-nitroindole and good inhibition by 7-azaindole fragments. Bio and chemiinformatics studies have shown that 7-azaindole compounds have better membrane permeability and enzyme inhibition properties. Molecular docking studies show that the amino acid residues 664-666 in the hinge region of the cytosolic domain of CSF1R to be the preferred region of binding for nitroindole and azaindole derivatives. Further optimization and biological analysis would identify these fragments as potential and promising leads as CSF1R inhibitors.
Assuntos
Indóis/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Biologia Computacional , Desenho de Fármacos , Humanos , Indóis/síntese química , Indóis/química , Simulação de Acoplamento Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Receptores Proteína Tirosina Quinases/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/química , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
Although the majority of adult tissues express only hexokinase 1 (HK1) for glycolysis, most cancers express hexokinase 2 (HK2) and many coexpress HK1 and HK2. In contrast to HK1+HK2+ cancers, HK1-HK2+ cancer subsets are sensitive to cytostasis induced by HK2shRNA knockdown and are also sensitive to synthetic lethality in response to the combination of HK2shRNA knockdown, an oxidative phosphorylation (OXPHOS) inhibitor diphenyleneiodonium (DPI), and a fatty acid oxidation (FAO) inhibitor perhexiline (PER). The majority of human multiple myeloma cell lines are HK1-HK2+. Here we describe an antisense oligonucleotide (ASO) directed against human HK2 (HK2-ASO1), which suppressed HK2 expression in human multiple myeloma cell cultures and human multiple myeloma mouse xenograft models. The HK2-ASO1/DPI/PER triple-combination achieved synthetic lethality in multiple myeloma cells in culture and prevented HK1-HK2+ multiple myeloma tumor xenograft progression. DPI was replaceable by the FDA-approved OXPHOS inhibitor metformin (MET), both for synthetic lethality in culture and for inhibition of tumor xenograft progression. In addition, we used an ASO targeting murine HK2 (mHK2-ASO1) to validate the safety of mHK2-ASO1/MET/PER combination therapy in mice bearing murine multiple myeloma tumors. HK2-ASO1 is the first agent that shows selective HK2 inhibition and therapeutic efficacy in cell culture and in animal models, supporting clinical development of this synthetically lethal combination as a therapy for HK1-HK2+ multiple myeloma. SIGNIFICANCE: A first-in-class HK2 antisense oligonucleotide suppresses HK2 expression in cell culture and in in vivo, presenting an effective, tolerated combination therapy for preventing progression of HK1-HK2+ multiple myeloma tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2748/F1.large.jpg.
Assuntos
Hexoquinase/genética , Mieloma Múltiplo/patologia , Oligonucleotídeos Antissenso/farmacologia , Mutações Sintéticas Letais , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Functional lysosomes mediate autophagy and macropinocytosis for nutrient acquisition. Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit high basal lysosomal activity, and inhibition of lysosome function suppresses PDAC cell proliferation and tumor growth. However, the codependencies induced by lysosomal inhibition in PDAC have not been systematically explored. We performed a comprehensive pharmacological inhibition screen of the protein kinome and found that replication stress response (RSR) inhibitors were synthetically lethal with chloroquine (CQ) in PDAC cells. CQ treatment reduced de novo nucleotide biosynthesis and induced replication stress. We found that CQ treatment caused mitochondrial dysfunction and depletion of aspartate, an essential precursor for de novo nucleotide synthesis, as an underlying mechanism. Supplementation with aspartate partially rescued the phenotypes induced by CQ. The synergy of CQ and the RSR inhibitor VE-822 was comprehensively validated in both 2D and 3D cultures of PDAC cell lines, a heterotypic spheroid culture with cancer-associated fibroblasts, and in vivo xenograft and syngeneic PDAC mouse models. These results indicate a codependency on functional lysosomes and RSR in PDAC and support the translational potential of the combination of CQ and RSR inhibitors.