RESUMO
Cancer cells must integrate multiple biosynthetic demands to drive indefinite proliferation. How these key cellular processes, such as metabolism and protein synthesis, crosstalk to fuel cancer cell growth is unknown. Here, we uncover the mechanism by which the Myc oncogene coordinates the production of the two most abundant classes of cellular macromolecules, proteins, and nucleic acids in cancer cells. We find that a single rate-limiting enzyme, phosphoribosyl-pyrophosphate synthetase 2 (PRPS2), promotes increased nucleotide biosynthesis in Myc-transformed cells. Remarkably, Prps2 couples protein and nucleotide biosynthesis through a specialized cis-regulatory element within the Prps2 5' UTR, which is controlled by the oncogene and translation initiation factor eIF4E downstream Myc activation. We demonstrate with a Prps2 knockout mouse that the nexus between protein and nucleotide biosynthesis controlled by PRPS2 is crucial for Myc-driven tumorigenesis. Together, these studies identify a translationally anchored anabolic circuit critical for cancer cell survival and an unexpected vulnerability for "undruggable" oncogenes, such as Myc. PAPERFLICK:
Assuntos
Carcinogênese , Nucleotídeos/biossíntese , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ribose-Fosfato Pirofosfoquinase/genética , Regiões 5' não Traduzidas , Animais , Linfócitos B/metabolismo , Sequência de Bases , Linfoma de Burkitt/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Células-Tronco Embrionárias , Fator de Iniciação 4E em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Células NIH 3T3 , Ribose-Fosfato Pirofosfoquinase/metabolismoRESUMO
Prostate cancer (PCa) is the second most common cancer type among American men and it is estimated that in 2023, 34,700 men will die from PCa. Since it can take a considerable amount of time for the disease to progress to clinically evident cancer, there is ample opportunity for effective chemopreventive strategies to be applied for the successful management of PCa progression. In the current study, we have developed a two-tiered metabolomics-based screen to identify synergistic combinations of phytochemicals for PCa chemoprevention. This involves an initial screen for ATP depletion in PCa cells followed by a targeted screen for blocking glutamine uptake in the same cells. One of the phytochemical combinations (enoxolone [ENO] + silibinin [SIL]), identified via this screen, was examined for effects on PCa cell survival, oncogenic signaling and tumor growth in vivo. This combination was found to synergistically reduce cell survival, colony formation and cell cycle progression of PCa cell lines to a greater extent than either agent alone. The combination of ENO and SIL also synergistically reduced tumor growth when administered ad libitum through the diet in a HMVP2 allograft PCa tumor model. Treatment with the combination also significantly reduced STAT3 and mTORC1 signaling pathways in mouse and human PCa cells while significantly reducing levels of critical cell cycle regulatory proteins, contributing to the synergistic inhibition of tumor growth observed. Collectively, the current results demonstrate a novel approach to identifying synergistic combinations of phytochemicals for chemoprevention of PCa and possibly other cancers.
Assuntos
Ácido Glicirretínico , Segunda Neoplasia Primária , Neoplasias da Próstata , Masculino , Humanos , Animais , Camundongos , Detecção Precoce de Câncer , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/prevenção & controle , Proteínas de Ciclo Celular , Linhagem Celular , Sobrevivência Celular , Linhagem Celular TumoralRESUMO
Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.
Assuntos
Cistationina gama-Liase/farmacologia , Metionina/antagonistas & inibidores , Mutagênese Sítio-Dirigida , Neoplasias da Próstata/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Cistationina gama-Liase/genética , Cistationina gama-Liase/isolamento & purificação , Cistationina gama-Liase/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Ensaios Enzimáticos , Humanos , Masculino , Metionina/sangue , Metionina/metabolismo , Camundongos , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias da Próstata/sangue , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Testes de Toxicidade Aguda , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Obesity is a major contributing factor to the pathogenesis of Type 2 diabetes. Multiple human genetics studies suggest that high activity of the low molecular weight protein tyrosine phosphatase (LMPTP) promotes metabolic syndrome in obesity. We reported that LMPTP is a critical promoter of insulin resistance in obesity by regulating liver insulin receptor signaling and that inhibition of LMPTP reverses obesity-associated diabetes in mice. Since LMPTP is expressed in adipose tissue but little is known about its function, here we examined the role of LMPTP in adipocyte biology. Using conditional knockout mice, we found that selective deletion of LMPTP in adipocytes impaired obesity-induced subcutaneous adipocyte hypertrophy. We assessed the role of LMPTP in adipogenesis in vitro, and found that LMPTP deletion or knockdown substantially impaired differentiation of primary preadipocytes and 3T3-L1 cells into adipocytes, respectively. Inhibition of LMPTP in 3T3-L1 preadipocytes also reduced adipogenesis and expression of proadipogenic transcription factors peroxisome proliferator activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha. Inhibition of LMPTP increased basal phosphorylation of platelet-derived growth factor receptor alpha (PDGFRα) on activation motif residue Y849 in 3T3-L1, resulting in increased activation of the mitogen-associated protein kinases p38 and c-Jun N-terminal kinase and increased PPARγ phosphorylation on inhibitory residue S82. Analysis of the metabolome of differentiating 3T3-L1 cells suggested that LMPTP inhibition decreased cell glucose utilization while enhancing mitochondrial respiration and nucleotide synthesis. In summary, we report a novel role for LMPTP as a key driver of adipocyte differentiation via control of PDGFRα signaling.
Assuntos
Adipócitos/metabolismo , Adipócitos/patologia , Adipogenia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Gordura Subcutânea/patologia , Células 3T3-L1 , Adipogenia/genética , Animais , Diferenciação Celular/genética , Respiração Celular , Tamanho Celular , Transporte de Elétrons , Deleção de Genes , Regulação da Expressão Gênica , Glucose/metabolismo , Glicólise , Hipertrofia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Metaboloma , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Modelos Biológicos , PPAR gama/metabolismo , Fosforilação , Fosfosserina/metabolismo , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Stereospecific recognition of metabolites plays a significant role in the detection of potential disease biomarkers thereby providing new insights in diagnosis and prognosis. D-Hdroxy/amino acids are recognized as potential biomarkers in several metabolic disorders. Despite continuous advances in metabolomics technologies, the simultaneous measurement of different classes of enantiomeric metabolites in a single analytical run remains challenging. Here, we develop a novel strategy for untargeted chiral metabolomics of hydroxy/amine groups (-OH/-NH2) containing metabolites, including all hydroxy acids (HAs) and amino acids (AAs), by chiral derivatization coupled with liquid chromatography-high resolution tandem mass spectrometry (LC-HR-MS/MS). Diacetyl-tartaric anhydride (DATAN) was used for the simultaneous derivatization of-OH/-NH2 containing metabolites as well as the resulting diastereomers, and all the derivatized metabolites were resolved in a single analytical run. Data independent MS/MS acquisition (DIA) was applied to positively identify DATAN-labeled metabolites based on reagent specific diagnostic fragment ions. We discriminated chiral from achiral metabolites based on the reversal of elution order of D and L isomers derivatized with the enantiomeric pair (±) of DATAN in an untargeted manner. Using the developed strategy, a library of 301 standards that consisted of 214 chiral and 87 achiral metabolites were separated and detected in a single analytical run. This approach was then applied to investigate the enantioselective metabolic profile of the bone marrow (BM) and peripheral blood (PB) plasma samples from patients with acute myeloid leukemia (AML) at diagnosis and following completion of the induction phase of chemotherapeutic treatment. The sensitivity and selectivity of the developed method enabled the detection of trace levels of the D-enantiomer of HAs and AAs in primary plasma patient samples. Several of these metabolites were significantly altered in response to chemotherapy. The developed LC-HR-MS method entails a valuable step forward in chiral metabolomics.
Assuntos
Metabolômica , Espectrometria de Massas em Tandem , Cromatografia Líquida , Humanos , Metaboloma , EstereoisomerismoRESUMO
Fatty acid profiling on gas chromatography-mass spectrometry (GC-MS) platforms is typically performed offline by manually derivatizing and analyzing small batches of samples. A GC-MS system with a fully integrated robotic autosampler can significantly improve sample handling, standardize data collection, and reduce the total hands-on time required for sample analysis. In this study, we report an optimized high-throughput GC-MS-based methodology that utilizes trimethyl sulfonium hydroxide (TMSH) as a derivatization reagent to convert fatty acids into fatty acid methyl esters. An automated online derivatization method was developed, in which the robotic autosampler derivatizes each sample individually and injects it into the GC-MS system in a high-throughput manner. This study investigated the robustness of automated TMSH derivatization by comparing fatty acid standards and lipid extracts, derivatized manually in batches and online automatically from four biological matrices. Automated derivatization improved reproducibility in 19 of 33 fatty acid standards, with nearly half of the 33 confirmed fatty acids in biological samples demonstrating improved reproducibility when compared to manually derivatized samples. In summary, we show that the online TMSH-based derivatization methodology is ideal for high-throughput fatty acid analysis, allowing rapid and efficient fatty acid profiling, with reduced sample handling, faster data acquisition, and, ultimately, improved data reproducibility.
RESUMO
Exploiting metabolic vulnerabilities of cancer cells with nontoxic, plant derived compounds constitutes a novel strategy for both chemoprevention and treatment. A high-throughput screening approach was used to evaluate a library of natural products to determine the most synergistic combination in precursor-B cell acute lymphoblast leukemia. Dimethylaminoparthenolide and shikonin effectively inhibited proliferation resulting in cell death in primary and immortalized leukemia cells, while having negligible effects on normal cells. Dimethylaminoparthenolide and shikonin have been shown separately to inhibit cell survival and proliferative signaling and activate tumor suppressors and proapoptotic pathways. Untargeted metabolomics and metabolic flux analysis with stable isotopically labeled glucose and glutamine exhibited a global shift in metabolism following treatment. Pathway analysis indicated significant differences in amino acid, antioxidant, tricarboxylic acid cycle, and nucleotide metabolism. Together, dimethylaminoparthenolide and shikonin reduced the shunting of glycolytic intermediates into the pentose phosphate pathway for biosynthetic purposes. Similarly, the incorporation of glutamine and glutamine-derived metabolites into purine and pyrimidine synthesis was inhibited by the combination of dimethylaminoparthenolide and shikonin, effectively impeding biosynthetic pathways critical for leukemia cell survival. This approach demonstrates that a synergistic pair of compounds with malignant cell specificity can effectively target metabolic pathways crucial to leukemia cell proliferation and induce apoptosis.
Assuntos
Proliferação de Células/efeitos dos fármacos , Naftoquinonas/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Sesquiterpenos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Criança , Ciclo do Ácido Cítrico/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Sinergismo Farmacológico , Glucose/metabolismo , Glutamina/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologiaRESUMO
Despite advances in surgery and adjuvant therapy, brain tumors represent one of the leading causes of cancer-related mortality and morbidity in both adults and children. Gliomas constitute about 60% of all cerebral tumors, showing varying degrees of malignancy. They are difficult to treat due to dismal prognosis and limited therapeutics. Metabolomics is the untargeted and targeted analyses of endogenous and exogenous small molecules, which charact erizes the phenotype of an individual. This emerging "omics" science provides functional readouts of cellular activity that contribute greatly to the understanding of cancer biology including brain tumor biology. Metabolites are highly informative as a direct signature of biochemical activity; therefore, metabolite profiling has become a promising approach for clinical diagnostics and prognostics. The metabolic alterations are well-recognized as one of the key hallmarks in monitoring disease progression, therapy, and revealing new molecular targets for effective therapeutic intervention. Taking advantage of the latest high-throughput analytical technologies, that is, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS), metabolomics is now a promising field for precision medicine and drug discovery. In the present report, we review the application of metabolomics and in vivo metabolic profiling in the context of adult gliomas and paediatric brain tumors. Analytical platforms such as high-resolution (HR) NMR, in vivo magnetic resonance spectroscopic imaging and high- and low-resolution MS are discussed. Moreover, the relevance of metabolic studies in the development of new therapeutic strategies for treatment of gliomas are reviewed.
Assuntos
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Glioma/metabolismo , Metaboloma , Metabolômica/métodos , Adulto , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Criança , Glioma/patologia , Humanos , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodosRESUMO
Metabolic reprogramming is increasingly being viewed as a hallmark of cancer. Accordingly, metabolic readouts can serve as biomarkers of response to therapy. The goal of this study was to investigate some of the MRS-detectable metabolic consequences of mitogen-activated protein kinase kinase (MEK) inhibition. We investigated PC3 prostate cancer, MCF-7 breast cancer and A375 melanoma cells, and determined that, consistent with previous studies, MRS-detectable levels of phosphocholine decreased significantly in all cell lines (to 63%, 50% and 18% of the control, respectively) following MEK inhibition with U0126. This effect was mediated by a decrease in the expression of choline kinase α, the enzyme that catalyzes the phosphorylation of choline. In contrast, the impact of MEK inhibition on glycolysis was cell line dependent. A375 cells, which express mutant BRAF, demonstrated significant decreases in glucose uptake (to 36% of control) and lactate production (to 42% of control) in line with positron emission tomography data. In contrast, in PC3 and MCF-7 cells, increases in glucose uptake (to 198% and 192% of control, respectively) and lactate production (to 177% and 212% of control, respectively) were observed, in line with a previous hyperpolarized (13) C MRS study. This effect is probably mediated by the activation of the phosphoinositide 3-kinase pathway and AMP-activated protein kinase. Our findings demonstrate the value of translatable non-invasive MRS methods for the provision of information on cellular metabolism as an indication of the activation of potential feedback loops following MEK inhibition.
Assuntos
Butadienos/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases Ativadas por AMP/fisiologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Glicólise , Humanos , Masculino , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilcolina/análise , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismoRESUMO
The prognosis for patients with pancreatic cancer is extremely poor, as evidenced by the disease's five-year survival rate of ~5%. New approaches are therefore urgently needed to improve detection, treatment, and monitoring of pancreatic cancer. MRS-detectable metabolic changes provide useful biomarkers for tumor detection and response-monitoring in other cancers. The goal of this study was to identify MRS-detectable biomarkers of pancreatic cancer that could enhance currently available imaging approaches. We used (1) H high-resolution magic angle spinning MRS to probe metabolite levels in pancreatic tissue samples from mouse models and patients. In mice, the levels of lipids dropped significantly in pancreata with lipopolysaccharide-induced inflammation, in pancreata with pre-cancerous metaplasia (4 week old p48-Cre;LSL-Kras(G12D) mice), and in pancreata with pancreatic intraepithelial neoplasia, which precedes invasive pancreatic cancer (8 week old p48-Cre LSL-Kras(G12D) mice), to 26 ± 19% (p = 0.03), 19 ± 16% (p = 0.04), and 26 ± 10% (p = 0.05) of controls, respectively. Lactate and taurine remained unchanged in inflammation and in pre-cancerous metaplasia but increased significantly in pancreatic intraepithelial neoplasia to 266 ± 61% (p = 0.0001) and 999 ± 174% (p < 0.00001) of controls, respectively. Importantly, analysis of patient biopsies was consistent with the mouse findings. Lipids dropped in pancreatitis and in invasive cancer biopsies to 29 ± 15% (p = 0.01) and 26 ± 38% (p = 0.02) of normal tissue. In addition, lactate and taurine levels remained unchanged in inflammation but rose in tumor samples to 244 ± 155% (p = 0.02) and 188 ± 67% (p = 0.02), respectively, compared with normal tissue. Based on these findings, we propose that a drop in lipid levels could serve to inform on pancreatitis and cancer-associated inflammation, whereas elevated lactate and taurine could serve to identify the presence of pancreatic intraepithelial neoplasia and invasive tumor. Our findings may help enhance current imaging methods to improve early pancreatic cancer detection and monitoring.
Assuntos
Carcinoma Ductal Pancreático/química , Lactatos/análise , Lipídeos/análise , Espectroscopia de Ressonância Magnética/métodos , Pâncreas/química , Neoplasias Pancreáticas/química , Pancreatite/metabolismo , Taurina/análise , Animais , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Diagnóstico Precoce , Genes ras , Humanos , Lipopolissacarídeos/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Pâncreas/patologia , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Pancreatite/induzido quimicamente , Pancreatite/diagnóstico , Pancreatite/patologia , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologiaRESUMO
The biological functions of the scaffold protein Ran Binding Protein 9 (RanBP9) remain elusive in macrophages or any other cell type where this protein is expressed together with its CTLH (C-terminal to LisH) complex partners. We have engineered a new mouse model, named RanBP9-TurnX, where RanBP9 fused to three copies of the HA tag (RanBP9-3xHA) can be turned into RanBP9-V5 tagged upon Cre-mediated recombination. We created this model to enable stringent biochemical studies at cell type specific level throughout the entire organism. Here, we have used this tool crossed with LysM-Cre transgenic mice to identify RanBP9 interactions in lung macrophages. We show that RanBP9-V5 and RanBP9-3xHA can be both co-immunoprecipitated with the known members of the CTLH complex from the same whole lung lysates. However, more than ninety percent of the proteins pulled down by RanBP9-V5 differ from those pulled-down by RanBP9-HA. The lung RanBP9-V5 associated proteome includes previously unknown interactions with macrophage-specific proteins as well as with players of the innate immune response, DNA damage response, metabolism, and mitochondrial function. This work provides the first lung specific RanBP9-associated interactome in physiological conditions and reveals that RanBP9 and the CTLH complex could be key regulators of macrophage bioenergetics and immune functions.
RESUMO
Sacituzumab Govitecan (SG) is an antibody-drug conjugate that has demonstrated efficacy in patients with TROP-2 expressing epithelial cancers. In a xenograft model of intracranial breast cancer, SG inhibited tumor growth and increased mouse survival. We conducted a prospective window-of-opportunity trial (NCT03995706) at the University of Texas Health Science Center at San Antonio to examine the intra-tumoral concentrations and intracranial activity of SG in patients undergoing craniotomy for breast cancer with brain metastases (BCBM) or recurrent glioblastoma (rGBM). We enrolled 25 patients aged ≥18 years diagnosed with BCBM and rGBM to receive a single intravenous dose of SG at 10 mg/kg given one day before resection and continued on days 1 and 8 of 21-day cycles following recovery. The PFS was 8 months and 2 months for BCBM and rGBM cohorts, respectively. The OS was 35.2 months and 9.5 months, respectively. Grade≥3 AE included neutropenia (28%), hypokalemia (8%), seizure (8%), thromboembolic event (8%), urinary tract infection (8%) and muscle weakness of the lower limb (8%). In post-surgical tissue, the median total SN-38 was 249.8 ng/g for BCBM and 104.5 ng/g for rGBM, thus fulfilling the primary endpoint. Biomarker analysis suggests delivery of payload by direct release at target site and that hypoxic changes do not drive indirect release. Secondary endpoint of OS was 35.2 months for the BCBM cohort and 9.5 months for rGBM. Non-planned exploratory endpoint of ORR was 38% for BCBM and 29%, respectively. Exploratory endpoint of Trop-2 expression was observed in 100% of BCBM and 78% of rGBM tumors. In conclusion, SG was found to be well tolerated with adequate penetration into intracranial tumors and promising preliminary activity within the CNS. Trial Registration: Trial (NCT03995706) enrolled at Clinical Trials.gov as Neuro/Sacituzumab Govitecan/Breast Brain Metastasis/Glioblastoma/Ph 0: https://clinicaltrials.gov/study/NCT03995706?cond=NCT03995706 .
Assuntos
Anticorpos Monoclonais Humanizados , Neoplasias Encefálicas , Neoplasias da Mama , Glioblastoma , Imunoconjugados , Recidiva Local de Neoplasia , Humanos , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Pessoa de Meia-Idade , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Humanizados/administração & dosagem , Adulto , Idoso , Imunoconjugados/uso terapêutico , Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , Estudos Prospectivos , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismoRESUMO
Protein tyrosine phosphatases (PTPs) play major roles in cancer and are emerging as therapeutic targets. Recent reports suggest low-molecular weight PTP (LMPTP)-encoded by the ACP1 gene-is overexpressed in prostate tumors. We found ACP1 up-regulated in human prostate tumors and ACP1 expression inversely correlated with overall survival. Using CRISPR-Cas9-generated LMPTP knockout C4-2B and MyC-CaP cells, we identified LMPTP as a critical promoter of prostate cancer (PCa) growth and bone metastasis. Through metabolomics, we found that LMPTP promotes PCa cell glutathione synthesis by dephosphorylating glutathione synthetase on inhibitory Tyr270. PCa cells lacking LMPTP showed reduced glutathione, enhanced activation of eukaryotic initiation factor 2-mediated stress response, and enhanced reactive oxygen species after exposure to taxane drugs. LMPTP inhibition slowed primary and bone metastatic prostate tumor growth in mice. These findings reveal a role for LMPTP as a critical promoter of PCa growth and metastasis and validate LMPTP inhibition as a therapeutic strategy for treating PCa through sensitization to oxidative stress.
Assuntos
Neoplasias da Próstata , Masculino , Humanos , Camundongos , Animais , Peso Molecular , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Tirosina , Proteínas Tirosina Fosfatases/metabolismoRESUMO
Alterations in cell metabolism are increasingly being recognized as a hallmark of cancer and are being exploited for the development of diagnostic tools and targeted therapeutics. Recently, ¹³C MRS-detectable hyperpolarized pyruvate to lactate conversion has been validated in models as a noninvasive imaging method for the detection of tumors and treatment response, and has successfully passed phase I clinical trials. To date, response to treatment has been associated with a decrease in hyperpolarized lactate production. In this study, we monitored the effect of treatment with the mitogen-activated protein kinase (MEK) inhibitor U0126 in prostate and breast cancer cells. Following treatment, we observed a 31% decrease in the flux of hyperpolarized ¹³C label in treated MCF-7 breast cancer cells relative to controls. In contrast, and unexpectedly, the flux increased to 167% in treated PC3 prostate cancer cells. To mechanistically explain these observations, we investigated treatment-induced changes in the different factors known to affect the pyruvate to lactate conversion. NADH (nicotinamide adenine dinucleotide, reduced form) levels remained unchanged, whereas lactate dehydrogenase expression and activity, as well as intracellular lactate, increased in both cell lines, providing an explanation for the elevated hyperpolarized lactate observed in PC3 cells. The expression of MCT1, which mediates pyruvate transport, decreased in treated MCF-7, but not PC3, cells. This identifies pyruvate transport as rate limiting in U0126-treated MCF-7 cells and explains the decrease in hyperpolarized lactate observed in these cells following treatment. Our findings highlight the complexity of interactions between MEK and metabolism, and the need for mechanistic validation before hyperpolarized ¹³C MRS can be used to monitor treatment-induced molecular responses.
Assuntos
Neoplasias da Mama/metabolismo , Butadienos/administração & dosagem , Ácido Láctico/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/administração & dosagem , Neoplasias da Próstata/metabolismo , Ácido Pirúvico/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Masculino , Neoplasias da Próstata/tratamento farmacológicoRESUMO
The human mitochondrial carrier family (MCF) consists of 53 members. Approximately one-fifth of them are still orphans of a function. Most mitochondrial transporters have been functionally characterized by reconstituting the bacterially expressed protein into liposomes and transport assays with radiolabeled compounds. The efficacy of this experimental approach is constrained to the commercial availability of the radiolabeled substrate to be used in the transport assays. A striking example is that of N-acetylglutamate (NAG), an essential regulator of the carbamoyl synthetase I activity and the entire urea cycle. Mammals cannot modulate mitochondrial NAG synthesis but can regulate the levels of NAG in the matrix by exporting it to the cytosol, where it is degraded. The mitochondrial NAG transporter is still unknown. Here, we report the generation of a yeast cell model suitable for identifying the putative mammalian mitochondrial NAG transporter. In yeast, the arginine biosynthesis starts in the mitochondria from NAG which is converted to ornithine that, once transported into cytosol, is metabolized to arginine. The deletion of ARG8 makes yeast cells unable to grow in the absence of arginine since they cannot synthetize ornithine but can still produce NAG. To make yeast cells dependent on a mitochondrial NAG exporter, we moved most of the yeast mitochondrial biosynthetic pathway to the cytosol by expressing four E. coli enzymes, argB-E, able to convert cytosolic NAG to ornithine. Although argB-E rescued the arginine auxotrophy of arg8∆ strain very poorly, the expression of the bacterial NAG synthase (argA), which would mimic the function of a putative NAG transporter increasing the cytosolic levels of NAG, fully rescued the growth defect of arg8∆ strain in the absence of arginine, demonstrating the potential suitability of the model generated.
Assuntos
Escherichia coli , Saccharomyces cerevisiae , Animais , Humanos , Saccharomyces cerevisiae/metabolismo , Escherichia coli/metabolismo , Mamíferos/metabolismo , Arginina/metabolismo , OrnitinaRESUMO
Somatic mutations are a major source of cancer development, and many driver mutations have been identified in protein coding regions. However, the function of mutations located in miRNA and their target binding sites throughout the human genome remains largely unknown. Here, we built detailed cancer-specific miRNA regulatory networks across 30 cancer types to systematically analyze the effect of mutations in miRNAs and their target sites in 3' untranslated region (3' UTR), coding sequence (CDS), and 5' UTR regions. A total of 3,518,261 mutations from 9,819 samples were mapped to miRNA-gene interactions (mGI). Mutations in miRNAs showed a mutually exclusive pattern with mutations in their target genes in almost all cancer types. A linear regression method identified 148 candidate driver mutations that can significantly perturb miRNA regulatory networks. Driver mutations in 3'UTRs played their roles by altering RNA binding energy and the expression of target genes. Finally, mutated driver gene targets in 3' UTRs were significantly downregulated in cancer and functioned as tumor suppressors during cancer progression, suggesting potential miRNA candidates with significant clinical implications. A user-friendly, open-access web portal (mGI-map) was developed to facilitate further use of this data resource. Together, these results will facilitate novel noncoding biomarker identification and therapeutic drug design targeting the miRNA regulatory networks. SIGNIFICANCE: A detailed miRNA-gene interaction map reveals extensive miRNA-mediated gene regulatory networks with mutation-induced perturbations across multiple cancers, serving as a resource for noncoding biomarker discovery and drug development.
Assuntos
MicroRNAs , Neoplasias , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias/genética , Mutação , Redes Reguladoras de Genes , Regiões 3' não Traduzidas/genéticaRESUMO
Malignancies can become reliant on glutamine as an alternative energy source and as a facilitator of aberrant DNA methylation, thus implicating glutaminase (GLS) as a potential therapeutic target. We demonstrate preclinical synergy of telaglenastat (CB-839), a selective GLS inhibitor, when combined with azacytidine (AZA), in vitro and in vivo, followed by a phase Ib/II study of the combination in patients with advanced MDS. Treatment with telaglenastat/AZA led to an ORR of 70% with CR/mCRs in 53% patients and a median overall survival of 11.6 months. scRNAseq and flow cytometry demonstrated a myeloid differentiation program at the stem cell level in clinical responders. Expression of non-canonical glutamine transporter, SLC38A1, was found to be overexpressed in MDS stem cells; was associated with clinical responses to telaglenastat/AZA and predictive of worse prognosis in a large MDS cohort. These data demonstrate the safety and efficacy of a combined metabolic and epigenetic approach in MDS.
RESUMO
Although targeting oxidative phosphorylation (OXPHOS) is a rational anticancer strategy, clinical benefit with OXPHOS inhibitors has yet to be achieved. Here we advanced IACS-010759, a highly potent and selective small-molecule complex I inhibitor, into two dose-escalation phase I trials in patients with relapsed/refractory acute myeloid leukemia (NCT02882321, n = 17) and advanced solid tumors (NCT03291938, n = 23). The primary endpoints were safety, tolerability, maximum tolerated dose and recommended phase 2 dose (RP2D) of IACS-010759. The PK, PD, and preliminary antitumor activities of IACS-010759 in patients were also evaluated as secondary endpoints in both clinical trials. IACS-010759 had a narrow therapeutic index with emergent dose-limiting toxicities, including elevated blood lactate and neurotoxicity, which obstructed efforts to maintain target exposure. Consequently no RP2D was established, only modest target inhibition and limited antitumor activity were observed at tolerated doses, and both trials were discontinued. Reverse translational studies in mice demonstrated that IACS-010759 induced behavioral and physiological changes indicative of peripheral neuropathy, which were minimized with the coadministration of a histone deacetylase 6 inhibitor. Additional studies are needed to elucidate the association between OXPHOS inhibition and neurotoxicity, and caution is warranted in the continued development of complex I inhibitors as antitumor agents.
Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Neoplasias , Animais , Camundongos , Antineoplásicos/efeitos adversos , Inibidores de Histona Desacetilases/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Neoplasias/patologia , Fosforilação Oxidativa , HumanosRESUMO
Glioblastomas (GBM) are the most common and aggressive form of primary malignant brain tumor in the adult population, and, despite modern therapies, patients often develop recurrent disease, and the disease remains incurable with median survival below 2 years. Resistance to bevacizumab is driven by hypoxia in the tumor and evofosfamide is a hypoxia-activated prodrug, which we tested in a phase 2, dual center (University of Texas Health Science Center in San Antonio and Dana Farber Cancer Institute) clinical trial after bevacizumab failure. Tumor hypoxic volume was quantified by 18F-misonidazole PET. To identify circulating metabolic biomarkers of tumor hypoxia in patients, we used a high-resolution liquid chromatography-mass spectrometry-based approach to profile blood metabolites and their specific enantiomeric forms using untargeted approaches. Moreover, to evaluate early response to treatment, we characterized changes in circulating metabolite levels during treatment with combined bevacizumab and evofosfamide in recurrent GBM after bevacizumab failure. Gamma aminobutyric acid, and glutamic acid as well as its enantiomeric form D-glutamic acid all inversely correlated with tumor hypoxia. Intermediates of the serine synthesis pathway, which is known to be modulated by hypoxia, also correlated with tumor hypoxia (phosphoserine and serine). Moreover, following treatment, lactic acid was modulated by treatment, likely in response to a hypoxia mediated modulation of oxidative vs glycolytic metabolism. In summary, although our results require further validation in larger patients' cohorts, we have identified candidate metabolic biomarkers that could evaluate the extent of tumor hypoxia and predict the benefit of combined bevacizumab and evofosfamide treatment in GBM following bevacizumab failure.
RESUMO
Drugs used in combination can synergize to increase efficacy, decrease toxicity, and prevent drug resistance. While conventional high-throughput screens that rely on univariate data are incredibly valuable to identify promising drug candidates, phenotypic screening methodologies could be beneficial to provide deep insight into the molecular response of drug combination with a likelihood of improved clinical outcomes. We developed a high-content metabolomics drug screening platform using stable isotope-tracer direct-infusion mass spectrometry that informs an algorithm to determine synergy from multivariate phenomics data. Using a cancer drug library, we validated the drug screening, integrating isotope-enriched metabolomics data and computational data mining, on a panel of prostate cell lines and verified the synergy between CB-839 and docetaxel both in vitro (three-dimensional model) and in vivo. The proposed unbiased metabolomics screening platform can be used to rapidly generate phenotype-informed datasets and quantify synergy for combinatorial drug discovery.