RESUMEN
Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.
Asunto(s)
Neoplasias de la Mama , Fibroblastos Asociados al Cáncer , Pirrolina Carboxilato Reductasas/metabolismo , Neoplasias de la Mama/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinogénesis/metabolismo , Carcinogénesis/patología , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Femenino , Glutamina/metabolismo , Humanos , Prolina , delta-1-Pirrolina-5-Carboxilato ReductasaRESUMEN
High levels of the anti-apoptotic BCL-2 family member MCL-1 are frequently found in breast cancer and, appropriately, BH3-mimetic drugs that specifically target MCL-1's function in apoptosis are in development as anti-cancer therapy. MCL-1 also has reported non-canonical roles that may be relevant in its tumour-promoting effect. Here we investigate the role of MCL-1 in clinically relevant breast cancer models and address whether the canonical role of MCL-1 in apoptosis, which can be targeted using BH3-mimetic drugs, is the major function for MCL-1 in breast cancer. We show that MCL-1 is essential in established tumours with genetic deletion inducing tumour regression and inhibition with the MCL-1-specific BH3-mimetic drug S63845 significantly impeding tumour growth. Importantly, we found that the anti-tumour functions achieved by MCL-1 deletion or inhibition were completely dependent on pro-apoptotic BAX/BAK. Interestingly, we find that MCL-1 is also critical for stem cell activity in human breast cancer cells and high MCL1 expression correlates with stemness markers in tumours. This strongly supports the idea that the key function of MCL-1 in breast cancer is through its anti-apoptotic function. This has important implications for the future use of MCL-1-specific BH3-mimetic drugs in breast cancer treatment.
Asunto(s)
Apoptosis/genética , Neoplasias de la Mama/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Animales , Femenino , Humanos , RatonesRESUMEN
The p53 tumor suppressor protein is a potent activator of proliferative arrest and cell death. In normal cells, this pathway is restrained by p53 protein degradation mediated by the E3-ubiquitin ligase activity of MDM2. Oncogenic stress releases p53 from MDM2 control, so activating the p53 response. However, many tumors that retain wild-type p53 inappropriately maintain the MDM2-p53 regulatory loop in order to continuously suppress p53 activity. We have shown previously that single point mutations in the human MDM2 RING finger domain prevent the interaction of MDM2 with the E2/ubiquitin complex, resulting in the loss of MDM2's E3 activity without preventing p53 binding. Here, we show that an analogous mouse MDM2 mutant (MDM2 I438K) restrains p53 sufficiently for normal growth but exhibits an enhanced stress response in vitro. In vivo, constitutive expression of MDM2 I438K leads to embryonic lethality that is rescued by p53 deletion, suggesting MDM2 I438K is not able to adequately control p53 function through development. However, the switch to I438K expression is tolerated in adult mice, sparing normal cells but allowing for an enhanced p53 response to DNA damage. Viewed as a proof of principle model for therapeutic development, our findings support an approach that would inhibit MDM2 E3 activity without preventing MDM2/p53 binding as a promising avenue for development of compounds to activate p53 in tumors with reduced on-target toxicities.
Asunto(s)
Desarrollo Embrionario/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Animales , Antineoplásicos Hormonales/farmacología , Proliferación Celular/genética , Células Cultivadas , Embrión de Mamíferos/enzimología , Activación Enzimática/efectos de los fármacos , Femenino , Masculino , Ratones , Mutación , Tamoxifeno/farmacologíaRESUMEN
Current nutritional recommendations are focused on energy, fat, carbohydrate, protein and vitamins. Less attention has been paid to the nutritional demand of one-carbon units for nucleotide and methionine synthesis. Here, we investigated the impact of sodium formate supplementation as a nutritional intervention to increase the dietary intake of one-carbon units. A cohort of six female and six male mice received 125 mM of sodium formate in the drinking water for three months. A control group of another six female and six male mice was also followed up for the same period of time. Tail vein blood samples were collected once a month and profiled with a haematology analyser. At the end of the study, blood and tissues were collected for metabolomics analysis and immune cell profiling. Formate supplementation had no significant physiological effect on male mice, except for a small decrease in body weight. Formate supplementation had no significant effect on the immune cell counts during the intervention or at the end of the study in either gender. In female mice, however, the body weight and spleen wet weight were significantly increased by formate supplementation, while the blood plasma levels of amino acids were decreased. Formate supplementation also increased the frequency of bifidobacteria, a probiotic bacterium, in the stools of female mice. We conclude that formate supplementation induces physiological changes in a gender-specific manner.
Asunto(s)
Aminoácidos/sangre , Peso Corporal/efectos de los fármacos , Suplementos Dietéticos , Formiatos/farmacología , Animales , Bifidobacterium/efectos de los fármacos , Bifidobacterium/metabolismo , Femenino , Formiatos/sangre , Microbioma Gastrointestinal , Sistema Inmunológico/metabolismo , Masculino , Ratones , Filogenia , Tamaño de la MuestraRESUMEN
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor prognosis and high rates of relapse. The lack of actionable targets for TNBC has contributed to the high mortality rates of this disease, and new candidate molecules for potential manipulation are urgently required. Here, we show that macrophage-stimulating protein (MSP) and its tyrosine kinase receptor, Recepteur d'origine nantais (RON), are potent drivers of cancer cell growth and tumor progression in a mouse model of TNBC driven by the loss of Trp53 and Brca1. After comparison of two genetically engineered mouse models of TNBC, we found that mammary tumors from K14-Cre;Brca1F/F ;Trp53F/F (KB1P) mice exhibit high endogenous levels of MSP and RON expression. We show that MSP stimulates serine/threonine kinase 1 and extracellular regulated MAPK activation as well as cancer cell growth in cell lines derived from the two mouse models, while genetic and pharmacological inhibition of RON prevents these effects. Similarly, KB1P tumor progression in mice was robustly attenuated by treatment with a RON inhibitor with accompanied reduction in the proliferation marker, Ki-67. Analysis of human gene expression data confirmed that the genes encoding MSP and RON are robustly expressed in human TNBC as well as other subsets of breast cancer. Our findings uncover a mouse model where MSP expression and RON expression are naturally increased, and they provide evidence that this receptor and its ligand are viable candidate molecules for targeted treatment of breast cancer.
Asunto(s)
Factor de Crecimiento de Hepatocito/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Neoplasias de la Mama Triple Negativas/patología , Animales , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Formate is a precursor for the de novo synthesis of purine and deoxythymidine nucleotides. Formate also interacts with energy metabolism by promoting the synthesis of adenine nucleotides. Here we use theoretical modelling together with metabolomics analysis to investigate the link between formate, nucleotide and energy metabolism. We uncover that endogenous or exogenous formate induces a metabolic switch from low to high adenine nucleotide levels, increasing the rate of glycolysis and repressing the AMPK activity. Formate also induces an increase in the pyrimidine precursor orotate and the urea cycle intermediate argininosuccinate, in agreement with the ATP-dependent activities of carbamoyl-phosphate and argininosuccinate synthetase. In vivo data for mouse and human cancers confirms the association between increased formate production, nucleotide and energy metabolism. Finally, the in vitro observations are recapitulated in mice following and intraperitoneal injection of formate. We conclude that formate is a potent regulator of purine, pyrimidine and energy metabolism.
Asunto(s)
Metabolismo Energético/efectos de los fármacos , Formiatos/farmacología , Nucleótidos/metabolismo , Adenosina Trifosfato/farmacología , Adenilato Quinasa/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones Endogámicos C57BL , Modelos Biológicos , Modelos Genéticos , Ácido Orótico/metabolismo , Pirimidinas/metabolismo , Ribonucleótidos/farmacologíaRESUMEN
BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. METHODS: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. RESULTS: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. CONCLUSIONS: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Neoplasias Cutáneas/patología , Animales , Biomarcadores de Tumor , Biopsia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Masculino , Mutación , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Formate overflow coupled to mitochondrial oxidative metabolism\ has been observed in cancer cell lines, but whether that takes place in the tumor microenvironment is not known. Here we report the observation of serine catabolism to formate in normal murine tissues, with a relative rate correlating with serine levels and the tissue oxidative state. Yet, serine catabolism to formate is increased in the transformed tissue of in vivo models of intestinal adenomas and mammary carcinomas. The increased serine catabolism to formate is associated with increased serum formate levels. Finally, we show that inhibition of formate production by genetic interference reduces cancer cell invasion and this phenotype can be rescued by exogenous formate. We conclude that increased formate overflow is a hallmark of oxidative cancers and that high formate levels promote invasion via a yet unknown mechanism.
Asunto(s)
Adenoma/metabolismo , Formiatos/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Intestinales/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Serina/metabolismo , Adenoma/genética , Adenoma/patología , Animales , Antimetabolitos Antineoplásicos/farmacología , Línea Celular Tumoral , Femenino , Formiatos/farmacología , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/metabolismo , Mucosa Intestinal/metabolismo , Neoplasias Intestinales/genética , Neoplasias Intestinales/patología , Intestinos/patología , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Glándulas Mamarias Animales/virología , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/virología , Virus del Tumor Mamario del Ratón/patogenicidad , Metotrexato/farmacología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción , Microambiente Tumoral/efectos de los fármacosRESUMEN
Analysis of publicly available genomic and gene expression data demonstrates that MCL1 expression is frequently elevated in breast cancer. Distinct from other pro-survival Bcl-2 family members, the short half-life of MCL-1 protein led us to investigate MCL-1 protein expression in a breast cancer tissue microarray and correlate this with clinical data. Here, we report associations between high MCL-1 and poor prognosis in specific subtypes of breast cancer including triple-negative breast cancer, an aggressive form that lacks targeted treatment options. Deletion of MCL-1 in the mammary epithelium of genetically engineered mice revealed an absolute requirement for MCL-1 in breast tumorigenesis. The clinical applicability of these findings was tested through a combination of approaches including knock-down or inhibition of MCL-1 to show triple-negative breast cancer cell line dependence on MCL-1 in vitro and in vivo. Our data demonstrate that high MCL-1 protein expression is associated with poor outcome in breast cancer and support the therapeutic targeting of MCL-1 in this disease.
Asunto(s)
Neoplasias de la Mama/metabolismo , Terapia Molecular Dirigida , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Animales , Neoplasias de la Mama/clasificación , Neoplasias de la Mama/patología , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Femenino , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Pronóstico , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The role of glutaminolysis in providing metabolites to support tumour growth is well-established, but the involvement of glutamine metabolism in invasive processes is yet to be elucidated. Here we show that normal mammary epithelial cells consume glutamine, but do not secrete glutamate. Indeed, low levels of extracellular glutamate are necessary to maintain epithelial homoeostasis, and provision of glutamate drives disruption of epithelial morphology and promotes key characteristics of the invasive phenotype such as lumen-filling and basement membrane disruption. By contrast, primary cultures of invasive breast cancer cells convert glutamine to glutamate which is released from the cell through the system Xc- antiporter to activate a metabotropic glutamate receptor. This contributes to the intrinsic aggressiveness of these cells by upregulating Rab27-dependent recycling of the transmembrane matrix metalloprotease, MT1-MMP to promote invasive behaviour leading to basement membrane disruption. These data indicate that acquisition of the ability to release glutamate is a key watershed in disease aggressiveness.
Asunto(s)
Neoplasias de la Mama/metabolismo , Ácido Glutámico/metabolismo , Glutamina/metabolismo , Glándulas Mamarias Humanas/metabolismo , Neoplasias Mamarias Animales/metabolismo , Sistema de Transporte de Aminoácidos y+/metabolismo , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Espacio Extracelular/metabolismo , Femenino , Homeostasis , Humanos , Neoplasias Mamarias Animales/patología , Metaloproteinasa 14 de la Matriz/metabolismo , Ratones , Invasividad Neoplásica , Receptores de Glutamato Metabotrópico/metabolismo , Regulación hacia Arriba , Proteínas rab27 de Unión a GTP/metabolismoRESUMEN
CD68+ tumor-associated macrophages (TAMs) are pro-tumorigenic, pro-angiogenic and are associated with decreased survival rates in patients with cancer, including breast cancer. Non-specific models of macrophage ablation reduce the number of TAMs and limit the development of mammary tumors. However, the lack of specificity and side effects associated with these models compromise their reliability. We hypothesized that specific and controlled macrophage depletion would provide precise data on the effects of reducing TAM numbers on tumor development. In this study, the MacLow mouse model of doxycycline-inducible and selective CD68+ macrophage depletion was crossed with the murine mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) mouse model of spontaneous ductal breast adenocarcinoma to generate the PyMT-MacLow line. In doxycycline-treated PyMT-MacLow mice, macrophage numbers were decreased in areas surrounding tumors by 43%. Reducing the number of macrophages by this level delayed tumor progression, generated less proliferative tumors, decreased the vascularization of carcinomas and down-regulated the expression of many pro-angiogenic genes. These results demonstrate that depleting CD68+ macrophages in an inducible and selective manner delays the development of mammary tumors and that the PyMT-MacLow model is a useful and unique tool for studying the role of TAMs in breast cancer.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Neoplasias de la Mama/inmunología , Modelos Animales de Enfermedad , Macrófagos/inmunología , Animales , Neoplasias de la Mama/patología , Doxiciclina/farmacología , Femenino , Humanos , Macrófagos/efectos de los fármacos , RatonesRESUMEN
Apoptosis represents a key anti-cancer therapeutic effector mechanism. During apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically kills cells even in the absence of caspase activity. Caspase activity can also have a variety of unwanted consequences that include DNA damage. We therefore investigated whether MOMP-induced caspase-independent cell death (CICD) might be a better way to kill cancer cells. We find that cells undergoing CICD display potent pro-inflammatory effects relative to apoptosis. Underlying this, MOMP was found to stimulate NF-κB activity through the downregulation of inhibitor of apoptosis proteins. Strikingly, engagement of CICD displays potent anti-tumorigenic effects, often promoting complete tumour regression in a manner dependent on intact immunity. Our data demonstrate that by activating NF-κB, MOMP can exert additional signalling functions besides triggering cell death. Moreover, they support a rationale for engaging caspase-independent cell death in cell-killing anti-cancer therapies.
Asunto(s)
Caspasas/metabolismo , Neoplasias del Colon/enzimología , Mediadores de Inflamación/metabolismo , Mitocondrias/enzimología , Membranas Mitocondriales/enzimología , FN-kappa B/metabolismo , Compuestos de Anilina/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Genotipo , Células HeLa , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Activación de Macrófagos , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Mitocondrias/efectos de los fármacos , Mitocondrias/inmunología , Mitocondrias/patología , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/inmunología , Membranas Mitocondriales/patología , FN-kappa B/deficiencia , Necrosis , Permeabilidad , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Sulfonamidas/farmacología , Factores de Tiempo , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Quinasa de Factor Nuclear kappa BRESUMEN
Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates ß-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis.
Asunto(s)
Comunicación Celular , Células Endoteliales/fisiología , Melanocitos/fisiología , Cadherinas/análisis , Línea Celular , Proteína 61 Rica en Cisteína/análisis , Regulación de la Expresión Génica , Humanos , Espectrometría de Masas , beta Catenina/análisisRESUMEN
The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion.
Asunto(s)
Canales de Cloruro/metabolismo , Progresión de la Enfermedad , Glutatión/metabolismo , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Femenino , Proteínas de Unión al GTP/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Endogámicos C57BL , Ratones Desnudos , Modelos Biológicos , Invasividad Neoplásica , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Oxidorreductasas/metabolismo , Unión Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteoma/metabolismo , Proteómica , Análisis de Supervivencia , Transglutaminasas/metabolismo , Resultado del TratamientoRESUMEN
Acetyl-CoA is a key metabolic intermediate with an important role in transcriptional regulation. The nuclear-cytosolic acetyl-CoA synthetase 2 (ACSS2) was found to sustain the growth of hypoxic tumor cells. It generates acetyl-CoA from acetate, but exactly which pathways it supports is not fully understood. Here, quantitative analysis of acetate metabolism reveals that ACSS2 fulfills distinct functions depending on its cellular location. Exogenous acetate uptake is controlled by expression of both ACSS2 and the mitochondrial ACSS1, and ACSS2 supports lipogenesis. The mitochondrial and lipogenic demand for two-carbon acetyl units considerably exceeds the uptake of exogenous acetate, leaving it to only sparingly contribute to histone acetylation. Surprisingly, oxygen and serum limitation increase nuclear localization of ACSS2. We find that nuclear ACSS2 recaptures acetate released from histone deacetylation for recycling by histone acetyltransferases. Our work provides evidence for limited equilibration between nuclear and cytosolic acetyl-CoA and demonstrates that ACSS2 retains acetate to maintain histone acetylation.
Asunto(s)
Acetato CoA Ligasa/metabolismo , Acetatos/metabolismo , Hipoxia de la Célula , Histonas/metabolismo , Acetato CoA Ligasa/antagonistas & inhibidores , Acetato CoA Ligasa/genética , Acetatos/química , Acetilcoenzima A/metabolismo , Acetilación , Isótopos de Carbono/química , Línea Celular Tumoral , Núcleo Celular/enzimología , Cromatografía Líquida de Alta Presión , Medios de Cultivo/química , Humanos , Espectrometría de Masas , Metaboloma , Microscopía Fluorescente , Mitocondrias/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Suero/químicaRESUMEN
Sildenafil, an inhibitor of the cGMP-degrading phosphodiesterase 5 that is used to treat erectile dysfunction, has been linked to an increased risk of melanoma. Here, we have examined the potential connection between cGMP-dependent signaling cascades and melanoma growth. Using a combination of biochemical assays and real-time monitoring of melanoma cells, we report a cGMP-dependent growth-promoting pathway in murine and human melanoma cells. We document that C-type natriuretic peptide (CNP), a ligand of the membrane-bound guanylate cyclase B, enhances the activity of cGMP-dependent protein kinase I (cGKI) in melanoma cells by increasing the intracellular levels of cGMP. Activation of this cGMP pathway promotes melanoma cell growth and migration in a p44/42 MAPK-dependent manner. Sildenafil treatment further increases intracellular cGMP concentrations, potentiating activation of this pathway. Collectively, our data identify this cGMP-cGKI pathway as the link between sildenafil usage and increased melanoma risk.
Asunto(s)
GMP Cíclico/metabolismo , Transducción de Señal/efectos de los fármacos , Citrato de Sildenafil/farmacología , Animales , Butadienos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/química , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Femenino , Humanos , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Péptido Natriurético Tipo-C/toxicidad , Nitrilos/farmacología , Inhibidores de Fosfodiesterasa 5/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Citrato de Sildenafil/uso terapéutico , Trasplante HomólogoRESUMEN
The cGMP-dependent protein kinase I (cGKI) is a key mediator of cGMP signaling, but the specific functions of its two isoforms, cGKIα and cGKIß, are poorly understood. Recent studies indicated a novel cGMP-independent role for cGKIα in redox sensing. To dissect the effects of oxidative stress on the cGKI isoforms, we used mouse embryonic fibroblasts and vascular smooth muscle cells (VSMCs) expressing both, one, or none of them. In cGKIα-expressing cells, but not in cells expressing only cGKIß, incubation with H2O2 induced the formation of a disulfide bond between the two identical subunits of the dimeric enzyme. Oxidation of cGKIα was associated with increased phosphorylation of its substrate, vasodilator-stimulated phosphoprotein. H2O2 did not stimulate cGMP production, indicating that it activates cGKIα directly via oxidation. Interestingly, there was a mutual influence of H2O2 and cGMP on cGKI activity and disulfide bond formation, respectively; preoxidation of the kinase with H2O2 slightly impaired its activation by cGMP, whereas preactivation of the enzyme with cGMP attenuated its oxidation by H2O2. To evaluate the functional relevance of the noncanonical H2O2-cGKIα pathway, we studied the regulation of the cytosolic Ca²âº concentration ([Ca²âº](i)). H2O2 suppressed norepinephrine-induced Ca²âº transients in cGKIα-expressing VSMCs and, to a lower extent, in VSMCs expressing only cGKIß or none of the isoforms. Thus, H2O2 lowers [Ca²âº](i) mainly via a cGKIα-dependent pathway. These results indicate that oxidative stress selectively targets the cGKIα isoform, which then modulates cellular processes in a cGMP-independent manner. A decrease in [Ca²âº](i) in VSMCs via activation of cGKIα might be a major mechanism of H2O2-induced vasodilation.
Asunto(s)
Calcio/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Citosol/metabolismo , Embrión de Mamíferos/enzimología , Fibroblastos/enzimología , Peróxido de Hidrógeno/farmacología , Músculo Liso Vascular/enzimología , Animales , Western Blotting , Células Cultivadas , GMP Cíclico/metabolismo , Disulfuros/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Oxidantes/farmacología , Transducción de SeñalRESUMEN
A growing body of evidence suggests that hydrogen sulfide (H2S) is a signaling molecule in mammalian cells. In the cardiovascular system, H2S enhances vasodilation and angiogenesis. H2S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (K(ATP)); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H2S-induced vasorelaxation. The effect of H2S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H2S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H2S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H2S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H2S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H2S production) were reduced in vessels of PKG-I knockout mice (PKG-Iâ»/â»). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-Iâ»/â», suggesting that there is a cross-talk between K(ATP) and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation.