RESUMEN
Urine sample analysis is irreplaceable as a non-invasive method for disease diagnosis and follow-up. However, in urine samples, non-degraded protein and RNA may be only found in urinary extracellular vesicles (uEVs). In recent years, various methods of uEV enrichment using low volumes of urine and unsophisticated equipment have been developed, with variable success. We compared the results of the differential ultracentrifugation procedure with 4 of these methods. The methods tested were a lectin-based purification, Exoquick (System Biosciences), Total Exosome Isolation from Invitrogen and an in-house modified procedure employing the Exosomal RNA Kit from Norgen Biotek Corp. The analysis of selected gene transcripts and protein markers of extracellular vesicles (EVs) revealed that each method isolates a different mixture of uEV protein markers. In our conditions, the extraction with Norgen's reagent achieved the best performance in terms of gene transcript and protein detection and reproducibility. By using this method, we were able to detect alterations of EVs protein markers in urine samples from prostate cancer adenoma patients. Taken together, our results show that the isolation of uEVs is feasible from small volumes of urine and avoiding ultracentrifugation, making easier the analysis in a clinical facility. However, caution should be taken in the selection of the enrichment method since they have a differential affinity for protein uEVs markers and by extension for different subpopulation of EVs.
RESUMEN
Prostate cancer is among the most frequent cancers in men, and despite its high rate of cure, the high number of cases results in an elevated mortality worldwide. Importantly, prostate cancer incidence is dramatically increasing in western societies in the past decades, suggesting that this type of tumor is exquisitely sensitive to lifestyle changes. Prostate cancer frequently exhibits alterations in the PTEN gene (inactivating mutations or gene deletions) or at the protein level (reduced protein expression or altered sub-cellular compartmentalization). The relevance of PTEN in this type of cancer is further supported by the fact that the sole deletion of PTEN in the murine prostate epithelium recapitulates many of the features of the human disease. In order to study the molecular alterations in prostate cancer, we need to overcome the methodological challenges that this tissue imposes. In this review we present protocols and methods, using PTEN as proof of concept, to study different molecular characteristics of prostate cancer.
Asunto(s)
Fosfohidrolasa PTEN/análisis , Fosfohidrolasa PTEN/biosíntesis , Neoplasias de la Próstata/metabolismo , Proteínas Supresoras de Tumor/análisis , Proteínas Supresoras de Tumor/biosíntesis , Animales , Humanos , Masculino , Ratones , Mutación/genética , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Supresoras de Tumor/genéticaRESUMEN
Zbtb7a has previously been described as a powerful proto-oncogene. Here we unexpectedly demonstrate that Zbtb7a has a critical oncosuppressive role in the prostate. Prostate-specific inactivation of Zbtb7a leads to a marked acceleration of Pten loss-driven prostate tumorigenesis through bypass of Pten loss-induced cellular senescence (PICS). We show that ZBTB7A physically interacts with SOX9 and functionally antagonizes its transcriptional activity on key target genes such as MIA, which is involved in tumor cell invasion, and H19, a long noncoding RNA precursor for an RB-targeting microRNA. Inactivation of Zbtb7a in vivo leads to Rb downregulation, PICS bypass and invasive prostate cancer. Notably, we found that ZBTB7A is genetically lost, as well as downregulated at both the mRNA and protein levels, in a subset of human advanced prostate cancers. Thus, we identify ZBTB7A as a context-dependent cancer gene that can act as an oncogene in some contexts but also has oncosuppressive-like activity in PTEN-null tumors.
Asunto(s)
Senescencia Celular/genética , Proteínas de Unión al ADN/fisiología , Genes Supresores de Tumor , Neoplasias de la Próstata/patología , Factor de Transcripción SOX9/genética , Factores de Transcripción/fisiología , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/genética , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Genes Supresores de Tumor/fisiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Invasividad Neoplásica , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Próstata/genética , Proto-Oncogenes Mas , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/fisiología , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cancer cells exhibit an aberrant metabolism that facilitates more efficient production of biomass and hence tumor growth and progression. However, the genetic cues modulating this metabolic switch remain largely undetermined. We identified a metabolic function for the promyelocytic leukemia (PML) gene, uncovering an unexpected role for this bona fide tumor suppressor in breast cancer cell survival. We found that PML acted as both a negative regulator of PPARγ coactivator 1A (PGC1A) acetylation and a potent activator of PPAR signaling and fatty acid oxidation. We further showed that PML promoted ATP production and inhibited anoikis. Importantly, PML expression allowed luminal filling in 3D basement membrane breast culture models, an effect that was reverted by the pharmacological inhibition of fatty acid oxidation. Additionally, immunohistochemical analysis of breast cancer biopsies revealed that PML was overexpressed in a subset of breast cancers and enriched in triple-negative cases. Indeed, PML expression in breast cancer correlated strikingly with reduced time to recurrence, a gene signature of poor prognosis, and activated PPAR signaling. These findings have important therapeutic implications, as PML and its key role in fatty acid oxidation metabolism are amenable to pharmacological suppression, a potential future mode of cancer prevention and treatment.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Acetilación , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Dieta Alta en Grasa/efectos adversos , Supervivencia sin Enfermedad , Ácidos Grasos/metabolismo , Femenino , Humanos , Estimación de Kaplan-Meier , Hígado/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Obesos , Proteínas Nucleares/genética , Obesidad/etiología , Obesidad/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidación-Reducción , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Proteína de la Leucemia Promielocítica , Procesamiento Proteico-Postraduccional , Transducción de Señal , Transactivadores/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Transcriptoma , Proteínas Supresoras de Tumor/genéticaRESUMEN
The promyelocytic leukaemia gene PML is a pleiotropic tumour suppressor. We have recently demonstrated that PML opposes mTOR-HIF1α-VEGF signalling in hypoxia. To determine the relevance of PML-mTOR antagonism in tumourigenesis, we have intercrossed Pml null mice with Tsc2 heterozygous mice, which develop kidney cysts and carcinomas exhibiting mTOR upregulation. We find that combined inactivation of Pml and Tsc2 results in aberrant TORC1 activity both in pre-tumoural kidneys as well as in kidney lesions. Such increase correlates with a marked acceleration in tumour progression, impacting on both the biology and histology of kidney carcinomas. Also, Pml inactivation decreases the rate of loss of heterozygosity (LOH) for the wt Tsc2 allele. Interestingly, however, aberrant TORC1 activity does not accelerate renal cystogenesis in Tsc2/Pml mutants. Our data demonstrate that activation of mTOR is critical for tumour progression, but not for tumour initiation in the kidney.
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Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Serina-Treonina Quinasas TOR/biosíntesis , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Carcinoma/patología , Cruzamientos Genéticos , Histocitoquímica , Inmunohistoquímica , Riñón/patología , Neoplasias Renales/patología , Imagen por Resonancia Magnética , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica , Radiografía Abdominal , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genéticaRESUMEN
Tumor cells exhibit aberrant metabolism characterized by high glycolysis even in the presence of oxygen. This metabolic reprogramming, known as the Warburg effect, provides tumor cells with the substrates required for biomass generation. Here, we show that the mitochondrial NAD-dependent deacetylase SIRT3 is a crucial regulator of the Warburg effect. Mechanistically, SIRT3 mediates metabolic reprogramming by destabilizing hypoxia-inducible factor-1α (HIF1α), a transcription factor that controls glycolytic gene expression. SIRT3 loss increases reactive oxygen species production, leading to HIF1α stabilization. SIRT3 expression is reduced in human breast cancers, and its loss correlates with the upregulation of HIF1α target genes. Finally, we find that SIRT3 overexpression represses glycolysis and proliferation in breast cancer cells, providing a metabolic mechanism for tumor suppression.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Metabolómica/métodos , Neoplasias/metabolismo , Sirtuina 3/metabolismo , Animales , Hipoxia de la Célula , Línea Celular Tumoral , Células Cultivadas , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Glucosa/metabolismo , Glucólisis , Células HEK293 , Humanos , Immunoblotting , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Ratones Desnudos , Neoplasias/genética , Neoplasias/patología , Estabilidad Proteica , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 3/genética , Trasplante HeterólogoRESUMEN
PTEN is a frequently mutated tumor suppressor gene that opposes the PI3K/AKT pathway through dephosphorylation of phosphoinositide-3,4,5-triphosphate. Recently, nuclear compartmentalization of PTEN was found as a key component of its tumor-suppressive activity; however its nuclear function remains poorly defined. Here we show that nuclear PTEN interacts with APC/C, promotes APC/C association with CDH1, and thereby enhances the tumor-suppressive activity of the APC-CDH1 complex. We find that nuclear exclusion but not phosphatase inactivation of PTEN impairs APC-CDH1. This nuclear function of PTEN provides a straightforward mechanistic explanation for the fail-safe cellular senescence response elicited by acute PTEN loss and the tumor-suppressive activity of catalytically inactive PTEN. Importantly, we demonstrate that PTEN mutant and PTEN null states are not synonymous as they are differentially sensitive to pharmacological inhibition of APC-CDH1 targets such as PLK1 and Aurora kinases. This finding identifies a strategy for cancer patient stratification and, thus, optimization of targeted therapies. PAPERCLIP:
Asunto(s)
Cadherinas/metabolismo , Senescencia Celular , Fosfohidrolasa PTEN/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Animales , Antígenos CD , Aurora Quinasas , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Masculino , Ratones , Trasplante de Neoplasias , Fosfohidrolasa PTEN/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Trasplante HeterólogoRESUMEN
Keloid scarring is a consequence of aberrant wound healing that leads to expansion of the scar beyond the confines of the skin injury. Keloid scars are characterized by excessive extracellular matrix disposition, prolonged proliferation of fibroblasts, increased angiogenesis, and inflammatory cell infiltration. There is no single satisfactory treatment for keloid, and it can lead to severe disfigurements and bodily dysfunction. Thus, clarification of the mechanisms underlying keloid formation, as well as those that prevent it from behaving as a malignant tumor, has significant consequences not only for treatment of keloid but also for the prevention of malignant tumor formation. Senescence is an irreversible form of growth arrest that has been shown to play a role, both in vitro and in vivo, in preventing malignant tumorigenesis upon oncogenic stress. In this study it is shown that fibroblasts embedded inside keloid scars proliferate at a slower rate compared with either those residing at the proliferative edges of the scar or normal fibroblasts. Likewise it is demonstrated that keloid fibroblasts exhibit a cell-cycle arrest with a G2/M DNA content and a higher rate of senescence. The results also indicate that levels of the tumor suppressor protein PML are higher in the active regions of keloid. The study therefore suggests that senescence is one possible mechanism by which keloid is maintained in a benign state. On this basis, "pro-senescence therapy" should be taken into consideration when designing treatment strategies for keloid.
RESUMEN
Cancer susceptibility has been attributed to at least one heterozygous genetic alteration in a tumor suppressor gene (TSG). It has been hypothesized that subtle variations in TSG expression can promote cancer development. However, this hypothesis has not yet been definitively supported in vivo. Pten is a TSG frequently lost in human cancer and mutated in inherited cancer-predisposition syndromes. Here we analyze Pten hypermorphic mice (Pten(hy/+)), expressing 80% normal levels of Pten. Pten(hy/+) mice develop a spectrum of tumors, with breast tumors occurring at the highest penetrance. All breast tumors analyzed here retained two intact copies of Pten and maintained Pten levels above heterozygosity. Notably, subtle downregulation of Pten altered the steady-state biology of the mammary tissues and the expression profiles of genes involved in cancer cell proliferation. We present an alterative working model for cancer development in which subtle reductions in the dose of TSGs predispose to tumorigenesis in a tissue-specific manner.
Asunto(s)
Neoplasias de la Mama/genética , Fosfohidrolasa PTEN/genética , Animales , Femenino , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Predisposición Genética a la Enfermedad/genética , Heterocigoto , Homocigoto , Humanos , Masculino , Neoplasias Mamarias Animales/genética , Ratones , Ratones Transgénicos , Modelos Genéticos , MutaciónRESUMEN
PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor that antagonizes signaling through the phosphatidylinositol 3-kinase-Akt pathway. We have demonstrated that subtle decreases in PTEN abundance can have critical consequences for tumorigenesis. Here, we used a computational approach to identify miR-22, miR-25, and miR-302 as three PTEN-targeting microRNA (miRNA) families found within nine genomic loci. We showed that miR-22 and the miR-106b~25 cluster are aberrantly overexpressed in human prostate cancer, correlate with abundance of the miRNA processing enzyme DICER, and potentiate cellular transformation both in vitro and in vivo. We demonstrated that the intronic miR-106b~25 cluster cooperates with its host gene MCM7 in cellular transformation both in vitro and in vivo, so that the concomitant overexpression of MCM7 and the miRNA cluster triggers prostatic intraepithelial neoplasia in transgenic mice. Therefore, the MCM7 gene locus delivers two simultaneous oncogenic insults when amplified or overexpressed in human cancer. Thus, we have uncovered a proto-oncogenic miRNA-dependent network for PTEN regulation and defined the MCM7 locus as a critical factor in initiating prostate tumorigenesis.
Asunto(s)
Proteínas de Ciclo Celular/genética , Transformación Celular Neoplásica , Proteínas de Unión al ADN/genética , Intrones , MicroARNs/genética , Familia de Multigenes , Proteínas Nucleares/genética , Fosfohidrolasa PTEN/genética , Proto-Oncogenes , Animales , Humanos , Masculino , Ratones , Ratones Transgénicos , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
Cellular senescence has been recently shown to have an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or by loss of tumour suppressor genes is thought to critically depend on induction of the p19(Arf)-p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumour-suppressive senescence response in mice and cells devoid of Skp2. Notably, Skp2 inactivation and oncogenic-stress-driven senescence neither elicit activation of the p19(Arf)-p53 pathway nor DNA damage, but instead depend on Atf4, p27 and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19(Arf)-p53 response is impaired, whereas a Skp2-SCF complex inhibitor can trigger cellular senescence in p53/Pten-deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof-of-principle evidence that pharmacological inhibition of Skp2 may represent a general approach for cancer prevention and therapy.
Asunto(s)
Transformación Celular Neoplásica , Senescencia Celular , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Factor de Transcripción Activador 4/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Animales , Transformación Celular Neoplásica/efectos de los fármacos , Células Cultivadas , Senescencia Celular/efectos de los fármacos , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibroblastos , Masculino , Ratones , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Próstata/citología , Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/prevención & control , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Quinasas Asociadas a Fase-S/antagonistas & inhibidores , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
One reported function of the tumor suppressor p19(Arf) is to stabilize p53, providing a critical checkpoint in the response to oncogenic insults. Acute loss of Pten leads to an increase in the abundance of p19(Arf), p53, and p21 proteins as part of a fail-safe senescence response. Here, we report that loss of p19(Arf) in prostate epithelium does not accelerate-but rather partially inhibits-the prostate cancer phenotype of Pten-deficient mice. Moreover, cellular senescence and a further decrease in the number of pre-neoplastic glands were observed in prostates of the Pten-p19(Arf) double-mutant mice. In both prostate epithelium and primary mouse embryo fibroblasts (MEFs), the increase in p53 protein abundance found upon loss of Pten was unaffected by the simultaneous loss of p19(Arf). However, in contrast to that in the prostate epithelium, p19(Arf) deficiency in MEFs lacking Pten abolished cell senescence and promoted hyperproliferation and transformation despite the unabated increase in p53 abundance. Consistent with the effect of p19(Arf) loss in Pten-deficient mouse prostate, we found that in human prostate cancers, loss of PTEN was not associated with loss of p14(ARF) (the human equivalent of mouse p19(Arf)). Collectively, these data reveal differential consequences of p19(Arf) inactivation in prostate cancer and MEFs upon Pten loss that are independent of the p53 pathway.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Fosfohidrolasa PTEN/metabolismo , Neoplasias de la Próstata/patología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Western Blotting , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Embrión de Mamíferos/citología , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Noqueados , Fosfohidrolasa PTEN/genética , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteína p14ARF Supresora de Tumor/genética , Proteína p14ARF Supresora de Tumor/metabolismoAsunto(s)
Autofagia/fisiología , Cannabinoides/metabolismo , Proteínas de Ciclo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Neoplasias , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Dronabinol/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Estrés Oxidativo , Transducción de Señal/fisiologíaRESUMEN
Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that delta(9)-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3-dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis. We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers.
Asunto(s)
Autofagia/efectos de los fármacos , Cannabinoides/farmacología , Retículo Endoplásmico/efectos de los fármacos , Glioma/patología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Autofagia/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cannabinoides/uso terapéutico , Caspasa 3/metabolismo , Proteínas de Ciclo Celular/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Transformada , Línea Celular Tumoral , Dronabinol/farmacología , Dronabinol/uso terapéutico , Retículo Endoplásmico/patología , Inhibidores Enzimáticos/farmacología , Factor 2 Eucariótico de Iniciación/metabolismo , Glioma/tratamiento farmacológico , Glioma/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Complejos Multiproteicos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Represoras/metabolismo , Proteínas Quinasas S6 Ribosómicas/metabolismo , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gliomas, one of the most malignant forms of cancer, exhibit high resistance to conventional therapies. Identification of the molecular mechanisms responsible for this resistance is therefore of great interest to improve the efficacy of the treatments against these tumors. Delta9-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the ability of these compounds to induce apoptosis of tumor cells. By analyzing the gene expression profile of two sub-clones of C6 glioma cells with different sensitivity to cannabinoid-induced apoptosis, we found a subset of genes with a marked differential expression in the two sub-clones. Furthermore, we identified the epidermal growth factor receptor ligand amphiregulin as a candidate factor to mediate the resistance of glioma cells to cannabinoid treatment. Amphiregulin was highly overexpressed in the cannabinoid-resistant cell line, both in culture and in tumor xenografts. Moreover, in vivo silencing of amphiregulin rendered the resistant tumors xenografts sensitive to cannabinoid antitumoral action. Amphiregulin expression was associated with increased extracellular signal-regulated kinase (ERK) activation, which mediated the resistance to THC by blunting the expression of p8 and TRB3-two genes involved in cannabinoid-induced apoptosis of glioma cells. Our findings therefore identify Amphirregulin as a factor for resistance of glioma cells to THC-induced apoptosis and contribute to unraveling the molecular bases underlying the emerging notion that targeted inhibition of the EGFR pathway can improve the efficacy of antitumoral therapies.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Dronabinol/farmacología , Glioma/tratamiento farmacológico , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Anfirregulina , Animales , Apoptosis/genética , Apoptosis/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/fisiología , Familia de Proteínas EGF , Activación Enzimática/genética , Activación Enzimática/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica , Glioma/genética , Glioma/patología , Glioma/fisiopatología , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Trasplante de Neoplasias , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The mammalian target of rapamycin (mTOR) is a crucial effector in a complex signaling network commonly disrupted in cancer. mTOR exerts its multiple functions in the context of two different multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Loss of the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) can hyperactivate mTOR through AKT and represents one of the most frequent events in human prostate cancer. We show here that conditional inactivation of mTor in the adult mouse prostate is seemingly inconsequential for this postmitotic tissue. Conversely, inactivation of mTor leads to a marked suppression of Pten loss-induced tumor initiation and progression in the prostate. This suppression is more pronounced than that elicited by the sole pharmacological abrogation of mTORC1. Acute inactivation of mTor in vitro also highlights the differential requirement of mTor function in proliferating and transformed cells. Collectively, our data constitute a strong rationale for developing specific mTOR inhibitors targeting both mTORC1 and mTORC2 for the treatment of tumors triggered by PTEN deficiency and aberrant mTOR signaling.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación Neoplásica de la Expresión Génica , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Serina-Treonina Quinasas TOR/fisiología , Animales , Línea Celular Transformada , Proliferación Celular , Transformación Celular Neoplásica , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Mitosis , Modelos Biológicos , Complejos Multiproteicos , Fosfohidrolasa PTEN/metabolismo , Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
Nuclear exclusion of the PTEN (phosphatase and tensin homologue deleted in chromosome 10) tumour suppressor has been associated with cancer progression. However, the mechanisms leading to this aberrant PTEN localization in human cancers are currently unknown. We have previously reported that ubiquitinylation of PTEN at specific lysine residues regulates its nuclear-cytoplasmic partitioning. Here we show that functional promyelocytic leukaemia protein (PML) nuclear bodies co-ordinate PTEN localization by opposing the action of a previously unknown PTEN-deubiquitinylating enzyme, herpesvirus-associated ubiquitin-specific protease (HAUSP, also known as USP7), and that the integrity of this molecular framework is required for PTEN to be able to enter the nucleus. We find that PTEN is aberrantly localized in acute promyelocytic leukaemia, in which PML function is disrupted by the PML-RARalpha fusion oncoprotein. Remarkably, treatment with drugs that trigger PML-RARalpha degradation, such as all-trans retinoic acid or arsenic trioxide, restore nuclear PTEN. We demonstrate that PML opposes the activity of HAUSP towards PTEN through a mechanism involving the adaptor protein DAXX (death domain-associated protein). In support of this paradigm, we show that HAUSP is overexpressed in human prostate cancer and is associated with PTEN nuclear exclusion. Thus, our results delineate a previously unknown PML-DAXX-HAUSP molecular network controlling PTEN deubiquitinylation and trafficking, which is perturbed by oncogenic cues in human cancer, in turn defining a new deubiquitinylation-dependent model for PTEN subcellular compartmentalization.
Asunto(s)
Proteínas Nucleares/metabolismo , Fosfohidrolasa PTEN/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Ubiquitinas/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteínas Co-Represoras , Fibroblastos , Humanos , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patología , Masculino , Ratones , Chaperonas Moleculares , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Tretinoina/farmacología , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética , Peptidasa Específica de Ubiquitina 7RESUMEN
Numerous studies have established a causal link between aberrant mammalian target of rapamycin (mTOR) activation and tumorigenesis, indicating that mTOR inhibition may have therapeutic potential. In this study, we show that rapamycin and its analogs activate the MAPK pathway in human cancer, in what represents a novel mTORC1-MAPK feedback loop. We found that tumor samples from patients with biopsy-accessible solid tumors of advanced disease treated with RAD001, a rapamycin derivative, showed an administration schedule-dependent increase in activation of the MAPK pathway. RAD001 treatment also led to MAPK activation in a mouse model of prostate cancer. We further show that rapamycin-induced MAPK activation occurs in both normal cells and cancer cells lines and that this feedback loop depends on an S6K-PI3K-Ras pathway. Significantly, pharmacological inhibition of the MAPK pathway enhanced the antitumoral effect of mTORC1 inhibition by rapamycin in cancer cells in vitro and in a xenograft mouse model. Taken together, our findings identify MAPK activation as a consequence of mTORC1 inhibition and underscore the potential of a combined therapeutic approach with mTORC1 and MAPK inhibitors, currently employed as single agents in the clinic, for the treatment of human cancers.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Neoplasias/enzimología , Factores de Transcripción/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Everolimus , Retroalimentación Fisiológica , Humanos , Inmunosupresores/farmacología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Neoplasias/metabolismo , Proteínas , Sirolimus/análogos & derivados , Sirolimus/farmacología , Serina-Treonina Quinasas TORRESUMEN
The mammalian target of rapamycin (mTOR) represents a critical signaling crossroad where pathways commonly disrupted in cancer converge. We report here that Rheb GTPase, the upstream activator of the mTOR complex 1 (mTORC1) is amplified in human prostate cancers. We demonstrate that Rheb overexpression promotes hyperplasia and a low-grade neoplastic phenotype in the mouse prostate while eliciting a concomitant senescence response and a negative feedback loop limiting Akt activation. Importantly, we show that Pten haploinsufficiency cooperates with Rheb overexpression to markedly promote prostate tumorigenesis. We conclude that Rheb acts as a proto-oncogene in the appropriate genetic milieu and signaling context.
Asunto(s)
Transformación Celular Neoplásica/patología , Proteínas de Unión al GTP Monoméricas/fisiología , Neuropéptidos/fisiología , Fosfohidrolasa PTEN/fisiología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factores de Transcripción/fisiología , Animales , Western Blotting , Línea Celular Tumoral , Aberraciones Cromosómicas , Humanos , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Transgénicos , Complejos Multiproteicos , Neoplasia Intraepitelial Prostática/etiología , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/etiología , Proteínas , Proto-Oncogenes Mas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína Homóloga de Ras Enriquecida en el Cerebro , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Serina-Treonina Quinasas TORRESUMEN
Cannabinoids, the active components of Cannabis sativa L. and their derivatives, inhibit tumor growth in laboratory animals by inducing apoptosis of tumor cells and impairing tumor angiogenesis. It has also been reported that these compounds inhibit tumor cell spreading, but the molecular targets of this cannabinoid action remain elusive. Here, we evaluated the effect of cannabinoids on matrix metalloproteinase (MMP) expression and its effect on tumor cell invasion. Local administration of Delta(9)-tetrahydrocannabinol (THC), the major active ingredient of cannabis, down-regulated MMP-2 expression in gliomas generated in mice, as determined by Western blot, immunofluorescence, and real-time quantitative PCR analyses. This cannabinoid-induced inhibition of MMP-2 expression in gliomas (a) was MMP-2-selective, as levels of other MMP family members were unaffected; (b) was mimicked by JWH-133, a CB(2) cannabinoid receptor-selective agonist that is devoid of psychoactive side effects; (c) was abrogated by fumonisin B1, a selective inhibitor of ceramide biosynthesis; and (d) was also evident in two patients with recurrent glioblastoma multiforme. THC inhibited MMP-2 expression and cell invasion in cultured glioma cells. Manipulation of MMP-2 expression by RNA interference and cDNA overexpression experiments proved that down-regulation of this MMP plays a critical role in THC-mediated inhibition of cell invasion. Cannabinoid-induced inhibition of MMP-2 expression and cell invasion was prevented by blocking ceramide biosynthesis and by knocking-down the expression of the stress protein p8. As MMP-2 up-regulation is associated with high progression and poor prognosis of gliomas and many other tumors, MMP-2 down-regulation constitutes a new hallmark of cannabinoid antitumoral activity.