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1.
Nat Commun ; 15(1): 5115, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38879607

RESUMEN

Neurofibromatosis Type II (NFII) is a genetic condition caused by loss of the NF2 gene, resulting in activation of the YAP/TAZ pathway and recurrent Schwann cell tumors, as well as meningiomas and ependymomas. Unfortunately, few pharmacological options are available for NFII. Here, we undertake a genome-wide CRISPR/Cas9 screen to search for synthetic-lethal genes that, when inhibited, cause death of NF2 mutant Schwann cells but not NF2 wildtype cells. We identify ACSL3 and G6PD as two synthetic-lethal partners for NF2, both involved in lipid biogenesis and cellular redox. We find that NF2 mutant Schwann cells are more oxidized than control cells, in part due to reduced expression of genes involved in NADPH generation such as ME1. Since G6PD and ME1 redundantly generate cytosolic NADPH, lack of either one is compatible with cell viability, but not down-regulation of both. Since genetic deficiency for G6PD is tolerated in the human population, G6PD could be a good pharmacological target for NFII.


Asunto(s)
Sistemas CRISPR-Cas , Coenzima A Ligasas , Glucosafosfato Deshidrogenasa , Neurofibromina 2 , Células de Schwann , Mutaciones Letales Sintéticas , Células de Schwann/metabolismo , Humanos , Glucosafosfato Deshidrogenasa/metabolismo , Glucosafosfato Deshidrogenasa/genética , Neurofibromina 2/metabolismo , Neurofibromina 2/genética , Coenzima A Ligasas/metabolismo , Coenzima A Ligasas/genética , Animales , Neurofibromatosis 2/metabolismo , Neurofibromatosis 2/genética , NADP/metabolismo , Ratones , Oxidación-Reducción
2.
Nat Metab ; 5(4): 660-676, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-37024754

RESUMEN

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is known to contain an active-site cysteine residue undergoing oxidation in response to hydrogen peroxide, leading to rapid inactivation of the enzyme. Here we show that human and mouse cells expressing a GAPDH mutant lacking this redox switch retain catalytic activity but are unable to stimulate the oxidative pentose phosphate pathway and enhance their reductive capacity. Specifically, we find that anchorage-independent growth of cells and spheroids is limited by an elevation of endogenous peroxide levels and is largely dependent on a functional GAPDH redox switch. Likewise, tumour growth in vivo is limited by peroxide stress and suppressed when the GAPDH redox switch is disabled in tumour cells. The induction of additional intratumoural oxidative stress by chemo- or radiotherapy synergized with the deactivation of the GAPDH redox switch. Mice lacking the GAPDH redox switch exhibit altered fatty acid metabolism in kidney and heart, apparently in compensation for the lack of the redox switch. Together, our findings demonstrate the physiological and pathophysiological relevance of oxidative GAPDH inactivation in mammals.


Asunto(s)
Cisteína , Gliceraldehído-3-Fosfato Deshidrogenasas , Humanos , Animales , Ratones , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Oxidación-Reducción , Cisteína/metabolismo , Estrés Oxidativo , Peróxido de Hidrógeno/farmacología , Mamíferos/metabolismo
3.
Science ; 378(6615): eabn5637, 2022 10 07.
Artículo en Inglés | MEDLINE | ID: mdl-36074822

RESUMEN

Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable because of a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer.


Asunto(s)
Aparato de Golgi , Enfermedades por Almacenamiento Lisosomal , Lisosomas , Proteínas , Animales , Aparato de Golgi/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lisosomas/metabolismo , Ratones , Transporte de Proteínas , Proteínas/genética , Proteínas/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo
4.
J Exp Clin Cancer Res ; 41(1): 190, 2022 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-35655310

RESUMEN

BACKGROUND: MicroRNAs (miRNAs) and isomiRs play important roles in tumorigenesis as essential regulators of gene expression. 5'isomiRs exhibit a shifted seed sequence compared to the canonical miRNA, resulting in different target spectra and thereby extending the phenotypic impact of the respective common pre-miRNA. However, for most miRNAs, expression and function of 5'isomiRs have not been studied in detail yet. Therefore, this study aims to investigate the functions of miRNAs and their 5'isomiRs. METHODS: The expression of 5'isomiRs was assessed in The Cancer Genome Atlas (TCGA) breast cancer patient dataset. Phenotypic effects of miR-183 overexpression in triple-negative breast cancer (TNBC) cell lines were investigated in vitro and in vivo by quantifying migration, proliferation, tumor growth and metastasis. Direct targeting of E2F1 by miR-183-5p|+2 was validated with a 3'UTR luciferase assay and linked to the phenotypes of isomiR overexpression. RESULTS: TCGA breast cancer patient data indicated that three variants of miR-183-5p are highly expressed and upregulated, namely miR-183-5p|0, miR-183-5p|+1 and miR-183-5p|+2. However, TNBC cell lines displayed reduced proliferation and invasion upon overexpression of pre-miR-183. While invasion was reduced individually by all three isomiRs, proliferation and cell cycle progression were specifically inhibited by overexpression of miR-183-5p|+2. Proteomic analysis revealed reduced expression of E2F target genes upon overexpression of this isomiR, which could be attributed to direct targeting of E2F1, specifically by miR-183-5p|+2. Knockdown of E2F1 partially phenocopied the effect of miR-183-5p|+2 overexpression on cell proliferation and cell cycle. Gene set enrichment analysis of TCGA and METABRIC patient data indicated that the activity of E2F strongly correlated with the expression of miR-183-5p, suggesting transcriptional regulation of the miRNA by a factor of the E2F family. Indeed, in vitro, expression of miR-183-5p was regulated by E2F1. Hence, miR-183-5p|+2 directly targeting E2F1 appears to be part of a negative feedback loop potentially fine-tuning its activity. CONCLUSIONS: This study demonstrates that 5'isomiRs originating from the same arm of the same pre-miRNA (i.e. pre-miR-183-5p) may exhibit different functions and thereby collectively contribute to the same phenotype. Here, one of three isomiRs was shown to counteract expression of the pre-miRNA by negatively regulating a transcriptional activator (i.e. E2F1). We speculate that this might be part of a regulatory mechanism to prevent uncontrolled cell proliferation, which is disabled during cancer progression.


Asunto(s)
MicroARNs , Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Retroalimentación , Humanos , MicroARNs/metabolismo , Proteómica , Neoplasias de la Mama Triple Negativas/metabolismo
5.
Stem Cell Reports ; 17(1): 143-158, 2022 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-34942088

RESUMEN

The genetic modification of stem cells (SCs) is typically achieved using integrating vectors, whose potential integrative genotoxicity and propensity for epigenetic silencing during differentiation limit their application. The genetic modification of cells should provide sustainable levels of transgene expression, without compromising the viability of a cell or its progeny. We developed nonviral, nonintegrating, and autonomously replicating minimally sized DNA nanovectors to persistently genetically modify SCs and their differentiated progeny without causing any molecular or genetic damage. These DNA vectors are capable of efficiently modifying murine and human pluripotent SCs with minimal impact and without differentiation-mediated transgene silencing or vector loss. We demonstrate that these vectors remain episomal and provide robust and sustained transgene expression during self-renewal and targeted differentiation of SCs both in vitro and in vivo through embryogenesis and differentiation into adult tissues, without damaging their phenotypic characteristics.


Asunto(s)
Diferenciación Celular , Expresión Génica , Vectores Genéticos/genética , Plásmidos/genética , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fibroblastos , Perfilación de la Expresión Génica , Humanos , Ratones , Transgenes
6.
Front Immunol ; 13: 1063313, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36591284

RESUMEN

Use of chimeric antigen receptor (CAR) T cells to treat B cell lymphoma and leukemia has been remarkably successful. Unfortunately, the therapeutic efficacy of CAR T cells against solid tumors is very limited, with immunosuppression by the pro-oxidative tumor microenvironment (TME) a major contributing factor. High levels of reactive oxygen species are well-tolerated by tumor cells due to their elevated expression of antioxidant proteins; however, this is not the case for T cells, which consequently become hypo-responsive. The aim of this study was to improve CAR T cell efficacy in solid tumors by empowering the antioxidant capacity of CAR T cells against the pro-oxidative TME. To this end, HER2-specific human CAR T cells stably expressing two antioxidant systems: thioredoxin-1 (TRX1), and glutaredoxin-1 (GRX1) were generated and characterized. Thereafter, antitumor functions of CAR T cells were evaluated under control or pro-oxidative conditions. To provide insights into the role of antioxidant systems, gene expression profiles as well as global protein oxidation were analyzed. Our results highlight that TRX1 is pivotal for T cell redox homeostasis. TRX1 expression allows CAR T cells to retain their cytolytic immune synapse formation, cytokine release, proliferation, and tumor cell-killing properties under pro-oxidative conditions. Evaluation of differentially expressed genes and the first comprehensive redoxosome analysis of T cells by mass spectrometry further clarified the underlying mechanisms. Taken together, enhancement of the key antioxidant TRX1 in human T cells opens possibilities to increase the efficacy of CAR T cell treatment against solid tumors.


Asunto(s)
Inmunoterapia Adoptiva , Neoplasias , Estrés Oxidativo , Linfocitos T , Microambiente Tumoral , Humanos , Antioxidantes/metabolismo , Inmunoterapia Adoptiva/métodos , Neoplasias/inmunología , Neoplasias/terapia , Oxidación-Reducción , Estrés Oxidativo/genética , Estrés Oxidativo/inmunología , Linfocitos T/inmunología , Tiorredoxinas/genética , Tiorredoxinas/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología
7.
Cell Rep ; 36(7): 109559, 2021 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-34407399

RESUMEN

Acute myeloid leukemia (AML) is a rapidly progressing cancer, for which chemotherapy remains standard treatment and additional therapeutic targets are requisite. Here, we show that AML cells secrete the stem cell growth factor R-spondin 2 (RSPO2) to promote their self-renewal and prevent cell differentiation. Although RSPO2 is a well-known WNT agonist, we reveal that it maintains AML self-renewal WNT independently, by inhibiting BMP receptor signaling. Autocrine RSPO2 signaling is also required to prevent differentiation and to promote self-renewal in normal hematopoietic stem cells as well as primary AML cells. Comprehensive datamining reveals that RSPO2 expression is elevated in patients with AML of poor prognosis. Consistently, inhibiting RSPO2 prolongs survival in AML mouse xenograft models. Our study indicates that in AML, RSPO2 acts as an autocrine BMP antagonist to promote cancer cell renewal and may serve as a marker for poor prognosis.


Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Transducción de Señal , Animales , Comunicación Autocrina/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Citarabina/farmacología , Células HEK293 , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Pronóstico , Factores de Riesgo , Transducción de Señal/efectos de los fármacos , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto
8.
Int J Cancer ; 148(8): 1993-2009, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33368291

RESUMEN

Uncontrolled proliferation and altered metabolic reprogramming are hallmarks of cancer. Active glycolysis and glutaminolysis are characteristic features of these hallmarks and required for tumorigenesis. A fine balance between cancer metabolism and autophagy is a prerequisite of homeostasis within cancer cells. Here we show that glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis, is highly upregulated in aggressive breast cancers, particularly the triple-negative breast cancer subtype. Abrogation of this enzyme results in decreased tricarboxylic acid cycle intermediates, which promotes the rewiring of glucose carbon atoms and alterations in nutrient levels. Concordantly, loss of GPT2 results in an impairment of mechanistic target of rapamycin complex 1 activity as well as the induction of autophagy. Furthermore, in vivo xenograft studies have shown that autophagy induction correlates with decreased tumor growth and that markers of induced autophagy correlate with low GPT2 levels in patient samples. Taken together, these findings indicate that cancer cells have a close network between metabolic and nutrient sensing pathways necessary to sustain tumorigenesis and that aminotransferase reactions play an important role in maintaining this balance.


Asunto(s)
Autofagia/genética , Regulación Neoplásica de la Expresión Génica , Transaminasas/genética , Neoplasias de la Mama Triple Negativas/genética , Carga Tumoral/genética , Animales , Sistemas CRISPR-Cas , Línea Celular Tumoral , Femenino , Técnicas de Inactivación de Genes , Humanos , Células MCF-7 , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Interferencia de ARN , Análisis de Supervivencia , Transaminasas/antagonistas & inhibidores , Transaminasas/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/terapia , Ensayos Antitumor por Modelo de Xenoinjerto/métodos
9.
Cancers (Basel) ; 12(10)2020 Oct 11.
Artículo en Inglés | MEDLINE | ID: mdl-33050633

RESUMEN

Breast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express Estrogen Receptor α account for around 70% of cases and are mostly treated with targeted endocrine therapy. However, depending on the degree of severity of the disease at diagnosis, 10 to 40% of these tumors eventually relapse due to resistance development. Even though recent novel approaches as the combination with CDK4/6 inhibitors increased the overall survival of relapsing patients, this remains relatively short and there is a urgent need to find alternative targetable pathways. In this study we profiled the early phases of the resistance development process to uncover drivers of this phenomenon. Time-resolved analysis revealed that ATF3, a member of the ATF/CREB family of transcription factors, acts as a novel regulator of the response to therapy via rewiring of central signaling processes towards the adaptation to endocrine treatment. ATF3 was found to be essential in controlling crucial processes such as proliferation, cell cycle, and apoptosis during the early response to treatment through the regulation of MAPK/AKT signaling pathways. Its essential role was confirmed in vivo in a mouse model, and elevated expression of ATF3 was verified in patient datasets, adding clinical relevance to our findings. This study proposes ATF3 as a novel mediator of endocrine resistance development in breast cancer and elucidates its role in the regulation of downstream pathways activities.

11.
Virology ; 538: 53-60, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31569015

RESUMEN

We have previously showed that a transgenic (Tg) mouse model with cytokeratin 14 promoter (K14)-driven expression of E6 and E7 from beta-3 HPV49 in the basal layer of the epidermis and of the mucosal epithelia of the digestive tract (K14 HPV49 E6/E7 Tg mice) are highly susceptible to upper digestive tract carcinogenesis upon exposure to 4-nitroquinoline 1-oxide (4NQO). Using whole-exome sequencing, we show that in K14 HPV49 E6/E7 Tg mice, development of 4NQO-induced cancers tightly correlates with the accumulation of somatic mutations in cancer-related genes. The mutational signature in 4NQO-treated mice was similar to the signature observed in humans exposed to tobacco smoking and tobacco chewing. Similar results were obtained with K14 Tg animals expressing mucosal high-risk HPV16 E6 and E7 oncogenes. Thus, beta-3 HPV49 share some functional similarities with HPV16 in Tg animals.


Asunto(s)
4-Nitroquinolina-1-Óxido/toxicidad , Betapapillomavirus/metabolismo , Neoplasias/genética , Nicotiana/efectos adversos , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Animales , Betapapillomavirus/genética , Susceptibilidad a Enfermedades , Femenino , Humanos , Ratones , Ratones Transgénicos , Mutación/efectos de los fármacos , Neoplasias/etiología , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus/genética
12.
BMC Cancer ; 19(1): 914, 2019 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-31519152

RESUMEN

BACKGROUND: NY-BR-1 has been described as a breast cancer associated differentiation antigen with intrinsic immunogenicity giving rise to endogenous T and B cell responses. The current study presents the first murine tumor model allowing functional investigation of NY-BR-1-specific immune responses in vivo. METHODS: A NY-BR-1 expressing tumor model was established in DR4tg mice based on heterotopic transplantation of stable transfectant clones derived from the murine H2 compatible breast cancer cell line EO771. Composition and phenotype of tumor infiltrating immune cells were analyzed by qPCR and FACS. MHC I binding affinity of candidate CTL epitopes predicted in silico was determined by FACS using the mutant cell line RMA-S. Frequencies of NY-BR-1 specific CTLs among splenocytes of immunized mice were quantified by FACS with an epitope loaded Db-dextramer. Functional CTL activity was determined by IFNγ catch or IFNγ ELISpot assays and statistical analysis was done applying the Mann Whitney test. Tumor protection experiments were performed by immunization of DR4tg mice with replication deficient recombinant adenovirus followed by s.c. challenge with NY-BR-1 expressing breast cancer cells. RESULTS: Our results show spontaneous accumulation of CD8+ T cells and F4/80+ myeloid cells preferentially in NY-BR-1 expressing tumors. Upon NY-BR-1-specific immunization experiments combined with in silico prediction and in vitro binding assays, the first NY-BR-1-specific H2-Db-restricted T cell epitope could be identified. Consequently, flow cytometric analysis with fluorochrome conjugated multimers showed enhanced frequencies of CD8+ T cells specific for the newly identified epitope in spleens of immunized mice. Moreover, immunization with Ad.NY-BR-1 resulted in partial protection against outgrowth of NY-BR-1 expressing tumors and promoted intratumoral accumulation of macrophages. CONCLUSION: This study introduces the first H2-Db-resctricted CD8+ T cell epitope-specific for the human breast cancer associated tumor antigen NY-BR-1. Our novel, partially humanized tumor model enables investigation of the interplay between HLA-DR4-restricted T cell responses and CTLs within their joint attack of NY-BR-1 expressing tumors.


Asunto(s)
Antígenos de Neoplasias/inmunología , Epítopos de Linfocito T/inmunología , Cadenas HLA-DRB1/genética , Neoplasias/etiología , Neoplasias/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Antígenos de Neoplasias/genética , Biomarcadores , Línea Celular Tumoral , Modelos Animales de Enfermedad , Cadenas HLA-DRB1/inmunología , Xenoinjertos , Humanos , Inmunización , Inmunofenotipificación , Leucocitos/inmunología , Leucocitos/metabolismo , Ratones , Ratones Transgénicos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo
13.
Blood ; 134(5): 445-455, 2019 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-31167801

RESUMEN

Therapeutic options for cutaneous T-cell lymphoma (CTCL) are limited and curative treatment regimens are not available. Thus, new targeted and well-tolerated therapeutic approaches are urgently needed. In this respect, we have recently shown that dimethyl fumerate (DMF) inhibits NF-κB acting as a survival factor in CTCL. Similarly, inhibition of the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) has been shown to induce cell death in CTCL especially when combined with histone deacetylase inhibitors. Therefore, we hypothesized that inhibition of Bcl-2 should potentiate NF-κB inhibition in a novel combination treatment of CTCL. We show that, in vitro, the Bcl-2 inhibitors ABT-199 and ABT-263 induced specific cell death in primary CD4+ cells from CTCL patients as well as in the CTCL cell line SeAx, but not in T cells of healthy donors nor in the CTCL cell line HH, which lacks Bcl-2. Combined treatment with ABT-199 and DMF caused synergistic cell death specifically in CTCL cells engaging 2 independent signaling pathways. To verify these findings in vivo, we performed combined ABT-199 and DMF treatment in a xenograft mouse model for CTCL. The combined treatment effectively reduced tumor growth and increased overall survival via synergistic induction of CTCL cell death and suppression of tumor cell proliferation. Essentially, the combination treatment was superior to ABT-199 monotherapy with respect to both efficacy and tolerability. To sum up, our data provide proof of principle for the therapeutic potential of combining Bcl-2 and NF-κB inhibitors in treating CTCL. Next, this potential should be explored further in a clinical study.


Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Linfoma Cutáneo de Células T/metabolismo , FN-kappa B/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Animales , Apoptosis , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Humanos , Linfoma Cutáneo de Células T/diagnóstico , Linfoma Cutáneo de Células T/tratamiento farmacológico , Linfoma Cutáneo de Células T/genética , Ratones , FN-kappa B/genética , Estadificación de Neoplasias , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Ensayos Antitumor por Modelo de Xenoinjerto
14.
Nat Commun ; 10(1): 2197, 2019 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-31097693

RESUMEN

In colorectal cancer (CRC), aberrant Wnt signalling is essential for tumorigenesis and maintenance of cancer stem cells. However, how other oncogenic pathways converge on Wnt signalling to modulate stem cell homeostasis in CRC currently remains poorly understood. Using large-scale compound screens in CRC, we identify MEK1/2 inhibitors as potent activators of Wnt/ß-catenin signalling. Targeting MEK increases Wnt activity in different CRC cell lines and murine intestine in vivo. Truncating mutations of APC generated by CRISPR/Cas9 strongly synergize with MEK inhibitors in enhancing Wnt responses in isogenic CRC models. Mechanistically, we demonstrate that MEK inhibition induces a rapid downregulation of AXIN1. Using patient-derived CRC organoids, we show that MEK inhibition leads to increased Wnt activity, elevated LGR5 levels and enrichment of gene signatures associated with stemness and cancer relapse. Our study demonstrates that clinically used MEK inhibitors inadvertently induce stem cell plasticity, revealing an unknown side effect of RAS pathway inhibition.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Antineoplásicos/uso terapéutico , Biopsia , Sistemas CRISPR-Cas/genética , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Plasticidad de la Célula/efectos de los fármacos , Neoplasias Colorrectales/patología , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Intestinos/citología , Intestinos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteómica , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/metabolismo
15.
J Proteome Res ; 18(3): 1352-1362, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30609375

RESUMEN

Hypoxia as well as metabolism are central hallmarks of cancer, and hypoxia-inducible factors (HIFs) and metabolic effectors are crucial elements in oxygen-compromised tumor environments. Knowledge of changes in the expression of metabolic proteins in response to HIF function could provide mechanistic insights into adaptation to hypoxic stress, tumorigenesis, and disease progression. We analyzed time-resolved alterations in metabolism-associated protein levels in response to different oxygen potentials across breast cancer cell lines. Effects on the cellular metabolism of both HIF-dependent and -independent processes were analyzed by reverse-phase protein array profiling and a custom statistical model. We revealed a strong induction of glucose transporter 1 (GLUT1) and lactate dehydrogenase A (LDHA) as well as reduced glutamate-ammonia ligase (GLUL) protein levels across all cell lines tested as consistent changes upon hypoxia induction. Low GLUL protein levels were correlated with aggressive molecular subtypes in breast cancer patient data sets and also with hypoxic tumor regions in a xenograft mouse tumor model. Moreover, low GLUL expression was associated with poor survival in breast cancer patients and with high HIF-1α-expressing patient subgroups. Our data reveal time-resolved changes in the regulation of metabolic proteins under oxygen-deprived conditions and elucidate GLUL as a strong responder to HIFs and the hypoxic environment.


Asunto(s)
Neoplasias de la Mama/genética , Glutamato-Amoníaco Ligasa/genética , Proteoma/genética , Proteómica , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Transportador de Glucosa de Tipo 1/genética , Xenoinjertos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , L-Lactato Deshidrogenasa/genética , Células MCF-7 , Ratones , Oxígeno/metabolismo , Hipoxia Tumoral
16.
Cell Discov ; 4: 37, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29977599

RESUMEN

Use of the diabetes type II drug Metformin is associated with a moderately lowered risk of cancer incidence in numerous tumor entities. Studying the molecular changes associated with the tumor-suppressive action of Metformin we found that the oncogene SOX4, which is upregulated in solid tumors and associated with poor prognosis, was induced by Wnt/ß-catenin signaling and blocked by Metformin. Wnt signaling inhibition by Metformin was surprisingly specific for cancer cells. Unraveling the underlying specificity, we identified Metformin and other Mitochondrial Complex I (MCI) inhibitors as inducers of intracellular acidification in cancer cells. We demonstrated that acidification triggers the unfolded protein response to induce the global transcriptional repressor DDIT3, known to block Wnt signaling. Moreover, our results suggest that intracellular acidification universally inhibits Wnt signaling. Based on these findings, we combined MCI inhibitors with H+ ionophores, to escalate cancer cells into intracellular hyper-acidification and ATP depletion. This treatment lowered intracellular pH both in vitro and in a mouse xenograft tumor model, depleted cellular ATP, blocked Wnt signaling, downregulated SOX4, and strongly decreased stemness and viability of cancer cells. Importantly, the inhibition of Wnt signaling occurred downstream of ß-catenin, encouraging applications in treatment of cancers caused by APC and ß-catenin mutations.

17.
PLoS Pathog ; 14(1): e1006783, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29324843

RESUMEN

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.


Asunto(s)
Carcinogénesis/efectos de la radiación , Neoplasias Inducidas por Radiación/metabolismo , Proteínas Oncogénicas Virales/metabolismo , Neoplasias Cutáneas/metabolismo , Piel/efectos de la radiación , Rayos Ultravioleta/efectos adversos , Proteínas Virales/metabolismo , Animales , Betapapillomavirus/metabolismo , Epidermis/metabolismo , Epidermis/patología , Epidermis/efectos de la radiación , Femenino , Eliminación de Gen , Genes p53/efectos de la radiación , Ratones , Ratones Transgénicos , Mutagénesis/efectos de la radiación , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Inducidas por Radiación/patología , Proteínas Oncogénicas Virales/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Proteínas Recombinantes/metabolismo , Piel/metabolismo , Piel/patología , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patología , Carga Tumoral/efectos de la radiación , Proteínas Virales/genética
18.
Cell Metab ; 26(6): 842-855.e5, 2017 Dec 05.
Artículo en Inglés | MEDLINE | ID: mdl-29056512

RESUMEN

Breast tumor recurrence and metastasis represent the main causes of cancer-related death in women, and treatments are still lacking. Here, we define the lipogenic enzyme acetyl-CoA carboxylase (ACC) 1 as a key player in breast cancer metastasis. ACC1 phosphorylation was increased in invading cells both in murine and human breast cancer, serving as a point of convergence for leptin and transforming growth factor (TGF) ß signaling. ACC1 phosphorylation was mediated by TGFß-activated kinase (TAK) 1, and ACC1 inhibition was indispensable for the elevation of cellular acetyl-CoA, the subsequent increase in Smad2 transcription factor acetylation and activation, and ultimately epithelial-mesenchymal transition and metastasis induction. ACC1 deficiency worsened tumor recurrence upon primary tumor resection in mice, and ACC1 phosphorylation levels correlated with metastatic potential in breast and lung cancer patients. Given the demonstrated effectiveness of anti-leptin receptor antibody treatment in halting ACC1-dependent tumor invasiveness, our work defines a "metabolocentric" approach in metastatic breast cancer therapy.


Asunto(s)
Acetil-CoA Carboxilasa/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias Pulmonares/secundario , Recurrencia Local de Neoplasia/patología , Acetil-CoA Carboxilasa/genética , Acetilación , Animales , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Leptina/metabolismo , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/metabolismo , Análisis de Matrices Tisulares
19.
Stem Cell Res Ther ; 8(1): 229, 2017 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-29037217

RESUMEN

BACKGROUND: Human induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties. METHODS: hiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes. RESULTS: hiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10-12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70-90 beats/min) and were triggered by spontaneous Ca2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed. CONCLUSION: We provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.


Asunto(s)
Relojes Biológicos , Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Miocitos Cardíacos/citología , Potenciales de Acción , Animales , Señalización del Calcio , Células Cultivadas , Proteína Homeótica Nkx-2.5/genética , Proteína Homeótica Nkx-2.5/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Contracción Miocárdica , Miocitos Cardíacos/clasificación , Ratas , Nodo Sinoatrial/citología , Nodo Sinoatrial/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo
20.
Cell Rep ; 20(6): 1422-1434, 2017 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-28793265

RESUMEN

Increased pro-inflammatory signaling is a hallmark of metabolic dysfunction in obesity and diabetes. Although both inflammatory and energy substrate handling processes represent critical layers of metabolic control, their molecular integration sites remain largely unknown. Here, we identify the heterodimerization interface between the α and ß subunits of transcription factor GA-binding protein (GAbp) as a negative target of tumor necrosis factor alpha (TNF-α) signaling. TNF-α prevented GAbpα and ß complex formation via reactive oxygen species (ROS), leading to the non-energy-dependent transcriptional inactivation of AMP-activated kinase (AMPK) ß1, which was identified as a direct hepatic GAbp target. Impairment of AMPKß1, in turn, elevated downstream cellular cholesterol biosynthesis, and hepatocyte-specific ablation of GAbpα induced systemic hypercholesterolemia and early macro-vascular lesion formation in mice. As GAbpα and AMPKß1 levels were also found to correlate in obese human patients, the ROS-GAbp-AMPK pathway may represent a key component of a hepato-vascular axis in diabetic long-term complications.


Asunto(s)
Aterosclerosis/metabolismo , Factor de Transcripción de la Proteína de Unión a GA/metabolismo , Hepatocitos/metabolismo , Hipercolesterolemia/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Aterosclerosis/etiología , Aterosclerosis/patología , Línea Celular , Células Cultivadas , Colesterol/metabolismo , Factor de Transcripción de la Proteína de Unión a GA/química , Hipercolesterolemia/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo
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