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Therapy resistance and metastasis, the most fatal steps in cancer, are often triggered by a (partial) activation of the epithelial-mesenchymal transition (EMT) programme. A mesenchymal phenotype predisposes to ferroptosis, a cell death pathway exerted by an iron and oxygen-radical-mediated peroxidation of phospholipids containing polyunsaturated fatty acids. We here show that various forms of EMT activation, including TGFß stimulation and acquired therapy resistance, increase ferroptosis susceptibility in cancer cells, which depends on the EMT transcription factor Zeb1. We demonstrate that Zeb1 increases the ratio of phospholipids containing pro-ferroptotic polyunsaturated fatty acids over cyto-protective monounsaturated fatty acids by modulating the differential expression of the underlying crucial enzymes stearoyl-Co-A desaturase 1 (SCD), fatty acid synthase (FASN), fatty acid desaturase 2 (FADS2), elongation of very long-chain fatty acid 5 (ELOVL5) and long-chain acyl-CoA synthetase 4 (ACSL4). Pharmacological inhibition of selected lipogenic enzymes (SCD and FADS2) allows the manipulation of ferroptosis sensitivity preferentially in high-Zeb1-expressing cancer cells. Our data are of potential translational relevance and suggest a combination of ferroptosis activators and SCD inhibitors for the treatment of aggressive cancers expressing high Zeb1.
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BACKGROUND: The identification of novel targets is of paramount importance to develop more effective drugs and improve the treatment of non-small cell lung cancer (NSCLC), the leading cause of cancer-related deaths worldwide. Since cells alter their metabolic rewiring during tumorigenesis and along cancer progression, targeting key metabolic players and metabolism-associated proteins represents a valuable approach with a high therapeutic potential. Metabolic fitness relies on the functionality of heat shock proteins (HSPs), molecular chaperones that facilitate the correct folding of metabolism enzymes and their assembly in macromolecular structures. METHODS: Gene fitness was determined by bioinformatics analysis from available datasets from genetic screenings. HSPD1 expression was evaluated by immunohistochemistry from formalin-fixed paraffin-embedded tissues from NSCLC patients. Real-time proliferation assays with and without cytotoxicity reagents, colony formation assays and cell cycle analyses were used to monitor growth and drug sensitivity of different NSCLC cells in vitro. In vivo growth was monitored with subcutaneous injections in immune-deficient mice. Cell metabolic activity was analyzed through extracellular metabolic flux analysis. Specific knockouts were introduced by CRISPR/Cas9. RESULTS: We show heat shock protein family D member 1 (HSPD1 or HSP60) as a survival gene ubiquitously expressed in NSCLC and associated with poor patients' prognosis. HSPD1 knockdown or its chemical disruption by the small molecule KHS101 induces a drastic breakdown of oxidative phosphorylation, and suppresses cell proliferation both in vitro and in vivo. By combining drug profiling with transcriptomics and through a whole-genome CRISPR/Cas9 screen, we demonstrate that HSPD1-targeted anti-cancer effects are dependent on oxidative phosphorylation and validated molecular determinants of KHS101 sensitivity, in particular, the creatine-transporter SLC6A8 and the subunit of the cytochrome c oxidase complex COX5B. CONCLUSIONS: These results highlight mitochondrial metabolism as an attractive target and HSPD1 as a potential theranostic marker for developing therapies to combat NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Chaperonina 60/metabolismo , Neoplasias Pulmonares/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Modelos Animales de Enfermedad , Humanos , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Ratones , Análisis de SupervivenciaRESUMEN
OBJECTIVE: X-linked inhibitor of apoptosis protein (XIAP) is a multifunctional protein with important functions in apoptosis, cellular differentiation and cytoskeletal organisation and is emerging as potential target for the treatment of various cancers. The aim of the current study was to investigate the role of XIAP in the pathogenesis of systemic sclerosis (SSc). METHODS: The expression of XIAP in human skin samples of patients with SSc and chronic graft versus host disease (cGvHD) and healthy individuals was analysed by quantitative PCR, immunofluorescence (IF) and western blot. XIAP was inactivated by siRNA-mediated knockdown and pharmacological inhibition. The effects of XIAP inactivation were analysed in cultured fibroblasts and in the fibrosis models bleomycin-induced and topoisomerase-I-(topoI)-induced fibrosis and in Wnt10b-transgenic mice. RESULTS: The expression of XIAP, but not of other inhibitor of apoptosis protein family members, was increased in fibroblasts in SSc and sclerodermatous cGvHD. Transforming growth factor beta (TGF-ß) induced the expression of XIAP in a SMAD3-dependent manner. Inactivation of XIAP reduced WNT-induced fibroblast activation and collagen release. Inhibition of XIAP also ameliorated fibrosis induced by bleomycin, topoI and overexpression of Wnt10b in well-tolerated doses. The profibrotic effects of XIAP were mediated via WNT/ß-catenin signalling. Inactivation of XIAP reduces binding of ß-catenin to TCF to in a TLE-dependent manner to block WNT/ß-catenin-dependent transcription. CONCLUSIONS: Our data characterise XIAP as a novel link between two core pathways of fibrosis. XIAP is overexpressed in SSc and cGvHD in a TGF-ß/SMAD3-dependent manner and in turn amplifies the profibrotic effects of WNT/ß-catenin signalling on fibroblasts via transducin-like enhancer of split 3. Targeted inactivation of XIAP inhibits the aberrant activation of fibroblasts in murine models of SSc.
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Esclerodermia Sistémica , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , beta Catenina , Animales , Bleomicina/farmacología , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibrosis , Humanos , Ratones , Esclerodermia Sistémica/patología , Piel/patología , Factor de Crecimiento Transformador beta/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , beta Catenina/metabolismoRESUMEN
Since their discovery, microRNAs (miRNAs) have been widely studied in almost every aspect of biology and medicine, leading to the identification of important gene regulation circuits and cellular mechanisms. However, investigations are generally focused on the analysis of their downstream targets and biological functions in overexpression and knockdown approaches, while miRNAs endogenous levels and activity remain poorly understood. Here, we used the cellular plasticity-regulating process of epithelial-to-mesenchymal transition (EMT) as a model to show the efficacy of a fluorescent sensor to separate cells with distinct EMT signatures, based on miR-200b/c activity. The system was further combined with a CRISPR-Cas9 screening platform to unbiasedly identify miR-200b/c upstream regulating genes. The sensor allows to infer miRNAs fundamental biological properties, as profiling of sorted cells indicated miR-200b/c as a molecular switch between EMT differentiation and proliferation, and suggested a role for metabolic enzymes in miR-200/EMT regulation. Analysis of miRNAs endogenous levels and activity for in vitro and in vivo applications could lead to a better understanding of their biological role in physiology and disease.
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Plasticidad de la Célula/genética , Neoplasias Colorrectales/genética , Transición Epitelial-Mesenquimal/genética , MicroARNs/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Neoplasias Colorrectales/patología , Células HCT116 , HumanosRESUMEN
BACKGROUND: Epithelial-to-mesenchymal transition (EMT) enhances motility, stemness, chemoresistance and metastasis. Little is known about how various pathways coordinate to elicit EMT's different functional aspects in non-small cell lung cancer (NSCLC). Thymidylate synthase (TS) has been previously correlated with EMT transcription factor ZEB1 in NSCLC and imparts resistance against anti-folate chemotherapy. In this study, we establish a functional correlation between TS, EMT, chemotherapy and metastasis and propose a network for TS mediated EMT. METHODS: Published datasets were analysed to evaluate the significance of TS in NSCLC fitness and prognosis. Promoter reporter assay was used to sort NSCLC cell lines in TSHIGH and TSLOW. Metastasis was assayed in a syngeneic mouse model. RESULTS: TS levels were prognostic and predicted chemotherapy response. Cell lines with higher TS promoter activity were more mesenchymal-like. RNA-seq identified EMT as one of the most differentially regulated pathways in connection to TS expression. EMT transcription factors HOXC6 and HMGA2 were identified as upstream regulator of TS, and AXL, SPARC and FOSL1 as downstream effectors. TS knock-down reduced the metastatic colonisation in vivo. CONCLUSION: These results establish TS as a theranostic NSCLC marker integrating survival, chemo-resistance and EMT, and identifies a regulatory network that could be targeted in EMT-driven NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Transición Epitelial-Mesenquimal/fisiología , Neoplasias Pulmonares/patología , Timidilato Sintasa/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Humanos , Neoplasias Pulmonares/enzimología , Ratones , FenotipoRESUMEN
Cancer cells frequently boost nucleotide metabolism (NM) to support their increased proliferation, but the consequences of elevated NM on tumor de-differentiation are mostly unexplored. Here, we identified a role for thymidylate synthase (TS), a NM enzyme and established drug target, in cancer cell de-differentiation and investigated its clinical significance in breast cancer (BC). In vitro, TS knockdown increased the population of CD24+ differentiated cells, and attenuated migration and sphere-formation. RNA-seq profiling indicated repression of epithelial-to-mesenchymal transition (EMT) signature genes upon TS knockdown, and TS-deficient cells showed an increased ability to invade and metastasize in vivo, consistent with the occurrence of a partial EMT phenotype. Mechanistically, TS enzymatic activity was found essential for maintenance of the EMT/stem-like state by fueling a dihydropyrimidine dehydrogenase-dependent pyrimidine catabolism. In patient tissues, TS levels were found significantly higher in poorly differentiated and in triple negative BC, and strongly correlated with worse prognosis. The present study provides the rationale to study in-depth the role of NM at the crossroads of proliferation and differentiation, and depicts new avenues for the design of novel drug combinations for the treatment of BC.
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Desdiferenciación Celular/fisiología , Timidilato Sintasa/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Antígeno CD24/metabolismo , Movimiento Celular , Proliferación Celular/fisiología , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Transición Epitelial-Mesenquimal/genética , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica/genética , Pronóstico , Pirimidinas/metabolismo , Esferoides Celulares , Timidilato Sintasa/genética , Células Tumorales CultivadasRESUMEN
Cancer cells alter their metabolism to support their malignant properties. In this study, we report that the glucose-transforming polyol pathway (PP) gene aldo-keto-reductase-1-member-B1 (AKR1B1) strongly correlates with epithelial-to-mesenchymal transition (EMT). This association was confirmed in samples from lung cancer patients and from an EMT-driven colon cancer mouse model with p53 deletion. In vitro, mesenchymal-like cancer cells showed increased AKR1B1 levels, and AKR1B1 knockdown was sufficient to revert EMT. An equivalent level of EMT suppression was measured by targeting the downstream enzyme sorbitol-dehydrogenase (SORD), further pointing at the involvement of the PP. Comparative RNA sequencing confirmed a profound alteration of EMT in PP-deficient cells, revealing a strong repression of TGFß signature genes. Excess glucose was found to promote EMT through autocrine TGFß stimulation, while PP-deficient cells were refractory to glucose-induced EMT. These data show that PP represents a molecular link between glucose metabolism, cancer differentiation, and aggressiveness, and may serve as a novel therapeutic target.Significance: A glucose-transforming pathway in TGFß-driven epithelial-to-mesenchymal transition provides novel mechanistic insights into the metabolic control of cancer differentiation. Cancer Res; 78(7); 1604-18. ©2018 AACR.
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Aldehído Reductasa/genética , Neoplasias del Colon/patología , Transición Epitelial-Mesenquimal/genética , L-Iditol 2-Deshidrogenasa/genética , Neoplasias Pulmonares/patología , Células A549 , Animales , Línea Celular Tumoral , Glucosa/metabolismo , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Células MCF-7 , Ratones , Interferencia de ARN , ARN Interferente Pequeño/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Thymidylate synthase (TS) is a fundamental enzyme of nucleotide metabolism and one of the oldest anti-cancer targets. Beginning from the analysis of gene array data from the NCI-60 panel of cancer cell lines, we identified a significant correlation at both gene and protein level between TS and the markers of epithelial-to-mesenchymal transition (EMT), a developmental process that allows cancer cells to acquire features of aggressiveness, like motility and chemoresistance. TS levels were found to be significantly augmented in mesenchymal-like compared to epithelial-like cancer cells, to be regulated by EMT induction, and to negatively correlate with micro-RNAs (miRNAs) usually expressed in epithelial-like cells and known to actively suppress EMT. Transfection of EMT-suppressing miRNAs reduced TS levels, and a specific role for miR-375 in targeting the TS 3'-untranslated region was identified. A particularly relevant association was found between TS and the powerful EMT driver ZEB1, the shRNA-mediated knockdown of which up-regulated miR-375 and reduced TS cellular levels. The TS-ZEB1 association was confirmed in clinical specimens from lung tumours and in a genetic mouse model of pancreatic cancer with ZEB1 deletion. Interestingly, TS itself appeared to have a regulatory role in EMT in cancer cells, as TS knockdown could directly reduce the EMT phenotype, the migratory ability of cells, the expression of stem-like markers, and chemoresistance. Taken together, these data indicate that the TS enzyme is functionally linked with EMT and cancer differentiation, with several potential translational implications. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Transición Epitelial-Mesenquimal , MicroARNs/genética , Neoplasias Pancreáticas/genética , Timidilato Sintasa/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Secuencia de Bases , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Persona de Mediana Edad , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , ARN Interferente Pequeño/genética , Timidilato Sintasa/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Antipsychotic drugs are effective for the treatment of schizophrenia. However, the functional consequences and subcellular sites of their accumulation in nervous tissue have remained elusive. Here, we investigated the role of the weak-base antipsychotics haloperidol, chlorpromazine, clozapine, and risperidone in synaptic vesicle recycling. Using multiple live-cell microscopic approaches and electron microscopy of rat hippocampal neurons as well as in vivo microdialysis experiments in chronically treated rats, we demonstrate the accumulation of the antipsychotic drugs in synaptic vesicles and their release upon neuronal activity, leading to a significant increase in extracellular drug concentrations. The secreted drugs exerted an autoinhibitory effect on vesicular exocytosis, which was promoted by the inhibition of voltage-gated sodium channels and depended on the stimulation intensity. Taken together, these results indicate that accumulated antipsychotic drugs recycle with synaptic vesicles and have a use-dependent, autoinhibitory effect on synaptic transmission.