RESUMEN
With age, hematopoietic stem cells (HSC) undergo changes in function, including reduced regenerative potential and loss of quiescence, which is accompanied by a significant expansion of the stem cell pool that can lead to haematological disorders. Elevated metabolic activity has been implicated in driving the HSC ageing phenotype. Here we show that nicotinamide riboside (NR), a form of vitamin B3, restores youthful metabolic capacity by modifying mitochondrial function in multiple ways including reduced expression of nuclear encoded metabolic pathway genes, damping of mitochondrial stress and a decrease in mitochondrial mass and network-size. Metabolic restoration is dependent on continuous NR supplementation and accompanied by a shift of the aged transcriptome towards the young HSC state, more youthful bone marrow cellular composition and an improved regenerative capacity in a transplant setting. Consequently, NR administration could support healthy ageing by re-establishing a more youthful hematopoietic system.
Asunto(s)
Envejecimiento , Células Madre Hematopoyéticas/efectos de los fármacos , NAD/metabolismo , Niacinamida/análogos & derivados , Compuestos de Piridinio/farmacología , Factores de Edad , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células Cultivadas , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Niacinamida/farmacología , Fosforilación Oxidativa/efectos de los fármacosRESUMEN
Protein products of the regenerating islet-derived (REG) gene family are important regulators of many cellular processes. Here we functionally characterise a non-protein coding product of the family, the long noncoding RNA (lncRNA) REG1CP that is transcribed from a DNA fragment at the family locus previously thought to be a pseudogene. REG1CP forms an RNA-DNA triplex with a homopurine stretch at the distal promoter of the REG3A gene, through which the DNA helicase FANCJ is tethered to the core promoter of REG3A where it unwinds double stranded DNA and facilitates a permissive state for glucocorticoid receptor α (GRα)-mediated REG3A transcription. As such, REG1CP promotes cancer cell proliferation and tumorigenicity and its upregulation is associated with poor outcome of patients. REG1CP is also transcriptionally inducible by GRα, indicative of feedforward regulation. These results reveal the function and regulation of REG1CP and suggest that REG1CP may constitute a target for cancer treatment.
Asunto(s)
Carcinogénesis/genética , Elementos de Facilitación Genéticos/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas Asociadas a Pancreatitis/genética , ARN Helicasas/genética , ARN Largo no Codificante/genética , Transcripción Genética , Biomarcadores de Tumor/genética , Línea Celular , Línea Celular Tumoral , ADN/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Células HT29 , Humanos , Neoplasias/genética , Neoplasias/patología , Proteínas Asociadas a Pancreatitis/metabolismo , Regiones Promotoras Genéticas/genética , ARN Helicasas/metabolismoRESUMEN
: Cancer cells in quiescence (G0 phase) are resistant to death, and re-entry of quiescent cancer cells into the cell-cycle plays an important role in cancer recurrence. Here we show that two p53-responsive miRNAs utilize distinct but complementary mechanisms to promote cancer cell quiescence by facilitating stabilization of p27. Purified quiescent B16 mouse melanoma cells expressed higher levels of miRNA-27b-3p and miRNA-455-3p relative to their proliferating counterparts. Induction of quiescence resulted in increased levels of these miRNAs in diverse types of human cancer cell lines. Inhibition of miRNA-27b-3p or miRNA-455-3p reduced, whereas its overexpression increased, the proportion of quiescent cells in the population, indicating that these miRNAs promote cancer cell quiescence. Accordingly, cancer xenografts bearing miRNA-27b-3p or miRNA-455-3p mimics were retarded in growth. miRNA-27b-3p targeted cyclin-dependent kinase regulatory subunit 1 (CKS1B), leading to reduction in p27 polyubiquitination mediated by S-phase kinase-associated protein 2 (Skp2). miRNA-455-3p targeted CDK2-associated cullin domain 1 (CAC1), which enhanced CDK2-mediated phosphorylation of p27 necessary for its polyubiquitination. Of note, the gene encoding miRNA-27b-3p was embedded in the intron of the chromosome 9 open reading frame 3 gene that was transcriptionally activated by p53. Similarly, the host gene of miRNA-455-3p, collagen alpha-1 (XXVII) chain, was also a p53 transcriptional target. Collectively, our results identify miRNA-27b-3p and miRNA-455-3p as important regulators of cancer cell quiescence in response to p53 and suggest that manipulating miRNA-27b-3p and miRNA-455-3p may constitute novel therapeutic avenues for improving outcomes of cancer treatment. SIGNIFICANCE: Two novel p53-responsive microRNAs whose distinct mechanisms of action both stabilize p27 to promote cell quiescence and may serve as therapeutic avenues for improving outcomes of cancer treatment.
Asunto(s)
Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , MicroARNs/genética , Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , Animales , Apoptosis/genética , Línea Celular Tumoral , Senescencia Celular/genética , Genes Reporteros , Genes cdc , Humanos , Ratones , Modelos Biológicos , Fosforilación , Interferencia de ARN , Proteínas Quinasas Asociadas a Fase-S/genética , Proteínas Quinasas Asociadas a Fase-S/metabolismoRESUMEN
The expression of CD47 on the cancer cell surface transmits "don't eat me" signalling that not only inhibits phagocytosis of cancer cells by phagocytes but also impairs anti-cancer T cell responses. Here we report that oncogenic activation of ERK plays an important role in transcriptional activation of CD47 through nuclear respiratory factor 1 (NRF-1) in melanoma cells. Treatment with BRAF/MEK inhibitors upregulated CD47 in cultured melanoma cells and fresh melanoma isolates. Similarly, melanoma cells selected for resistance to the BRAF inhibitor vemurafenib expressed higher levels of CD47. The increase in CD47 expression was mediated by ERK signalling, as it was associated with rebound activation of ERK and co-knockdown of ERK1/2 by siRNA diminished upregulation of CD47 in melanoma cells after exposure to BRAF/MEK inhibitors. Furthermore, ERK1/2 knockdown also reduced the constitutive expression of CD47 in melanoma cells. We identified a DNA fragment that was enriched with the consensus binding sites for NRF-1 and was transcriptionally responsive to BRAF/MEK inhibitor treatment. Knockdown of NRF-1 inhibited the increase in CD47, indicating that NRF-1 has a critical role in transcriptional activation of CD47 by ERK signalling. Functional studies showed that melanoma cells resistant to vemurafenib were more susceptible to macrophage phagocytosis when CD47 was blocked. So these results suggest that NRF-1-mediated regulation of CD47 expression is a novel mechanism by which ERK signalling promotes the pathogenesis of melanoma, and that the combination of CD47 blockade and BRAF/MEK inhibitors may be a useful approach for improving their therapeutic efficacy.
RESUMEN
MTH1 helps prevent misincorporation of ROS-damaged dNTPs into genomic DNA; however, there is little understanding of how MTH1 itself is regulated. Here, we report that MTH1 is regulated by polyubiquitination mediated by the E3 ligase Skp2. In melanoma cells, MTH1 was upregulated commonly mainly due to its improved stability caused by K63-linked polyubiquitination. Although Skp2 along with other components of the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex was physically associated with MTH1, blocking the SCF function ablated MTH1 ubiquitination and expression. Conversely, overexpressing Skp2-elevated levels of MTH1 associated with an increase in its K63-linked ubiquitination. In melanoma cell lines and patient specimens, we observed a positive correlation of Skp2 and MTH1 expression. Mechanistic investigations showed that Skp2 limited DNA damage and apoptosis triggered by oxidative stress and that MAPK upregulated Skp2 and MTH1 to render cells more resistant to such stress. Collectively, our findings identify Skp2-mediated K63-linked polyubiquitination as a critical regulatory mechanism responsible for MTH1 upregulation in melanoma, with potential implications to target the MAPK/Skp2/MTH1 pathway to improve its treatment. Cancer Res; 77(22); 6226-39. ©2017 AACR.
Asunto(s)
Enzimas Reparadoras del ADN/genética , Melanoma/genética , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Western Blotting , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Enzimas Reparadoras del ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Sistema de Señalización de MAP Quinasas/genética , Melanoma/metabolismo , Melanoma/patología , Monoéster Fosfórico Hidrolasas/metabolismo , Poliubiquitina/metabolismo , Estabilidad Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas Asociadas a Fase-S/metabolismo , UbiquitinaciónRESUMEN
Oncogenic mutations of KRAS pose a great challenge in the treatment of colorectal cancer. Here we report that mutant KRAS colon cancer cells are nevertheless more susceptible to apoptosis induced by the HSP90 inhibitor AUY922 than those carrying wild-type KRAS. Although AUY922 inhibited HSP90 activity with comparable potency in colon cancer cells irrespective of their KRAS mutational statuses, those with mutant KRAS were markedly more sensitive to AUY922-induced apoptosis. This was associated with upregulation of the BH3-only proteins Bim, Bik, and PUMA. However, only Bim appeared essential, in that knockdown of Bim abolished, whereas knockdown of Bik or PUMA only moderately attenuated apoptosis induced by AUY922. Mechanistic investigations revealed that endoplasmic reticulum (ER) stress was responsible for AUY922-induced upregulation of Bim, which was inhibited by a chemical chaperone or overexpression of GRP78. Conversely, siRNA knockdown of GRP78 or XBP-1 enhanced AUY922-induced apoptosis. Remarkably, AUY922 inhibited the growth of mutant KRAS colon cancer xenografts through activation of Bim that was similarly associated with ER stress. Taken together, these results suggest that AUY922 is a promising drug in the treatment of mutant KRAS colon cancers, and the agents that enhance the apoptosis-inducing potential of Bim may be useful to improve the therapeutic efficacy.
Asunto(s)
Antineoplásicos/farmacología , Proteína 11 Similar a Bcl2/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Isoxazoles/farmacología , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Resorcinoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Chaperón BiP del Retículo Endoplásmico , Expresión Génica , Proteínas de Choque Térmico/antagonistas & inhibidores , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Carga Tumoral/efectos de los fármacos , Carga Tumoral/genética , Proteína 1 de Unión a la X-Box/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncogenic mutations of BRAF occur in approximately 10% of colon cancers and are associated with their resistance to clinically available therapeutic drugs and poor prognosis of the patients. Here we report that colon cancer cells with mutant BRAF are also resistant to the heat shock protein 90 (HSP90) inhibitor AUY922, and that this is caused by rebound activation of ERK and Akt. Although AUY922 triggered rapid reduction in ERK and Akt activation in both wild-type and mutant BRAF colon cancer cells, activation of ERK and Akt rebounded shortly in the latter leading to resistance of the cells to AUY922-induced apoptosis. Reactivation of ERK was associated with the persistent expression of mutant BRAF, which, despite being a client of HSP90, was only partially degraded by AUY922, whereas reactivation of Akt was related to the activity of the HSP90 co-chaperone, cell division cycle 37 (CDC37), in that knockdown of CDC37 inhibited Akt reactivation in mutant colon cancer cells treated with AUY922. In support, as a HSP90 client protein, Akt was only diminished by AUY922 in wild-type but not mutant BRAF colon cancer cells. Collectively, these results reveal that reactivation of ERK and Akt associated respectively with the activity of mutant BRAF and CDC37 renders mutant BRAF colon cancer cells resistant to AUY922, with implications of co-targeting mutant BRAF and/or CDC37 and HSP90 in the treatment of mutant BRAF colon cancers.
Asunto(s)
Neoplasias del Colon/metabolismo , Resistencia a Antineoplásicos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Isoxazoles/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Resorcinoles/química , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Chaperoninas/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mutación , Proteínas Proto-Oncogénicas B-raf/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The effect of MTH1 inhibition on cancer cell survival has been elusive. Here we report that although silencing of MTH1 does not affect survival of melanoma cells, TH588, one of the first-in-class MTH1 inhibitors, kills melanoma cells through apoptosis independently of its inhibitory effect on MTH1. Induction of apoptosis by TH588 was not alleviated by MTH1 overexpression or introduction of the bacterial homolog of MTH1 that has 8-oxodGTPase activity but cannot be inhibited by TH588, indicating that MTH1 inhibition is not the cause of TH588-induced killing of melanoma cells. Although knockdown of MTH1 did not impinge on the viability of melanoma cells, it rendered melanoma cells sensitive to apoptosis induced by the oxidative stress inducer elesclomol. Of note, treatment with elesclomol also enhanced TH588-induced apoptosis, whereas a reactive oxygen species scavenger or an antioxidant attenuated the apoptosis triggered by TH588. Indeed, the sensitivity of melanoma cells to TH588 was correlated with endogenous levels of reactive oxygen species. Collectively, these results indicate that the cytotoxicity of TH588 toward melanoma cells is not associated with its inhibitory effect on MTH1, although it is mediated by cellular production of ROS.
Asunto(s)
Apoptosis/efectos de los fármacos , Melanoma/tratamiento farmacológico , Estrés Oxidativo , Pirimidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Melanoma/metabolismo , Melanoma/patologíaRESUMEN
It has been previously shown that the inhibition of mitogen activated protein kinase kinase (MEK) contributes to apoptosis and suppression of different cancer cells. Correspondingly, a number of MEK1/2 inhibitors have been designed and evaluated since 2001. However, they did not satisfy essential pharmacokinetic (PK) and pharmacodynamic (PD) properties thus, almost most of them were terminated in pre-clinical or clinical studies. This study aims to design new specific MEK1/2 inhibitors with improved PK/PD profiles to be used as alternative cancer medications. In first part of this study, a comprehensive screening, for the first time, was done on well-known MEK1/2 inhibitors using a number of computational programs such as AutoDock Tools 4.2 (ADT) and AutoDock Vina. Therefore a valuable training dataset as well as a reliable pharmacophore model were provided which were then used to design new inhibitors. According to the results of training dataset, Trametinib was determined as the best inhibitor provided, so far. So, Trametinib was used as the lead structure to design new inhibitors in this study. In second part of this investigation, a set of new allosteric MEK1/2 inhibitors were designed significantly improving the binding energy as well as the ADMET properties, suggesting more specific and stable ligand-receptor complexes. Consequently, the structures 14 and 15 of our inhibitors, as the most potent structures, are great substituents for Trametinib to be used and evaluated in clinical trials as alternative cancer drugs.