RESUMEN
MicroRNAs (miRNAs) regulate diverse cancer hallmarks through sequence-specific regulation of gene expression, so genetic variability in their seed sequences or target sites could be responsible for cancer initiation or progression. While several efforts have been made to predict the locations of single nucleotide variants (SNVs) at miRNA target sites and associate them with cancer risk and susceptibility, there have been few direct assessments of SNVs in both mature miRNAs and their target sites to assess their impact on miRNA function in cancers. Using genome-wide target capture of miRNAs and miRNA-binding sites followed by deep sequencing in prostate cancer cell lines, here we identified prostate cancer-specific SNVs in mature miRNAs and their target binding sites. SNV rs9860655 in the mature sequence of miR-570 was not present in benign prostate hyperplasia (BPH) tissue or cell lines but was detectable in clinical prostate cancer tissue samples and adjacent normal tissue. SLC45A3 (prostein), a putative oncogene target of miR-1178, was highly upregulated in PC3 cells harboring an miR-1178 seed sequence SNV. Finally, systematic assessment of losses and gains of miRNA targets through 3'UTR SNVs revealed SNV-associated changes in target oncogene and tumor suppressor gene expression that might be associated with prostate carcinogenesis. Further work is required to systematically assess the functional effects of miRNA SNVs.
Asunto(s)
Carcinogénesis/genética , MicroARNs/genética , Proteínas de Transporte de Monosacáridos/genética , Neoplasias de la Próstata/genética , Sitios de Unión/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Variación Genética/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Polimorfismo de Nucleótido Simple/genética , Neoplasias de la Próstata/patología , Proteínas de Unión al ARN/genéticaRESUMEN
MicroRNAs (miRs) are important posttranscriptional regulators of cell fate in both normal and disease states. miR-211 has previously been shown to be a direct regulator of metabolism in BRAFV600E-mutant melanoma cells in vitro. Here, we report that miR-211 expression promotes the aggressive growth of BRAFV600E-mutant melanoma xenografts in vivo. miR-211 promoted proliferation through the posttranscriptional activation of extracellular signal-regulated kinase (ERK) 5 signaling, which has recently been implicated in the resistance to BRAF and MAPK/ERK kinase inhibitors. We therefore examined whether miR-211 similarly modulated melanoma resistance to the BRAF inhibitor vemurafenib and the MAPK/ERK kinase inhibitor cobimetinib. Consistent with this model, miR-211 expression increased melanoma cell resistance to both the inhibitors, and this resistance was associated with an increased ERK5 phosphorylation. miR-211 mediates these effects by directly inhibiting the expression of DUSP6, an ERK5 pathway-specific phosphatase and now shown to be an miR-211 target gene. These results dissect the role of the miR-211-DUSP6-ERK5 axis in melanoma tumor growth and suggest a mechanism for the development of drug-resistant tumors and a target for overcoming resistance.
Asunto(s)
Resistencia a Antineoplásicos/genética , Fosfatasa 6 de Especificidad Dual/genética , Melanoma/tratamiento farmacológico , MicroARNs/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Animales , Azetidinas/farmacología , Azetidinas/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/genética , Fosfatasa 6 de Especificidad Dual/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Sistema de Señalización de MAP Quinasas/genética , Melanoma/genética , Melanoma/patología , Ratones , Proteína Quinasa 7 Activada por Mitógenos/genética , Mutación , Fosforilación/genética , Piperidinas/farmacología , Piperidinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Vemurafenib/farmacología , Vemurafenib/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Sensitive and specific diagnostic and prognostic biomarkers for prostate cancer (PCa) are urgently needed. Urine samples are a non-invasive means to obtain abundant and readily accessible "liquid biopsies". Herein we used urine liquid biopsies to identify and characterize a novel group of urine-enriched RNAs and metabolites in patients with PCa and normal individuals with or without benign prostatic disease. Differentially expressed RNAs were identified in urine samples by deep sequencing and metabolites in urine were measured by mass spectrometry. mRNA and metabolite profiles were distinct in patients with benign and malignant disease. Integrated analysis of urinary gene expression and metabolite signatures unveiled an aberrant glutamate metabolism and tricarboxylic acid (TCA) cycle node in prostate cancer-derived cells. Functional validation supported a role for glutamate metabolism and glutamate oxaloacetate transaminase 1 (GOT1)-dependent redox balance in PCa, which could be exploited for novel biomarkers and therapies. In this study, we discovered cancer-specific changes in urinary RNAs and metabolites, paving the way for the development of sensitive and specific urinary PCa diagnostic biomarkers either alone or in combination. Our methodology was based on single void urine samples (i.e., without prostatic massage). The integrated analysis of metabolomic and transcriptomic data from these liquid biopsies revealed a glutamate metabolism and tricarboxylic acid cycle node that was specific to prostate-derived cancer cells and cancer-specific metabolic changes in urine.
Asunto(s)
Biomarcadores de Tumor/orina , Neoplasias de la Próstata/orina , ARN Mensajero/orina , Ciclo del Ácido Cítrico , Ácido Glutámico/metabolismo , Humanos , Biopsia Líquida , Masculino , Próstata/metabolismo , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/genéticaRESUMEN
The long non-coding RNA GAS5 has been shown to modulate cancer proliferation in numerous human cancer systems and has been correlated with successful patient outcome. Our examination of GAS5 in neuroblastoma has revealed robust expression in both MYCN-amplified and non-amplified cell lines. Knockdown of GAS5 In vitro resulted in defects in cell proliferation, apoptosis, and induced cell cycle arrest. Further analysis of GAS5 clones revealed multiple novel splice variants, two of which inversely modulated with MYCN status. Complementation studies of the variants post-knockdown of GAS5 indicated alternate phenotypes, with one variant (FL) considerably enhancing cell proliferation by rescuing cell cycle arrest and the other (C2) driving apoptosis, suggesting a unique role for each in neuroblastoma cancer physiology. Global sequencing and ELISA arrays revealed that the loss of GAS5 induced p53, BRCA1, and GADD45A, which appeared to modulate cell cycle arrest in concert. Complementation with only the FL GAS5 clone could rescue cell cycle arrest, stabilizing HDM2, and leading to the loss of p53. Together, these data offer novel therapeutic targets in the form of lncRNA splice variants for separate challenges against cancer growth and cell death.
Asunto(s)
Empalme Alternativo , Apoptosis , Proteína BRCA1/genética , Biomarcadores de Tumor/genética , Puntos de Control del Ciclo Celular , Proliferación Celular , Neuroblastoma/genética , ARN Largo no Codificante/genética , Proteína p53 Supresora de Tumor/genética , Proteína BRCA1/metabolismo , Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Interferencia de ARN , ARN Largo no Codificante/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Molecular mechanisms by which long noncoding RNA (lncRNA) molecules may influence cancerous condition are poorly understood. The aberrant expression of SPRIGHTLY lncRNA, encoded within the drosophila gene homolog Sprouty-4 intron, is correlated with a variety of cancers, including human melanomas. We demonstrate by SHAPE-seq and dChIRP that SPRIGHTLY RNA secondary structure has a core pseudoknotted domain. This lncRNA interacts with the intronic regions of six pre-mRNAs: SOX5, SMYD3, SND1, MEOX2, DCTN6, and RASAL2, all of which have cancer-related functions. Hemizygous knockout of SPRIGHTLY by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 in melanoma cells significantly decreases SPRIGHTLY lncRNA levels, simultaneously decreases the levels of its interacting pre-mRNA molecules, and decreases anchorage-independent growth rate of cells and the rate of in vivo tumor growth in mouse xenografts. These results provide the first demonstration of an lncRNA's three-dimensional coordinating role in facilitating cancer-related gene expression in human melanomas.
Asunto(s)
Precursores del ARN/genética , ARN Largo no Codificante/genética , Animales , Sistemas CRISPR-Cas/genética , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Humanos , Melanoma/genética , Ratones , Ratones SCIDRESUMEN
MicroRNA 211 (miR-211) negatively regulates genes that drive invasion of metastatic melanoma. Compared to normal human melanocytes, miR-211 expression is significantly reduced or absent in nonpigmented melanoma cells and lost during human melanoma progression. To investigate the molecular mechanism of its tumor suppressor function, miR-211 was ectopically expressed in nonpigmented melanoma cells. Ectopic expression of miR-211 reduced hypoxia-inducible factor 1α (HIF-1α) protein levels and decreased cell growth during hypoxia. HIF-1α protein loss was correlated with the downregulation of a miR-211 target gene, pyruvate dehydrogenase kinase 4 (PDK4). We present evidence that resumption of miR-211-mediated downregulation of PDK4 in melanoma cells causes inhibition of invasion by nonpigmented melanomas via HIF-1α protein destabilization. Thus, the tumor suppressor miR-211 acts as a metabolic switch, and its loss is expected to promote cancer hallmarks in human melanomas. Melanoma, one of the deadliest forms of skin cancer, kills nearly 10,000 people in the United States per year. We had previously shown that a small noncoding RNA, termed miR-211, suppresses invasion and the growth of aggressive melanoma cells. The results presented here support the hypothesis that miR-211 loss in melanoma cells causes abnormal regulation of energy metabolism, which in turn allows cancer cells to survive under low oxygen concentrations-a condition that generally kills normal cells. These findings highlight a novel mechanism of melanoma formation: miR-211 is a molecular switch that is turned off in melanoma cells, raising the hope that in the future we might be able to turn the switch back on, thus providing a better treatment option for melanoma.
Asunto(s)
Melanoma/metabolismo , MicroARNs/fisiología , Hipoxia de la Célula , Línea Celular Tumoral , Movimiento Celular , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Melanocitos/metabolismo , Melanoma/genética , Mitocondrias/metabolismo , Invasividad Neoplásica , Consumo de Oxígeno , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Receptores de Estrógenos/metabolismoRESUMEN
FNDC3B was recently identified in an oncogenomic screen for amplified oncogenes in hepatocellular carcinoma. It is located at 3q26 and is amplified in over 20% of cancers, usually as part of a broad amplified region encompassing the entire 3q arm. Consistent with an oncogenic role in multiple cancer types, we show here that overexpression of FNDC3B is capable of malignantly transforming mammary and kidney epithelial cells in addition to hepatocytes. To explore how FNDC3B transforms cells, we determined the cellular localization of its gene product and the cancer pathways that it activates. We found that the FNDC3B oncoprotein localizes to the Golgi network, and that its correct localization is essential for its transforming function. We found that overexpression of FNDC3B induces the epithelial-to-mesenchymal transition (EMT) and activates several cancer pathways, including PI3-kinase/Akt, Rb1 and TGFß signaling. For TGFß signaling, we analyzed the point in the pathway at which FNDC3B operates and obtained evidence that it induces expression of all three TGFß ligands and also promotes TGFBR1 cell-surface localization. We found that RNAi-mediated knockdown of FNDC3B in cancer cells with 3q amplification suppressed their clonogenicity and tumorigenicity, but that the same RNAi knockdown had no effect on single-copy 3q cancer cells. These results indicate that FNDC3B is an important oncogenic driver gene of the 3q amplicon, adding to the growing list of oncogenic drivers within this commonly amplified region.
Asunto(s)
Transformación Celular Neoplásica , Cromosomas Humanos Par 3/genética , Fibronectinas/genética , Regulación Neoplásica de la Expresión Génica , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Cromosomas Humanos Par 3/metabolismo , Transición Epitelial-Mesenquimal , Fibronectinas/metabolismo , Dosificación de Gen , Técnicas de Silenciamiento del Gen , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Aparato de Golgi/metabolismo , Humanos , Inmunoprecipitación , Oncogenes , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Transducción de Señal , Activación Transcripcional , Transfección , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
RNAi screening holds the promise of systemizing the search for combination therapeutic strategies. Here we performed a pooled shRNA library screen to look for promising targets to inhibit in combination with inhibition of the mitotic regulator polo-like kinase (PLK1). The library contained ~4,500 shRNAs targeting various signaling and cancer-related genes and was screened in four lung cancer cell lines using both high (IC80) and low (IC20) amounts of the PLK1 inhibitor GSK461364. The relative abundance of cells containing individual shRNAs following drug treatment was determined by microarray analysis, using the mock treatment replicates as the normalizing reference. Overall, the inferred influences of individual shRNAs in both high and low drug treatment were remarkably similar in all four cell lines and involved a large percentage of the library. To investigate which functional categories of shRNAs were most prominent in influencing drug response, we used statistical analysis of microarrays (SAM) in combination with a filter for genes that had two or more concordant shRNAs. The most significant functional categories that came out of this analysis included receptor tyrosine kinases and nuclear hormone receptors. Through individual validation experiments, we determined that the two shRNAs from the library targeting the nuclear retinoic acid receptor gene RARA did indeed silence RARA expression and as predicted conferred resistance to GSK461364. This led us to test whether activation of RARA receptor with retinoids could sensitize cells to GSK461364. We found that retinoids did increase the drug sensitivity and enhanced the ability of PLK1 inhibition to induce mitotic arrest and apoptosis. These results suggest that retinoids could be used to enhance the effectiveness of GSK461364 and provide further evidence that RNAi screens can be effective tools to identify combination target strategies.