RESUMEN
This study was conducted to investigate the possibility that TP53 mRNA is variably expressed in chronic lymphocytic leukaemia (CLL) and that under-expression is associated with TP53 dysfunction and adverse outcome. Although TP53 mRNA levels did indeed vary among the 104 CLL samples examined, this variability resulted primarily from over-expression of TP53 mRNA in 18 samples, all of which lacked TP53 deletion/mutation. These patients had higher lymphocyte counts and shorter overall and treatment-free survival times compared to cases with low TP53 mRNA expression and no TP53 deletion/mutation. Furthermore, TP53 mRNA levels did not correlate with levels of TP53 protein or its transcriptional target CDKN1A. We speculated that the adverse outcome associated with TP53 mRNA over-expression might reflect variation in levels of MIR15A and MIR16-1, which are encoded on chromosome 13q14 and target TP53 and some oncogenes including BCL2. In keeping with our hypothesis, 13q14 copy number and levels of MIR15A/MIR16-1 correlated positively with one another but negatively with levels of TP53 mRNA and BCL2 mRNA. Our findings support a model in which loss of MIR15A/MIR16-1 at chromosome 13q14 results in adverse outcome due to de-repression of oncogenes such as BCL2, and up-regulation of TP53 mRNA as a bystander effect.
Asunto(s)
Cromosomas Humanos Par 13/genética , Regulación Leucémica de la Expresión Génica , Genes bcl-2 , Genes p53 , Leucemia Linfocítica Crónica de Células B/genética , MicroARNs/fisiología , Proteínas de Neoplasias/genética , ARN Mensajero/biosíntesis , ARN Neoplásico/fisiología , Anciano , Cromatografía Líquida de Alta Presión , Cromosomas Humanos Par 17/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Progresión de la Enfermedad , Femenino , Dosificación de Gen , Regulación Leucémica de la Expresión Génica/genética , Humanos , Hibridación Fluorescente in Situ , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/mortalidad , Masculino , MicroARNs/genética , Persona de Mediana Edad , Proteínas de Neoplasias/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
Breast cancer brain metastasis (BCBM) is an area of unmet clinical need. MicroRNAs (miRNAs) have been linked to the metastatic process in breast cancer (BC). In this study, we aim to determine differentially-expressed miRNAs utilising primary BCs that did not relapse (BCNR, n = 12), primaries that relapsed (BCR) and their paired (n = 40 pairs) brain metastases (BM) using the NanoString™ nCounter™ miRNA Expression Assays. Significance analysis of microarrays identified 58 and 11 differentially-expressed miRNAs between BCNR vs BCR and BCR vs BM respectively and pathway analysis revealed enrichment for genes involved in invasion and metastasis. Four miRNAs, miR-132-3p, miR-199a-5p, miR-150-5p and miR-155-5p, were differentially-expressed within both cohorts (BCNR-BCR, BCR-BM) and receiver-operating characteristic curve analysis (p = 0.00137) and Kaplan-Meier survival method (p = 0.0029, brain metastasis-free survival; p = 0.0007, overall survival) demonstrated their potential use as prognostic markers. Ingenuity pathway enrichment linked them to the MET oncogene, and the cMET protein was overexpressed in the BCR (p < 0.0001) and BM (p = 0.0008) cases, compared to the BCNRs. The 4-miRNAs panel identified in this study could be potentially used to distinguish BC patients with an increased risk of developing BCBM and provide potential novel therapeutic targets, whereas cMET-targeting warrants further investigation in the treatment of BCBM.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Neoplasias de la Mama/mortalidad , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Biomarcadores de Tumor/genética , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Perfilación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Invasividad Neoplásica , Recurrencia Local de Neoplasia , Distribución Normal , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente Principal , Pronóstico , Curva ROC , Riesgo , Resultado del TratamientoRESUMEN
Molecular analysis of paired tumours highlights the limitations of the current clinical criteria for identifying second primary tumours. At present the finding of identical novel microsatellite alleles in paired lesions provides a "gold standard" marker for establishing clonal origin. However, these aberrations occur at low frequency and other methods for determining clonality have been proposed. In the present study we have applied 3 molecular tests to establish whether it is possible to combine the results obtained with the different approaches to provide information about the likely origin of a second tumour when novel alleles are not found. Our findings provide substantive molecular evidence that a proportion of second tumours are recurrences of an index lesion and suggest that the finding of concordant allelic imbalance at two or more loci at two different chromosome arms together with concordant p53 mutations might provide a useful surrogate. We briefly review other published reports and emphasis the need to plan treatment to eliminate precursor lesions in the field rather than focusing on the visible primary lesion and the 1-2 cm of surrounding mucosa traditionally considered to be "at risk".
Asunto(s)
Carcinoma de Células Escamosas/genética , Repeticiones de Microsatélite/genética , Neoplasias de la Boca/genética , Neoplasias Primarias Múltiples/genética , Neoplasias Primarias Secundarias/genética , Anciano , Carcinoma de Células Escamosas/virología , Cromosomas Humanos X/genética , Femenino , Genes p53/genética , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/virología , Neoplasias Primarias Múltiples/virología , Neoplasias Primarias Secundarias/virología , Papillomaviridae/aislamiento & purificación , Polimorfismo Genético/genéticaRESUMEN
Bi-directional communication with the microenvironment is essential for homing and survival of cancer cells with implications for disease biology and behaviour. In chronic lymphocytic leukemia (CLL), the role of the microenvironment on malignant cell behaviour is well described. However, how CLL cells engage and recruit nurturing cells is poorly characterised. Here we demonstrate that CLL cells secrete exosomes that are nanovesicles originating from the fusion of multivesicular bodies with the plasma membrane, to shuttle proteins, lipids, microRNAs (miR) and mRNAs to recipient cells. We characterise and confirm the size (50-100 nm) and identity of the CLL-derived exosomes by Electron microscopy (EM), Atomic force microscopy (AFM), flow cytometry and western blotting using both exosome- and CLL-specific markers. Incubation of CLL-exosomes, derived either from cell culture supernatants or from patient plasma, with human stromal cells shows that they are readily taken up into endosomes, and induce expression of genes such as c-fos and ATM as well as enhance proliferation of recipient HS-5 cells. Furthermore, we show that CLL exosomes encapsulate abundant small RNAs and are enriched in certain miRs and specifically hsa-miR-202-3p. We suggest that such specific packaging of miR-202-3p into exosomes results in enhanced expression of 'suppressor of fused' (Sufu), a Hedgehog (Hh) signalling intermediate, in the parental CLL cells. Thus, our data show that CLL cells secrete exosomes that alter the transcriptome and behaviour of recipient cells. Such communication with microenvironment is likely to have an important role in CLL disease biology.
Asunto(s)
Exosomas/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Células del Estroma/metabolismo , Transcriptoma , Transporte Biológico , Comunicación Celular , Línea Celular Tumoral , Proliferación Celular , Endosomas/metabolismo , Exosomas/ultraestructura , Perfilación de la Expresión Génica , Humanos , MicroARNs/química , Interferencia de ARN , ARN Mensajero/química , ARN Mensajero/genética , Proteínas Represoras/genética , Microambiente TumoralRESUMEN
Signals through the B-cell antigen receptor (BCR) are important for the survival of chronic lymphocytic leukemia (CLL) cells. Therefore, factors that influence these signals have important pathophysiological roles in this disease. One key mediator of BCR signaling is protein kinase C beta (PKCbeta), which regulates the activation of I-kappaB kinases and the deactivation of Bruton tyrosine kinase within the signaling pathways initiated by BCR engagement. The present study demonstrates that overexpression of the PKCbetaII isoform is a feature of CLL cells and that activity of this enzyme strongly correlates with CLL cell response to BCR engagement. Thus, intracellular Ca2+ release and increases in cell survival after BCR cross-linking were significantly greater in CLL patients with low levels than in CLL patients with high levels of active PKCbetaII. Furthermore, BCR-induced Ca2+ fluxes could be restored in CLL patients with high levels of active PKCbetaII by pretreating the cells with the PKCbeta-specific inhibitor LY379196. Conversely, BCR-mediated intracellular Ca2+ release could be inhibited in CLL cells with low levels of active PKCbetaII by pretreatment with the PKC agonist bryostatin. Taken together, these results demonstrate that overexpressed active PKCbetaII plays a role in the regulation and outcome of BCR signals that can be important for the progression of CLL.