RESUMEN
The NAD+-dependent deacetylase SIRT1 can be oncogenic or tumor suppressive depending on the tissue. Little is known about the role of SIRT1 in non-small cell lung carcinoma (NSCLC), one of the deadliest cancers, that is frequently associated with mutated K-RAS Therefore, we investigated the effect of SIRT1 on K-RAS-driven lung carcinogenesis. We report that SIRT1 protein levels are downregulated by oncogenic K-RAS in a MEK and PI3K-dependent manner in mouse embryo fibroblasts (MEFs), and in human lung adenocarcinoma cell lines. Furthermore, Sirt1 overexpression in mice delays the appearance of K-RasG12V-driven lung adenocarcinomas, reducing the number and size of carcinomas at the time of death and extending survival. Consistently, lower levels of SIRT1 are associated with worse prognosis in human NSCLCs. Mechanistically, analysis of mouse Sirt1-Tg pneumocytes, isolated shortly after K-RasG12V activation, reveals that Sirt1 overexpression alters pathways involved in tumor development: proliferation, apoptosis, or extracellular matrix organization. Our work demonstrates a tumor suppressive role of SIRT1 in the development of K-RAS-driven lung adenocarcinomas in mice and humans, suggesting that the SIRT1-K-RAS axis could be a therapeutic target for NSCLCs.
Asunto(s)
Adenocarcinoma del Pulmón/metabolismo , Carcinogénesis/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Sirtuina 1/metabolismo , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/patología , Células Epiteliales Alveolares , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Células Cultivadas , Regulación hacia Abajo , Fibroblastos/metabolismo , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Terapia Molecular Dirigida , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Supervivencia sin Progresión , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Myopodin is a cytoskeleton protein that shuttles to the nucleus depending on the cellular differentiation and stress. It has shown tumor suppressor functions. Myopodin methylation status was useful for staging bladder and colon tumors and predicting clinical outcome. To our knowledge, myopodin has not been tested in kidney cancer to date. The purpose of this study was to evaluate whether myopodin methylation status could be clinically useful in renal cancer (1) as a prognostic biomarker and 2) as a predictive factor of response to antiangiogenic therapy in patients with metastatic disease. Methylation-specific polymerase chain reactions (MS-PCR) were used to evaluate myopodin methylation in 88 kidney tumors. These belonged to patients with localized disease and no evidence of disease during follow-up (n = 25) (group 1), and 63 patients under antiangiogenic therapy (sunitinib, sorafenib, pazopanib, and temsirolimus), from which group 2 had non-metastatic disease at diagnosis (n = 32), and group 3 showed metastatic disease at diagnosis (n = 31). Univariate and multivariate Cox analyses were utilized to assess outcome and response to antiangiogenic agents taking progression, disease-specific survival, and overall survival as clinical endpoints. Myopodin was methylated in 50 out of the 88 kidney tumors (56.8 %). Among the 88 cases analyzed, 10 of them recurred (11.4 %), 51 progressed (57.9 %), and 40 died of disease (45.4 %). Myopodin methylation status correlated to MSKCC Risk score (p = 0.050) and the presence of distant metastasis (p = 0.039). Taking all patients, an unmethylated myopodin identified patients with shorter progression-free survival, disease-specific survival, and overall survival. Using also in univariate and multivariate models, an unmethylated myopodin predicted response to antiangiogenic therapy (groups 2 and 3) using progression-free survival, disease-specific, and overall survival as clinical endpoints. Myopodin was revealed hypermethylated in kidney cancer. Myopodin methylation status identified which patients showed a more aggressive clinical behavior and predicted antiangiogenic response. These observations support the clinical utility of an unmethylated myopodin as a prognostic and predictive biomarker in kidney cancer.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Biomarcadores de Tumor/genética , Carcinoma Papilar/genética , Carcinoma de Células Renales/genética , Metilación de ADN , Neoplasias Renales/genética , Proteínas de Microfilamentos/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Carcinoma Papilar/tratamiento farmacológico , Carcinoma Papilar/secundario , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/secundario , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Pronóstico , Tasa de SupervivenciaRESUMEN
Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.
Asunto(s)
Metilación de ADN , Línea Celular , Transformación Celular Neoplásica/genética , Análisis por Conglomerados , Islas de CpG , Epigenómica/métodos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Neoplasias/genéticaRESUMEN
miRNAs are small noncoding RNAs with critical roles in a large variety of biological processes such as development and tumorigenesis. miRNA expression profiling has been reported to be a powerful tool to classify tissue samples, including cancers, based on their developmental lineage. In this study, we have profiled the expression of miRNAs in bladder carcinoma in situ (CIS) and distinct cell compartments of the normal bladder, namely umbrella and basal-intermediate urothelial cells, as well as the muscularis propria. We identified several miRNAs differentially expressed between umbrella and basal-intermediate cells (miR-133a, miR-139-3p, miR-142-3p, miR-199b-5p, and miR-221). In situ hybridization confirmed the expression of miR-133a and miR-139-3p in umbrella cells, and miR-142-3p in basal-intermediate cells. Strikingly, miRNA expression levels of CIS most closely resembled the miRNA profile of umbrella cells. Finally, we examined well-established umbrella and basal-intermediate cell immunohistochemical biomarkers in an independent series of CIS samples. Again, this analysis revealed the significant expression of umbrella-specific markers in CIS when compared to non-CIS lesions. Overall, our studies represent a comprehensive and accurate description of the different miRNAs expressed in CIS tumors and three distinct histological areas of the urinary bladder. Notably, this study provides evidence of the possible origin relationship between CIS and normal umbrella cells.
Asunto(s)
Carcinoma in Situ/genética , Carcinoma in Situ/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patología , Adolescente , Adulto , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Separación Celular , Análisis por Conglomerados , Humanos , Inmunohistoquímica , Captura por Microdisección con Láser , Masculino , MicroARNs/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Reproducibilidad de los Resultados , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Urotelio/metabolismo , Urotelio/patologíaRESUMEN
Transcripts from the four genes encoding cyclin D1, MCM7, TRIM29, and UBE2C have previously been included in gene expression signatures for outcome prediction in stage Ta/T1 urothelial carcinomas. We investigated the prognostic value of the protein expressions in Ta/T1 urothelial carcinomas patients. We used four different tissue microarrays (TMAs) with a total of 859 Ta/T1 urothelial carcinomas from Danish, Swedish, Spanish, and Taiwanese patient cohorts with long-term follow-up. Protein expression was measured by IHC, and antibody specificity was validated by Western blotting. We found the expression of cyclin D1, MCM7, TRIM29, and UBE2C to be significantly associated with progression to muscle-invasive bladder cancer (log-rank test; P < 0.001) in the Danish training cohort (n = 283). Multivariate Cox regression analysis identified cyclin D1 (P = 0.003), TRIM29 (P = 0.001), and UBE2C (P < 0.001) as independent prognostic markers. The prognostic value of the four proteins was validated in a joint validation cohort from Sweden, Spain, and Taiwan (n = 576). Computer-assisted image analysis of the prognostic markers produced results comparable to those obtained by manual scoring. Finally, a four-protein maximum-likelihood classifier was trained on the Danish training cohort and applied to the validation cohort. The four protein markers may help optimize treatment of patients with Ta/T1 bladder cancer. Additional prospective studies are needed for further validation of their clinical relevance.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Dinamarca , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Componente 7 del Complejo de Mantenimiento de Minicromosoma , Análisis Multivariante , Músculos/patología , Invasividad Neoplásica , Modelos de Riesgos Proporcionales , Reproducibilidad de los Resultados , Factores de Riesgo , España , Suecia , Taiwán , Adulto JovenRESUMEN
DNA methylation of tumor suppressor genes (TSGs) represents a frequent and early epigenetic event with potential applications for cancer detection and disease evolution. Our aim was to examine the stratification and prognostic biomarker role of the methylation of a novel panel of TSGs in bladder cancer. The methylation status of 18 TSGs was evaluated in bladder cancer cells (n=14) and paraffin-embedded primary bladder tumors (n=61), using a methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). Recurrence, progression, and disease-specific survival were analyzed using univariate and multivariate Cox models. PRDM2, HLTF, ID4, DLC1, BNIP3, H2AFX, CACNA1G, TGIF, and CACNA1A were discovered methylated in bladder cancer. The methylation of RUNX3 (p=0.026), TWIST1 (p=0.009), SFRP4 (p=0.002), and CCND2 (p=0.027) correlated to tumor stage. Univariate analyses indicated prognostic associations for recurrence (DLC1, SFRP5, H2AFX, CACNA1G), progression (DLC1, SFRP5, CACNA1G), disease-specific (PRDM2, DLC1, SFRP5, CACNA1G, and TIMP3), and overall survival (SFRP5 and TIMP3). In multivariate analyses, several TSGs remained as independent prognosticators for recurrence (SFRP5, H2AFX), progression (CACNA1G), and disease-specific survival (SFRP5). Thus, a novel set of TSGs was identified, frequently methylated in bladder cancer cells and tumors. TSG methylation allowed histopathologic and outcome stratification using paraffin-embedded tumors. This is clinically relevant by offering a strategy for the management of patients affected with uroepithelial neoplasias in pathology routine laboratories.
Asunto(s)
Metilación de ADN , Genes Supresores de Tumor , Neoplasias de la Vejiga Urinaria/genética , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adhesión en Parafina , Pronóstico , Modelos de Riesgos Proporcionales , Neoplasias de la Vejiga Urinaria/mortalidad , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Of the most important clinical needs for bladder cancer (BC) management is the identification of biomarkers for disease aggressiveness. Urine is a "gold mine" for biomarker discovery, nevertheless, with multiple proteins being in low amounts, urine proteomics becomes challenging. In the present study we applied a fractionation strategy of urinary proteins based on the use of immobilized metal affinity chromatography for the discovery of biomarkers for aggressive BC. Urine samples from patients with non invasive (two pools) and invasive (two pools) BC were subjected to immobilized metal affinity chromatography fractionation and eluted proteins analyzed by 1D-SDS-PAGE, band excision and liquid chromatography tandem MS. Among the identified proteins, multiple corresponded to proteins with affinity for metals and/or reported to be phosphorylated and included proteins with demonstrated association with BC such as MMP9, fibrinogen forms, and clusterin. In agreement to the immobilized metal affinity chromatography results, aminopeptidase N, profilin 1, and myeloblastin were further found to be differentially expressed in urine from patients with invasive compared with non invasive BC and benign controls, by Western blot or Elisa analysis, nevertheless exhibiting high interindividual variability. By tissue microarray analysis, profilin 1 was found to have a marked decrease of expression in the epithelial cells of the invasive (T2+) versus high risk non invasive (T1G3) tumors with occasional expression in stroma; importantly, this pattern strongly correlated with poor prognosis and increased mortality. The functional relevance of profilin 1 was investigated in the T24 BC cells where blockage of the protein by the use of antibodies resulted in decreased cell motility with concomitant decrease in actin polymerization. Collectively, our study involves the application of a fractionation method of urinary proteins and as one main result of this analysis reveals the association of profilin 1 with BC paving the way for its further investigation in BC stratification.
Asunto(s)
Biomarcadores de Tumor/orina , Profilinas/orina , Neoplasias de la Vejiga Urinaria/orina , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Antígenos CD13/orina , Cromatografía de Afinidad , Cromatografía Liquida , Células Epiteliales/metabolismo , Humanos , Persona de Mediana Edad , Mieloblastina/orina , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/orina , Profilinas/metabolismo , Células del Estroma/metabolismo , Espectrometría de Masas en Tándem , Vejiga Urinaria/metabolismo , Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Array-CGH represents a comprehensive tool to discover genomic disease alterations that could potentially be applied to body fluids. In this report, we aimed at applying array-CGH to urinary samples to characterize bladder cancer. METHODS: Urinary DNA from bladder cancer patients and controls were hybridized on 44K oligonucleotide arrays. Validation analyses of identified regions and candidates included fluorescent in situ hybridization (FISH) and immunohistochemistry in an independent set of bladder tumors spotted on custom-made tissue arrays (n = 181). RESULTS: Quality control of array-CGH provided high reproducibility in dilution experiments and when comparing reference pools. The most frequent genomic alterations (minimal recurrent regions) among bladder cancer urinary specimens included gains at 1q and 5p, and losses at 10p and 11p. Supervised hierarchical clustering identified the gain at 1q23.3-q24.1 significantly correlated to stage (p = 0.011), and grade (p = 0.002). The amplification and overexpression of Prefoldin (PFND2), a selected candidate mapping to 1q23.3-q24.1, correlated to increasing stage and tumor grade by means of custom-designed and optimized FISH (p = 0.013 and p = 0.023, respectively), and immunohistochemistry (p ≤0.0005 and p = 0.011, respectively), in an independent set of bladder tumors included in tissue arrays. Moreover, PFND2 overexpression was significantly associated with poor disease-specific survival (p ≤0.0005). PFND2 was amplified and overexpressed in bladder tumors belonging to patients providing urinary specimens where 1q23.3q24.1 amplification was detected by array-CGH. CONCLUSIONS: Genomic profiles of urinary DNA mirrowed bladder tumors. Molecular profiling of urinary DNA using array-CGH contributed to further characterize genomic alterations involved in bladder cancer progression. PFND2 was identified as a tumor stratification and clinical outcome prognostic biomarker for bladder cancer patients.
Asunto(s)
Hibridación Genómica Comparativa , ADN/orina , Regulación Neoplásica de la Expresión Génica , Chaperonas Moleculares/genética , Neoplasias de la Vejiga Urinaria/genética , Biomarcadores/metabolismo , Biomarcadores de Tumor , Cromosomas Humanos Par 1 , Análisis por Conglomerados , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Reproducibilidad de los Resultados , Neoplasias de la Vejiga Urinaria/orinaRESUMEN
Altered microRNA (miRNA) expression may occur early in bladder cancer and may play a role in carcinogenesis and tumor behavior. We evaluated whether alterations in miRNA expression could improve disease stratification and outcome prognosis in bladder tumors and noninvasive diagnosis in urinary samples. miR-143, miR-222, and miR-452 expression levels were analyzed by quantitative RT-PCR (RT-qPCR) in paired urinary and matching tumors and in two independent prospective series of tumors and urinary specimens. Differential expression of miR-143, miR-222, and miR-452 in urine were verified by in situ hybridization in matching tumors. Tumor miRNA expression by RT-qPCR correlated with tumor grade, size, and presence of carcinoma in situ for miR-222, recurrence (miR-222 and miR-143), progression (miR-222 and miR-143), disease-specific survival (miR-222), and overall survival (miR-222). Protein expression patterns of potential miRNA targets, including vascular endothelial growth factor, BCL2, v-erb-b2 erythroblastic leukemia viral oncogene (ERBB) homolog 3, and ERBB4, were evaluated by IHC in tissue arrays containing tumors for which miRNAs were assessed by RT-qPCR. Target expression correlated with expression of their predicted regulatory miRNAs, recurrence (ERBB3), progression (ERBB4), disease-specific survival (ERBB3 and ERBB4), and overall survival (ERBB3 and ERBB4). Furthermore, RT-qPCR of miR-452 (area under the curve, 0.848) and miR-222 (area under the curve, 0.718) in urine provided high accuracies for bladder cancer diagnosis. Thus, bladder tumors were characterized by changes in miRNA expression that could aid in tumor stratification and clinical outcome prognosis, and miRNAs were detected in urinary specimens for noninvasive diagnosis.
Asunto(s)
Biomarcadores de Tumor/metabolismo , MicroARNs/metabolismo , Neoplasias de la Vejiga Urinaria/diagnóstico , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/orina , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Masculino , MicroARNs/genética , MicroARNs/orina , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Pronóstico , Estudios Prospectivos , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Neoplásico/orina , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Análisis de Supervivencia , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Bladder cancer is a common cancer with particularly high recurrence after transurethral resection. In this study, we investigated the prognostic value of the protein expression of cathepsin E, maspin, polo-like kinase 1 (Plk1), and survivin in patients with stage Ta and T1 urothelial carcinomas. Transcripts from the four genes encoding these proteins were previously included in gene expression signatures for outcome prediction for Ta/T1 bladder cancer. We used three different tissue microarrays with 693 non-muscle invasive urothelial carcinomas from Danish, Swedish, and Spanish patient cohorts with long-term follow-up. Protein expression was measured by immunohistochemistry, and antibody specificity was validated by Western blotting. In the Danish patient cohort, we found the expression of cathepsin E, maspin, Plk1, and survivin to be significantly associated with progression to stage T2 to T4 bladder cancer (for each marker: log-rank test; P < 0.001). Multivariate Cox regression analysis identified cathepsin E (P < 0.001), Plk1 (P = 0.021), maspin (P = 0.001), and survivin (P = 0.001) as independent prognostic markers. Furthermore, maspin, survivin, and cathepsin E expression significantly subgrouped patients already stratified by European Organization for Research and Treatment of Cancer risk scores. Finally, we successfully validated the results in tumors from 410 patients from both Sweden and Spain. We conclude that all four protein markers may have prognostic value in non-muscle invasive bladder cancer for guiding optimal treatment of patients. Additional prospective studies are needed for further validation of the clinical relevance of this marker panel.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Vejiga Urinaria/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Catepsina E/metabolismo , Proteínas de Ciclo Celular/metabolismo , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/metabolismo , Pronóstico , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Serpinas/metabolismo , Survivin , Neoplasias de la Vejiga Urinaria/patología , Quinasa Tipo Polo 1RESUMEN
PURPOSE: Changes in DNA methylation of tumor suppressor genes early in carcinogenesis represent potential indicators of cancer detection and disease evolution. We examined the diagnostic, stratification and prognostic biomarker roles in urine of the methylation of a novel panel of tumor suppressor genes in bladder cancer. MATERIAL AND METHODS: We evaluated the methylation of 18 tumor suppressor genes in 2 prospective, independent sets of urine samples (training set of 120 preparations and validation set of 128) from patients with bladder cancer (170) and controls (78) using methylation specific multiplex ligation-dependent probe amplification. Diagnostic performance was evaluated with ROC curves. Recurrence, progression and disease specific survival were analyzed using univariate and multivariate Cox models. RESULTS: PRDM2, HLTF, ID4, DLC1, BNIP3, H2AFX, CACNA1G, TGIF and CACNA1A were methylated in bladder cancer. CCND2, SCGB3A1, BNIP3, ID4 and RUNX3 were the most frequently methylated tumor suppressor genes in each urine set. Methylation of several tumor suppressor genes correlated with clinicopathological variables, such as stage, tumor grade, focality or age. ROC analysis revealed significant diagnostic accuracy for RUNX3 and CACNA1A in the training set, and for RUNX3 and ID4 in the validation set. On univariate and multivariate analysis CACNA1A methylation correlated with recurrence in the training set, while in the validation set PRDM2 and BNIP3 were significantly associated with recurrence and disease specific survival, respectively. CONCLUSIONS: Tumor suppressor gene methylation allowed for histopathological and clinical stratification. Urine methylation has noninvasive usefulness not only for diagnostic assessment but also as independent bladder cancer prognosticators.
Asunto(s)
Genes Supresores de Tumor , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/orina , Metilación de ADN , Progresión de la Enfermedad , Humanos , Invasividad Neoplásica , Reacción en Cadena de la Polimerasa , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Curva ROC , Reproducibilidad de los Resultados , Tasa de Supervivencia , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
KISS1 is a metastasis suppressor lost in several solid malignancies. We evaluated the clinical relevance of KiSS-1 methylation and its protein expression in colorectal cancer. The epigenetic silencing of KiSS-1 by hypermethylation was tested in colon cancer cells (n = 5) before and after azacytidine treatment. KiSS-1 methylation was evaluated by methylation-specific PCR in colorectal cancer cells, and normal, benign, and tumor tissues (n = 352) were grouped in a training set (n = 62) and two independent validation cohorts (n = 100 and n = 190). KiSS-1 protein expression was analyzed by immunohistochemistry on tissue arrays. KiSS-1 hypermethylation correlated with transcript and protein expression loss, being increased in vitro by azacytidine. Methylation rates were 53.1, 70.0, and 80.0 % in the training and validation sets, respectively. In the training set, KiSS-1 methylation rendered a diagnostic accuracy of 72.7 % (p = 0.002). Combination of KiSS-1 methylation and serum CEA (p = 0.001) increased the prognostic utility of CEA alone (p = 0.022). In the first validation set, KiSS-1 methylation correlated with tumor grade (p = 0.011), predicted recurrence (p = 0.009), metastasis (p = 0.004), disease-free (p = 0.034), and overall survival (p = 0.015). In the second validation cohort, KiSS-1 methylation predicted disease-specific survival (p = 0.030). In the training set, cytoplasmic KiSS-1 expression was significantly higher in nonneoplastic biopsies as compared to colorectal tumors (p < 0.0005). In the validation set, loss of cytoplasmic expression correlated with tumor stage (p = 0.007), grade (p = 0.035), recurrence (p = 0.017), and disease-specific survival (p = 0.022). KiSS-1 was revealed epigenetically modified in colorectal cancer. The diagnostic and prognostic utility of KiSS-1 methylation and expression patterns suggests their assessment for the clinical management of colorectal cancer patients.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Metilación de ADN , Silenciador del Gen , Kisspeptinas/genética , Adulto , Anciano , Anciano de 80 o más Años , Azacitidina/farmacología , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Neoplasias Colorrectales/diagnóstico , Progresión de la Enfermedad , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Kisspeptinas/metabolismo , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , PronósticoRESUMEN
Prostate cancer screening based on prostate-specific antigen (PSA) testing has been a matter of controversy. Although screening for prostate cancer was effective in reducing mortality, it resulted in overdiagnosis, which translated into unnecessary treatments and numerous adverse effects. As a result, recommendations from scientific societies became increasingly restrictive. In the recent years, new approaches to prostate cancer screening have been proposed. These new approaches are aimed at solving the controversy between widespread screening vs. no screening, and reconsidering PSA testing as a screening tool with a good benefit/risk balance. In this context, the European Association of Urology submitted a proposal to the European Commission for prostate cancer screening to be performed as a function of baseline PSA concentrations. The European Commission recently recommended the implementation of organized prostate cancer screening programs for men aged ≤70 years based on PSA values in combination with follow-up magnetic resonance imaging.
RESUMEN
The TP63 gene, a member of the TP53 tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform-specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors.
Asunto(s)
Progresión de la Enfermedad , Fosfoproteínas/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Urotelio/embriología , Urotelio/metabolismo , Animales , Especificidad de Anticuerpos/inmunología , Línea Celular Tumoral , Humanos , Ratones , Modelos Biológicos , Proteínas Mutantes/metabolismo , Invasividad Neoplásica , Fenotipo , Fosfoproteínas/deficiencia , Isoformas de Proteínas/metabolismo , Reproducibilidad de los Resultados , Transactivadores/deficiencia , Factores de Transcripción , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/metabolismo , Urotelio/anomalías , Urotelio/patologíaRESUMEN
KISS1 is a metastasis suppressor gene that is lost in several malignancies, including bladder cancer. We tested the epigenetic silencing hypothesis and evaluated the biological influence of KISS1 methylation on its expression and clinical relevance in bladder cancer. KISS1 hypermethylation was frequent in bladder cancer cells analyzed by methylation-specific PCR and bisulfite sequencing and was associated with low gene expression, being restored in vitro by demethylating azacytidine. Hypermethylation was also frequently observed in a large series of bladder tumors (83.1%, n = 804). KISS1 methylation was associated with increasing stage (P = 0.001) and tumor grade (P = 0.010). KISS1 methylation was associated with low KISS1 transcript expression by quantitative RT-PCR (P = 0.037). KISS1 transcript expression was also associated with histopathological tumor stage (P < 0.0005). Low transcript expression alone (P = 0.003) or combined with methylation (P = 0.019) was associated with poor disease-specific survival (n = 205). KISS1 transcript expression remained an independent prognosticator in multivariate analyses (P = 0.017). KISS1 hypermethylation was identified in bladder cancer, providing a potential mechanistic explanation (epigenetic silencing) for the observed loss of KISS1 in uroepithelial malignancies. Associations of KISS1 methylation and its expression with histopathological variables and poor survival suggest the utility of incorporating KISS1 measurement using paraffin-embedded material for tumor stratification and clinical outcome prognosis of patients with uroepithelial neoplasias.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Kisspeptinas/genética , Neoplasias de la Vejiga Urinaria/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Islas de CpG , Metilación de ADN , Supervivencia sin Enfermedad , Epigénesis Genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Metástasis de la Neoplasia , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN , Resultado del Tratamiento , Neoplasias de la Vejiga Urinaria/terapiaRESUMEN
A compelling body of evidences sustains the importance of epigenetic mechanisms in the development and progression of cancer. Assessing the epigenetic component of bladder tumors is strongly improving our understanding of their biology and clinical behavior. In terms of DNA methylation, cancer cells show genome-wide hypomethylation and site-specific CpG island promoter hypermethylation. In the context of other epigenetic alterations, this review will focus on the hypermethylation of CpG islands in promoter regions, as the most widely described epigenetic modification in bladder cancer. CpG islands hypermethylation is believed to be critical in the transcriptional silencing and regulation of tumor suppressor and crucial cancer genes involved in the major molecular pathways controlling bladder cancer development and progression. In particular, several biological pathways of frequently methylated genes include cell cycle, DNA repair, apoptosis, and invasion, among others. Furthermore, translational aspects of bladder cancer methylomes described to date will be discussed towards their potential application as bladder cancer biomarkers. Several tissue methylation signatures and individual candidates have been evidenced, that could potentially stratify tumors histopathologically, and discriminate patients in terms of their clinical outcome. Tumor methylation profiles could also be detected in urinary specimens showing a promising role as non-invasive markers for cancer diagnosis towards an early detection and potentially for the surveillance of bladder cancer patients in a near future. However, the epigenomic exploration of bladder cancer has only just begun. Genome-scale DNA methylation profiling studies will further highlight the relevance of the epigenetic component to gain knowledge of bladder cancer biology and identify those profiles and candidates better correlating with clinical behavior.
Asunto(s)
Metilación de ADN , Neoplasias/genética , Neoplasias de la Vejiga Urinaria/genética , Apoptosis , Biomarcadores de Tumor , Línea Celular Tumoral , Islas de CpG , Reparación del ADN , Progresión de la Enfermedad , Epigénesis Genética , Silenciador del Gen , Humanos , Modelos Genéticos , Invasividad Neoplásica , Neoplasias/diagnóstico , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
Myopodin is an actin-binding protein believed to play a tumor suppressor role in several solid neoplasias. We evaluated the potential differential myopodin methylation and expression and their clinical relevance in colon cancer. The epigenetic silencing of myopodin by hypermethylation was tested in colon cancer cells (n = 5) before and after azacitidine treatment. Myopodin methylation status was evaluated by methylation-specific PCR in colon cancer cells and colorectal tissues (n = 210) grouped in a training set (n = 62) and two independent validation series (n = 100 and n = 48) collected at independent clinical settings. Myopodin expression patterns were analyzed by immunohistochemistry on tissue arrays. Myopodin hypermethylation correlated with gene and protein expression loss, being increased in vitro by azacitidine. Myopodin was frequently methylated in colon cancer cells (four out of five). Methylation rates were 90.3%, 70.0%, and 47.8% in the training and validation sets, respectively. Myopodin methylation rendered a diagnostic accuracy of 83.9% (p < 0.0005). Cytoplasmic myopodin expression was significantly higher in non-neoplastic biopsies compared to colon tumors (p < 0.0005). Loss of myopodin expression correlated with increasing tumor stage (p = 0.011), methylation (p = 0.005), and poor overall survival (p = 0.003). In the first validation set (n = 100), myopodin methylation predicted disease-free (p = 0.046) and overall survival (p = 0.031). In the second validation cohort, myopodin methylation and protein expression patterns predicted disease-specific (p = 0.012 and p = 0.001, respectively) and overall survival (p = 0.009 and p = 0.043, respectively). Thus, myopodin was revealed to be epigenetically modified in colon cancer. The diagnostic and prognostic clinical utility of myopodin methylation and expression patterns suggest considering their assessment for the clinical management of colon cancer patients.
Asunto(s)
Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/genética , Adulto , Anciano , Anciano de 80 o más Años , Azacitidina/farmacología , Secuencia de Bases , Biomarcadores de Tumor , Neoplasias del Colon/diagnóstico , Islas de CpG , Metilación de ADN , Supervivencia sin Enfermedad , Epigénesis Genética , Femenino , Humanos , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodosRESUMEN
KiSS-1 is a metastasis suppressor gene reported to be involved in the progression of several solid neoplasias. The loss of KiSS-1 gene expression has been shown to be inversely correlated with increasing tumor stage, distant metastases, and poor overall survival in bladder tumors. To identify the molecular pathways associated with the metastasis suppressor role of KiSS-1 in bladder cancer, we carried out a proteomics analysis of bladder cancer cells (EJ138) transiently transfected with a vector encompassing the full-length KiSS-1 gene using an iTRAQ (isobaric tags for relative and absolute quantitation) approach. Protein extracts collected after 24- and 48-h transfection were fractionated and cleaved with trypsin, and the resulting peptides were labeled with iTRAQ reagents. The labeled peptides were separated by strong cation exchange and reversed phase LC and analyzed by MALDI-TOF/TOF MS. Three software packages were utilized for data analysis: ProteinPilot for identification and quantification of differentially expressed proteins, Protein Center for gene ontology analysis, and Ingenuity Pathways Analysis to provide insight into biological networks. Comparative analysis among transfected, mock, and empty vector-exposed cells identified 1529 proteins with high confidence (>99%) showing high correlation rates among replicates (70%). The involvement of the identified proteins in biological networks served to characterize molecular pathways associated with KiSS-1 expression and to select critical candidates for verification analyses by Western blot using independent transfected replicates. As part of complementary clinical validation strategies, immunohistochemical analyses of proteins regulated by KiSS-1, such as Filamin A, were performed on bladder tumors spotted onto tissue microarrays (n = 280). In summary, our study not only served to uncover molecular mechanisms associated with the metastasis suppressor role of KiSS-1 in bladder cancer but also to reveal the biomarker role of Filamin A in bladder cancer progression and clinical outcome.
Asunto(s)
Cromatografía Liquida/métodos , Proteínas de Neoplasias/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Transfección , Proteínas Supresoras de Tumor/genética , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Humanos , Inmunohistoquímica , Kisspeptinas , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The evaluation of biomarkers in bodily fluids necessitates the development of robust methods to quantify proteins in a complex background, using large sets of samples. The ability to multiplex numerous analytes in a single assay expedites the process. Liquid chromatography-mass spectrometry (LC-MS) analyses performed in selected reaction monitoring (SRM) in conjunction with stable isotope dilution MS present an effective way to detect and quantify biomarker candidates in bodily fluids. The strategy presented involves an initial qualification of predefined sets of proteins in urine. The technique was applied to detect and quantify peptides in urine samples as surrogates for a few endogenous proteins. Multiplexed assays were developed to analyze proteins associated with bladder cancer; a few exogenous proteins were added as internal standards. The sample preparation and the analytical protocols were optimized to ensure reproducibility, analytical precision, and quantification limits in the low nanogram per milliliter range. Analyses were performed using known amounts of isotopically labeled peptides. Systematic replication of the measurements indicated intra-assay and inter-assay variability, with CVs in the range of 10%. The differences measured for two targeted proteins were correlated with their level of expression in the corresponding tumors using immunohistochemistry.
Asunto(s)
Péptidos/orina , Proteómica/normas , Secuencia de Aminoácidos , Análisis de Varianza , Biomarcadores de Tumor/orina , Estudios de Casos y Controles , Cromatografía Liquida , Clusterina/orina , Interpretación Estadística de Datos , Humanos , Receptores de Hialuranos/orina , Inmunohistoquímica , Datos de Secuencia Molecular , Péptidos/química , Péptidos/normas , Proteinuria/orina , Proteómica/métodos , Proteómica/estadística & datos numéricos , Control de Calidad , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/normas , Espectrometría de Masas en Tándem/estadística & datos numéricos , Neoplasias de la Vejiga Urinaria/orina , Orina/químicaRESUMEN
There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers.