RESUMEN
CONTEXT: Urothelial carcinoma (UC) is common and highly recurrent. Diagnosis and follow-up involve invasive cystoscopies. OBJECTIVE: To evaluate H19 RNA in urine cells as diagnostic tool for UC. MATERIALS AND METHODS: RT-PCR analysis of urine samples from healthy volunteers and UC patients. RESULTS: H19 RNA was unequivocally detected in the urine of 90.5% of patients and 25.9% of controls. H19 copies were three orders of magnitude higher in patients. Receiver operating characteristic analysis showed an area under the curve of 0.933. CONCLUSIONS: This pilot study shows that urinary cell H19 is a highly sensitive test for UC and pending verification could transform patient management.
Asunto(s)
ARN Largo no Codificante/orina , Neoplasias Urológicas/diagnóstico , Urotelio/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/orina , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proyectos Piloto , Reacción en Cadena de la Polimerasa , Curva ROC , Neoplasias Urológicas/orinaRESUMEN
The oncofetal H19 gene transcribes a long non-coding RNA(lncRNA) that is essential for tumor growth. Here we found that numerous established inducers of epithelial to mesenchymal transition(EMT) also induced H19/miR-675 expression. Both TGF-ß and hypoxia concomitantly induced H19 and miR-675 with the induction of EMT markers. We identified the PI3K/AKT pathway mediating the inductions of Slug, H19 RNA and miR-675 in response to TGF-ß treatment, while Slug induction depended on H19 RNA. In the EMT induced multidrug resistance model, H19 level was also induced. In a mouse breast cancer model, H19 expression was tightly correlated with metastatic potential. In patients, we detected high H19 expression in all common metastatic sites tested, regardless of tumor primary origin. H19 RNA suppressed the expression of E-cadherin protein. H19 up-regulated Slug expression concomitant with the suppression of E-cadherin protein through a mechanism that involved miR-675. Slug also up-regulated H19 expression and activated its promoter. Altogether, these results may support the existence of a positive feedback loop between Slug and H19/miR-675, that regulates E-cadherin expression. H19 RNA enhanced the invasive potential of cancer cells in vitro and enhanced tumor metastasis in vivo. Additionally, H19 knockdown attenuated the scattering and tumorigenic effects of HGF/SF. Our results present novel mechanistic insights into a critical role for H19 RNA in tumor progression and indicate a previously unknown link between H19/miR-675, Slug and E-cadherin in the regulation of cancer cell EMT programs.
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Neoplasias de la Mama/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , ARN Largo no Codificante/genética , Factores de Transcripción/genética , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/genética , Cadherinas/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos/genética , Retroalimentación Fisiológica , Femenino , Humanos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , MicroARNs/metabolismo , Metástasis de la Neoplasia , Oxígeno/metabolismo , Oxígeno/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Largo no Codificante/metabolismo , Transducción de Señal , Factores de Transcripción de la Familia Snail , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Expression of the imprinted H19 gene is remarkably elevated in a large number of human cancers. Recently, we reported that H19 RNA is up-regulated in hypoxic stress and furthermore, it possesses oncogenic properties. However, the underlying mechanism(s) of these phenomena remain(s) unknown. Here we demonstrate a tight correlation between H19 RNA elevation by hypoxia and the status of the p53 tumor suppressor. Wild type p53 (p53(wt)) prevents the induction of H19 upon hypoxia, and upon its reconstitution in p53(null) cells. The last case is accompanied by a decrease in cell viability. The p53 effect is nuclear and seems independent of its tetramerization. Furthermore, using knockdown and over-expression approaches we identified HIF1-alpha as a critical factor that is responsible for H19 induction upon hypoxia. Knocking down HIF1-alpha abolishes H19 RNA induction, while its over-expression significantly enhances the H19 elevation in p53(null) hypoxic cells. In p53(wt) hypoxic cells simultaneous suppression of p53 and over-expression of HIF1-alpha are needed to induce H19 significantly, while each treatment separately resulting in a mild induction, indicating that the molecular mechanism of p53 suppression effect on H19 may at least in part involve interfering with HIF1-alpha activity. In vivo a significant increase in H19 expression occurred in tumors derived from p53(null) cells but not in p53(wt) cells. Taken together, our results indicate that a functional link exists between p53, HIF1-alpha and H19 that determines H19 elevation in hypoxic cancer cells. We suggest that this linkage plays a role in tumor development.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Hipoxia/metabolismo , Neoplasias/metabolismo , ARN no Traducido/genética , Proteína p53 Supresora de Tumor/fisiología , Animales , Western Blotting , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Desnudos , Neoplasias/genética , Neoplasias/patología , ARN Largo no Codificante , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , ARN no Traducido/antagonistas & inhibidores , ARN no Traducido/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Ovarian cancer ascites fluid (OCAF), contains malignant cells, is usually present in women with an advanced stage disease and currently has no effective therapy. Hence, we developed a new therapy strategy to target the expression of diphtheria toxin gene under the control of H19 regulatory sequences in ovarian tumor cells. H19 RNA is present at high levels in human cancer tissues (including ovarian cancer), while existing at a nearly undetectable level in the surrounding normal tissue. METHODS: H19 gene expression was tested in cells from OCAF by the in-situ hybridization technique (ISH) using an H19 RNA probe. The therapeutic potential of the toxin vector DTA-H19 was tested in ovarian carcinoma cell lines and in a heterotopic animal model for ovarian cancer. RESULTS: H19 RNA was detected in 90% of patients with OCAF as determined by ISH. Intratumoral injection of DTA-H19 into ectopically developed tumors caused 40% inhibition of tumor growth. CONCLUSION: These observations may be the first step towards a major breakthrough in the treatment of human OCAF, while the effect in solid tumors required further investigation. It should enable us to identify likely non-responders in advance, and to treat patients who are resistant to all known therapies, thereby avoiding treatment failure.
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Marcación de Gen , Terapia Genética/métodos , Neoplasias Ováricas/terapia , Plásmidos , ARN no Traducido/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Ascitis/patología , Ascitis/terapia , Línea Celular Tumoral , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Ratones , Ratones Desnudos , Neoplasias Ováricas/patología , Plásmidos/genética , Plásmidos/metabolismo , ARN Largo no Codificante , ARN no Traducido/metabolismoRESUMEN
BACKGROUND: The H19-IGF2 locus is either highly expressed and/or shows aberrant allelic pattern of expression in a large array of human cancers, while rarely expressed in the corresponding normal tissue. Preclinical, clinical studies and human compassionate using a DNA plasmid containing H19 and/or IGF2-P4 regulatory sequences that drive the expression of an intracellular toxin [diphtheria toxin A-fragment (DTA)] have demonstrated promising results in several types of carcinomas. Recently we reported that a single construct that expresses DTA under the control of both H19 and IGF2 P4 promoters showed superior efficacy in vitro as well as in vivo, in comparison to a single promoter construct in bladder carcinoma. Here we extended this approach to glioblastoma and tested the antitumor efficacy of the double promoter DTA-expressing vector (H19-DTA-P4-DTA) in vitro as well as in heterotopic animal model. H19 gene expression was tested by in-situ hybridization (ISH) and by quantitative Real-Time PCR (qRT-PCR) in samples of diffuse glioma. METHODS: IGF2-P4 gene expression was tested by qRT-PCR as well. RESULTS: Both H19 and IGF2-P4 transcripts were highly expressed in high grade gliomas. Furthermore, significant H19 expression in other types of primary brain tumors as well as in brain metastases was detected by ISH. Both A172 and U87 human glioblastoma cell lines showed high expression of IGF2-P4 while the A172 cell line showed high expression of H19 RNA as well. H19-DTA-P4-DTA exhibited superior cytotoxic activity compared to the single promoter expression vectors, in U87 and A172 glioblastoma cell lines in vitro and showed antitumoral efficacy in heterotopic glioblastoma animal model. CONCLUSIONS: Our findings indicate antitumoral efficacy against glioblastoma of the targeted double promoter vector H19-DTA-P4-DTA, both in-vitro and in-vivo. Thus, its test in orthotopic animal model of glioblastoma as well as in clinical trials is warranted.
RESUMEN
INTRODUCTION: Ovarian cancer ascitic fluid, which contains malignant cells, is usually present in women with an advanced stage disease. There are currently no effective therapies for the treatment of ovarian cancer ascitic fluid. We developed a new therapeutic strategy to target expression of the diphtheria toxin fragment A gene in ovarian tumor cells under the control of H19 regulatory sequences. CASE PRESENTATION: A 64-year-old Caucasian woman was diagnosed with a stage IIIc epithelial ovarian cancer. She suffered from progressive disease, accumulation of malignant ascites that needed to be drained weekly, abdominal pain, vomiting, anorexia and severe weakness. Infusion of the diphtheria toxin A chain-H19 plasmid into the peritoneum of our patient resulted in complete resolution of the ascites with minimum adverse events. CONCLUSION: On the basis of this preliminary experience, we are currently conducting an extensive Phase I study on a larger number of patients in order to assess the safety and preliminary efficacy of this novel patient-oriented treatment approach.
RESUMEN
BACKGROUND: Mutations and epigenetic aberrant signaling of growth factors pathways contribute to carcinogenesis. Recent studies reveal that non-coding RNAs are controllers of gene expression. H19 is an imprinted gene that demonstrates maternal monoallelic expression without a protein product; although its expression is shut off in most tissues postnatally, it is re-activated during adult tissue regeneration and tumorigenesis. Moreover, H19 is highly expressed in liver metastasis derived from a range of carcinomas. The objective of this study is to explore the role of H19 in carcinogenesis, and to determine its identification as an anti-tumor target. METHODOLOGY/PRINCIPLE FINDINGS: By controlling oxygen pressure during tumor cell growth and H19 expression levels, we investigated the role of H19 expression in vitro and in vivo in hepatocellular (HCC) and bladder carcinoma. Hypoxia upregulates the level of H19 RNA. Ablations of tumorigenicity of HCC and bladder carcinomas in vivo are seen by H19 knockdown which also significantly abrogates anchorage-independent growth after hypoxia recovery, while ectopic H19 expression enhances tumorigenic potential of carcinoma cells in vivo. Knocking-down H19 message in hypoxic stress severely diminishes p57(kip2) induction. We identified a number of potential downstream targets of H19 RNA, including angiogenin and FGF18. CONCLUSIONS: H19 RNA harbors pro-tumorigenic properties, thus the H19 gene behaves as an oncogene and may serve as a potential new target for anti-tumor therapy.
Asunto(s)
División Celular/genética , ARN no Traducido/fisiología , Neoplasias de la Vejiga Urinaria/patología , Animales , Secuencia de Bases , Hipoxia de la Célula , Línea Celular Tumoral , Cartilla de ADN , Técnicas de Silenciamiento del Gen , Humanos , Hibridación in Situ , Ratones , Ratones Desnudos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Largo no Codificante , ARN Interferente Pequeño , ARN no Traducido/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Vejiga Urinaria/genéticaRESUMEN
Real-time PCR technology is highly advantageous for gene studies based on the genetic nature of the transferred material. Urine and blood samples were collected before and after treatment. Treatment of bladder carcinoma patients with plasmid constructs expressing the diphtheria toxin gene was monitored. Detection range from 5x10(6) copies to Asunto(s)
ADN/análisis
, Terapia Genética
, Plásmidos/uso terapéutico
, Reacción en Cadena de la Polimerasa/métodos
, Neoplasias de la Vejiga Urinaria/terapia
, ADN/sangre
, ADN/orina
, Dosificación de Gen
, Humanos