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Validación Y Estudio De La Implicación De Los Genes Adrbk2, Adrb1, Adra2b, Axin 2, Atp6v1c1 Y Atp6v0e En El Carcinoma Oral De

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Validación Y Estudio De La Implicación De Los Genes Adrbk2, Adrb1, Adra2b, Axin 2, Atp6v1c1 Y Atp6v0e En El Carcinoma Oral De FACULTADE DE MEDICINA E ODONTOLOXÍA Departamento de Estomatoloxía VALIDACIÓN Y ESTUDIO DE LA IMPLICACIÓN DE LOS GENES ADRBK2, ADRB1, ADRA2B, AXIN 2, ATP6V1C1 Y ATP6V0E EN EL CARCINOMA ORAL DE CÉLULAS ESCAMOSAS MEDIANTE PCR CUANTITATIVA EN TIEMPO REAL TESIS DOCTORAL EVA MARÍA OTERO REY Santiago de Compostela, enero de 2006 FACULTAD DE MEDICINA Y ODONTOLOGÍA DEPARTAMENTO DE ESTOMATOLOGÍA VALIDACIÓN Y ESTUDIO DE LA IMPLICACIÓN DE LOS GENES ADRBK2, ADRB1, ADRA2B, AXIN 2, ATP6V1C1 Y ATP6V0E EN EL CARCINOMA ORAL DE CÉLULAS ESCAMOSAS MEDIANTE PCR CUANTITATIVA EN TIEMPO REAL Autora: EVA MARÍA OTERO REY Santiago de Compostela, 20 de Enero de 2006. D. ABEL GARCÍA GARCÍA, Profesor Titular de Cirugía Oral y Maxilofacial de la Facultad de Medicina y Odontología de la Universidad de Santiago de Compostela; D. FRANCISCO BARROS ANGUEIRA, Doctor en Biología y Jefe de Laboratorio de la Fundación Pública Galega de Medicina Xenómica del Sergas; y D. JOSÉ MANUEL SOMOZA MARTÍN, Doctor en Odontología de la Universidad de Santiago de Compostela, CERTIFICAN: Que la presente Tesis Doctoral titulada “VALIDACIÓN Y ESTUDIO DE LA IMPLICACIÓN DE LOS GENES ADRBK2, ADRB1, ADRA2B, AXIN 2, ATP6V1C1 Y ATP6V0E EN EL CARCINOMA ORAL DE CÉLULAS ESCAMOSAS MEDIANTE PCR CUANTITATIVA EN TIEMPO REAL”, ha sido elaborada por Dña. EVA MARÍA OTERO REY bajo nuestra dirección, y hallándose concluida, autorizamos su presentación a fin de que pueda ser defendida ante el Tribunal correspondiente. Y para que así conste, se expide la presente certificación en Santiago de Compostela, a 20 de Enero de 2006. Prof. Dr. D. Abel García García Dr. D. Francisco Barros Angueira Dr. D. José Manuel Somoza Martín El presente trabajo de investigación ha sido realizado en el contexto del proyecto de investigación: EXPRESIÓN GENÉTICA DIFERENCIAL EN CÉLULAS EPITELIALES DE LESIONES PREMALIGNAS Y CARCINOMAS EPIDERMOIDES DE LA CAVIDAD ORAL Financiación: Xunta de Galicia (PGIDT01PXI20804PR) Director del proyecto e investigador principal: Prof Abel García García (Universidad de Santiago de Compostela) AGRADECIMIENTOS: Al Profesor Abel García García, por ser el promotor de esta investigación y por la amistad, confianza y cariño que me ha demostrado a lo largo de estos años. Al Dr. Francisco Barros Angueira, por su inestimable ayuda y aportación a esta Tesis Doctoral y por su dedicación a la investigación. Al Dr. José Manuel Somoza Martín, por su apoyo en este y otros trabajos realizados, así como por su paciencia y ayuda constante. Al Profesor José Manuel Gándara Rey, por su gran aportación docente a mi formación y por su continua dedicación a la enseñanza. Al Profesor Andrés Blanco Carrión, una de las mejores personas que conozco y con la que tengo la suerte de trabajar día a día. A Manuel Peñamaría Mallón, compañero y amigo, que ha estado siempre dispuesto a prestar su ayuda de forma desinteresada. A todos mis compañeros del Máster de Medicina Oral, Cirugía Oral e Implantología y del Máster de Odontología Práctica Diaria de la Universidad de Santiago de Compostela. A Sonia, por saber escuchar y aconsejarme en cada momento. A Oky, por tener la suerte de tener a mi lado una persona que me entiende y me apoya día a día. A mis padres, por guiar mis pasos con su ejemplo ÍNDICE ÍNDICE JUSTIFICACIÓN 15 I. INTRODUCCIÓN 21 1.- Carcinoma oral de células escamosas 23 1.1. Concepto 23 1.2. Epidemiología 24 1.3. Etiopatogenia 25 1.4. Clínica 27 1.5. Diagnóstico 28 1.5.1. Diagnóstico histológico y ultraestructural 28 1.5.2. Inmunohistoquímica 31 1.5.3. Técnicas de biología molecular 32 1.6. Estadiaje 33 1.7. Pronóstico 35 1.8. Tratamiento 37 99 ÍNDICE 2.- Bases moleculares del cáncer 41 2.1. La maquinaria de proliferación celular 41 2.1.1. Ciclo celular 42 2.1.2. Regulación del ciclo celular 44 2.2. Oncogenes, genes supresores tumorales 46 2.3. Adaptaciones celulares, lesión y muerte celular 51 2.4. La maquinaria de muerte celular 59 2.4.1. Apoptosis 60 2.4.2. Genes reguladores de la apoptosis 61 2.5. Diferencias entre las células normales y las células cancerosas 62 2.6. Carcinogénesis 64 2.7. Angiogénesis 66 2.8. Técnicas de biología molecular 68 3.- Bases moleculares del carcinoma oral de células escamosas 75 3.1. Carcinoma oral de células escamosas y microarrays de ADN 76 3.2. Genes sobreexpresados en el carcinoma oral de células escamosas según un estudio con el array Atlas Glass Human 3.8 I Microarray 78 10 10 ÍNDICE 4.- Validación de los resultados obtenidos con microarrays de ADN 107 4.1. PCR cuantitativa en tiempo real 108 II. HIPÓTESIS DE TRABAJO Y OBJETIVOS 113 III. MATERIAL Y MÉTODO 117 1.- Selección de pacientes 119 1.1. Edad y sexo 119 1.2. Consumo de tabaco 119 1.3. Localización del tumor 120 1.4. Estadio del tumor 120 2.- Selección de los genes a estudio 121 3.- Metodología de trabajo 122 3.1. Obtención de las muestras 122 3.2. Procesado de las muestras 123 3.3. Cuantificación por PCR en tiempo real 124 4.- Normalización de datos 127 5.- Análisis del nivel de expresión 129 6.- Estudio estadístico 129 11 ÍNDICE IV. RESULTADOS 131 1.- Estudio de los genes 133 1.1.- ADRBK2 (adrenergic, beta, receptor kinase 2) 133 1.2.-ADRB1 (adrenergic, beta-1-, receptor) 135 1.3.-ADRA2B (adrenergic, alpha-2B-, receptor) 137 1.4.- AXIN 2 139 1.5.- ATP6V1C1(ATPase, H+ transporting, lysosomal 42KDa, V1 subunit C, isoform 1) 141 1.6.-ATP6V0E (ATPase, H+ transporting, lysosomal 9KDa, V0 subunit e) 143 2.- Normalización de datos 145 2.1.- ADRBK2 (adrenergic, beta, receptor kinase 2) 145 2.2.-ADRB1 (adrenergic, beta-1-, receptor) 146 2.3.-ADRA2B (adrenergic, alpha-2B-, receptor) 147 2.4.-AXIN 2 148 2.5.-ATP6V1C1(ATPase, H+ transporting, lysosomal 42KDa, V1 subunit C, isoform 1) 149 2.6.-ATP6V0E (ATPase, H+ transporting, lysosomal 9KDa, V0 subunit e) 150 12 12 ÍNDICE 3.- Análisis del nivel de expresión 151 V. DISCUSIÓN 155 VI. CONCLUSIONES 177 VII. BIBLIOGRAFÍA 181 VIII. ANEXOS 209 13 JUSTIFICACIÓN 15 16 JUSTIFICACIÓN Cada año más de 300.000 nuevos casos de carcinoma oral de células escamosas (COCE) son diagnosticados en todo el mundo (1). De éstos, aproximadamente 50.000 casos ocurren en Estados Unidos y Europa (2). En España, la incidencia del cáncer oral es de 12 a 15 casos/100.000 habitantes/año en varones y de 2 a 4 casos/100.000 habitantes/año en mujeres, y representa entre el 2 y el 3% de todas las muertes por cáncer en nuestro país (3). Estudios epidemiológicos recientes (4) han demostrado un aumento de la mortalidad anual por cáncer oral, de un 25% para los hombres y de un 9% para las mujeres, durante el período de 1975-1994, lo que hace que el cáncer oral deba ser considerado un problema de salud pública en España (5). Desafortunadamente, los rasgos clínico-patológicos de la enfermedad son la única medida disponible para predecir su pronóstico, guiar el tratamiento y aconsejar a los pacientes. El tratamiento convencional, ya sea con cirugía, radioterapia y/o quimioterapia, está asociado con una elevada morbilidad afectando el habla, la deglución y, en general, la calidad de vida del paciente. Pese a los avances en estas intervenciones, la recurrencia de la enfermedad se observa en más del 50% de los pacientes con altas tasas de mortalidad asociada (6). Esto viene acompañado del hecho de que mientras nuestra habilidad para controlar localmente la enfermedad ha progresado, la tasa total de supervivencia a los 5 años no ha mejorado significativamente en las pasadas tres décadas. El COCE, como enfermedad neoplásica de etiología multifactorial, es una de las enfermedades más heterogéneas. Aunque existen diversos factores de 17 JUSTIFICACIÓN riesgo, los más importantes son el tabaco y el alcohol, potenciando su acción cuando se consumen conjuntamente. Sin embargo, pese a los esfuerzos para identificar biomarcadores, ningún marcador específico puede ser usado con confianza en la detección precoz o como indicador de prognosis. El estadiaje de la enfermedad se basa en la actualidad únicamente en los hallazgos clínicos de localización, con atención a complicaciones locales, regionales y a distancia. Aunque el estadiaje tiene un fuerte valor predictivo con respecto a la probabilidad de supervivencia, los problemas asociados a su aplicación ponen en evidencia la necesidad de un refinamiento del sistema y de la identificación de factores pronósticos adicionales. Son las limitaciones de las características clínicas en pacientes con cáncer oral las que han llevado a la búsqueda de biomarcadores de pronóstico por parte de diversos grupos de investigación. Inicialmente los esfuerzos se centraron en marcadores individuales, como p53, queratinas o p16, pero su utilidad es limitada dada su penetrancia incompleta y expresión variable (7). Es por ello que los esfuerzos recientes se han centrado en el análisis de los cambios de expresión génica global con la esperanza de identificar cambios asociados con la carcinogénesis en estos tumores, y de identificar firmas genéticas específicas que permitan predecir las consecuencias de la enfermedad (8). El proyecto Genoma Humano ha sido el catalizador para el desarrollo de tecnologías de alta productividad que han permitido mapear y secuenciar genomas complejos. En los últimos años tales tecnologías se han extendido hasta permitir la medida de la expresión relativa de miles de genes simultáneamente en un único experimento. La plataforma con mayor responsabilidad en esta nueva frontera de la investigación genómica es la de microarrays de ADN, también conocida como chip de ADN o chip génico. Actualmente existe una gran variedad de tipos de microarrays de ADN en el mercado, pero todos se basan en el principio de tomar una librería representativa de fragmentos de ADN de un 18 18 JUSTIFICACIÓN genoma complejo e imprimir dichos productos a intervalos regularmente espaciados sobre una superficie sólida.
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