{"id":113522,"date":"2012-05-12T00:00:00","date_gmt":"2012-05-12T00:00:00","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/new-oncogenic-drivers-in-hepatocellular-carcinoma-role-of-the-rna-binding-protein-hu-antigen-r\/"},"modified":"2012-05-12T00:00:00","modified_gmt":"2012-05-12T00:00:00","slug":"new-oncogenic-drivers-in-hepatocellular-carcinoma-role-of-the-rna-binding-protein-hu-antigen-r","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/bioquimica-molecular\/new-oncogenic-drivers-in-hepatocellular-carcinoma-role-of-the-rna-binding-protein-hu-antigen-r\/","title":{"rendered":"New oncogenic drivers in hepatocellular carcinoma: role of the rna binding protein hu antigen r"},"content":{"rendered":"<h2>Tesis doctoral de <strong> David Fernandez Ramos <\/strong><\/h2>\n<p>Resumenla enfermedad hep\u00e1tica cr\u00f3nica es una de las principales causas de mortalidad en humanos.Comprende enfermedades con diferentes etiolog\u00edas como la infecci\u00f3n por los virus de la hepatitis b y c,toxinas, consumo de alcohol y drogas, enfermedades autoinmunes y hereditarias y la enfermedad del h\u00edgadograso no alcoh\u00f3lica (ehgna). La ehgna es una de las principales enfermedades hep\u00e1ticas cr\u00f3nicas en lospa\u00edses desarrollados, asociada con los factores de riesgo del s\u00edndrome metab\u00f3lico (obesidad, resistencia a lainsulina, dislipidemia e hipertensi\u00f3n), e incluyendo alteraciones que van desde la esteatosis hasta laesteatohepatitis no alcoh\u00f3lica (ehna), que en algunos casos puede ir acompa\u00f1ada de fibrosis. Los pacientesde ehna con presencia de fibrosis pueden llegar a desarrollar carcinoma hepatocelular (chc).El chc representa la tercera causa de muerte por c\u00e1ncer, y la primera causa de muerte en pacientescirr\u00f3ticos. El chc es el c\u00e1ncer m\u00e1s prevalente en la poblaci\u00f3n, con mal pron\u00f3stico incluso en los pa\u00edsesdesarrollados. Su etiolog\u00eda es diversa, con los virus de la hepatitis b y c, el alcoholismo, la aflatoxina b1 y elehgna como principales factores de riesgo. Entre los factores moleculares involucrados en la progresi\u00f3ndesde ehgna a chc podemos encontrar el estr\u00e9s oxidativo, el metabolismo de la metionina y la disfunci\u00f3nde p53. Numerosos estudios han mostrado que los pacientes cirr\u00f3ticos con alto riesgo de desarrollar chcpresentan una desregulaci\u00f3n del metabolismo de la metionina y unos niveles anormales de sadenosilmetionina(same).La s-adenosylmetionina es el principal donador biol\u00f3gico de grupos metilo, y el h\u00edgado aparece como elprincipal responsable de su homeostasis. Same juega un papel fundamental en la proliferaci\u00f3n de loshepatocitos y su diferenciaci\u00f3n, y en la apoptosis de c\u00e9lulas tumorales. En el h\u00edgado same es capaz deinhibir la activaci\u00f3n de la ruta lkb1\/ampk\/enos y evitar la translocaci\u00f3n al citoplasma de hur, una prote\u00ednade uni\u00f3n al arn, siendo ambos mecanismos importantes para la proliferaci\u00f3n de los hepatocitos y laregeneraci\u00f3n hep\u00e1tica. Por tanto, los niveles de same deben estar estrechamente regulados. Las dosprincipales enzimas responsables de la s\u00edntesis y el catabolismo de same son la metioninaadenosiltransferasa (mat) y la glicina n-metiltransferasa (gnmt), respectivamente. La enzima mat est\u00e1codificada por dos genes, mat1a y mat2a. Mat1a codifica para la formaci\u00f3n de las enzimas mat i y matiii, y mat2a codifica para mat ii. Mat1a se expresa en el h\u00edgado adulto y diferenciado, mientras quemat2a se expresa en el h\u00edgado fetal y en proliferaci\u00f3n. Durante el desarrollo del h\u00edgado hay un cambio en laexpresi\u00f3n desde mat2a hasta mat1a, y durante la proliferaci\u00f3n y la desdiferenciaci\u00f3n hep\u00e1ticas y durantela transformaci\u00f3n maligna, los niveles de expresi\u00f3n de mat1a se reducen junto con un aumento en laexpresi\u00f3n de mat2a. La regulaci\u00f3n de la expresi\u00f3n de mat1a y mat2a se ha relacionado con la metilaci\u00f3nde los promotores y la acetilaci\u00f3n de las histonas asociadas a ellos.Por otra parte, la enzima gnmt, responsable de la catabolizaci\u00f3n de same, est\u00e1 presente en grandescantidades en el h\u00edgado, y muy reducida en tumores hep\u00e1ticos y prost\u00e1ticos. Se han encontrado individuoscon mutaciones en gnmt que espont\u00e1neamente desarrollan enfermedad hep\u00e1tica. La enzima gnmt regulael ratio same a sah, el cual es considerado como el \u00edndice del potencial de transmetilaci\u00f3n de la c\u00e9lula. Ladesregulaci\u00f3n de este ratio puede resultar en metilaciones aberrantes.Con el fin de estudiar las implicaciones de la desregulaci\u00f3n del nivel de same en el h\u00edgado, segeneraron dos modelos de ratones knockout, mat1a-ko y gnmt-ko, caracterizados por niveles de samecr\u00f3nicamente disminuidos y elevados, respectivamente. El rat\u00f3n mat1a-ko presenta estr\u00e9s oxidativo ydesarrolla esteatosis y ehna a los 8 meses, y finalmente chc. El rat\u00f3n gnmt-ko desarrolla esteatosis,fibrosis y chc. La observaci\u00f3n de que tanto los altos como los bajos niveles de same provocan unapatolog\u00eda similar subrayan la importancia del mantenimiento de la homeostasis de same.La regulaci\u00f3n de los niveles de same est\u00e1 relacionada con la regulaci\u00f3n de la expresi\u00f3n de los arnmde mat1a y mat2a. La explicaci\u00f3n de dicha regulaci\u00f3n por la metilaci\u00f3n de los promotores y la acetilaci\u00f3n32 resumen\/summaryde las histonas no explica por completo los cambios entre mat1a y mat2a. Adem\u00e1s, la conversi\u00f3n de lametionina en same es capaz de regular la tasa de recambio del arnm de mat2a.El principal objetivo de este estudio es la identificaci\u00f3n de nuevos mecanismos implicados en laproliferaci\u00f3n, diferenciaci\u00f3n y desdiferenciaci\u00f3n de los hepatocitos, la regeneraci\u00f3n hep\u00e1tica y latransformaci\u00f3n maligna, en relaci\u00f3n con same. Nuestra hip\u00f3tesis consiste en la existencia de una regulaci\u00f3npost-transcripcional de los arnm de mat1a y mat2a mediante prote\u00ednas de uni\u00f3n al arn, estabilizando ydesestabilizando dichos arnm. Nuestros datos indican que la prote\u00edna de uni\u00f3n al arn hur se une alarnm de mat2a estabiliz\u00e1ndolo, y la prote\u00edna de uni\u00f3n al arn auf1 se une al arnm de mat1adesestabiliz\u00e1ndolo. Asimismo, la metilaci\u00f3n de hur por same cambia su funcionalidad, de forma que se uneal arnm de mat2a desestabiliz\u00e1ndolo o inhibiendo su traducci\u00f3n. Los niveles de metil-hur\/hur y auf1var\u00edan durante el desarrollo del h\u00edgado, la desdiferenciaci\u00f3n de los hepatocitos y la transformaci\u00f3n maligna,regulando los niveles de los arnm de mat2a y mat1a. Asimismo, el modelo de rat\u00f3n gnmt-ko, con unnivel de same elevado cr\u00f3nicamente, presenta una desregulaci\u00f3n de mat2a por metil-hur\/hur.Como el rat\u00f3n gnmt-ko presenta desregulaci\u00f3n de mat2a y se caracteriza por altos niveles de same,estudiamos su respuesta regenerativa tras hepatectom\u00eda parcial. Encontramos que el rat\u00f3n gnmt-kopresenta una alta mortalidad tras la hepatectom\u00eda parcial, junto con la inhibici\u00f3n de la ruta lkb1\/ampk\/enosy de la translocaci\u00f3n de hur al citoplasma, procesos fundamentales para la normal proliferaci\u00f3n yregeneraci\u00f3n hep\u00e1tica. Adem\u00e1s, el bloqueo de la fosforilaci\u00f3n de ampk promueve la activaci\u00f3n basal delfactor de transcripci\u00f3n nf\u00c2\u00bfb junto con la p\u00e9rdida de la capacidad de activaci\u00f3n de nf\u00c2\u00bfb en respuesta atnf\u00c2\u00bf, as\u00ed como el bloqueo de la expresi\u00f3n de inos tras la hepatectom\u00eda parcial.De acuerdo con nuestros resultados, la prote\u00edna de uni\u00f3n al arn hur es fundamental para laproliferaci\u00f3n de los hepatocitos, la diferenciaci\u00f3n hep\u00e1tica y la transformaci\u00f3n maligna. La regulaci\u00f3n de sufunci\u00f3n est\u00e1 relacionada con la localizaci\u00f3n subcelular, la fosforilaci\u00f3n, la metilaci\u00f3n y su abundanciaproteica. En concreto, su abundancia est\u00e1 regulada por ubiquitinizaci\u00f3n, pero los mecanismos responsablesde la estabilizaci\u00f3n de la prote\u00edna son desconocidos. El estudio de la regulaci\u00f3n de la estabilidad de laprote\u00edna hur en chc y c\u00e1ncer de colon nos llev\u00f3 a descubrir la existencia de una estabilizaci\u00f3n mediante laneddilizaci\u00f3n de hur. Mediante el an\u00e1lisis de la abundancia de hur en l\u00edneas celulares de chc y c\u00e1ncerde colon y en muestras humanas de chc y met\u00e1stasis de colon al h\u00edgado, concluimos que mdm2 neddilizahur en el citoplasma, promoviendo su localizaci\u00f3n nuclear y protegi\u00e9ndolo de la degradaci\u00f3n por elproeasoma. El an\u00e1lisis mutacional de la prote\u00edna hur nos permiti\u00f3 localizar las lisinas en las que tiene lugaresta modificaci\u00f3n post-traduccional.En conclusi\u00f3n, nuestros resultados descubren un muevo mecanismo de regulaci\u00f3n post-transcripcionalde mat1a y mat2a, subrayando la importancia de la homeostasis de same en la proliferaci\u00f3n,diferenciaci\u00f3n y transformaci\u00f3n maligna en el h\u00edgado. Adem\u00e1s, el descubrimiento de un nuevo mecanismo deregulaci\u00f3n de la abundancia de hur en chc y c\u00e1ncer de colon a trav\u00e9s de la neddilizaci\u00f3n mediada pormdm2, abre nuevas v\u00edas para el tratamiento de estas enfermedades.Resumen\/summary 33summarythe chronic liver disease is one of the main causes of mortality in humans. It comprises illnesses withetiologies such as hepatitis b and c virus infection, toxins, alcohol and drugs consumption, autoimmune andhereditary diseases and non-alcoholic fatty liver disease (nafld). Nafld is one of the main chronic liverdiseases in developed countries, associated with the metabolic syndrome risk factors (obesity, insulinresistance, dyslipidemia and hypertension), and including alterations from steatosis to non-alcoholicsteatohepatitis (nash), in some cases accompanied by fibrosis. Nafld patients of nash with fibrosis canfinally develop hepatocellular carcinoma (hcc).Hcc represents the third leading cause of cancer death globally, and the first cause of death in cirrhoticpatients. Hcc is the most prevalent cancer in the population, with a poor prognosis even in the developedcountries. The etiology is diverse, with hepatitis b and c virus, alcoholism, aflatoxin b1 and nafld as themain risk factors. Among the molecular factors involved in nafld progression to hcc we can find theoxidative stress, the methionine metabolism and the impairment of p53. In particular, several studies haveshown that human patients with liver cirrhosis at a high risk of hcc development present impairment inmethionine metabolism and abnormal s-adenosylmethionine (same) levels.Same is the main methyl donor in the cell, being the liver the principal responsible of its homeostasis.Same plays a critical role in hepatocyte proliferation, differentiation and tumoral cells apoptosis. In the liversame is able to inhibit the activation of the lkb1\/ampk\/enos pathway, and avoid the translocation of therna binding protein (rbp) hur, which are important mechanisms for hepatocyte proliferation and liverregeneration. Therefore, same levels must be tightly regulated. The two main enzymes in same synthesisand catabolism are methionine adenosyltransferase (mat) and glycine n-methyltransferase (gnmt),respectively. The mat enzymes are codified by two genes, mat1a and mat2a. Mat1a encodes for mat iand mat iii enzymes, and mat2a codifies for mat ii. Mat1a is expressed in the adult and differentiatedliver, whereas mat2a is expressed in fetal and proliferating liver. During liver development, there is a switchfrom mat2a to mat1a expression, and during liver de-differentiation, proliferation and malignanttransformation, mat1a levels decrease together with an increase of mat2a levels. This regulation of mat1aand mat2a expression is related with promoter methylation and histone acetylation.On the other hand, the same catabolizing enzyme gnmt, is present in large amounts in the liver, andhighly reduced in liver and prostate tumors. Individuals with gnmt mutations spontaneously develop liverdisease. Gnmt enzyme regulates the same to sah ratio, which is considered the index of thetransmethylation potential of the cell. The impairment of this ratio can result into aberrant methylation patterns.In order to study the implications of the impairment of same regulation in the liver, two knockout modelswere developed, mat1a-ko and gnmt-ko, characterized by chronic deficiency and excess of same levels,respectively. The mat1a-ko mice present oxidative stress, and develop steatosis and nash at 8 month, andhcc. The gnmt-ko mice develop steatosis, fibrosis and finally hcc. The observations that both low andhigh same levels lead to similar pathology highlight the importance of the same homeostasis.The regulation of same levels involves mat1a and mat2a mrna expression regulation. The regulationof their expression based on the promoter methylation and histone acetylation does not completely explain thechanges between mat1a and mat2a. In addition, methionine conversion into same regulates mat2amrna turnover.The main objective of this study is to identify new mechanisms implicated in the hepatocyte proliferation,differentiation and dedifferentiation, liver regeneration and malignant transformation, in relation with same.We hypothesize that there is a post-transcriptional regulation of mat1a and mat2a mrnas that involverbps, which bind to mrnas stabilizing or destabilizing them. Our data indicate that the rbp hur binds tomat2a mrna stabilizing it, and the rbp auf1 binds to and destabilizes mat1a mrna. Importantly, samemethylates hur promoting mat2a mrna destabilization or inhibition of the translation. The levels of methyl-hur\/hur and auf1 vary during liver development, hepatocyte de-differentiation and malignant34 resumen\/summarytransformation, thus regulating the levels of mat2a and mat1a mrnas. The knockout mouse model gnmtko,with chronically elevated same levels, also presents an impairment in mat2a mrna regulation bymethyl-hur\/hur.As the gnmt-ko mice presents dysregulation of mat2a and is characterized by high same levels, westudied its regenerative response after partial hepatectomy (ph). We found that gnmt-ko mice present highmortality after ph, together with the inhibition of the lkb1\/ampk\/enos pathway and the translocation of hurto the cytoplasm, processes fundamental for the normal liver proliferation and regeneration. In addition, theblockade of ampk phosphorylation promotes nf\u00c2\u00bfb basal activation and lack of tnf\u00c2\u00bf-induced nf\u00c2\u00bfbactivation and inos expression after ph. All these results show the impairment in the liver regeneration of thegnmt-ko mice.The rbp hur appears, according with our results, as fundamental in the hepatocyte proliferation, liverdifferentiation and malignant transformation processes. The regulation of its function is related with hursubcellular localization, phosphorylation, methylation and protein abundance. In particular, hur abundance isregulated by ubiquitination, but the mechanisms leading to hur stability are not known. We studied theregulation of hur protein stability in hcc and colon cancer, and we found that hur is stabilized byneddylation. By studying hur abundance in hcc and colon cancer cell lines, and in human hcc andmetastatic colon cancer samples, we conclude that mdm2 neddylates hur in the cytosol, promoting itsnuclear localization and protecting it from the proteasomal degradation. The mutational analysis of hurprotein allowed us to map the lysines involved in this post-translational modification.In conclusion, our results uncover a new regulatory post-transcriptional mechanism for mat1a andmat2a, highlighting the importance of same homeostasis in the proliferation, differentiation and malignanttransformation of the liver. In addition, the finding of a new mechanism for the regulation of hur abundance inhcc and colon cancer through mdm2-mediated neddylation opens a new field in the treatment of thesemalignancies.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>New oncogenic drivers in hepatocellular carcinoma: role of the rna binding protein hu antigen r<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 New oncogenic drivers in hepatocellular carcinoma: role of the rna binding protein hu antigen r <\/li>\n<li><strong>Autor:<\/strong>\u00a0 David Fernandez Ramos <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Pa\u00eds vasco\/euskal herriko unibertsitatea<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 05\/12\/2012<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>M\u00aa Luz Martinez Chantar<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: Mar\u00eda  isabel Fabregat romero <\/li>\n<li>mat\u00edas Antonio \u00e1vila zaragoz\u00e1 (vocal)<\/li>\n<li>albert Morales mu\u00f1oz (vocal)<\/li>\n<li>irene D\u00edaz moreno (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de David Fernandez Ramos Resumenla enfermedad hep\u00e1tica cr\u00f3nica es una de las principales causas de mortalidad en humanos.Comprende 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