Academic literature on the topic 'SOXB1 Transcription Factors'

The transcription factor SOX1 is a key regulator of neural stem cell development, acting to keep neural stem cells (NSCs) in an undifferentiated state. Postnatal expression of Sox1 is typically confined to the central nervous system (CNS), however, its expression in non-neural tissues has recently been implicated in tumorigenesis. The mechanism through which SOX1 may exert its function is not fully understood, and studies have mainly focused on changes in SOX1 expression at a transcriptional level, while its post-translational regulation remains undetermined. To investigate this, data were extracted from different publicly available databases and analysed to search for putative SOX1 post-translational modifications (PTMs). Results were compared to PTMs associated with SOX2 in order to identify potentially key PTM motifs common to these SOXB1 proteins, and mapped on SOX1 domain structural models. This approach identified several putative acetylation, phosphorylation, glycosylation and sumoylation sites within known functional domains of SOX1. In particular, a novel SOXB1 motif (xKSExSxxP) was identified within the SOX1 protein, which was also found in other unrelated proteins, most of which were transcription factors. These results also highlighted potential phospho-sumoyl switches within this SOXB1 motif identified in SOX1, which could regulate its transcriptional activity. This analysis indicates different types of PTMs within SOX1, which may influence its regulatory role as a transcription factor, by bringing changes to its DNA binding capacities and its interactions with partner proteins. These results provide new research avenues for future investigations on the mechanisms regulating SOX1 activity, which could inform its roles in the contexts of neural stem cell development and cancer.

SOX2 is a key transcription factor in embryonic development representing a universal marker of pluripotent stem cells. Based on the functional redundancy and overlapping expression patterns of SOXB1 subgroup members during development, the goal of this study has been to analyze if some aspects of regulation of expression are preserved between human SOX2 and SOX3 genes. Thus, we have tested several transcription factors previously demonstrated to play roles in controlling SOX3 gene activity for potential participation in the regulation of SOX2 gene expression in NT2/D1 cells. Here we report on the activation of SOX2 expression by ubiquitous transcription factors (NF-Y, Sp1 and MAZ), TALE family members (Pbx1 and Meis1), as well as liganded RXR?. Elucidating components involved in the regulation of SOX gene expression represent a valuable contribution in unraveling the regulatory networks operating in pluripotent embryonic cells.

From a differential display designed to isolate genes that are down-regulated upon differentiation of the central nervous system in Danio rerio embryos, we isolated d-asb11 (ankyrin repeat and suppressor of cytokine signaling box–containing protein 11). Knockdown of the d-Asb11 protein altered the expression of neural precursor genes sox2 and sox3 and resulted in an initial relative increase in proneural cell numbers. This was reflected by neurogenin1 expansion followed by premature neuronal differentiation, as demonstrated by HuC labeling and resulting in reduced size of the definitive neuronal compartment. Forced misexpression of d-asb11 was capable of ectopically inducing sox2 while it diminished or entirely abolished neurogenesis. Overexpression of d-Asb11 in both a pluripotent and a neural-committed progenitor cell line resulted in the stimulus-induced inhibition of terminal neuronal differentiation and enhanced proliferation. We conclude that d-Asb11 is a novel regulator of the neuronal progenitor compartment size by maintaining the neural precursors in the proliferating undifferentiated state possibly through the control of SoxB1 transcription factors.

Abstract GnRH is a pivotal hypothalamic neurohormone governing reproduction and sexual development. Because transcriptional regulation is crucial for the spatial and temporal expression of the GnRH gene, a region approximately 3.0 kb upstream of the mammalian GnRH promoter has been extensive studied. In the present study, we demonstrate a transcription-enhancer located in the first intron (intron A) region of the GnRH gene. This transcriptional enhancer harbors putative sex-determining region Y-related high-mobility-group box (SOX) family transcription factor-binding sites, which are well conserved across many mammalian species. The class-C SOX member proteins (SOX-C) (SOX4 and SOX11) specifically augment this transcriptional activation by binding to these SOX-binding sites. In accordance, SOX11 is highly enriched in immortalized GnRH-producing GT1-1 cells, and suppression of its expression significantly decreases GnRH gene expression as well as GnRH secretion. Chromatin immunoprecipitation shows that endogenous SOX-C factors recognize and bind to the intronic enhancer in GT1-1 cells and the hypothalamus. Accompanying immunohistochemical analysis demonstrates that SOX4 or SOX11 are highly expressed in the majority of hypothalamic GnRH neurons in adult mice. Taken together, these findings demonstrate that SOX-C transcription factors function as important transcriptional regulators of cell type-specific GnRH gene expression by acting on the intronic transcriptional enhancer.

ABSTRACT Intermediate-filament Nestin and group B1 SOX transcription factors (SOX1/2/3) are often employed as markers for neural primordium, suggesting their regulatory link. We have identified adjacent and essential SOX and POU factor binding sites in the Nestin neural enhancer. The 30-bp sequence of the enhancer including these sites (Nes30) showed a nervous system-specific and SOX-POU-dependent enhancer activity in multimeric forms in transfection assays and was utilized in assessing the specificity of the synergism; combinations of either group B1 or group C SOX (SOX11) with class III POU proved effective. In embryonic day 13.5 mouse spinal cord, Nestin was expressed in the cells with nuclei in the ventricular and subventricular zones. SOX1/2/3 expression was confined to the nuclei of the ventricular zone; SOX11 localized to the nuclei of both subventricular (high-level expression) and intermediate (low-level expression) zones. Class III POU (Brn2) was expressed at high levels, localizing to the nucleus in the ventricular and subventricular zones; moderate expression was observed in the intermediate zone, distributed in the cytoplasm. These data support the model that synergic interactions between group B1/C SOX and class III POU within the nucleus determine Nestin expression. Evidence also suggests that such interactions are involved in the regulation of neural primordial cells.

Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming. First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.

Recent breakthroughs in creating induced pluripotent stem cells (iPS cells) provide alternative means to obtain embryonic stem (ES) cell-like cells without destroying embryos by introducing four reprogramming factors (Oct3/4, Sox2, and Klf4/c-Myc or Nanog/Lin28) into somatic cells. However, the molecular basis of reprogramming is largely unknown. To address this question, we employed microRNAs, small molecules, and conducted genome-wide RNAi screen, to investigate the regulatory mechanisms of reprogramming. First we showed that depleting miR-21 and miR-29a enhances reprogramming in mouse embryonic fibroblasts (MEFs). We also showed that p53 and ERK1/2 pathways are regulated by miR-21 and miR-29a and function in reprogramming. Second, we showed that computational chemical biology combined with genomic analysis can be used to identify small molecules regulating reprogramming. We discovered that the NSAID Nabumetone and the anti-cancer drug OHTM could replace Sox2 during reprogramming. Nabumetone could also replace c-Myc or Sox2 without compromising self-renewal and pluripotency of derived iPS cells. To identify the cell-fate determinants during reprogramming, we integrated a genome-wide RNAi screen with transcriptome analysis to dissect the molecular requirements in reprogramming. We found that extensive interactions of embryonic stem cell core circuitry regulators are established in mature iPS cells, including Utf1, Nr6a1, Tdgf1, Gsc, Fgf10, T, Chrd, Dppa3, Fgf17, Eomes, Foxa2. Remarkably, genes with non-differential change play the most critical roles in the transitions of reprogramming. Functional validation showed that some genes act as essential or barrier roles to reprogramming. We also identified several genes required for maintaining ES cell properties. Altogether, our results demonstrate the significance of miRNA function in regulating multiple signaling networks involved in reprogramming. And our work further advanced the reprogramming field by identifying several new key modulators.

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin lymphomas associated with poor prognosis and frequent relapses. Recently, the neuronal transcription factor SOX11 has been identified as a very specific biomarker for MCL. SOX11 is constantly overexpressed in virtually all aggressive MCLs, and at lower levels in a subgroup of Burkitt lymphoma and acute lymphoblastic leukemia but not in other lymphoid neoplasms. SOX11 was found exclusively overexpressed in conventional MCL and totally absent in normal lymphoid cells or in MCL patients with indolent clinical course and prolonged survival. Although SOX11 function and potential target genes in lymphoid cells are poorly known, the highly specific expression in aggressive MCL suggests that it may be an important element in the development and progression of this tumor. Two publications resulted from our studies and compose this thesis. In paper I, we have studied the molecular mechanisms leading to the aberrant expression of SOX11 in aggressive MCL. MCL is one of the lymphoid neoplasms with highest number of genetic aberrations but none involving the SOX11 genomic region at chromosome 2p25. As no chromosomal changes affecting SOX11 locus were identified in MCL, we hypothesized that epigenetic events could lead to SOX11 aberrant overexpression. We performed a comprehensive SOX11 gene expression and epigenetic studies. We observed that SOX11 expression was associated with unmehtylated DNA and presence of activating histone marks (H3K9/14Ac and H3K4me3) in embryonic stem cells and some aggressive B-cell neoplasms, including MCL. Conversely, the loss of SOX11 expression in adult stem cells, normal hematopoietic cells and other lymphoid neoplasms was associated with the presence of silencing histone marks H3K9me2 and H3K27me3 with or without simultaneous DNA methylation. We concluded that the pathogenic role of SOX11 is associated with its de novo expression in some aggressive lymphoid malignancies, which is mediated by a shift from inactivating to activating histone modifications. In paper II, we have focused on uncovering putative biological functions of SOX11 in aggressive MCL. Using chromatin immunoprecipitation microarray analysis combined with gene expression profiling upon SOX11 knockdown, we identified target genes and transcriptional programs regulated by SOX11 including the block of mature B-cell differentiation, modulation of cell cycle, apoptosis, and stem cell development. PAX5 stood out as one of the major SOX11 direct target genes. SOX11 silencing downregulates PAX5, induces BLIMP1 expression, and promotes the shift from a mature B-cell into the initial plasmacytic differentiation phenotype in both MCL primary tumor cells and an in vitro model. Our results suggested that SOX11 contributes to tumor development by altering the terminal B-cell differentiation program of MCL cells. Moreover, we have demonstrated the tumorigenic ability of SOX11 in vivo, using a xenotransplant model of MCL in CB17-SCID mice. The significant reduction on tumor growth of the SOX11-silenced cells compared to the growth of control MCL cells in the xenograft experiments highlighted the implication of SOX11 expression in the aggressive behavior of this lymphoma. Overall our results demonstrated that SOX11 can act as oncogene in MCL.
El linfoma de células del manto (LCM) es un subtipo agresivo de linfoma non Hodgkin asociado a un mal pronóstico y recaídas frecuentes. Recientemente, el factor de transcripción neuronal SOX11 se ha identificado como un marcador muy específico de LCM. SOX11 se encuentra sobreexpresado constantemente en todos los LCM agresivos y en niveles más bajos en un subgrupo de Burkitt linfoma (BL) y leucemia linfoblástica aguda (LLA) aunque no en otros neoplasmas linfoides. SOX11 se encontró exclusivamente sobreexpresado en LMC convencionales y totalmente ausente en células linfoides normales o en pacientes LMC con un curso clínico indolente y una supervivencia prolongada. Aunque la función de SOX11 y sus genes potenciales dianas aún se desconocen, su elevada expresión específica en LCM sugiere que puede ser un elemento importante en la progresión y desarrollo de este tumor. Se han obtenidos dos publicaciones de los estudios que componen esta tesis. En el primer artículo hemos estudiado los mecanismos moleculares responsables de la expresión aberrante de SOX11 en LCM agresivo. El artículo I proporciona una caracterización exhaustiva de los mecanismos epigenéticos que conducen a la desregulación de SOX11 en estas neoplasias linfoides. En general se observó una significativa correlación inversa entre la metilación del promotor de SOX11 y la expresión de dicho gen. Sin embargo, en muchas muestras (células madre embrionarias, ESC, o adultas, células B normales y algunos LCM indolentes, leucemia linfática crónica y linfoma folicular) la expresión de SOX11 se vió reprimida a pesar de su estado no metilado. Estos hallazgos nos sugieren que la expresión de SOX11 no depende exclusivamente del estado de metilación del ADN del gen y nos llevó a estudiar mecanismos epigenéticos alternativos. Hemos observado que la expresión de SOX11 se asocia con la presencia de marcas de activación de las histonas (H3K9/14Ac y H3K4me3) en las células madre embrionarias y algunas neoplasias de células B agresivas. Por el contrario, en las muestras que no expresan SOX11, incluidas las células madre adultas, las células hematopoyéticas normales y diversas neoplasias linfoides, se observó que el promotor de SOX11 mostraba un enriquecimiento en las marcas de silenciamiento H3K9me2 y H3K27me3. El silenciamiento de SOX11 en líneas celulares fue revertido por el inhibidor de la histona deacetilasa (SAHA) pero no por el inhibidor de la ADN metiltransferasa (AZA). Estos datos indican que, como SOX11 no se expresa en las células linfoides normales, no metiladas, lo más probable es que la hipermetilación del ADN en algunos tumores sin expresión de SOX11 sea funcionalmente inerte, y podría estar asociada con la reducción de la plasticidad epigenética en células tumorales. También observamos que la expresión de novo de SOX11 se asocia con las neoplasias linfoides agresivas como el LCM, algunos subtipos de B-LLA y algunos casos de BL siendo este efecto mediado por un “switch” entre inactivación y activación de las modificaciones de las histonas. Además, como SOX11 se expresa fuertemente en las ESC, los datos sugieren que la expresión de SOX11 podría estar asociada con la adquisición de características de la cromatina similares a la de las células madre. En el artículo II nos hemos centrado en identificar las posibles funciones biológicas de SOX11 en LCM. Combinando análisis de microarray de immunopecipitación de cromatina y perfil de expresión diferencial después de silenciar SOX11, hemos identificado genes dianas y programas transcriptionales regulados por SOX11, incluyendo el bloqueo de la diferenciación de la célula B madura, la modulación de ciclo celular, apoptosis y desarrollo de célula madre. PAX5 destacó como uno de los genes diana directos de SOX11 más significativo. El silenciamiento de SOX11 downregula PAX5, induce la expresión de BLIMP1 y promueve el cambio de fenotipo de célula B madura a la diferenciación inicial plasmacítica, tanto en células tumorales primarias de LCM como en un modelo en vitro. Nuestros resultados sugieren que SOX11 contribuye al desarrollo tumoral mediante la modulación del programa de diferenciación terminal de célula B en LCM. Además, hemos demostrado la habilidad tumorigénica de SOX11 en vivo mediante un modelo de xenotrasplante de LCM en ratones CB17-SCID. La reducción significativa del crecimiento tumoral de las células con expresión de SOX11 silenciada comparada con el crecimiento de células de LCM con elevados niveles de expresión de SOX11 de los xenotrasplantes, demuestra la implicación de la expresión de SOX11 en el comportamiento agresivo de este linfoma. En conclusión, nuestros resultados demuestran que SOX11 puede actuar como oncogén en LCM.

Mantle cell lymphoma (MCL) is an aggressive lymphoid neoplasm derived from mature B cells genetically characterized by the presence of the t(11;14)(q13;q32) translocation causing cyclin D1 overexpression and therefore cell cycle deregulation. However, other additional secondary genetic alterations also contribute to MCL pathogenesis. Recent studies identified a distinct subgroup of tumors with an indolent clinical behavior even without chemotherapy that tend to present with non-nodal, leukemic disease instead of extensive nodal infiltration. Moreover, recent molecular studies have identified SRY (Sex determining region-Y)-like HMG box-11 (SOX11) as one of the best characterized discriminatory genes between these two clinical subtypes of MCL, being almost exclusively overexpressed in the aggressive presentation of the disease. SOX11 is a high mobility group (HMG) transcription factor (TF) characterized by its critical involvement in embryonic development and neuronal cell differentiation. Although not apparently expressed in any normal cell subtype of the lymphoid system, it has been recently identified as playing a major role in several solid tumors and some subtypes of aggressive lymphomas. However, SOX11 oncogenic mechanisms contributing to the development and progression of these diseases are largely unknown. Although SOX11 function and potential target genes in lymphoid cells are poorly known, its high specific expression in aggressive MCL suggests that it may be an important element in the development and progression of this disease. Two publications resulted from our studies and compose this thesis. In paper I, we initiated the molecular characterization and elucidation of the SOX11-regulated transcriptional program in MCL. We performed an integrative analysis coupling data from human genome-wide promoter analysis by SOX11 Chromatin Immunoprecipitation (ChIP)-chip experiments and gene expression profiling (GEP) upon SOX11 silencing in MCL cell lines, and identified target genes and transcriptional programs regulated by SOX11 including the block of mature B cell differentiation, modulation of cell cycle, apoptosis, and stem cell development. Paired box protein 5 (PAX5) stood out as one of SOX11 major direct target genes. We found that SOX11 silencing downregulated PAX5, induced B lymphocyte induced maturation protein 1 (BLIMP1) expression, and promoted the shift from a mature B cell into the initial plasmacytic differentiation phenotype in both MCL primary tumor cells and an in vitro model. We also demonstrated the oncogenic implication of SOX11 expression in the aggressive behavior of MCL as SOX11-knockdown derived tumors displayed a significant reduction on tumor growth compared to SOX11-positive tumors in subcutaneous MCL xenografts. Although our results suggested that SOX11 contributed to tumor development by altering the terminal B cell differentiation program of MCL cells, the specific SOX11-regulated mechanisms promoting the oncogenic and rapid tumor growth of aggressive MCL still remained to be elucidated. Therefore, in paper II, we further characterized the potential SOX11-regulated oncogenic mechanisms by integrating our ChIP-chip data with further analyses including GEP derived from the xenograft SOX11-positive and silenced tumors. Gene ontology analysis of SOX11 target genes and gene set enrichment analyses (GSEA) of the differentially expressed genes in xenografts, human primary tumors and MCL cell lines revealed that blood vessel development and angiogenic gene signatures were significantly enriched in SOX11-positive cells. Consequently, we performed in vitro angiogenic assays and observed that conditioned media derived from SOX11-positive cells induced higher tube formation, proliferation and migration of endothelial cells, corroborated by a prominent higher density of microvessels in human primary tumors and mouse xenografts. Strikingly, gene and protein expression profiling revealed that SOX11-modulation of angiogenesis in MCL was mediated by the upregulation of the pro-angiogencic factor Platelet-derived growth factor-A (PDGFA). Inhibition of the PDGFA pathway impaired angiogenesis development both in vitro and in vivo, and also MCL tumor growth in vivo, representing a promising novel therapeutic strategy for the treatment of aggressive MCL. Overall, we have unraveled and characterized, for the first time, the oncogenic role of SOX11 and the tumorigenic pathways it orchestrates in MCL. Importantly, the elucidation of molecular oncogenic mechanisms that are deregulated by SOX11 in MCL will aid the management and stratification of MCL patients and lead to the development of new therapeutic strategies for this aggressive disease.
Les neoplàsies limfoides són un grup heterogeni de tumors caracteritzats per un event genètic inicial i l’acumulació d’alteracions moleculars secundàries que condicionen la progressió tumoral. Els mecanismes genètics que condueixen a alteracions genètiques primàries són principalment les translocacions cromosòmiques que, generalment, resulten en l’activació d’un proto-oncogen que comporta l’alteració de la proliferació, l’apoptosi o la via de diferenciació de les cèl·lules B. El limfoma de cèl·lules del mantell (LCM) és una neoplàsia limfoide caracteritzada per una proliferació alta de limfòcits B madurs, i és considerada un dels limfomes no Hodgkin (LNH) més agressius amb una supervivència mitjana de 3-4 anys. L’evolució clínica acostuma a ser molt agressiva amb poca resposta al tractament i freqüent recidiva; són pocs els pacients que es curen amb les teràpies actuals. Tot i així, observacions clíniques recents han identificat alguns LCM amb un curs clínic indolent de la malaltia i una llarga supervivència dels pacients, inclòs sense la necessitat de tractament. Estudis d’expressió gènica diferencial han identificat que SOX11, un factor de transcripció neuronal, és un dels gens més ben caracteritzats per diferenciar els subtipus de LCM agressius i indolents ja que se sobre-expressa en els LCM agressius però no en els indolents. SOX11 pertany a la família dels gens SOX que codifiquen per possibles reguladors transcripcionals que desenvolupen funcions importants en diferents processos del desenvolupament. La funció de SOX11 i molts altres membres de la superfamília de proteïnes SOX actualment no es coneix, tot i que es creu que la funció de les proteïnes SOX és, almenys, en part estructural permetent que altres factors de transcripció s’uneixin al solc format a l’ADN i/o reunint elements reguladors, el que facilita la formació de complexes proteics. SOX11 se sobre-expressa constantment en gairebé tots els tumors de LCM agressius, a nivells inferiors en un subgrup de limfomes de Burkitt i limfoblàstics, però no en altres neoplàsies limfoides, en cèl·lules limfoides normals ni en pacients de LCM que segueixen una llarga evolució clínica indolent. Per tant, SOX11 ha estat identificat com a biomarcador en els tumors de LCM i possible factor pronòstic. Tot i que la funció de SOX11 i els seus possibles gens diana en cèl·lules limfoides es desconeixen, la seva alta expressió en els LCM agressius suggereix que SOX11 pot ser un element important en el desenvolupament i progressió d’aquest tumor. Per tant, els objectius d’aquest projecte de tesi doctoral són identificar, caracteritzar i validar les possibles vies oncogèniques regulades per SOX11 per obtenir un millor i major coneixement de la patogènesi del LCM, i trobar possibles candidats per a teràpies específiques per aquest tumor. Per realitzar aquests objectius hem estudiat els gens directament regulats per SOX11 mitjançant immunoprecipitació de cromatina,així com el fenotip associat a la pèrdua de l’expressió de SOX11 en línies cel·lulars de LCM establement transduïdes (model in vitro). A més a més, també hem estudiat l’habilitat tumorigènica de SOX11 mitjançant el xenotransplantament subcutani de les línies cel·lulars generades in vitro en ratolins immunodeficients (SCID) (model in vivo). Un cop hem identificat els possibles gens diana i les vies oncogèniques regulades per SOX11 mitjançant els models in vitro i in vivo, els hem validat en tumors humans primaris del LCM. S’han obtingut dos treballs d’investigació dels estudis que composen aquesta tesi doctoral, publicats a la prestigiosa revista d’hematologia Blood els anys 2013 i 2014. En ambdós treballs hem estudiat els mecanismes moleculars regulats per SOX11 responsables de la patogènesi i el desenvolupament maligne del LCM. El primer treball es centra en la caracterització del bloqueig del desenvolupament dels limfòcits B madurs degut a la sobre-expressió de SOX11 en el LCM, suggerint un diferent desenvolupament en la formació dels subtipus de LCM agressius i indolents. A més, també demostrem que SOX11 és indispensable pel creixement del LCM ja que tumors SOX11-negatius inoculats en ratolins SCID presenten un creixement menor comparat amb els tumors SOX11-positius. En el segon treball hem identificat, per primera vegada, la funció oncogènica de SOX11 en el LCM ja que regula processos angiogènics responsables del major creixement de les cèl·lules SOX11-positives. D’altra banda també mostrem noves aproximacions terapèutiques per aquest tipus de tumor que ajudarien a una millor estratificació i classificació dels pacients de LCM. Al primer article ens hem centrat en identificar les possibles funcions biològiques de SOX11 en el LCM. Combinant anàlisis de microarray d’immunoprecipitació de cromatina i perfil d’expressió diferencial després de silenciar l’expressió de SOX11, hem identificat gens diana i programes transcripcionals regulats per SOX11, incloent el bloqueig de la diferenciació de la cèl·lula B madura, la modulació del cicle cel·lular, l’apoptosi i el desenvolupament de cèl·lules mare. PAX5 apareix com un dels gens diana directes més significatius de SOX11. El silenciament de SOX11 disminueix l’expressió de PAX5, indueix la de BLIMP1 i promou el canvi de fenotip de cèl·lula B madura a la diferenciació plasmacítica inicial, tant en cèl·lules tumorals primàries del LCM com en un model in vitro. Els nostres resultats suggereixen que SOX11 contribueix al desenvolupament tumoral mitjançant la modulació del programa de la diferenciació terminal de la cèl·lula B en el LCM. A més, hem demostrat l’habilitat tumorigènica de SOX11 in vivo mitjançant un model de xenotransplantament de LCM en ratolins SCID. La reducció significativa del creixement tumoral de les cèl·lules amb expressió de SOX11 silenciada comparada amb el creixement de les cèl·lules de LCM amb alts nivells d’expressió de SOX11 dels xenotransplantaments, demostra la implicació de l’expressió de SOX11 en el comportament agressiu d’aquest limfoma. SOX11 se sobre-expressa en diferents tumors sòlids i en la majoria de LCM agressius. En el primer article constituent d’aquesta tesi doctoral hem demostrat que el silenciament de l’expressió de SOX11 redueix el creixement tumoral en un model de xenotransplantament, consistent amb el curs clínic indolent dels pacients del LCM SOX11-negatius. Tot i així, els mecanismes oncogènics directament regulats per SOX11 encara no s’han descrit. Per tant, en el segon treball ens hem centrat en la identificació i caracterització molecular de dits mecanismes. Hem observat que els tumors dels xenotransplantaments murins així com de pacients de LCM SOX11-positius estan enriquits en vies gèniques relacionades amb l’angiogènesi, i presenten una major densitat de microvasos comparada amb els tumors derivats dels xenotransplantaments i els tumors humans primaris SOX11-negatius. A més, el medi cel·lular condicionat derivat de cèl·lules SOX11-positives indueix una major proliferació, migració, formació de tubs, i activació de vies de senyalització angiogèniques en cèl·lules endotelials in vitro. Hem identificat el factor pro-angiogènic PDGFA com un gen diana directe de SOX11 en cèl·lules de LCM, la inhibició del qual elimina els efectes angiogènics accentuats en cèl·lules endotelials in vitro. Per altra banda, PDGFA està sobre-expressat en cèl·lules SOX11-positives tant en cultius cel·lulars de LCM, xenotransplantaments com en pacients de LCM. In vivo, el tractament dels xenotransplantaments SOX11-positius amb el fàrmac imatinib, un inhibidor del receptor de PDGFA, disminueix la neo-vascularització i el creixement tumoral de dites cèl·lules, equilibrant-lo al creixement tumoral de les cèl·lules SOX11-negatives. El nostre estudi identifica, per primera vegada, que SOX11 regula senyals de supervivència angiogèniques en el LCM, demostrant la seva implicació oncogènica en el comportament i progressió agressius d’aquest tumor maligne. A més a més, recalca la via SOX11-PDGFA com una possible diana terapèutica pel subtipus agressiu del LCM, una malaltia en la que encara actualment hi ha poques opcions terapèutiques duradores i complertes. En general, les conclusions d’aquest treball de tesi doctoral han estat les següents: 1. Identificació dels gens diana i les vies transcripcionals regulades per SOX11 en el LCM mitjançant experiments in vitro i in vivo. 2. Caracterització del bloqueig de la diferenciació dels limfòcits B madurs com a mecanisme del desenvolupament dels diferents subtipus de LCM agressius i indolents. Dit bloqueig està mediat per la sobre-expressió de PAX5 mitjançant SOX11. 3. Demostració de la implicació oncogènica de SOX11 en la patogènesi del LCM. 4. Caracterització de la regulació de processos angiogènics per part de SOX11 com a mecanisme oncogènic responsable del creixement tumoral del LCM agressiu. Dita regulació està mediada per la sobre-expressió de PDGFA mitjançant SOX11. 5. Identificació de possibles noves dianes terapèutiques en el LCM.

Introdução: As células-tronco apresentam capacidade de proliferação, autorrenovação, potencial de diferenciação e já foram descritas na hipófise estando envolvidas na renovação celular e regulação homeostática, porém pouco se sabe sobre o seu perfil de expressão nos quadros de hipopituitarismo. Dentre os marcadores de células-tronco descritos previamente na hipófise, destacam-se os genes Sox2, Nanog, Nestina, Cd44 e Oct4. Outro marcador, o gene Nr2e1 (Tlx), encontrado em células-tronco neuronais, apresenta-se elevado durante a embriogênese e na vida adulta no cérebro de camundongos, mas, até o momento, não foi caracterizado na hipófise. Objetivo: Analisar a imunolocalização do SOX2 e o padrão de expressão de marcadores de células-tronco/progenitoras, fatores de transcrição precoce, marcadores de apoptose e proliferação celular na hipófise de três linhagens de camundongos com hipopituitarismo de causa genética por alteração em fatores precoces de diferenciação glandular, as linhagens Ames (Prop1) e Snell (Pou1f1), e por fator tardio de conjugação dos hormônios glicoproteicos, a linhagem alfaGSU, nocaute do gene Cga. Material e Métodos: Foram coletadas hipófises nos tempos P0 (ao nascimento), P7 (final da primeira onda de crescimento glandular), 4 semanas (4S-período da puberdade) e 8 semanas (8S-vida adulta). Nas três linhagens de animais, realizou-se imuno-histoquímica com SOX2 e RT-qPCR com os marcadores de células-tronco/progenitoras Sox2, Nanog, Nestina, Cd44, Oct4 e Nr2e1, fatores de transcrição precoces (Hesx1, Hes1 e Otx2), fator de proliferação celular (Ki67), fatores de diferenciação celular (S100beta e Sox9) e marcadores de apoptose (Caspases 3 e 7). A quantificação relativa dos genes-alvo nos animais mutantes teve como calibrador os seus respectivos selvagens. Resultados: A imunolocalização do SOX2 foi observada na zona que circunda a fenda de Rathke (camada marginal) e em nichos difusos pela glândula nas três linhagens estudadas. Na linhagem alfaGSU, evidenciou-se uma redução de Nanog, Nr2e1, Oct4, e Hesx1 em 4S e de Nestina em 8S. Na linhagem Snell, observou-se aumento na expressão de Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e aumento de Sox2, Cd44, Hesx1, Otx2 e Sox9 em 8S, associado à redução de Ki67 em ambos os períodos. Na linhagem Ames, evidenciou-se aumento de Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta e Sox9 em 4S e 8S. O gene Nr2e1 esteve hiperexpresso em todos os tempos. Houve redução do Ki67 em 4S. As caspases 3 e 7 não se apresentaram alteradas em nenhuma linhagem e/ou tempo. Discussão e conclusão: O padrão de imunolocalização de SOX2 encontrado nas três linhagens estudadas foi semelhante ao descrito em animais sem hipopituitarismo. A evidência da presença do Nr2e1 o coloca como um novo marcador de células-tronco/progenitoras na hipófise. A expressão elevada dos marcadores de células-tronco/progenitoras nas linhagens Ames e Snell sugere que a ausência dos fatores de transcrição precoces não permitiria que a célula tronco/progenitora iniciasse o processo de diferenciação celular, enquanto o oposto ocorreria na linhagem alfaGSU. Adicionalmente, estes achados justificam a hipoplasia hipofisária observada em animais com defeitos em fatores de transcrição expressos no início da diferenciação hipofisária, nos quais o acúmulo de células-tronco pode ser um indicador da indiferenciação hipofisária
Introduction: The role of stem cells, with their capacity for proliferation, self-renewal, and differentiation, has already been described in the cell turnover and homeostatic regulation of the pituitary gland. However, little is known about the expression profiles of these markers in hypopituitarism. Among the stem cell markers previously described in the pituitary include the genes for Sox2, Nanog, nestin, CD44 and Oct4. Another gene marker, Nr2e1 (Tlx), found in neural stem cells, is highly expressed during embryogenesis and adulthood, but so far has not been characterized in the pituitary. Objective: To analyze the immunohistochemical profile of SOX2, as well as the pattern of expression of various markers of stem/progenitor cells, early transcription factors, apoptosis factors and cell proliferation in three pituitary strains of mice with a genetic cause of hypopituitarism. Strains studied with hypopituitarism due to changes in factors of precocious glandular differentiation, include the Ames (Prop1) and Snell (Pou1f1) lineages; hypopituitarism due to the delayed conjugation of glycoprotein hormones include the alfaGSU strain, which is caused by the knockout of the Cga gene. Material and Methods: We collected pituitaries at four time points including P0 (birth), P7 (considered the end of the first wave of growth glandular), 4 weeks (4S - puberty period) and 8 weeks (8S - adulthood). All three strains were subjected to immunohistochemical analysis of SOX2 and RT-qPCR of markers of stem/progenitor cells Sox2, Nanog, Nestin, Cd44, Oct4 and Nr2e1, early transcription factors (Hesx1, Otx2 and Hes1), cell proliferation (Ki67), cell differentiation factors (S100beta and Sox9) and apoptosis (caspases 3 and 7) markers. Relative quantification of target genes in mutant animals was normalized to their respective wild type littermate. Results: The immunolocalization of SOX2 was observed in the area surrounding the Rathke cleft (marginal layer), as well as in diffuse niches throughout the gland in all three strains studied. The alfaGSU strain showed a reduction of Nanog, Nr2e1, Oct4 and Hesx1 at 4S, and Nestin at 8S. The Snell mice exhibited an increase of expression in Sox2, Nanog, Cd44, Nr2e1, Hesx1, Hes1, Otx2, S100beta and Sox9 in at 4S and increased Sox2, Cd44, Hesx1, Otx2 and Sox9 at 8S, associated with the reduction of Ki67 in both periods. The Ames strain showed an increase of Sox2, Nanog, Cd44, Hesx1, Hes1, Otx2, S100beta and Sox9 at 4S and 8S; the gene Nr2e1 was over expressed at all times; and there was reduction in Ki67 at 4S. Caspases 3 and 7 had not changed in any strain, at any time. Discussion and Conclusion: The pattern of immunolocalization of SOX2 found in the three strains studied was similar to that described in animals without hypopituitarism. The presence of Nr2e1 in our study suggests it as a new marker of stem/progenitor cells in the pituitary. The high expression of markers of stem/progenitor cells in the Ames and Snell strains suggests that the absence of early transcription factors Prop1 and Pou1f1 do not allow the stem/ progenitors cells to start the process of cell differentiation, while the opposite occurs in the alfaGSU lineage. Additionally, these findings explain the pituitary hypoplasia observed in animals with defects in early transcription factors, as indicated by the accumulation of stem cells in the Snell and Ames lineages, preventing the initiation of pituitary differentiation