L'atrophie musculaire bulbo-spinale (SBMA) est une dégénérescence lente et progressive des motoneurones causée par l'élongation du triplet nucléotidique (CAG) dans le gène codant pour le récepteur aux androgènes (RA) localisé sur le chromosome X. Dans la SBMA, ce récepteur à extension polyglutaminique (polyQ) pathogène s'accumule de manière ligand dépendante dans le cytoplasme sous forme d'agrégats mais également dans le noyau y créant des corps d'inclusions nucléaires considérés comme la marque identitaire histologique, dont le caractère cytotoxique est aujourd'hui remis en question. Nous avons développé un modèle SBMA in vitro basé sur l'expression inductible d'un RA51Q dans la lignée hybride NSC34, qui est comparé au modèle normal NSC34 exprimant un RA contenant 20Q. Nous avons démontré que l'expression du RA51Q entraîne une diminution de la viabilité ainsi qu'une altération de la croissance neuritique sans formation d'agrégats insolubles dans le noyau ou le cytoplasme des cellules. Le RA en tant que membre de la superfamille des récepteurs nucléaires est un facteur de transcription mais peut également induire des voies de signalisation non génomiques via sa localisation membranaire. Après avoir montré une localisation du RA20Q et du RA51Q dans les « lipid rafts », nous avons corrélé la diminution de la viabilité et de la pousse neuritique induite par le RA51Q à une altération de la signalisation cellulaire non génomique. Les résultats obtenus mettent en évidence une dérégulation des voies de signalisation PI3K/Akt et JNK/c-jun induite par l'expression du RA muté dans notre modèle SBMA. / Spinal Bulbar Muscular Atrophy (SBMA) is a progressive inherited motoneuron disease caused by the expansion of a trinucleotide (CAG) repeat in the gene coding for the androgen receptor (AR) located on the X chromosome. This rare disease causes muscle weaknesses, hypotonia, hyporeflexia, fasciculations of facial muscles in male patients. The androgen-dependent formation of cytoplasmic aggregates and nuclear inclusions are pathological hallmarks of this polyglutamine disease but their potential neurotoxicity is still under debate. We developed a SBMA model based on a doxycycline-inducible AR51Q expression system in the NSC34 hybrid cell line. We have shown that the expression of the mutated AR leads to a reduced viability and to an alteration of neurite outgrowth compared to cells expressing the normal AR20Q. The AR belongs to the nuclear receptor superfamily of transcription factors. However, recent data have put in evidence a membrane localization of AR initiating non-genomic signaling pathways. Because we have not observed insoluble aggregates, reduced viability and neurite outgrowth could not be correlated to AR aggregation. We hypothesized that motoneuron death is not only due to aggregate formation but also to the alteration of AR signaling pathways. We focused on a correlation between the AR localization in lipid rafts and the observed phenotypes. Our results highlight the deregulation of PI3K/Akt and JNK/c-jun signaling pathways induced by the expression of AR51Q in our SBMA model.

La voie de signalisation des androgènes, à travers le récepteur aux androgènes (AR), joue un rôle important dans le développement et la fonction de la prostate, ainsi que dans l’initiation et la progression du cancer de la prostate. La découverte de nouveaux effecteurs de la signalisation androgènes-AR permettra une meilleure compréhension de ces mécanismes. MiR-135a a été identifié comme un gène cible de la voie de signalisation androgènes-AR. Après stimulation androgénique, AR active directement la transcription du gène miR-135a2, en se fixant sur un élément de réponse aux androgènes dans la région promotrice.Une surexpression de miR-135a inhibe la migration et l’invasion de cellules prostatiques cancéreuses, en régulant négativement l’expression des protéines ROCK1 et ROCK2, deux gènes cibles de miR-135a nouvellement identifiés.De plus, miR-135a cible et régule négativement l’expression du facteur de transcription FOXN3, capable de moduler l’activité transcriptionnelle de AR et la prolifération cellulaire dépendante des androgènes.L’étude fonctionnelle de miR-135a suggère donc qu’il puisse être impliqué dans la progression du cancer de la prostate, en régulant la formation des métastases et la signalisation androgénique. L’expression de miR-135a, dans le tissu tumoral par rapport au tissu sain adjacent, de prostatectomies de patients, est inversement corrélée aux paramètres d’agressivité de la maladie, suggérant qu’il puisse être utilisé comme marqueur de pronostic du cancer de la prostate.Ces résultats font de miR-135a un nouvel effecteur de la voie de signalisation de AR, pouvant contribuer à la progression du cancer de la prostate. / Androgens signaling through the androgen receptor (AR) is critical for normal prostate development and function, as well as prostate cancer initiation and progression. The discovery of new effectors of androgens-AR pathway will allow a better understanding of these mechanisms.MiR-135a has been identified as a target gene in androgen-AR signaling pathway. After androgen stimulation, AR directly activates the transcription of miR-135a2 gene by binding to an androgen response element in the promoter region.Ectopic expression of miR-135a was found to induce morphological modification leading to an inhibition of migration and invasion in prostate cancer cells, by down-regulating ROCK1 and ROCK 2 expression, two newly identified miR-135a target genes.Moreover, miR-135a targets and downregulates the expression of the transcription factor FOXN3, able to modulate AR transcriptional activity and androgen-mediated cell proliferation.Thus, functional study of miR-135a suggests that it could be implicated in prostate cancer progression, by regulating metastases formation and androgen signaling.MiR-135a expression level in surgical cancerous speciments normalized to pair-matched normal counterpart tissues was inversely correlated with aggressivity parameters of the disease, suggesting that it could be used as a candidate prognostic marker in human prostate cancer.These results define miR-135a as a novel effector in androgens-AR signaling, which may contribute to prostate cancer progression.

Retinoide sind Steroide, Derivate des Vitamin A, die ihre Wirkung durch Interaktion mit Retinoid-Rezeptoren, lokalisiert im Zellkern, entfalten. Diese Retinoid-Rezeptoren, weisen funktionelle und strukturelle Ähnlichkeiten mit dem Androgenrezeptor auf. Untersuchungen an Zellkulturen zeigen, daß der Effekt von Retinoiden auf das Zellwachstum von Prostata-Epithelzellen und Prostatakarzinomzellen keiner einfachen Kinetik folgt, sondern neben der Abhängigkeit von der Retinoid-Dosis, auch von der Zellinie, insbesondere deren Androgenrezeptor abhängt. So zeigten LNCaP-Zellen Unterschiede in der Zellproliferation im Vergleich zu PC3 oder NRP154 (Prostatakarzinom Ratte) und NRP152 (Prostataepithel Ratte). Northern-blots mit Poly(A)RNA von verschiedenen Prostata-Zellinien (benigne und maligne) nach Behandlung mit Retinoic acid (RA) bestätigte dosisabhängige Unterschiede in der Expression der Androgenrezeptor (AR)-mRNA.Umgekehrt verursachte die Behandlung mit Testosteron in verschiedenen Prostata-Zellinien (benigne und maligne) Unterschiede in der Expression der Retinoid-Rezeptor-mRNA für RAR( und RAR(. Die Ergebnisse unterstützen somit die Hypothese einer Interaktion von Retinoiden und Androgenen mit deren respektiven Rezeptoren. Untersuchungen an humanem Prostatagewebe bestätigten Unterschiede in der Expression von RAR(mRNA und RAR(mRNA mittels RT-PCR. Durch immunhistochemische Untersuchungen an humanem Prostatagewebe mit Antikörpern gegen RAR ( und RAR( konnten deren Lokalisation und Expression nachgewiesen werden. Dabei zeigte sich wiederum eine erhöhte Immunreaktivität von RAR( beim Prostatakarzinom, im Gegensatz zu RAR(, das bei benignem Prostataepithelium eine deutlich stärkere Immunreaktivität aufwies. Zusammenfassend belegen unsere Untersuchungen, daß Retinoide einen meist wachstumshemmenden Effekt auf Prostatakarzinomzellinien verursachen, der wahrscheinlich neben Bindung an Retinoid-Rezeptoren (RARs`) durch Interaktion mit dem Androgenrezeptor (AR), vermittelt durch Hemmung verschiedener membrangebundener Zellproteine und Rezeptoren, wie EGF-R verursacht wird. / Retinoids are steroids, derivatives of vitamine A, that excert their activities by interaction with the retinoic acid receptors (RARs') located in the cell nucleus. The RARs possess structural and functional similarities to the androgen receptor (AR). Investigations in cell cultures demonstrated that the effect of retinoic acid (RA) on cell proliferation is dependent not only on the RA dosage, but also on the androgen receptor status of the cell line. LNCaP cells showed a difference in cell proliferation when treated with RA, as opposed to PC3 or NRP154 (rat prostate cancer cell line) and NRP152 (rat prostate epithelial cell line). Northern blots with Poly(A)RNA from different prostate cell lines (benigne and maligne, with different androgen dependency) when treated with different concentrations of RA, demonstrated a dose-dependent expression of the androgen receptor (AR)mRNA. Conversely resulted the treatment with different concentrations of testosterone to different expressions of the RAR-mRNAs. The results, therefore, support the hypothesis of an interaction of retinoids and androgen with their respective receptors. Investigations with human prostate tissue (malignant and benign) confirms differences in RAR-expression byRT-PCR and immunhistochemistry. Again, prostate cancer showed an overexpression of RAR(, whereas benign prostate tissue demonstrated an overexpression of RAR(. Our investigations, in summary, demonstrate the overall inhibitory effect of retinoids with respect to cell proliferation in prostate cancer cell lines. There is evidence , that this biologic effect is not only triggert by interaction of retinoids and androgens with their own receptors, but occurs by cross-interaction of retinoids with the androgen receptor and androgens with the RARs. Furthermore, the biologic effect of retinoids on cell growth is dependent on membrane bound receptors, such as EGF-R.

Recentemente, tem sido demonstrado que a maioria dos RNAs transcritos em células humanas são RNAs não-codificadores de proteínas (ncRNAs) originados de íntrons ou regiões intergênicas. Em trabalhos anteriores realizados por nosso grupo, foram descritos longos ncRNAs transcritos de regiões intrônicas de genes codificadores e cuja expressão foi correlacionada ao grau de diferenciação de tumores de próstata, apontando para a relevância fisiológica desta classe de transcritos. Apesar de sua abundância, as propriedades, funções e regulação da grande maioria dos ncRNAs ainda não foram elucidadas. O objetivo do presente trabalho foi investigar a biossíntese de ncRNAs intrônicos em células humanas, primordialmente a contribuição da RNA Polimerase II (RNAP II), bem como aspectos de sua regulação. Primeiramente, o modelo de regulação da expressão gênica por hormônio andrógeno foi utilizado para avaliação da participação direta de um fator de transcrição de RNAP II, o Receptor de Andrógeno (AR), na modulação da transcrição de ncRNAs intrônicos. Utilizando-se a técnica de imunoprecipitação da cromatina, foi detectada a ligação do AR ao elemento de resposta a andrógeno (ARE) presente em um possível promotor de um transcrito intrônico antisenso (derivado do locus Myo5A), cuja expressão é aumentada em células da linhagem LNCaP tratadas com o hormônio. A ligação ao ARE foi induzida pelo tratamento, sugerindo que o efeito do andrógeno na expressão do ncRNA é mediado pelo AR. Em uma segunda abordagem, o efeito da inibição da transcrição por RNAP II com α-amanitina por 24 h em células LNCaP foi avaliado com o uso de microarranjos de oligonucleotídeos representando transcritos total ou parcialmente intrônicos, além de éxons de genes codificadores. A expressão de menos de 20 % dos transcritos intrônicos foi afetada, fração significativamente menor que a observada para os transcritos exônicos (40 %). Ainda que a maioria dos ncRNAs intrônicos diferencialmente expressos tenha sua abundância diminuída, interessantemente, 13 a 16 % foram aumentados, contrastando com aproximadamente 2 a 3 % de exônicos que aumentaram. Os resultados obtidos neste trabalho indicam que a RNAP II atua na transcrição de ncRNAs intrônicos, mas que uma fração considerável pode ser transcrita por outra RNA Polimerase. / It has been recently shown that the bulk of the transcription in human cells is comprised of non-protein-coding RNAs (or noncoding RNAs - ncRNAs) transcribed from introns and intergenic regions of the genome. Previous work from our group has demonstrated that expression of long intronic ncRNAs can be correlated to the degree of prostate tumor differentiation, underscoring the physiological relevance of these transcripts. However, the properties, functions, and regulation of this huge population of ncRNAs remain largely unknown. The present work aimed to investigate the biosynthesis of intronic ncRNAs and aspects of its regulation in human cells, focusing on the contribution of RNA Polymerase II (RNAP II). Initially, the model of regulation of gene expression by androgen hormone was used in order to evaluate the participation of the RNAP II transcription factor Androgen Receptor (AR) in the transcriptional regulation of intronic ncRNAs. Chromatin immunoprecipitation experiments revealed the binding of the AR in an androgen response element (ARE) present in a putative promoter driving the expression of an antisense intronic transcript in Myo5A locus in LNCaP cells. The interaction occurred in an androgen-inducible fashion, along with the up-regulation of the transcript, suggesting that hormone activation occurred in a direct manner mediated by the AR. In a different approach, the effect of RNAP II inhibition with α-amanitin for 24 h in LNCaP cells was analyzed using an oligoarray representing totally and partially intronic transcripts, as well as exons of proteincoding genes. The expression of less than 20 % of the intronic transcripts was affected by the treatment, contrasting to a significantly higher fraction observed for exonic messages (40 %). Moreover, most differentially expressed intronic transcripts were down-regulated, but strikingly 13 to 16 % were up-regulated in cells with blocked RNAP II, while this fraction for exonic transcripts was about 2 %. The results described here demonstrate that RNAP II in fact plays a role in intronic transcription in human cells, but also highlight that another transcriptional system may account for the biogenesis of a fraction of intronic ncRNAs.

Introdução: O homem transexual (HT) é um indivíduo de sexo genético feminino, com fenótipo feminino normal, que deseja viver e ser aceito como um membro do sexo masculino. O tratamento hormonal que é realizado no processo de redesignação sexual nesses pacientes consiste na administração de testosterona nas suas diversas apresentações, mais comumente ésteres de testosterona de curta ou longa ação. O tratamento hormonal visa induzir virilização, através da produção de um padrão masculino de crescimento dos pelos faciais e corporais, aumento da massa muscular e interrupção dos ciclos menstruais. O efeito da terapia androgênica na saúde cardiovascular de HT é pouco conhecido, principalmente em relação às repercussões em longo prazo. O HT representa um modelo ideal e único para a avaliação das ações da testosterona exógena administrada em doses suprafisiológicas em um organismo geneticamente feminino. Alguns estudos de farmacogenética demonstraram a influência da repetição CAG do gene do receptor androgênico (RA) nos efeitos observados durante terapia com testosterona em homens hipogonádicos e a maioria dos estudos confirmou a modulação desses polimorfismos sobre fatores de risco cardiovascular. Objetivos: avaliar em HT em tratamento androgênico a prevalência de fatores clássicos de risco cardiovascular e as propriedades estruturais e funcionais dos vasos arteriais; correlacionar a distribuição alélica do microssatélite CAG RA com a ocorrência de comorbidades e com as propriedades estruturais e funcionais dos vasos arteriais; comparar os valores das propriedades estruturais e funcionais dos vasos arteriais de HT com uma população controle (feminina e masculina). Pacientes: 46 pacientes com diagnóstico de HT (faixa etária 42 ± 10 anos) acompanhados no Ambulatório da Unidade de Disforia de Gênero do HCFMUSP e em tratamento com ésteres de testosterona há pelo menos um ano (variação de 1 a 38 anos) foram selecionados para o estudo. Métodos: Parâmetros clínicos (IMC, circunferência abdominal, relação cintura quadril, pressão arterial e pressão de pulso, composição corporal por bioimpedância), a presença de comorbidades (hipertensão arterial, dislipidemia, diabetes mellitus, obesidade) e vícios (tabagismo, etilismo e uso de drogas ilícitas), dados laboratoriais (hematócrito, glicemia de jejum, insulina, índice HOMA IR, hemoglobina glicada, colesterol total, HDL colesterol, LDL colesterol, triglicerídeos e creatinina) e parâmetros vasculares (espessura íntima média da carótida, diâmetro da carótida, percentual da variação sisto-diastólica da carótida e velocidade de onda de pulso dos vasos arteriais) foram avaliados no grupo de HT. Os mesmos parâmetros vasculares também foram avaliados em controles saudáveis masculinos e femininos pareados para idade e IMC com os HT. A distribuição alélica do microssatélite CAG RA foi avaliada em 44 HT através da análise do produto amplificado da região de repetições CAG do exon 1 do gene do RA, utilizando o software GeneMapper. Resultados e Conclusões: Neste grupo de HT em terapia com ésteres de testosterona observamos uma prevalência de dislipidemia de 42%, hipertensão arterial sistêmica de 35%, obesidade de 30%, diabetes de 4% e tabagismo de 20%. HT em tratamento androgênico apresentaram maior velocidade de onda de pulso carotídeo-femoral do que controles masculinos, mas não do que controles femininos, embora no subgrupo >= 42 anos os HT tenham apresentado maior VOP do que controles masculinos e femininos. Não houve diferença de diâmetro, distensão relativa e espessura íntima média carotídea entre HT e controles. Maior diâmetro, maior espessura íntima média e menor distensão relativa da carótida foram observados em HT obesos e hipertensos; e maior velocidade de onda de pulso aórtica em HT hipertensos. Os parâmetros correlacionados à medida funcional da artéria aorta foram a idade, o tempo de tratamento androgênico e a relação cintura-quadril, enquanto que as propriedades estruturais e funcionais da carótida se correlacionaram com idade, parâmetros antropométricos e glicêmicos. Não houve influência do trato CAG RA na comparação entre os HT com e sem comorbidades metabólicas. Repetições CAG RA curtas se associaram com níveis significativamente mais elevados de glicemia de jejum, insulina basal e HOMA IR. Em relação aos parâmetros antropométricos, pressóricos, lipídicos e arteriais, não foi identificada associação com o número de repetições CAG RA. Estes achados sugerem um potencial efeito deletério da terapia androgênica prolongada sobre os vasos arteriais e a necessidade de medidas preventivas em HT / Introduction: Transgender men (TM) are 46, XX individuals, with normal female phenotype, who desire to live and be accepted as a male member. Testosterone esters are used in sex reassignment therapy to induce virilization and to adapt the body to the male identity. The effects of androgen therapy on TM cardiovascular function are poorly known, particularly with regard to long-term androgen treatment. TM represents a good model for evaluation of high-dose exogenous testosterone action in biological women. Pharmacogenetic studies have demonstrated the influence of CAG polymorphic tract of the androgen receptor gene (AR) on the androgenic effects observed during testosterone therapy in hypogonadal men, and most studies confirmed the modulation of these polymorphisms on cardiovascular risk factors. Objective: to evaluate the prevalence of cardiovascular risk factors and the structural and functional properties of large arteries in TM on long-term cross sex hormone therapy compared to a male and female healthy control group; to correlate the allelic distribution of CAG AR polymorphic tract with the cardiovascular comorbidities and the structural and functional properties of large arteries in TM. Patients: Forty-six patients with a diagnosis of TM (42 ± 10 years old), followed at the Gender Dysphoria Unit-HCFMUSP, receiving cross-sex hormone treatment with testosterone esters for at least one year (ranging from 1 to 38 years) were selected for the study. Methods: Clinical parameters (BMI, waist circumference, waist-to-hip ratio, blood pressure, pulse pressure, body fat percentage), the presence of cardiovascular comorbidities (hypertension, dyslipidemia, diabetes mellitus, obesity) and addictions (smoking, alcohol and drug abuse), laboratory parameters (hematocrit, fasting plasma glucose, basal insulin, HOMA IR index, glycated hemoglobin, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides and creatinine) and vascular parameters (carotid intima-media thickness, carotid diameter, carotid relative distensibility and aortic pulse wave velocity - PWV) were evaluated in the TM group. The same vascular parameters were also evaluated in healthy male and female control group, matched for age and BMI. The allelic distribution of the CAG AR polymorphic tract was evaluated in 44 TM using the GeneMapper software. Results and Conclusions: In the TM group, we observed dyslipidemia in 42%, hypertension in 35%, obesity in 30%, diabetes in 4% and smoking habit in 20%. The mean aortic PWV values in TM was higher than in male healthy controls (p=0.005), but not than in female controls (p=0.640). When categorized by age, considering the median age, TM >= 42 years had higher aortic PWV measures than male (p < 0.001) and female (p = 0.024) controls, regardless of their arterial blood pressure values. There was no difference in carotid diameter, carotid relative distensibility and carotid intima-media thickness between TM and controls. Obese and hypertensive TM presented significantly higher values of carotid diameter and carotid intima-media thickness, and lower values of carotid relative distensibility than healthy transgenders. Hypertensive TM showed higher aortic PWV values than non-hypertensive TM. The aortic stiffness correlated significantly and positively with age, androgen treatment duration and waist-to-hip ratio in TM. Properties of the carotid artery correlated with age, anthropometric parameters and glycemic parameters in TM. Shorter CAG polymorphic tracts of TM were associated with higher levels of fasting plasma glucose, basal insulin and HOMA IR index. There was no influence of the CAG polymorphic tract of TM on the presence of cardiovascular comorbidities, anthropometric, pressure, lipid and arterial parameters. These findings suggest a potential deleterious effect of the long-term testosterone therapy on vessels and the need for preventive measures in TM

Le récepteur des androgènes (RA) est la principale cible thérapeutique du cancer de la prostate (CaP) métastatique. Bien que cette thérapie soit initialement efficace, les effets sont transitoires. De nombreux mécanismes peuvent expliquer la progression du CaP vers un stade de résistance à la castration, telles les modifications du RA. Des données récentes ont montré que les variants constitutifs RA-Q641X et RA-V7, caractérisés par la perte du domaine de liaison au ligand, étaient associés à l’expression de marqueurs mésenchymateux. L’étude de la régulation de la N-cadhérine a mis en évidence que si le RA sauvage et les variants constitutifs se liaient tous deux aux éléments de réponse du gène codant, seuls les derniers étaient associés à une augmentation de l’acétylation de l’histone H4, marque positive de la transcription. Le RNA-seq a révélé que leur expression était aussi corrélée à la régulation de sets de gènes spécifiques incluant des facteurs de transcription dont certains ont déjà été caractérisés en cancérologie.En ce qui concerne le RA-T576A, porteur d’une mutation faux-sens, les données ont révélé une séquence consensus de liaison à l’ADN moins conservée pour le RA sauvage que pour ce mutant et l’importance du 11ème nucléotide des éléments de réponse. De plus, cette mutation a semblé impacter le transcriptome du RA. Ce travail met en évidence le comportement distinct des variants du RA et aide à mieux comprendre leurs modes d’action en décrivant leurs activités transcriptionnelles. / The androgen receptor (AR) is the main therapeutic target in metastatic prostate cancer (PCa). Although this therapy is initially effective, the effects are transient. Many mechanisms can explain PCa progression toward castration resistance including abnormalities in the AR. Recent data have shown that constitutive AR (e.g AR-Q641X and AR-V7), which have lost the ligand binding domain, were associated with the induction of mesenchymal marker expression. The study of N-cadherin regulation highlighted that while both constitutive AR and wild type AR bound to response elements located in the encoding gene, only the AR variants were associated with an increase of H4 acetylation, a positive transcription mark. RNA-seq revealed that their expression was also correlated to specific sets of genes regulation, including transcription factors and genes involved in migration, AR regulation, and therapeutic resistance.Concerning AR-T576A, which hold a missense mutation, data revealed a less conserved consensus sequence for the wild type AR than for this mutant and highlighted the importance of the 11th nucleotide of the response element for AR recruitment to DNA. Plus, this mutation seemed to impair AR transcriptome. This work highlights the distinct AR variants’ behavior and helps to understand their mode of action by depicting their transcriptional landscapes.

Die im Rahmen dieser Arbeit durchgeführten Untersuchungen zum Einfluss der Wachstumsfaktoren IGF-I, EGF und dem Zytokin IL-6 auf den Androgenrezeptor-Signalweg zeigten in verschiedenen Prostatakarzinom-zelllinien schon nach zwei Stunden eine deutliche Degradation des Androgenrezeptor-Proteins. Die ausschließlich auf Protein-Ebene stattfindende, Wachstumsfaktor-induzierte negative Regulation des Androgenrezeptors konnte durch einen schnellen Androgeneffekt wieder aufgehoben werden. Mittels Luziferase-Reportergen-Assays wurde kein Einfluss der Wachstums-faktorwirkung auf die transkriptionelle Aktivität des Androgenrezeptors nachgewiesen. Darüber hinaus konnte eine signifikant reprimierende Wirkung durch IGF-I und EGF in Kombination mit geringen Mengen DHT beobachtet werden. Weitere Resultate dieser Arbeit deuten auf einen, durch den PI3-Kinase-Signalweg vermittelten, proteasomalen Abbauprozess des Rezeptors hin. Da die Suppression der downstream gelegenen Proteinkinase Akt keine Veränderung hinsichtlich der Degradation aufwies, konzentrierte sich die weiterführende Arbeit auf eine mögliche direkte Regulation des Androgen-rezeptors durch die PI3-Kinase. Unter Verwendung von rekombinanten GST-Fusionsproteinen konnte in Interaktionsstudien unter in vitro Bedingungen eine Phosphotyrosin-unabhängige Bindung zwischen der C-SH2-Domäne der p85-Untereinheit der PI3-Kinase und dem N- und C-Terminus des Androgenrezeptors nachgewiesen werden. Durch die nähere Charakterisierung dieser Bindungsbereiche mit Hilfe von Peptidarrays und anschließenden Alanin-Substitutionen war es möglich, für den N-Terminus 18, für den C-Terminus des Androgenrezeptors 6 und für die p85-C-SH2-Domäne der PI3-Kinase 11 Aminosäuren zu identifizieren. Die durch gezielte Punktmutagenese an diesen Aminosäurepositionen hergestellten Androgenrezeptor-Einzel- und -Mehrfachmutanten wiesen in Bindungsstudien dennoch Interaktion zur PI3-Kinase auf. Eine von Anderson und Kollegen postulierte Phosphotyrosin-unabhängige Bindung der SH2-Domänen der p85-Untereinheit der PI3-Kinase durch sogenannte „basic-X-basic“-Motive wurde ebenfalls in Interaktionstests zwischen der PI3-Kinase und dem Androgenrezeptor überprüft. Aufgrund der Tatsache, dass einige der identifizierten Aminosäuren auf dem Androgenrezeptor Teil eines „basic-X-basic“-Bindungsmotives sind, wurden Kombinationsmutanten generiert, die sowohl im N-Terminus als auch im C﷓Terminus des Androgenrezeptors ein bzw. zwei zerstörte „basic-X-basic“-Motive enthielten. Untersuchungen zum Bindungsverhalten dieser Mutanten zeigten zwar weiterhin Interaktion zur p85-C-SH2-Domäne der PI3-Kinase, jedoch der durch Western-blot-Analyse überprüfte IGF-I-induzierte Degradationseffekt des Androgenrezeptor-Proteins konnte mit zwei der verwendeten Androgenrezeptor-Kombinationsmutanten nicht mehr beobachtet werden.

Η επιθηλιακή προς μεσεγχυματική μετατροπή είναι ένα φαινόμενο που πιθανότατα εμπλέκεται στην παθογένεια του καρκίνου του λάρυγγα. Η ΕΜΤ εξελισσόμενη μέσα από δαιδαλώδη μονοπάτια μεταγωγής σήματος καταλήγει να προσδώσει στο καρκινικό κύτταρο δομικά και λειτουργικά χαρακτηριστικά που το καθιστούν ικανό να μπορεί να διεισδύει στους ιστούς και να μεθίσταται. Κεντρικό μόριο στα μοριακά μονοπάτια που διαμεσολαβούν την ΕΜΤ στον καρκίνο του λάρυγγα είναι μια κινάση, η ILK, που δέχεται σήματα από τις ιντεγκρίνες και τους υποδοχείς αυξητικών παραγόντων. Στους επιθηλιακούς καρκίνους αναφέρεται η εμπλοκή της σε λειτουργίες όπως ρύθμιση του κυτταρικού κύκλου, αποφυγή της απόπτωσης, νεοαγγειογένεση, απώλεια των δομών συνοχής του κυττάρου, έκφραση μεταλλοπρωτεασών και αναδιαμόρφωση του κυτταροσκελετού. Στο λαρυγγικό καρκίνο όμως κρίσιμα φαινόμενα για τον μεταστατικό-επιθετικό χαρακτήρα των κυττάρων, όπως, η εξαφάνιση των E-cadherin, η μετακίνηση των β-catenin στον πυρήνα και η συσχέτιση μεταξύ τους, που διαπιστώθηκαν, δεν βρέθηκε να συνδέονται με την υπερέκφραση της ILK καθιστώντας προφανώς άλλους μηχανισμούς υπεύθυνους για την επιτέλεση αυτών των λειτουργιών. Ιδιαίτερα ενδιαφέρουσα όμως ήταν και η εντόπιση της ILK στον πυρήνα των κυττάρων του καρκίνου του λάρυγγα δίνοντας μια νέα προοπτική στην έρευνα για τον ρόλο της ILK στον καρκίνο. Στο μονοπάτι μεταγωγής σήματος της ILK στο λαρυγγικό καρκίνο βρέθηκε πως συμμετέχει και η ενεργοποιημένη Akt με την οποία επίσης σχετίζονται πολλές κρίσιμες λειτουργίες για το καρκινικό κύτταρο. Ωστόσο η p-Akt στο λαρυγγικό καρκίνο φαίνεται πως έχει κάποιο ρόλο αντίθετο με την λειτουργία του καρκινικού κυττάρου καθώς χαρακτηρίζει όγκους καλύτερης διαφοροποίησης. Ο λαρυγγικός καρκίνος τέλος διαπιστώθηκε πως χαρακτηρίζεται από την έκφραση υποδοχέων ανδρογόνων και οιστρογόνων καθιστώντας πολύ πιθανό το ρόλο αυτών των μορίων στην παθογένεια της νόσου. Ενδιαφέρουσα για την πιθανότητα εμπλοκής των υποδοχέων στερεοειδών ορμονών του φύλου στην ΕΜΤ ίσως να είναι η συσχέτιση των υποδοχέων ανδρογόνων και οιστρογόνων με την ILK και p-Akt αντίστοιχα. Οι υποδοχείς οιστρογόνων μάλιστα χαρακτηρίζοντας όγκους λάρυγγα αρχικών σταδίων ίσως θα μπορούσε να αποδειχτεί μόριο με προγνωστική αξία αλλά και θεραπευτικός στόχος. Τέλος η μελέτη της έκφρασης της ILK, της p-Akt και των υποδοχέων στεροειδών ορμονών του φύλου δεν ανέδειξε μια διαφορετική έκφραση μεταξύ υπεργλωττιδικών και γλωττιδικών καρκίνων του λάρυγγος ώστε να υποστηρίξει την ύπαρξη ενός μοριακού υποβάθρου στην παρατήρηση της ανόμοιας κλινικής συμπεριφοράς μεταξύ όγκων από τις δύο αυτές ανατομικά διακριτές περιοχές. / Epithelial to mesenchymal transition (EMT) is a process possibly implicated in the pathogenesis of laryngeal cancer. EMT is a type of epithelial cell plasticity which is characterized by long lasting phenotypic and molecular modifications of the epithelial cell as a result of a transforming procedure leading to a cell of mesenchymal type. This molecular procedure seems to be pivotal for the metastasis of epithelial cancers and its attribution to the epithelial cells is the gain of structural and functional traits which enable them to invade the tissues and metastaze. In the current study ILK expression, which is a central molecule in the signal transduction pathways of EMT, seems to be implicated in human laryngeal carcinoma. Furthermore, localization of ILK in the nucleus possibly suggests novel nuclear functions of ILK. In addition, enhanced ILK expression in laryngeal carcinoma correlates with activation of Akt. Moreover, while activated Act seems to characterize well differentiated tumors, loss of E-cadherin and activation of β-catenin were correlated with high grade carcinomas. Finally, in laryngeal cancer, mechanisms other than ILK overexpression seem to account for downregulation of E-cadherin and activation of β-catenin. Additionaly this study concluded that estrogen and androgen receptors are expressed in laryngeal carcinomas, indicating their possible role in the pathogenesis of this disease. It is interesting that the receptors of gender-related steroid hormones could have a possible relation to epithelial to mesenchymal transition phenomenon since a correlation between androgen and estrogen receptors with ILK and p-Akt respectively, was revealed. Moreover estrogen receptors characterize primary TMN-stage laryngeal carcinomas. This can be very important as it makes a prospect of using ER as a prognostic factor but even more as a novel hormonal therapy for laryngeal carcinomas. Finally the study of molecules like ILK, p-Akt, estrogen and androgen receptors did not reveal any differentiantal expression between glotic and supraglotic laryngeal carcinomas in order to support the existence of a molecular background, at least as far as those molecules are concerned, to the distinct clinical outcome of those different anatomically-derived laryngeal carcinomas. Metastasis is a rapid development in the ominous course of cancer. The effort to interpret the molecular basis of this phenomenon is not a subject of simple academic interest since the exploit of the molecular mechanisms so as to gain the control of metastasis could be the ‘‘bet’’ for a futuristic ‘‘molecular surgery’’.

No description available.

The enzymes CYP27A1 and CYP7B1 are widely expressed in various human tissues and perform catalytic reactions in cholesterol homeostasis and endocrine signaling. We have investigated the metabolism of a synthetic oxysterol. In this study, we show that CYP27A1 is the enzyme responsible for a 28-hydroxylation of this oxysterol and that the rate of CYP27A1-mediated metabolism is relatively slow. This may give an explanation for the prolonged inhibitory effects on cholesterol biosynthesis that have been shown for this oxysterol. The current study contributes to the knowledge of synthetically produced oxysterols and their potential use as cholesterol lowering drugs. In two studies we investigated CYP7B1-mediated metabolism of different sex hormones. Our data indicate that CYP7B1 may carry out a previously unknown catalytic reaction involving an androgen. Taken together the data suggest that varying steroid concentrations in cells and tissues may be important for CYP7B1-dependent metabolism of sex hormones and sex hormone precursors. CYP7B1-mediated hydroxylation of sex hormones may influence the cellular levels of these steroids and may be a potential pathway for elimination of the steroids from the cell. Some known CYP7B1 substrates are agonists for ERα and ERβ but the reported role(s) of CYP7B1 for ER action are not fully understood. In the last study we investigated the role(s) of CYP7B1-mediated metabolism for ER-mediated action. Our data indicate that CYP7B1-mediated conversion of steroids that affect ER-mediated response into their 7α-hydroxymetabolites will result in loss of action. This indicates that CYP7B1 may have an important role for regulation of ER-mediated processes in the body. In summary, results from this thesis contribute to the knowledge on the metabolism of synthetic oxysterols of potential use as cholesterol lowering drugs and the role(s) of CYP7B1-mediated metabolism for processes related to the functions of sex hormones. / Disputationsordförande;Professor Eva Brittebo, Inst. för Biovetenskap, Avd. för Toxikologi, Uppsala Universitet, UppsalaBetygsnämndens ledamöten; Docent Lena Ekström, Inst. för Laboratoriemedicin, Avd. för Klinisk Kemi, Karolinska Universitetssjukhuset, HuddingeDocent Ulf Diczfaluzy, Inst. för Laboratoriemedicin, Avd. för Klinisk Kemi, Karolinska Universitetssjukhuset, HuddingeProfessor Agneta Oskarsson, Inst. BVF, Avd. för farmakologi och toxikologi, SLU, Uppsala