O melanoma cutâneo é um tipo de tumor formado a partir dos melanócitos, células de origem neuroectodérmica que habitam a epiderme, sendo responsáveis por sua pigmentação. Embora este tumor seja o tipo menos frequente de câncer de pele, ele está associado com altas taxas de mortalidade, principalmente devido a seu comportamento agressivo (alta capacidade metastática) e quimiorresistência aos tratamentos (quimioterapia e radioterapia). Os processos da quimiorresistência em melanoma ainda não são totalmente conhecidos. Estudos prévios de nosso grupo evidenciaram que a expressão da proteína Proibitina (PHB) encontra-se aumentada em células de melanoma frente à exposição a certos quimioterápicos, executando sua função de molécula anti-apoptótica, quando localizada no citoplasma, e/ou supressora tumoral quando no núcleo. Adicionalmente, foi visto que a repressão de PHB sensibiliza as células de melanoma, enquanto a sua superexpressão protege da morte celular induzida por cisplatina. Além disso, estudos de associação genótipo-fenótipo revelaram que o alelo menos frequente/raro do SNP rs6917 (C1703T) na região 3\'UTR do gene da PHB foi associado com o risco aumentado de desenvolvimento do melanoma. O objetivo do nosso trabalho foi avaliar o envolvimento do SNP rs6917 da 3\'UTR do gene PHB em linhagens de melanoma humano e sua resposta celular frente ao tratamento com agentes quimioterápicos indutores de estresse celular como temozolomida, cisplatina e vemurafenib. Para avaliar a contribuição do polimorfismo rs6917 no desenvolvimento de melanoma, foi desenvolvido um estudo tipo caso-controle numa população brasileira. O estudo analisou 198 pacientes com melanoma e 200 controles. Em ensaios in vitro as linhagens celulares de melanoma humano SK-Mel 05 e UACC-62 (BRAFV600E mutadas) foram transfectadas com as variantes polimórficas UTR/C e UTR/T clonadas no plasmideo pmirGLO Dual-Luciferase nos sítios de restição NheI/XhoI. A Geneticina (G418) foi utilizada para seleção estável das células, ensaios de western Blot, qRT-PCR e luminiscencia confirmaram a expresão do transgene. As diferentes doses de cisplatina, temozolomida e vemurafenib foram definidas para o tratamento das células. Para avaliar a morte celular foi realizada a técnica de citometria de fluxo após incorporação com iodeto de propidio. Após os tratamentos, foram realizados ensaios clonogênicos e de imunofluorescencia para determinar sobrevivencia celular e localização subcelular da proibitina, respectivamente. Nossos resultados revelaram que variáveis clinicas como a presença de cabelos claros (loiros ou ruivos), mais de 20 pintas, exposição solar intermitente, queimaduras solares na infância e adolescência são fatores de risco para o aparecimento de melanoma. Nos casos, os portadores do alelo T mostraram um risco aumentado em 5,6 vezes para o desenvolvimento de melanoma de crescimento vertical em comparação com os pacientes com genótipo CC. Ensaios in vitro, mostraram que células de melanoma humano superexpressando o alelo raro 3\'UTR/T após tratamento com cisplatina, temozolomida e vemurafenib apresentavam um fenotipo mais proliferativo e clonogênico com menor morte celular quando comparadas com as células 3\'UTR/C e vetor vazio. Ensaios de Western blot e bioluminescência mostraram, respectivamente, um aumento na expressão e atividade do gene da luciferase nas células 3\'UTR/T. Estes resultados mostram que o SNP rs6917 modula a expressão de PHB e que o alelo raro T representa um polimorfismo funcional promovendo a quimiorresistência em melanoma / Melanoma is a type of skin tumor formed from melanocytes, cells of neuroectodermal origin that inhabit the epidermis, being responsible for its pigmentation. Although its low incidence, melanoma is associated with high rates of mortality due to its resistance to chemotherapy. The mechanisms involved in melanoma chemoresistance are not well fully understood yet, therefore, the comprehension of this phenomenon may be useful for the development of new treatment strategies. Previous results from our laboratory showed that Prohibitin (PHB), a protein with diverse functions including regulation of cell cycle progression and apoptosis, was overexpressed in human melanoma cells lines when exposed to high-doses of the chemotherapy drug, cisplatin. PHB knockdown sensitized melanoma cells, meanwhile PHB recombinant overexpression protected melanoma cells to cisplatin-induced cell death. Studies of genotype-phenotype association revealed that the Single Nucleotide Polymorphism rs6917 at the portion 3\'UTR of the PHB gene showed an association with some risk factors for the development of melanoma. The aim of this study was to investigate if the SNP rs6917 affects PHB protein function by modulating cell proliferation and chemoresistance in human melanoma cell lines when exposed to stress inductors agents such as cisplatin, temozolomide and vemurafenib. A hospital-based case-control study was carried out in a Brazilian sample to evaluate the contribution of rs6917 polymorphism in melanoma development. The study comprised 198 melanoma patients and 200 controls. For invitro assays SK-Mel 05 and UACC-62 human melanoma cell lines (both BRAFV600E mutated) were used. The 852bp of PHB-3\'UTR harboring wild-type (3\'UTR/C) or the rare allele (3\'UTR/T) were cloned at pmirGLO Dual-Luciferase plasmid at NheI/XhoI restriction sites and stable cell lines were generated. Geneticin (G418) was used for stable selection, qRT-PCR, Western Blot and luciferase assays confirmed the transgene expression. Different doses of cisplatin, temozolomide and vemurafenib were defined to treat the cells. Cell death was evaluated using flow cytometry after propidium iodide incorporation. Cell survival was assessed by clonogenic assay after cells undergone treatment. Our results revealed that clinical variables like presence of light hair, more than 20 moles, intermintent sun expossure and sunburns at childhood are risk factors for melanoma development. We also showed that T allele carriers have a 5,6 times increased risk to develop vertical growth melanoma in comparison with the CC genotype patients. In vitro assays with transfected melanoma cells, SK-Mel 05 and UACC-62, overexpressing the rare allele 3\'UTR/T showed a more proliferative and clonogenical phenotype and less induced cell death after cisplatin, temozolomide and vemurafenib treatment when compared to cells overexpressing the wild-type allele 3\'UTR/C and empty vector. Westernblot and bioluminiscence assays showed respectively an increase in the expresion and activity of the luciferase gene of the 3\'UTR/T cells in comparison with the 3\'UTR/C and empty vector cells. All together these results showed that the SNP rs6917 modulates the expression of PHB and that the rare allele T represents a functional polymorphism by promoting a chemoresistance phenotype in melanoma

O melanoma cutâneo é um tipo de tumor formado a partir dos melanócitos, células de origem neuroectodérmica que habitam a epiderme, sendo responsáveis por sua pigmentação. Embora este tumor seja o tipo menos frequente de câncer de pele, ele está associado com altas taxas de mortalidade, principalmente devido a seu comportamento agressivo (alta capacidade metastática) e quimiorresistência aos tratamentos (quimioterapia e radioterapia). Os processos da quimiorresistência em melanoma ainda não são totalmente conhecidos. Estudos prévios de nosso grupo evidenciaram que a expressão da proteína Proibitina (PHB) encontra-se aumentada em células de melanoma frente à exposição a certos quimioterápicos, executando sua função de molécula anti-apoptótica, quando localizada no citoplasma, e/ou supressora tumoral quando no núcleo. Adicionalmente, foi visto que a repressão de PHB sensibiliza as células de melanoma, enquanto a sua superexpressão protege da morte celular induzida por cisplatina. Além disso, estudos de associação genótipo-fenótipo revelaram que o alelo menos frequente/raro do SNP rs6917 (C1703T) na região 3\'UTR do gene da PHB foi associado com o risco aumentado de desenvolvimento do melanoma. O objetivo do nosso trabalho foi avaliar o envolvimento do SNP rs6917 da 3\'UTR do gene PHB em linhagens de melanoma humano e sua resposta celular frente ao tratamento com agentes quimioterápicos indutores de estresse celular como temozolomida, cisplatina e vemurafenib. Para avaliar a contribuição do polimorfismo rs6917 no desenvolvimento de melanoma, foi desenvolvido um estudo tipo caso-controle numa população brasileira. O estudo analisou 198 pacientes com melanoma e 200 controles. Em ensaios in vitro as linhagens celulares de melanoma humano SK-Mel 05 e UACC-62 (BRAFV600E mutadas) foram transfectadas com as variantes polimórficas UTR/C e UTR/T clonadas no plasmideo pmirGLO Dual-Luciferase nos sítios de restição NheI/XhoI. A Geneticina (G418) foi utilizada para seleção estável das células, ensaios de western Blot, qRT-PCR e luminiscencia confirmaram a expresão do transgene. As diferentes doses de cisplatina, temozolomida e vemurafenib foram definidas para o tratamento das células. Para avaliar a morte celular foi realizada a técnica de citometria de fluxo após incorporação com iodeto de propidio. Após os tratamentos, foram realizados ensaios clonogênicos e de imunofluorescencia para determinar sobrevivencia celular e localização subcelular da proibitina, respectivamente. Nossos resultados revelaram que variáveis clinicas como a presença de cabelos claros (loiros ou ruivos), mais de 20 pintas, exposição solar intermitente, queimaduras solares na infância e adolescência são fatores de risco para o aparecimento de melanoma. Nos casos, os portadores do alelo T mostraram um risco aumentado em 5,6 vezes para o desenvolvimento de melanoma de crescimento vertical em comparação com os pacientes com genótipo CC. Ensaios in vitro, mostraram que células de melanoma humano superexpressando o alelo raro 3\'UTR/T após tratamento com cisplatina, temozolomida e vemurafenib apresentavam um fenotipo mais proliferativo e clonogênico com menor morte celular quando comparadas com as células 3\'UTR/C e vetor vazio. Ensaios de Western blot e bioluminescência mostraram, respectivamente, um aumento na expressão e atividade do gene da luciferase nas células 3\'UTR/T. Estes resultados mostram que o SNP rs6917 modula a expressão de PHB e que o alelo raro T representa um polimorfismo funcional promovendo a quimiorresistência em melanoma / Melanoma is a type of skin tumor formed from melanocytes, cells of neuroectodermal origin that inhabit the epidermis, being responsible for its pigmentation. Although its low incidence, melanoma is associated with high rates of mortality due to its resistance to chemotherapy. The mechanisms involved in melanoma chemoresistance are not well fully understood yet, therefore, the comprehension of this phenomenon may be useful for the development of new treatment strategies. Previous results from our laboratory showed that Prohibitin (PHB), a protein with diverse functions including regulation of cell cycle progression and apoptosis, was overexpressed in human melanoma cells lines when exposed to high-doses of the chemotherapy drug, cisplatin. PHB knockdown sensitized melanoma cells, meanwhile PHB recombinant overexpression protected melanoma cells to cisplatin-induced cell death. Studies of genotype-phenotype association revealed that the Single Nucleotide Polymorphism rs6917 at the portion 3\'UTR of the PHB gene showed an association with some risk factors for the development of melanoma. The aim of this study was to investigate if the SNP rs6917 affects PHB protein function by modulating cell proliferation and chemoresistance in human melanoma cell lines when exposed to stress inductors agents such as cisplatin, temozolomide and vemurafenib. A hospital-based case-control study was carried out in a Brazilian sample to evaluate the contribution of rs6917 polymorphism in melanoma development. The study comprised 198 melanoma patients and 200 controls. For invitro assays SK-Mel 05 and UACC-62 human melanoma cell lines (both BRAFV600E mutated) were used. The 852bp of PHB-3\'UTR harboring wild-type (3\'UTR/C) or the rare allele (3\'UTR/T) were cloned at pmirGLO Dual-Luciferase plasmid at NheI/XhoI restriction sites and stable cell lines were generated. Geneticin (G418) was used for stable selection, qRT-PCR, Western Blot and luciferase assays confirmed the transgene expression. Different doses of cisplatin, temozolomide and vemurafenib were defined to treat the cells. Cell death was evaluated using flow cytometry after propidium iodide incorporation. Cell survival was assessed by clonogenic assay after cells undergone treatment. Our results revealed that clinical variables like presence of light hair, more than 20 moles, intermintent sun expossure and sunburns at childhood are risk factors for melanoma development. We also showed that T allele carriers have a 5,6 times increased risk to develop vertical growth melanoma in comparison with the CC genotype patients. In vitro assays with transfected melanoma cells, SK-Mel 05 and UACC-62, overexpressing the rare allele 3\'UTR/T showed a more proliferative and clonogenical phenotype and less induced cell death after cisplatin, temozolomide and vemurafenib treatment when compared to cells overexpressing the wild-type allele 3\'UTR/C and empty vector. Westernblot and bioluminiscence assays showed respectively an increase in the expresion and activity of the luciferase gene of the 3\'UTR/T cells in comparison with the 3\'UTR/C and empty vector cells. All together these results showed that the SNP rs6917 modulates the expression of PHB and that the rare allele T represents a functional polymorphism by promoting a chemoresistance phenotype in melanoma

Le transport actif de sodium par les cellules épithéliales alvéolaires est le principal mécanisme impliqué dans la régulation du niveau de liquide dans le poumon distal. Le canal épithélial sodique (ENaC) exprimé par les cellules épithéliales alvéolaires est essentiel à la résorption du liquide des poumons à la naissance ainsi que la résolution de l'œdème pulmonaire chez l'adulte. L'activité et l'expression du canal ENaC sont modulées par de nombreux stress pathophysiologiques. L'inflammation pulmonaire constitue un facteur important dans l'inhibition de l'expression du canal ENaC et pourrait favoriser la formation d'œdème pulmonaire. Nous avons précédemment démontré que différentes cytokines pro-inflammatoires, ainsi que les lipopolysaccharides (LPS) de Pseudomonas aeruginosa, inhibent l'expression de l'ARNm αENaC par des mécanismes de régulation transcriptionnelle et post-transcriptionnelle. Ces résultats suggèrent que les mécanismes qui modulent la stabilité des ARNm αENaC pourraient jouer un rôle important dans la régulation du niveau d’expression du transcrit en condition inflammatoire. Le principal objectif de mes travaux était de caractériser les mécanismes de modulation de l’ARNm αENaC dans les cellules épithéliales alvéolaires lors de différents stress pathophysiologiques et déterminer si cette modulation pouvait s’expliquer en partie par une régulation de la stabilité du transcrit. Mes travaux montrent que les LPS et la cycloheximide inhibent l’expression de l’ARNm αENaC de façon similaire via l’activation des voies de signalisation des MAPK ERK1/2 et p38. Cependant, les mécanismes de modulation de l’expression de l'ARNm αENaC sont différents puisque les LPS répriment la transcription du gène, alors que la cycloheximide diminuerait la stabilité du transcrit via des mécanismes post-transcriptionnels impliquant la région 3' non traduite (3'UTR) de l'ARNm αENaC. Pour mieux étudier le rôle du 3'UTR dans ce processus, nous avons développé un modèle Tet-Off nous permettant de mesurer la demi-vie de l’ARNm αENaC indépendamment de l’utilisation d’un inhibiteur de la transcription comme l'actinomycine D (Act. D). Nous avons montré que la demi-vie de l’ARNm αENaC était de 100min, un temps beaucoup plus court que celui rapporté dans la littérature. Nous avons démontré que l’Act. D a un effet stabilisateur important sur l’ARNm αENaC et qu’il ne peut être utilisé pour évaluer la stabilité du transcrit. À l’aide de différents mutants de délétion, nous avons entrepris de déterminer la nature des régions du 3’UTR impliquées dans la modulation de la stabilité du transcrit. Nous avons trouvé que le 3’UTR joue un rôle à la fois de stabilisation (région 3’UTR proximale) et de déstabilisation (région 3’UTR distale) du transcrit. Notre système nous a finalement permis de confirmer que la diminution de l’ARNm αENaC observée en présence de TNF-α s’expliquait en partie par une diminution importante de la stabilité du transcrit induite par cette cytokine. Enfin, nous avons identifié la nature des protéines pouvant se lier au 3’UTR de l’ARNm αENaC et déterminé lesquelles pouvaient moduler la stabilité du transcrit. Des trois protéines candidates trouvées, nous avons confirmé que la surexpression de DHX36 et TIAL1 diminue le niveau de transcrit par un mécanisme impliquant la stabilité du messager. Les travaux présentés ici montrent la complexité des voies de signalisation induites par différents stress sur les cellules épithéliales alvéolaires et montrent comment la stabilité de l’ARNm αENaC et en particulier, les séquences du 3’UTR jouent un rôle important dans la modulation du niveau de transcrit. Le modèle Tet-Off que nous avons développé permet d’estimer le temps de demi-vie réel de l’ARNm αENaC et montre que le 3’UTR du messager joue un rôle complexe dans la stabilisation du messager en condition de base ainsi qu’en condition pro-inflammatoire. Enfin, nous avons identifié deux protéines liant l’ARNm qui pourraient jouer un rôle important dans la modulation de la stabilité du transcrit. / The epithelial sodium channel (ENaC) expressed in alveolar epithelial cells plays a major role for lung liquid clearance at birth and lung edema resorption in adulthood. The expression and activity of ENaC are inhibited by many pathophysiological stress that could have an impact in the clinical outcome of acute respiratory distress syndrome (ARDS). Pulmonary inflammation is an important factor in this inhibition that may promote or sustain pulmonary edema. We have previously shown that pro-inflammatory cytokines and lipopolysaccharide (LPS) from Pseudomonas aeruginosa inhibit αENaC mRNA expression by transcriptional and post-transcriptional mechanisms, suggesting that a modulation of αENaC mRNA stability could play a role in this process. The main objective of the present work was to characterize how different pathophysiological stress affect αENaC mRNA expression in alveolar epithelial cells and determine whether this modulation could be explained in part by regulating the stability of the transcript. Our study shows that LPS and cycloheximide decrease the level of αENaC mRNA with a similar time course and via the activation of the MAPK ERK1/2 and p38 signaling pathways. Despite similarities, there were important differences in the mechanisms involved in the modulation of αENaC mRNA expression. While LPS repress αENaC mRNA transcription, cycloheximide triggers post-transcriptional mechanisms involving the 3' untranslated region (3'UTR) of αENaC mRNA. To further study the role of αENaC 3'UTR in this process, we developed a Tet-Off model that allows us to measure the half-life of αENaC mRNA regardless of the use of a transcription inhibitor such as actinomycin D (Act. D). Using this system, we showed a 100 min half-life for αENaC mRNA, a much shorter time then the one reported for this mRNA using Act. D. We showed that Act. D has an important stabilizing effect on αENaC mRNA and cannot be used to assess the stability of the transcript. Using deletion mutants of the αENaC 3'UTR region, we determined how different portions of 3'UTR were important in modulating stability of the transcript. We found that the 3'UTR has dual functions, with portions important to promote stabilization (proximal 3'UTR) and others that strongly destabilize (distal 3'UTR) the transcript. Our system also allowed us to confirm that the decreased expression of αENaC mRNA induced by TNF-α results in part by a decreased stability of the mRNA. Finally, we identified several RNA-binding proteins that interact specifically with αENaC 3'UTR and determined if these proteins had an impact on transcript stability. Surexpression of two of these proteins in alveolar epithelial cells, DHX36 and TIAL1 was able to decrease the level of αENaC mRNA via a downregulation of mRNA stability. The work presented here shows the complexity of the signal transduction pathways elicited by different pathological stress conditions in alveolar epithelial cells and is the first to show that αENaC mRNA stability elicited by sequences in 3’UTR plays an important role in modulating the level of the transcript. The Tet-Off model that we developed allows to accurately estimate the half-life of αENaC mRNA and shows that the 3’UTR portion of the mRNA plays a complex role in the modulation of transcript stability in basal and pro-inflammatory conditions. Finally, we identified two putative RNA-binding proteins able to specifically recognize αENaC 3’UTR and modulate the transcript stability.

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