Doctor of Philosophy (PhD) / The incidence of melanoma has risen dramatically during the past three decades, yet there has been little improvement in effective treatments for this intractable and aggressive disease. Melanoma tumours are notoriously resistant to apoptosis, a cell suicide program that is activated by most cancer therapies. This thesis explores the role of the melanoma susceptibility gene product p14ARF in promoting cell cycle arrest and apoptosis, in order to resolve the impact of this tumour suppressor in melanomagenesis and melanoma susceptibility. The p14ARF tumour suppressor gene is mutated in almost half of all cancers, and germline mutations in p14ARF confer a greatly increased risk of developing melanoma. The primary function of p14ARF is to relay oncogenic signals to p53, a central regulator of cellular response to stress. There is conflicting evidence regarding the role of p14ARF in promoting apoptosis. Much of the current evidence is based on murine studies, which may not translate accurately to humans due to important differences in animal physiology and the primary sequence and functions of the mouse and human ARF proteins. Furthermore, results from previous studies are often compounded by supra-physiological expression of p14ARF, and are complicated by the fact that p14ARF shares its genomic sequence with the p16INK4a tumour suppressor gene. This study demonstrates that p14ARF expression in human cancer and primary cell lines promotes rapid p53-dependent cell cycle arrest, rather than apoptosis. As p14ARF expression did not induce apoptosis, we investigated if p14ARF could modulate the sensitivity of a cell to apoptosis induced by cytotoxic agents. Using a p14ARF-inducible U2OS osteosarcoma cell line model, we examined the impact of p14ARF expression on the apoptotic response of the cell to a panel of thirteen cytotoxic agents. p14ARF expression increased apoptosis caused by a sub-set of agents, including trichostatin A, sodium butyrate, DRB, Adriamycin and UVB radiation. p14ARF-mediated chemosensitivity was p53- and caspase-dependent, and involved the loss of mitochondrial potential. While loss of mitochondrial potential was dependent on p53, it was not blocked by caspase inhibition, demonstrating that caspases play a role downstream of mitochondrial depolarisation. Inhibition of individual components of the apoptotic program showed that p14ARF-mediated chemosensitivity was not strictly dependent on the pro-apoptotic Bax or Fas proteins. We also investigated whether p14ARF could sensitise melanoma to chemotherapeutics in vivo. We investigated the expression level of p14ARF, p16INK4a and MITFm and mutation status of B-RAF, N-RAS and PTEN in melanomas from 30 patients that had undergone isolated limb infusion - a palliative therapeutic strategy that results in much higher response rates than systemic treatment. Expression of p14ARF did not predict response to the drugs actinomycin D and melphalan . Instead, high expression of p16INK4a and presence of activating N-RAS mutation were independent predictors of response to high doses of these chemotherapeutic drugs. This work suggests that p14ARF analogues may be beneficial adjuncts in cancer therapy, but are unlikely to be effective as single agents. Additionally, p14ARF mimetics will only be effective in tumours with intact p53 signalling. Melanomas frequently carry functional p53, and may be susceptible to this mode of treatment providing the apoptotic pathway downstream of p53 is intact or can be restored.

Doctor of Philosophy (PhD) / The incidence of melanoma has risen dramatically during the past three decades, yet there has been little improvement in effective treatments for this intractable and aggressive disease. Melanoma tumours are notoriously resistant to apoptosis, a cell suicide program that is activated by most cancer therapies. This thesis explores the role of the melanoma susceptibility gene product p14ARF in promoting cell cycle arrest and apoptosis, in order to resolve the impact of this tumour suppressor in melanomagenesis and melanoma susceptibility. The p14ARF tumour suppressor gene is mutated in almost half of all cancers, and germline mutations in p14ARF confer a greatly increased risk of developing melanoma. The primary function of p14ARF is to relay oncogenic signals to p53, a central regulator of cellular response to stress. There is conflicting evidence regarding the role of p14ARF in promoting apoptosis. Much of the current evidence is based on murine studies, which may not translate accurately to humans due to important differences in animal physiology and the primary sequence and functions of the mouse and human ARF proteins. Furthermore, results from previous studies are often compounded by supra-physiological expression of p14ARF, and are complicated by the fact that p14ARF shares its genomic sequence with the p16INK4a tumour suppressor gene. This study demonstrates that p14ARF expression in human cancer and primary cell lines promotes rapid p53-dependent cell cycle arrest, rather than apoptosis. As p14ARF expression did not induce apoptosis, we investigated if p14ARF could modulate the sensitivity of a cell to apoptosis induced by cytotoxic agents. Using a p14ARF-inducible U2OS osteosarcoma cell line model, we examined the impact of p14ARF expression on the apoptotic response of the cell to a panel of thirteen cytotoxic agents. p14ARF expression increased apoptosis caused by a sub-set of agents, including trichostatin A, sodium butyrate, DRB, Adriamycin and UVB radiation. p14ARF-mediated chemosensitivity was p53- and caspase-dependent, and involved the loss of mitochondrial potential. While loss of mitochondrial potential was dependent on p53, it was not blocked by caspase inhibition, demonstrating that caspases play a role downstream of mitochondrial depolarisation. Inhibition of individual components of the apoptotic program showed that p14ARF-mediated chemosensitivity was not strictly dependent on the pro-apoptotic Bax or Fas proteins. We also investigated whether p14ARF could sensitise melanoma to chemotherapeutics in vivo. We investigated the expression level of p14ARF, p16INK4a and MITFm and mutation status of B-RAF, N-RAS and PTEN in melanomas from 30 patients that had undergone isolated limb infusion - a palliative therapeutic strategy that results in much higher response rates than systemic treatment. Expression of p14ARF did not predict response to the drugs actinomycin D and melphalan . Instead, high expression of p16INK4a and presence of activating N-RAS mutation were independent predictors of response to high doses of these chemotherapeutic drugs. This work suggests that p14ARF analogues may be beneficial adjuncts in cancer therapy, but are unlikely to be effective as single agents. Additionally, p14ARF mimetics will only be effective in tumours with intact p53 signalling. Melanomas frequently carry functional p53, and may be susceptible to this mode of treatment providing the apoptotic pathway downstream of p53 is intact or can be restored.

L'EGFR est un récepteur transmembranaire à activité tyrosine kinase (TK) qui transduit des signaux de prolifération et de survie cellulaire. Dans les cancers du poumon, son activité est fréquemment dérégulée par surexpression et/ou par mutation au niveau de son domaine TK. Ces mutations sont principalement de deux types (EGFR-L858R et EGFR-Del19) et sont dites activatrices car elles induisent une activation constitutive des signalisations oncogéniques de l'EGFR. Elles sont aussi un facteur prédictif de réponse aux EGFR-TKIs qui inhibent spécifiquement ce récepteur. P14ARF est un suppresseur de tumeur qui restreint la prolifération cellulaire et maintient la stabilité génomique. Nous avons décrit son inactivation dans les cancers du poumon et démontré que son expression freine leur développement. Nos résultats récents montrent que l'expression de p14ARF est inhibée dans une très grande majorité d'adénocarcinomes pulmonaires présentant une mutation activatrice de l'EGFR. Sur la base de ces résultats nous avons émis l'hypothèse que l'inhibition de l'expression de p14ARF contribuerait à l'expansion clonale des tumeurs porteuses d'un EGFR muté. P14ARF pourrait ainsi être un frein à l'activité oncogénique de l'EGFR.Dans différents modèles d'adénocarcinomes pulmonaires exprimant un mutant EGFR-L858R nous montrons que l'expression transitoire de p14ARF active une signalisation pro-apoptotique dépendante de STAT3 et de Bcl2. En retour, l'EGFR inhibe l'expression de p14ARF et bloque ses fonctions pro-apoptotiques. Nous montrons aussi que l'activation de l'EGFR (sauvage ou muté) inhibe l'expression de p14ARF à un niveau transcriptionnel. Ceci implique une translocation nucléaire de l'EGFR contrôlée par les PI3Ks de classe III (Vps34) et la fixation de l'EGFR sur le promoteur de ARF. L'ensemble de ces travaux identifie pour la première fois un lien fonctionnel entre les voies de signalisation de l'EGFR et de p14ARF. Ils mettent en évidence un nouveau mécanisme de progression tumorale par lequel une signalisation nucléaire de l'EGFR inactive le suppresseur de tumeur p14ARF afin de permettre la croissance tumorale. / EGFR is a transmembrane tyrosine kinase (TK) receptor which activates proliferative and survival signals. In lung cancer, its activity is frequently deregulated by overexpression and/or mutation in its TK domain. These mutations are mainly of two types (L858R and Del19) and are called « driver mutations » because they induce constitutive activation of EGFR oncogenic signaling. They also represent a predictive responsive factor to EGFR TKIs that specifically inhibit this receptor. P14ARF is a tumor suppressor that restricts cellular proliferation and maintains genomic stability. We described its inactivation in lung cancer and demonstrated that its expression inhibits their development. Our recent results show that the expression of p14ARF is inhibited in a majority of lung adenocarcinomas expressing an activating EGFR mutation. Based on these results we hypothesized that inhibition of p14ARF expression contributes to clonal expansion of mutated EGFR-bearing tumor. P14ARF could be a break to EGFR oncogenic activity.In different models of lung adenocarcinoma expressing a L858R EGFR mutant we show that transient expression of p14ARF activates a pro-apoptotic STAT3/Bcl-2-dependent signaling pathway. In turn, EGFR inhibits the expression of p14ARF and blocks its pro-apoptotic function. We also show that EGFR (wild type or mutated) activation inhibits the expression of p14ARF at the transcriptional level. This implies a nuclear translocation of EGFR controlled by Class III PI3K (Vps34) and its fixation to the ARF promoter. This work identifies for the first time a functional link between EGFR and p14ARF signaling pathways. They highlight a new mechanism of tumor progression by which a nuclear EGFR signaling inactivates the tumor suppressor p14ARF to allow tumor growth.

Tumour initiation and progression occur due to oncogenic mutations that also contribute to therapeutic resistance in many human tumours. Mutations activating the "PI3K/AKT" signalling pathway and inactivating the "TP53" tumour suppressor gene are common mechanisms that cancer cells require to proliferate and escape pre-programmed cell death. p53 mutant (p53mut) tumours not only fail to respond to DNA damaging therapy, but are also described to promote therapeutic resistance by dominant negative suppression of p53 dependent promoter activity. Our work identifies the crucial interaction between the PI3K/AKT pathway and p53 mutations that regulate treatment sensitivity in tumours. Using a combination of in vitro and in vivo techniques we demonstrate that AKT inhibition promotes reduced cellular levels of p53mut via a novel Nucleophosmin 1 (NPM) mediated regulation of the tumour suppressor p14ARF and promotes re-engagement of cell cycle arrest, senescence and increased sensitivity to ionising radiation in both in vivo and in vitro systems. We show that the PI3K/AKT pathway plays an important role in the regulation of p53mut and inhibitors of this pathway can re-sensitise treatment resistant tumours. This has helped us to simultaneously highlight the cohort of patients where the greatest efficacy may be achieved in clinical practise. We further show that the AKT mediated regulation of NPM that we describe in solid tumours is relevant in Acute Myeloid Leukaemia (AML) with mutated NPM, albeit showing physiologically different effects. This further highlights the necessity for rational treatment planning with the newer targeted agents that inhibit specific signalling pathways in AML patients.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic cancer resistant to current treatments. A better understanding of the genetic basis of PDAC progression is urgently needed to improve treatment options. The ARF tumor suppressor is inactivated in ~45% of PDAC. My thesis lab identified a new, uncharacterized ARF binding protein, Partner of ARF isoform 1A (Parf-1A). This thesis explores the hypothesis that Parf-1A plays an important role in PDAC and ARF tumor suppressor signaling Initial studies sought to develop a novel mouse xenograft model of PDAC metastasis that would expedite testing of putative PDAC genes. Human PDAC cell lines stably expressing luciferase were generated and introduced by intracardiac injection into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored non-invasively by bioluminescence imaging and found to recapitulate PDAC tumor formation and metastatic distribution. The model was validated by the ability of ARF to suppress PDAC cancer cell migration in vitro and reduce tumor cell colonization in vivo; establishing a new bioluminescent mouse model for rapidly assessing the significance of suspected PDAC genes. Using human PDAC cell lines and tumor specimens, we investigated the role and significance of Parf-1A to PDAC. RNAi analyses demonstrated Parf-1A is required for PDAC cell survival, proliferation and resistance to the PDAC therapeutic, oxaliplatin. PDAC cells are ARF-null; therefore these tumor promoting activities of Parf-1A were independent of ARF. Notably, immunohistochemical analyses of Parf-1A in human PDAC tumors showed Parf-1A expression is a prognostic marker of poor survival in PDAC patients. These data suggest Parf-1A is a novel biomarker of PDAC and potential target for anticancer therapy. Other studies tested how Parf-1A influenced ARF signaling. Parf-1A depletion and overexpression showed it inhibits ARF anti-proliferative activity by mobilizing ARF from the nucleus (where it is functional) into the cytoplasm. These data show Parf-1A is a new inhibitor of ARF. Considered with findings that Parf-1A can act independent of ARF to promote PDAC tumorigenesis, such results suggest Parf-1A is a novel oncoprotein that acts through multiple pathways to facilitate tumorigenesis. Thus, Parf-1A may have broad relevance to many types of human cancers.

O melanoma é um dos tipos de câncer de pele cuja frequência tem crescido nos últimos anos e apresentado elevada taxa de mortalidade, apesar de ter reduzida prevalência. Mesmo havendo um considerável avanço nas propostas terapêuticas nos últimos anos, ainda se vê necessário o desenvolvimento de novas abordagens, sendo a terapia gênica uma promissora possibilidade para tal. Utilizando vetores adenovirais com promotor responsivo à p53 (PGTx beta) para a transferência gênica de p19Arf (proteína supressora de tumor) e interferon-beta (citocina imunomodulatória) em células de melanoma murino com o gene Trp53 selvagem, o nosso grupo demonstrou previamente que a combinação dos dois genes, mas não o tratamento individual, promove efeito citotóxico sinérgico com a liberação de marcadores de morte imunogênica, in vitro; e significativa redução da progressão tumoral acompanhada de uma forte resposta imunológica de linfócitos T CD4+ e CD8+, células NK e neutrófilos contra desafios tumorais, in vivo. Porém, como a translação para modelos de melanomas humanos ainda estava em estágio inicial, ainda não haviam sido confirmamos se esses benefícios também seriam recapitulados. Observações inicias sugeriam que apenas a transferência gênica de interferon-beta seja suficiente para induzir morte celular em linhagens humanas portadoras de TP53 selvagem, sem ainda terem sido identificado o efeito da transferência de p14ARF e nem a necessidade de p53 endógeno para a resposta. Dessa forma, o presente projeto buscou avaliar os efeitos antitumorais provocados pela terapia gênica combinada de p14ARF e interferon-beta em modelos de melanoma humano utilizando linhagens com e sem a via da p53 integra. Para isso, foram utilizadas diferentes linhagens celulares com TP53 selvagem ou com distintas mutações e também foram construídos vetores adenovirais com o promotor constitutivo CMV, tornando assim possível a expressão dos transgenes de maneira independente do status do TP53 endógeno. O presente trabalho revelou que a transferência combinada do interferon-beta e p14ARF revelou vantagem quanto ao estímulo citotóxico e regulação negativa na dinâmica da população em ambas as linhagens UACC-62 e SK-Mel-29, independentemente do estado da via da p53. Na avaliação dos mecanismos de morte foi observado que ambas a linhagens apresentaram marcação positiva para marcadores da via da apoptose, porém com possível participação de outras modalidades de morte-celular, como a necrose, para a linhagem com o TP53 mutado (SK-Mel-29). Além disso, mostramos que os tratamentos potencialmente induzem vias de morte com caráter imunogênico pela secreção de ATP e exposição da calreticulina, sendo este último marcador mais significantemente observado mediante o tratamento combinado. Assim, recapitulamos o benefício observado em modelo murino para a transferência gênica do interferon-beta e p14ARF em modelo de melanoma humano, e investigamos marcadores importantes à translação da proposta terapêutica para o melanoma / Melanoma is one of the types of skin cancer whose frequency has grown in the last years and presents a high mortality rate, despite its low prevalence. Although there has been considerable progress in therapeutic proposals in recent years, it is still necessary to develop new approaches, being gene therapy a promising possibility for this. With the use of adenoviral vectors with a p53 responsive promoter (PGTx beta) for the gene transfer of p19Arf (tumor suppressor protein) and interferon-beta (immunomodulatory cytokine) in murine melanoma cells bearing wild-type Trp53 gene, our group previously demonstrated that the combination of the two genes, but not individual treatment, promotes a synergistic cytotoxic effect with the release of immunogenic death markers in vitro; and significant reduction of tumor progression with a strong immune response mediated by CD4+ and CD8+ T lymphocytes, NK cells and neutrophils in tumor challenges in vivo. However, as the translation for human melanoma models was still at an early stage, it still was not possible to confirm whether these benefits would also be recapitulated in a human model. Initial observations suggested that interferon-beta gene transfer is sufficient to induce cell death in wild-type TP53-bearing human melanoma cell lines, with the effect of p14ARF gene transfer and the role for endogenous p53 in this response yet to be investigated. Thus, the present work aimed to evaluate the antitumor effects induced upon the combined gene transfer of p14ARF and interferon-beta in human melanoma cell lines with and without a functional p53 pathway. For this, different cell lines bearing wild-type TP53 or with different mutations were used and adenoviral vectors with the constitutive CMV promoter were also constructed, making possible the expression of the transgenes independently of the endogenous TP53 status. The present work showed that the combined transfer of interferon-beta and p14ARF was advantageous in cytotoxic stimulation and negative regulation in population dynamics for both cell lines UACC-62 and SK-Mel-29, regardless of p53 pathway status. In the evaluation of the triggered cell death mechanisms it was observed that both cell lines presented positive markers of the apoptosis pathway, but with possible participation of other cell death mechanism, such as necrosis, for the mutated TP53 cell line SK-Mel-29. In addition, we showed that the treatments potentially induced cell death pathways with immunogenic features including the secretion of ATP and calreticulin exposure, being the latter marker more significantly presented after the combined treatment. Thus, we recapitulated the benefit observed in murine model for the gene transfer of interferon-beta and p14ARF in the model of human melanoma, and investigated important markers for the translation of the melanoma therapeutic proposal

O melanoma é um dos tipos de câncer de pele cuja frequência tem crescido nos últimos anos e apresentado elevada taxa de mortalidade, apesar de ter reduzida prevalência. Mesmo havendo um considerável avanço nas propostas terapêuticas nos últimos anos, ainda se vê necessário o desenvolvimento de novas abordagens, sendo a terapia gênica uma promissora possibilidade para tal. Utilizando vetores adenovirais com promotor responsivo à p53 (PGTx beta) para a transferência gênica de p19Arf (proteína supressora de tumor) e interferon-beta (citocina imunomodulatória) em células de melanoma murino com o gene Trp53 selvagem, o nosso grupo demonstrou previamente que a combinação dos dois genes, mas não o tratamento individual, promove efeito citotóxico sinérgico com a liberação de marcadores de morte imunogênica, in vitro; e significativa redução da progressão tumoral acompanhada de uma forte resposta imunológica de linfócitos T CD4+ e CD8+, células NK e neutrófilos contra desafios tumorais, in vivo. Porém, como a translação para modelos de melanomas humanos ainda estava em estágio inicial, ainda não haviam sido confirmamos se esses benefícios também seriam recapitulados. Observações inicias sugeriam que apenas a transferência gênica de interferon-beta seja suficiente para induzir morte celular em linhagens humanas portadoras de TP53 selvagem, sem ainda terem sido identificado o efeito da transferência de p14ARF e nem a necessidade de p53 endógeno para a resposta. Dessa forma, o presente projeto buscou avaliar os efeitos antitumorais provocados pela terapia gênica combinada de p14ARF e interferon-beta em modelos de melanoma humano utilizando linhagens com e sem a via da p53 integra. Para isso, foram utilizadas diferentes linhagens celulares com TP53 selvagem ou com distintas mutações e também foram construídos vetores adenovirais com o promotor constitutivo CMV, tornando assim possível a expressão dos transgenes de maneira independente do status do TP53 endógeno. O presente trabalho revelou que a transferência combinada do interferon-beta e p14ARF revelou vantagem quanto ao estímulo citotóxico e regulação negativa na dinâmica da população em ambas as linhagens UACC-62 e SK-Mel-29, independentemente do estado da via da p53. Na avaliação dos mecanismos de morte foi observado que ambas a linhagens apresentaram marcação positiva para marcadores da via da apoptose, porém com possível participação de outras modalidades de morte-celular, como a necrose, para a linhagem com o TP53 mutado (SK-Mel-29). Além disso, mostramos que os tratamentos potencialmente induzem vias de morte com caráter imunogênico pela secreção de ATP e exposição da calreticulina, sendo este último marcador mais significantemente observado mediante o tratamento combinado. Assim, recapitulamos o benefício observado em modelo murino para a transferência gênica do interferon-beta e p14ARF em modelo de melanoma humano, e investigamos marcadores importantes à translação da proposta terapêutica para o melanoma / Melanoma is one of the types of skin cancer whose frequency has grown in the last years and presents a high mortality rate, despite its low prevalence. Although there has been considerable progress in therapeutic proposals in recent years, it is still necessary to develop new approaches, being gene therapy a promising possibility for this. With the use of adenoviral vectors with a p53 responsive promoter (PGTx beta) for the gene transfer of p19Arf (tumor suppressor protein) and interferon-beta (immunomodulatory cytokine) in murine melanoma cells bearing wild-type Trp53 gene, our group previously demonstrated that the combination of the two genes, but not individual treatment, promotes a synergistic cytotoxic effect with the release of immunogenic death markers in vitro; and significant reduction of tumor progression with a strong immune response mediated by CD4+ and CD8+ T lymphocytes, NK cells and neutrophils in tumor challenges in vivo. However, as the translation for human melanoma models was still at an early stage, it still was not possible to confirm whether these benefits would also be recapitulated in a human model. Initial observations suggested that interferon-beta gene transfer is sufficient to induce cell death in wild-type TP53-bearing human melanoma cell lines, with the effect of p14ARF gene transfer and the role for endogenous p53 in this response yet to be investigated. Thus, the present work aimed to evaluate the antitumor effects induced upon the combined gene transfer of p14ARF and interferon-beta in human melanoma cell lines with and without a functional p53 pathway. For this, different cell lines bearing wild-type TP53 or with different mutations were used and adenoviral vectors with the constitutive CMV promoter were also constructed, making possible the expression of the transgenes independently of the endogenous TP53 status. The present work showed that the combined transfer of interferon-beta and p14ARF was advantageous in cytotoxic stimulation and negative regulation in population dynamics for both cell lines UACC-62 and SK-Mel-29, regardless of p53 pathway status. In the evaluation of the triggered cell death mechanisms it was observed that both cell lines presented positive markers of the apoptosis pathway, but with possible participation of other cell death mechanism, such as necrosis, for the mutated TP53 cell line SK-Mel-29. In addition, we showed that the treatments potentially induced cell death pathways with immunogenic features including the secretion of ATP and calreticulin exposure, being the latter marker more significantly presented after the combined treatment. Thus, we recapitulated the benefit observed in murine model for the gene transfer of interferon-beta and p14ARF in the model of human melanoma, and investigated important markers for the translation of the melanoma therapeutic proposal

Ao longo dos processos de imortalização e transformação maligna, as células adquirem inúmeras alterações genéticas, que são causadas por fatores endógenos e exógenos como agentes biológicos e a geração de espécies reativas de oxigênio. Neste trabalho, uma linhagem celular espontaneamente transformada foi clonada a partir de explantes de embriões de camundongos C57bl/6. Esta linhagem mostrou-se produtora de pigmento escuro; a análise citoquímica e ultraestrutural permitiu caracterizar a linhagem como tendo origem melanocítica. A linhagem, denominada Mgal3, mostrou-se tumorigênica quando implantada no tecido subcutâneo de animais singenéicos, apresentando capacidade de disseminação linfática, dando origem a metástases em linfonodos, o que permitiu caracteriza-la como uma linhagem de melanoma murino. O processo de transformação deste melanoma caracterizou-se pela expressão de genes retrovirais endógenos, com expressão do antígeno associado a melanoma (MAA), reconhecido pelo anticorpo monoclonal MM2-9B6; ausência de mutações nos exons 5 a 8 do gene supressor de tumor TP53; e, silenciamento do gene CDKN2a, que codifica duas proteínas que atuam em redes de supressão de tumores, p16INK4a e p19ARF. A perda de expressão de pelo menos um destes produtos gênicos parece associada a mecanismos epigenéticos, uma vez que o tratamento de Mgal3 com o inibidor de DNA metiltransferase 5-Aza-2-deoxicitidina, restaurou a transcrição de pelo menos um dos transcritos do gene CDKN2a. Da mesma forma, observamos que o gene LGALS3, que codifica a lectina animal galectina-3 também é silenciado nesta linhagem, mostrando que esta molécula não está associada à manutenção desta célula transformada em condições de cultivo. / A novel murine melanoma cell line named Mgal3 was generated from embryo explants. This cell line gave rise to metastatic tumors when injected subcutaneously in C57bl/6 mice. Tumor histogenesis was determined at the cytochemical (Fontana Masson staining), immunohistochemical (staining with anti-HMB45 and anti-S100) and ultrastructural levels. Mgal3 produces high amounts of retroviral C particles and was recognized by the mAb MM2-9B6, which reacts with a melanoma associated antigen derived from the envelope of the ecotropic retrovirus MelArv. No mutations were found in TP53 exons 5-8, however loss of CDKN2a expression was observed. Treatment of Mgal3 with the demethylating agent azadeoxycytidine indicated that at least one of the genes encoded at the CDKN2a locus was silenced by promoter hypermethylation. Furthermore, this cell line did not express the animal lectin, galectin-3. The galectin-3 gene promoter seemed to be hypermethylated, since treatment of Mgal3 with azadeoxycytidine led to the de novo expression of the lectin.

Contrôle de la signalisation oncogénique du mutant L858R de l'EGFR par la protéine suppresseur de tumeur p14ARF dans les adénocarcinomes pulmonaires. Le récepteur à l'EGF (EGFR) est un oncogène puissant impliqué dans le développement des cancers du poumon. Dans ces cancers, la présence de mutations activatrices de l'EGFR (majoritairement L858R et Del19) est un facteur prédictif de réponse aux agents pharmacologiques qui ciblent spécifiquement ce récepteur (EGFR-TKI). Cependant, l'association entre réponse thérapeutique et mutation est plus complexe que prévue, soulignant la nécessité d'approfondir la compréhension des mécanismes moléculaires impliqués dans développement de ces cancers. Nous avons précédemment montré que la quasi-totalité des tumeurs pulmonaires avec des mutations activatrices de l'EGFR présente une expression faible ou indétectable de la protéine suppressive de tumeur p14ARF. Ces résultats nous ont conduites à émettre l'hypothèse que l'expression de p14ARF était un frein essentiel à l'expansion clonale de ces cellules. Nous décrivons pour la première fois une relation fonctionnelle entre p14ARF et du mutant L858R de l'EGFR dans laquelle p14ARF inhibe la croissance de cellules exprimant ce mutant en induisant leur apoptose. Les effets suppresseurs de tumeur de p14ARF impliquent une fonction pro-apoptotique originale de STAT3 qui conduit à l'inhibition de l'expression de la protéine anti-apoptotique Bcl-2. De plus, nous montrons que les cellules EGFR-L858R maintiennent leur avantage de croissance en inhibant l'expression de p14ARF et la signalisation pro-apoptotique STAT3/Bcl-2 qui en découle. Nos résultats identifient également p14ARF comme une nouvelle cible transcriptionnelle de STAT3, mettant ainsi en évidence une boucle de rétrocontrôle positif entre ces deux protéines qui pourrait entretenir la signalisation pro-apoptotique médiée par STAT3. Sur la base de ces résultats, nous suggérons que la réactivation de la voie p14ARF/STAT3/Bcl-2 pourrait être une nouvelle stratégie thérapeutique dans le traitement de ces cancers. / Control of EGFR-L858R oncogenic signaling pathway by the p14ARF tumor suppressor in lung adenocarcinoma. The EGF receptor (EGFR) is a strong oncogene involved in lung carcinogenesis. In these cancers, sensitivity to inhibitors of the EGFR tyrosine kinase activity (EGFR-TKI) has been shown to be related to the presence of activating mutations in the TK domain of EGFR (mainly L858R and Del19). However, the association between mutations and responsiveness to EGFR-TKI based treatment is more complex than previously envisioned, underlying the pressing need to study thoroughly the molecular mechanisms of lung cancer growth. We previously showed that almost all lung cancer with EGFR activated mutations has very low or undetectable levels of the p14ARF tumor suppressor protein. These results led us to postulate that expression of p14ARF is an efficient break against clonal proliferation of these cells. We report for the first time a relationship between p14ARF and mutant EGFR-L858R in which p14ARF inhibits the growth of EGFR-L858R expressing cells by inducing apoptosis. The p14ARF tumor suppressor effects involve an original STAT3 pro-apoptotic function that drives the inhibition of the anti-apoptotic Bcl-2 protein. Moreover, we show that the EGFR-L858R mutant maintains their survival and proliferation characteristics by inhibiting p14ARF expression and consequently the STAT3/Bcl-2 pro-apoptotic pathway. Our results also identify p14ARF as a new transcriptional target of STAT3, therefore providing evidence of a positive feed-back loop that could maintain STAT3 pro-apoptotic pathway. Based on these data, we suggest that manipulation of this pathway could be a therapeutic strategy for lung cancer treatment.

Ao longo dos processos de imortalização e transformação maligna, as células adquirem inúmeras alterações genéticas, que são causadas por fatores endógenos e exógenos como agentes biológicos e a geração de espécies reativas de oxigênio. Neste trabalho, uma linhagem celular espontaneamente transformada foi clonada a partir de explantes de embriões de camundongos C57bl/6. Esta linhagem mostrou-se produtora de pigmento escuro; a análise citoquímica e ultraestrutural permitiu caracterizar a linhagem como tendo origem melanocítica. A linhagem, denominada Mgal3, mostrou-se tumorigênica quando implantada no tecido subcutâneo de animais singenéicos, apresentando capacidade de disseminação linfática, dando origem a metástases em linfonodos, o que permitiu caracteriza-la como uma linhagem de melanoma murino. O processo de transformação deste melanoma caracterizou-se pela expressão de genes retrovirais endógenos, com expressão do antígeno associado a melanoma (MAA), reconhecido pelo anticorpo monoclonal MM2-9B6; ausência de mutações nos exons 5 a 8 do gene supressor de tumor TP53; e, silenciamento do gene CDKN2a, que codifica duas proteínas que atuam em redes de supressão de tumores, p16INK4a e p19ARF. A perda de expressão de pelo menos um destes produtos gênicos parece associada a mecanismos epigenéticos, uma vez que o tratamento de Mgal3 com o inibidor de DNA metiltransferase 5-Aza-2-deoxicitidina, restaurou a transcrição de pelo menos um dos transcritos do gene CDKN2a. Da mesma forma, observamos que o gene LGALS3, que codifica a lectina animal galectina-3 também é silenciado nesta linhagem, mostrando que esta molécula não está associada à manutenção desta célula transformada em condições de cultivo. / A novel murine melanoma cell line named Mgal3 was generated from embryo explants. This cell line gave rise to metastatic tumors when injected subcutaneously in C57bl/6 mice. Tumor histogenesis was determined at the cytochemical (Fontana Masson staining), immunohistochemical (staining with anti-HMB45 and anti-S100) and ultrastructural levels. Mgal3 produces high amounts of retroviral C particles and was recognized by the mAb MM2-9B6, which reacts with a melanoma associated antigen derived from the envelope of the ecotropic retrovirus MelArv. No mutations were found in TP53 exons 5-8, however loss of CDKN2a expression was observed. Treatment of Mgal3 with the demethylating agent azadeoxycytidine indicated that at least one of the genes encoded at the CDKN2a locus was silenced by promoter hypermethylation. Furthermore, this cell line did not express the animal lectin, galectin-3. The galectin-3 gene promoter seemed to be hypermethylated, since treatment of Mgal3 with azadeoxycytidine led to the de novo expression of the lectin.