Lung cancer is the leading cause of cancer related deaths in the United States, with an estimated 158, 040 deaths in 2015, accounting for 27% of all cancer deaths. Recent research has identified several important molecular driver oncogenes, including epidermal growth factor receptor (EGFR). EGFR is encoded by exons 18-21, each of which harbor specific mutations within the tyrosine kinase domain. These mutations can drive cell growth, proliferation, and survival, resulting in the formation of non-small cell lung cancer. The development of EGFR tyrosine kinase inhibitors, allows the targeting of these specific mutations without the toxicity normally associated with standard chemotherapy. Unfortunately, inevitably resistance to therapy manifests, requiring a change in therapy and adding complexity to treatment decision making for clinicians and patients alike. Through a comprehensive examination of current literature, this review will establish a standard for first line, targeted treatment for specific genetic mutations within the EGFR gene, as well as address treatment options once resistance to first-line therapy inevitably develops.

Les gliomes sont les tumeurs cérébrales primitives les plus fréquentes de l’adulte. Le glioblastome (grade IV) en est la forme la plus agressive, caractérisé par sa résistance aux traitements actuels (chirurgie, chimiothérapie et radiothérapie). La mortalité de cette pathologie est quasi constante (survie médiane de 15 mois), ce qui justifie l’importance de découvrir de nouvelles cibles thérapeutiques. Le challenge est d'arriver à identifier des marqueurs spécifiques pour proposer un schéma thérapeutique alignant des stratégies de thérapies ciblées qui vont améliorer la prise en charge clinique, la survie globale et la survie sans progression des patients atteints de ces pathologies. Deux axes sont au centre des recherches fondamentales, translationnelles et cliniques. Le premier axe se définit autour du développement de molécules inhibitrices des voies de signalisation et le second autour du concept de cellules souches tumorales (CST) de glioblastomes (GBM) découvertes récemment dans le cerveau et qui révolutionnent la conception de la transformation tumorale.ASPM (Abnormal Spindle Like Microcéphaly Associated) est une cible candidate pertinente susceptible de participer au développement des gliomes (Horvath et al., 2007 ; Hagmann et al., 2008). Cette protéine régule la prolifération des neuroblastes, elle est fortement exprimée au stade embryonnaire, mais, reste faiblement exprimée dans le cerveau adulte. Par ailleurs, ASPM est impliquée dans divers processus de cancérisation (surexprimée dans les cancers du sein, du foie et du cerveau…), toute fois, le mécanisme responsable de cette dérégulation n’est pas encore bien caractérisé.Nos études menées sur une série de 169 gliomes humains, sélectionnés à partir de notre cohorte de patients, montrent que le gène ASPM est un marqueur de la progression vers la malignité, les grades les plus élevés exprimant le plus fortement ASPM. En outre, nous avons également montré que le niveau des transcrits d’ASPM est augmenté dans les récidives de gliomes et qu’en in vitro, ASPM contrôle la formation des gliomasphères (CST de GBM) avec une augmentation de l’expression de ses transcrits dans les cultures in vitro au fil des passages. En continuité de ces observations, nous avons alors développé un sh-miR-RNA spécifique d’ASPM permettant l’extinction post-transcriptionnelle de ce gène. Les résultats obtenus in vitro montrent que la perte d’expression d’ASPM conduit à un arrêt de la prolifération et aboutit à une mort cellulaire massive.Actuellement, des modèles de greffe de gliomasphères chez la souris (orthotopique) sont en cours de développement pour confirmer les effets observés in vitro et vérifier in vivo la validité de notre approche thérapeutique. En perspective, nous tenterons d’étudier les effets du silencing d’ASPM sur la voie de signalisation la plus dérégulée (pRB / E2F ou PI3K / AKT). Enfin, nous étudierons le rôle potentiel de cette protéine dans le contrôle du cycle cellulaire, et, in fine la mise en évidence de ses partenaires… / Glioblastoma (GBM) is the most frequent and aggressive form of primary brain tumors in adults; it is characterized by its resistance to current treatments (surgery, chemotherapy and radiotherapy). The prognosis is grim with a median survival of only 15 months underlining the importance to develop new therapeutic strategies. The recent development of the “tumor stem cell” (TSC) concept in hemopathies has been secondarily applied to gliomas with the identification of subpopulations of GBM cells which express neural stem cell markers and fulfill the criteria for stemness. Some evidences also suggest that this subpopulation could play a primary role in resistance to radio- and chemotherapy.ASPM (Abnormal Spindle Like Microcephaly Associated) is a protein regulating the proliferation of neuroblasts, highly expressed in the embryonic stage but weakly expressed in the adult brain. Preliminary reports suggesting that it could be involved in the development of gliomas (Horvath et al., 2007, Hagemann et al., 2008) prompted us to analyze further the role of this protein, focusing on its potential as a relevant candidate therapeutic target. In a series of 175 gliomas samples of various grades, we found that ASPM mRNA expression was strongly correlated with increasing tumor grade. We also found that ASPM expression increased at recurrence when compared to the initial lesion. Subsequently, we could demonstrate in vitro and in vivo that ASPM expression also increased over serial passages in gliomaspheres and in a mouse glioma xenograft model. In a therapeutic perspective, the effect of lentivirus-mediated shRNA post-transcriptional silencing of ASPM was evaluated in two different gliomasphere models and a dramatic proliferation arrest and cell death was observed. Taken together, these data suggest that ASPM is involved in the malignant progression of gliomas, possibly through expansion of a cancer stem cell compartment, and could be an attractive therapeutic target in glioblastoma multiforme.Another potential candidate tumor stem cell target in glioma is the sonic hedgehog pathway (hedgehog-Gli) which is required for GBM growth and stem cell expansion. In a collaborative study, it was found that NANOG, a transcription factor critically involved with self-renewal of undifferentiated embryonic stem cells, modulates gliomasphere clonogenicity, CD133+ stem cell behavior and proliferation. NANOG was regulated by hedgehog-Gli signalling and was essential for GBM tumourigenicity in orthotopic xenografts suggesting that it could also be a useful potential therapeutic target.Conclusions: Accumulating evidences suggest that tumor stem cells play an important role in the oncogenesis of gliomas and in their resistance to treatment. Our data support this concept and suggest that specific stemness markers may become useful targets to improve treatment of this devastating disease.

Embora o melanoma represente apenas 4% das neoplasias malignas da pele, é considerado a mais grave por ser altamente etal. Em virtude da via MAPK (Mitogen activated protein kinase) estar intimamente ligada ao descontrole da proliferação celular, especialmente em melanoma, esta via se tornou um alvo para o desenvolvimento de terapias direcionadas a oncogenes, como os potentes quimioterápicos Vemurafenibe (inibidor da mutação V600E em BRAF - BRAFV600E) e Trametinibe (inibidor de MEK). Cada vez mais, melhores taxas de respostas vêm sendo alcançadas com os novos medicamentos, porém a maioria dos pacientes está sujeita a recidivas após 7 meses de tratamento devido ao desenvolvimento de quimioresistência, justificando a constante busca por novos compostos terapêuticos. Dados de nosso laboratório indicam que 4-nerolidilcatecol (4-NC) induz aumento na expressão de p53, produção de ROS e dano ao DNA, culminando em apoptose dependente de caspase-3 em células de melanoma por ser um inibidor proteassomal. Além disto, o 4-nerolidilcatecol (4- NC) demonstrou efeito inibitório na proliferação de células de melanoma em modelo de cultura organotípica de pele. Desta forma, este projeto visa avaliar a possibilidade de superação da quimioresistência aos inibidores de BRAF e de MEK, utilizando terapias combinatórias com 4-NC em células de melanoma humano resistentes a estes inibidores. Primeiramente, linhagens celulares de melanoma resistentes aos inibidores de BRAF (R) e BRAF/MEK (DR) foram geradas a partir de células parentais BRAF mutadas (P) e caracterizadas por MTT, microscopia de fluorescência e western blotting. Estas células foram submetidas ao tratamento com 4-NC que apresentou citotoxicidade na concentração de 30µM, inibição de formação de colônias e diminuição na invasão em modelos in vitro de culturas 2D e 3D em todas as linhagens estudadas (P, R e DR). O 4-NC foi ainda capaz de induzir estresse de retículo endoplasmático com indução de apoptose. Visando a explorar o efeito terapêutico in vivo do 4-NC, outro estudo foi conduzido em animais submetidos a enxerto xenográfico com células parentais de melanoma NRAS mutadas. Após desenvolvimento do tumor, os animais foram tratados 3 vezes por semana durante 3 semanas com 4-NC (10 mg/kg) por via i.p. O 4-NC foi capaz de inibir em até 4 vezes o crescimento dos tumores xenográficos (4/10) quando comparado com os controles, com remissão completa do tumor em um animal. A expressão de p53 e PARP clivada foi aumentada nos tumores dos animais tratados, sugerindo apoptose. A expressão gênica de MMP2 e MMP14 estava diminuída nas mesmas amostras, demonstrando o papel do 4-NC na inibição da invasão do melanoma in vivo. Finalmente, a toxicidade sistêmica do 4-NC foi avaliada nas mesmas doses empregadas no ensaio in vivo de tumorigênese. A baixa toxicidade observada nos ensaios toxicológicos com tratamentos sub-crônicos com 4-NC e a citotoxicidade demonstrada em modelos xenográficos nos leva a considerar este composto como promissor para estudos futuros e sua aplicação no tratamento do melanoma cutâneo humano, incluindo pacientes resistentes aos inibidores de BRAF e MEK. / Melanoma accounts for only 4% of malignant neoplasms of the skin, but is considered the most serious because it is highly deadly. Because the MAPK (Mitogen activated protein kinase) pathway is closely linked to the lack of control of cell proliferation, especially in melanoma, this pathway has become a target for the development of oncogene-targeted therapies, such as the potent chemotherapeutic agents Vemurafenib (V600E mutation inhibitor in BRAF - BRAFV600E) and Trametinib (MEK inhibitor). Increasingly, better response rates have been achieved with the new drugs, but most patients are subject to relapses after 7 months of treatment due to several mechanisms, which justify the constant search for new therapeutic compounds. Data from our laboratory indicate that 4-nerolidylcatechol (4-NC) induces increased p53 expression, ROS production and DNA damage, culminating in caspase-3 dependent apoptosis in melanoma cells. The 4-NC compound demonstrated an inhibitory effect on melanoma cell proliferation in an organotypic skin culture model. Thus, this project aims to evaluate the possibility of overcoming the existing chemoresistance to BRAF and MEK inhibitors, using 4-NC combinatory therapies in human melanoma cells resistant to these inhibitors. Firstly, melanoma cell lines resistant to BRAF (R) and BRAF / MEK (DR) inhibitors were generated from naive cells mutated BRAF (N) and characterized by MTT, fluorescence microscopy and western blotting. These cells were submitted to 4-NC treatment that showed cytotoxicity with 30 µM, inhibition of colony formation and decrease in invasion in 2D and 3D in vitro models in all cell line studied (N, R and DR). Furthermore, 4-NC was able to induce endoplasmic reticulum stress with apoptosis induction. In order to explore the in vivo therapeutic effect of 4-NC, an additional study was conducted using xenograft model with NRAS-mutated melanoma cell line. After tumor development, the animals were treated 3 times per week for 3 weeks with 4-NC (10 mg / kg) by i.p. injection. 4-NC was able to inhibit up to 4- fold the growth of xenograft tumors (4/10) when compared to controls, with complete tumor remission in one animal. Cleaved PARP and p53 expression were increased in the tumors of treated animals, suggesting apoptosis. MMP2 and MMP14 gene expression were decreased in the same samples, demonstrating the role of 4-NC in inhibiting melanoma invasion in vivo. Finally, the systemic toxicity of 4-NC was evaluated at the same doses employed in the in vivo tumorigenesis assay. The low toxicity observed in the toxicological assays with sub-chronic 4- NC treatments and the demonstrated cytotoxicity in xenograft models leads us to consider this compound as promising for future studies and its application in the treatment of cutaneous human melanoma, including patients resistants to BRAF and MEK inhibitors.

Abstract Cancer has been a big issue in the medical science. Many scientists in the world have been trying so hard to find the effective drugs or therapies for different kinds of cancers. However, cancer is till so hard to cure. Until these days targeted therapies show up and give cancer patient a new hope to cure cancer. Targeted therapies are drugs which can block the growth and spread of cancer by interfering with specific molecules involved in tumor growth and progression. By focusing on molecular and cellular changes that are specific to cancer, targeted therapies are more effective than other types of treatment, including chemotherapy and radiotherapy, and less harmful to normal cells. Targeted therapies have been known by reducing side effects and improving quality life as well. Therefore, many drug manufactures have been investing money on investigating targeted therapies. Target therapy market is rapidly expending, and the competition among the drug manufactures has been more and fiercer. The key or critical success factors to the business of target cancer drugs are the most important issue for the drug manufactures to get the most profit from target drugs. The key success factors can be analyzed by sell strategy, research, development strategy, price strategy and government policy. This paper is trying to find the key success factors for business of target cancer drugs by deep interviewing the top sells managers in the industry and the top doctors in the hospital. Then these interview contents are analyzed by SWOT (strength, weakness, opportunity and threat) to find the key success factors for business of target cancer drugs. With these key success factors, hopefully it can help the drug manufactures make good business on target cancer drug market. Keywords: target drug, target therapy, key success factor, critical success factor, SWOT, deep interview.

Submitted by PAULO CÉSAR JARK null (paulocjark@hotmail.com) on 2016-12-12T17:43:46Z No. of bitstreams: 1 TESE PAULO JARK IMPRESSÃO.pdf: 1837097 bytes, checksum: e5756c844b29f7062a50211bad6f5b0a (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-12-15T15:04:37Z (GMT) No. of bitstreams: 1 jark_pc_dr_jabo.pdf: 1837097 bytes, checksum: e5756c844b29f7062a50211bad6f5b0a (MD5) / Made available in DSpace on 2016-12-15T15:04:37Z (GMT). No. of bitstreams: 1 jark_pc_dr_jabo.pdf: 1837097 bytes, checksum: e5756c844b29f7062a50211bad6f5b0a (MD5) Previous issue date: 2016-11-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A via Janus Kinase (JAK) e do transdutor de sinal e ativador de transcrição (STAT) desempenham papéis importantes na patogênese de neoplasias hematopoiéticas. A ativação da via JAK2/STAT3 promove o crescimento e sobrevivência celular em uma variedade de linfomas humanos. Há uma necessidade de compreender a participação da via JAK2/STAT3 em linfomas caninos difusos de grandes células B e do potencial terapêutico dos inibidores de JAK no tratamento dessa doença. O objetivo do presente estudo foi avaliar a expressão de JAK2-STAT3 em linfomas difusos de grandes células B e o impacto do uso de inibidores de JAK2 como AZD1480 e CYT387 no crescimento in vitro dessa linhagem tumoral. Foi realizada técnica de imuno-histoquímica com os anticorpos anti-STAT3 e anti-STAT3 fosforilado (p-STAT3) em linfonodos acometidos por linfoma difuso de grandes células B e comparado à linfonodos normais e reativos. Para avaliação do efeito terapêutico dos inibidores de JAK2 (AZD1480 e CYT387) foi realizado ensaio de viabilidade celular pelo método de azul de tripan utilizando linhagens celulares de linfoma difuso de grandes células B (CLBL-1) e análise de apoptose por citometria de fluxo utilizando o sistema Annexin V. Houve aumento significativo na expressão de STAT3 e p-STAT3 em linfomas difusos de grandes células B em comparação com linfonodos normais. Ambos os fármacos inibiram o crescimento celular em proporções dependentes da dose administrada e houve um aumento significativo nas taxas de apoptose das células tratadas com inibidores de JAK-2 em comparação ao grupo controle tratado com DMSO. Este é o primeiro estudo a avaliar a via JAK2/STAT3 em linfomas difusos de grandes céluslas B canino e esses dados permitem compreender e explorar o potencial terapêutico dos inibidores de JAK permitindo estudos futuros da eficácia clínica desses fármacos na oncologia veterinária / The Janus Kinase (JAK) and signal transducer and activator of transcription (STAT) pathway play important roles in the pathogenesis of hematologic malignancies. Activated JAK2-STAT3 signaling pathway promotes the growth and survival of a variety of lymphomas in human. There is a great demand for understanding JAK-STAT pathway in canine diffuse large B cell lymphoma (DLBCLs) and evaluating the therapeutic potential of JAK inhibitors. Our study aims to evaluate the expression of JAK2-STAT3 pathway in canine DLBCLs and to assess the impact of AZD1480 and CYT387, two novel JAK inhibitors, on canine DLBCL cell growth. Immunohistochemistry was performed in canine DLBCLs, normal and reactive lymph nodes with primary antibodies against STAT3 and phosphorylated STAT3 (p-STAT3). To evaluate the therapeutic effect of novel JAK inhibitors, canine DLBCL cell line CLBL-1 was treated with either AZD1480 or CYT387 and trypan blue viability assay was performed post treatment. There was a significant increase in expression of STAT3 and pSTAT3 in canine DLBCLs compared with the normal lymph node. Both AZD1480 and CYT387 inhibited canine DLBCL cells in a dose dependent manner. This is the first study to evaluate the JAK2/STAT3 pathway in canine DLBCLs. The knowledge of JAK2-STAT3 activity in canine DLBCLs enables us to understand and explore the therapeutic potential of JAK inhibitors. The dose dependent cell growth inhibition by novel JAK inhibitors in this study will lead into the future studies of the underlying mechanism

Embora o melanoma represente apenas 4% das neoplasias malignas da pele, é considerado a mais grave por ser altamente etal. Em virtude da via MAPK (Mitogen activated protein kinase) estar intimamente ligada ao descontrole da proliferação celular, especialmente em melanoma, esta via se tornou um alvo para o desenvolvimento de terapias direcionadas a oncogenes, como os potentes quimioterápicos Vemurafenibe (inibidor da mutação V600E em BRAF - BRAFV600E) e Trametinibe (inibidor de MEK). Cada vez mais, melhores taxas de respostas vêm sendo alcançadas com os novos medicamentos, porém a maioria dos pacientes está sujeita a recidivas após 7 meses de tratamento devido ao desenvolvimento de quimioresistência, justificando a constante busca por novos compostos terapêuticos. Dados de nosso laboratório indicam que 4-nerolidilcatecol (4-NC) induz aumento na expressão de p53, produção de ROS e dano ao DNA, culminando em apoptose dependente de caspase-3 em células de melanoma por ser um inibidor proteassomal. Além disto, o 4-nerolidilcatecol (4- NC) demonstrou efeito inibitório na proliferação de células de melanoma em modelo de cultura organotípica de pele. Desta forma, este projeto visa avaliar a possibilidade de superação da quimioresistência aos inibidores de BRAF e de MEK, utilizando terapias combinatórias com 4-NC em células de melanoma humano resistentes a estes inibidores. Primeiramente, linhagens celulares de melanoma resistentes aos inibidores de BRAF (R) e BRAF/MEK (DR) foram geradas a partir de células parentais BRAF mutadas (P) e caracterizadas por MTT, microscopia de fluorescência e western blotting. Estas células foram submetidas ao tratamento com 4-NC que apresentou citotoxicidade na concentração de 30µM, inibição de formação de colônias e diminuição na invasão em modelos in vitro de culturas 2D e 3D em todas as linhagens estudadas (P, R e DR). O 4-NC foi ainda capaz de induzir estresse de retículo endoplasmático com indução de apoptose. Visando a explorar o efeito terapêutico in vivo do 4-NC, outro estudo foi conduzido em animais submetidos a enxerto xenográfico com células parentais de melanoma NRAS mutadas. Após desenvolvimento do tumor, os animais foram tratados 3 vezes por semana durante 3 semanas com 4-NC (10 mg/kg) por via i.p. O 4-NC foi capaz de inibir em até 4 vezes o crescimento dos tumores xenográficos (4/10) quando comparado com os controles, com remissão completa do tumor em um animal. A expressão de p53 e PARP clivada foi aumentada nos tumores dos animais tratados, sugerindo apoptose. A expressão gênica de MMP2 e MMP14 estava diminuída nas mesmas amostras, demonstrando o papel do 4-NC na inibição da invasão do melanoma in vivo. Finalmente, a toxicidade sistêmica do 4-NC foi avaliada nas mesmas doses empregadas no ensaio in vivo de tumorigênese. A baixa toxicidade observada nos ensaios toxicológicos com tratamentos sub-crônicos com 4-NC e a citotoxicidade demonstrada em modelos xenográficos nos leva a considerar este composto como promissor para estudos futuros e sua aplicação no tratamento do melanoma cutâneo humano, incluindo pacientes resistentes aos inibidores de BRAF e MEK. / Melanoma accounts for only 4% of malignant neoplasms of the skin, but is considered the most serious because it is highly deadly. Because the MAPK (Mitogen activated protein kinase) pathway is closely linked to the lack of control of cell proliferation, especially in melanoma, this pathway has become a target for the development of oncogene-targeted therapies, such as the potent chemotherapeutic agents Vemurafenib (V600E mutation inhibitor in BRAF - BRAFV600E) and Trametinib (MEK inhibitor). Increasingly, better response rates have been achieved with the new drugs, but most patients are subject to relapses after 7 months of treatment due to several mechanisms, which justify the constant search for new therapeutic compounds. Data from our laboratory indicate that 4-nerolidylcatechol (4-NC) induces increased p53 expression, ROS production and DNA damage, culminating in caspase-3 dependent apoptosis in melanoma cells. The 4-NC compound demonstrated an inhibitory effect on melanoma cell proliferation in an organotypic skin culture model. Thus, this project aims to evaluate the possibility of overcoming the existing chemoresistance to BRAF and MEK inhibitors, using 4-NC combinatory therapies in human melanoma cells resistant to these inhibitors. Firstly, melanoma cell lines resistant to BRAF (R) and BRAF / MEK (DR) inhibitors were generated from naive cells mutated BRAF (N) and characterized by MTT, fluorescence microscopy and western blotting. These cells were submitted to 4-NC treatment that showed cytotoxicity with 30 µM, inhibition of colony formation and decrease in invasion in 2D and 3D in vitro models in all cell line studied (N, R and DR). Furthermore, 4-NC was able to induce endoplasmic reticulum stress with apoptosis induction. In order to explore the in vivo therapeutic effect of 4-NC, an additional study was conducted using xenograft model with NRAS-mutated melanoma cell line. After tumor development, the animals were treated 3 times per week for 3 weeks with 4-NC (10 mg / kg) by i.p. injection. 4-NC was able to inhibit up to 4- fold the growth of xenograft tumors (4/10) when compared to controls, with complete tumor remission in one animal. Cleaved PARP and p53 expression were increased in the tumors of treated animals, suggesting apoptosis. MMP2 and MMP14 gene expression were decreased in the same samples, demonstrating the role of 4-NC in inhibiting melanoma invasion in vivo. Finally, the systemic toxicity of 4-NC was evaluated at the same doses employed in the in vivo tumorigenesis assay. The low toxicity observed in the toxicological assays with sub-chronic 4- NC treatments and the demonstrated cytotoxicity in xenograft models leads us to consider this compound as promising for future studies and its application in the treatment of cutaneous human melanoma, including patients resistants to BRAF and MEK inhibitors.

La transition épithélio-mésenchymateuse (EMT) confère un phénotype agressif aux cancers du sein avec la perte de leurs jonctions épithéliales intercellulaires, leur polarité et identité, et l’acquisition des caractéristiques mésenchymateuses accompagnées de l’invasion et la résistance à la chimiothérapie. Cependant, les mécanismes et les signaux associés qui régissent ces dysfonctionnements au cours de l’EMT des cancers du sein demeurent encore mal connus. Dans cette étude, nous avons entrepris de comparer deux modèles génétiques des cellules MCF-7 du cancer du sein en EMT induite par l’expression constitutive de Snail ou l’invalidation de WISP-2. Nous avons trouvé que les répresseurs des E-cadhérines tels que Slug, Zeb1/2, Twist et Snail étaient surexprimés dans les cellules en EMT avec l’induction d’un phénotype triple-négatif (TN), la perte du récepteur aux oestrogènes, GPR30, et le gain du pouvoir invasif dans le collagène de type I. De plus,l’expression ectopique de Snail dans les cellules transfectées supprime aussi les transcrits de WISP-2. L’EMT est accompagnée de la dominance de plusieurs voies de signalisation proinvasives impliquant les protéines GαGβγ, PKCα, AKT et l’induction de c-Jun. Nous avons mis en évidence l’activation du cycle cellulaire et l’inhibition de l’axe p27/cyclindependent kinase CDK3 responsable de la promotion de la croissance cellulaire. De manière intéressante, le traitement des cellules en EMT avec les inhibiteurs des GαGβγ (BIM-46187, Gallein), PKCα (Gö6976, MT477, sh-RNAs) et de la cascade de signalisation PI3K/AKT (wortmannin) réduit leur potentiel invasif. Par contre, les inhibiteurs sélectifs des récepteurs HER et des voies de signalisation MAPK/SAPK n’ont aucun effet sur les deux modèles d’EMT signant leur indépendance à ces voies oncogéniques. L’ensemble de nos résultats suggèrent que les protagonistes de signalisation représentés par les GαGβγ, PKCα et PI3K/AKT peuvent être considérés comme de potentiels biomarqueurs prédictifs et des cibles thérapeutiques dans la prise en charge des cancers du sein TN en EMT. / The epithelial-to-mesenchymal transition (EMT) confers an aggressive subtype to breast cancers (BC) following disruption of intercellular junctions, epithelial cell polarity, induction of mesenchymal traits, invasive growth and chemotherapy resistance. However, the mechanisms underlying the EMT and its associated signaling dysfunctions in BC are still poorly understood. Here, we have undertaken a comparative study in two genetic models of MCF-7 breast cancer cells induced to EMT by WISP-2 silencing and Snail transformation.We report that the E-cadherin repressors Slug, Zeb1/2 and Twist were overexpressed in these EMT cells. They induced a triple negative phenotype (loss of estrogen ERα and progesterone PRA/PRB receptors, no HER2 amplification), combined with loss of the GPR30 ER and promotion of invasive growth in collagen gels. Ectopic Snail expression suppressed WISP-2 transcripts and down-regulated WISP-2 gene promoter expression in transfected cells. The EMT caused dominance of several proinvasive pathways including GαGβγ subunits, PKCα, AKT and c-Jun induction, coupled with growth responses (more cells at S and G2/M phases of the cell cycle), in line with inhibition of the p27kip1/cyclin-dependent kinase CDK3 cascade. RNA interference or selective inhibitors targeting GαGβγ subunits (BIM-46187, gallein), PKCα (Gö6976, MT477, sh-RNAs) and PI3K-AKT (wortmannin) alleviated the invasive phenotype. In contrast, MCF-7 cells in EMT showed signalingindependence to inhibitors of HER family tyrosine kinases and the mitogen- and stressactivated protein kinases. Our study suggests that the signaling protagonists GαGβγ, PKCα and PI3K-AKT are promising candidates as predictive molecular biomarkers and therapeutic targets in the management of clinical BC in EMT.

La formation de nouveaux vaisseaux sanguins ou angiogenèse soutient la croissance tumorale en fournissant l'oxygène et les nutriments qui lui sont nécessaires. Le rôle clé du facteur de croissance de l'endothélium vasculaire VEGF dans ce processus a suscité le développement de stratégies anti-angiogéniques pour le traitement du cancer. Cependant, des travaux précliniques et des données cliniques suggèrent l'émergence de résistances aux anti-angiogéniques, en raison notamment de la redondance des facteurs de croissance pro-angiogéniques. Il est donc nécessaire de développer des stratégies alternatives plus efficaces. En 2010, notre laboratoire a apporté la preuve de concept d'une thérapie anti-tumorale et anti-angiogénique innovante basée la dégradation des ARNm à demi-vie courte par la protéine à doigts de zinc TIS11b. Néanmoins, l'instabilité de la protéine thérapeutique a entravé la caractérisation plus détaillée de cette stratégie. Dans ce contexte, l'objectif majeur de ma thèse était l'optimisation de la stabilité et de l'activité de TIS11b et l'évaluation de son efficacité thérapeutique. Pour cela, nous avons généré une nouvelle protéine TIS11b génétiquement modifiée sur la base d'études biochimiques et moléculaires. Notamment, nous avons observé que la phosphorylation de la sérine 334 située dans le domaine C-terminal de TIS11b augmente de façon très significative la stabilité de la protéine et potentialise son activité déstabilisatrice de l'ARNm du VEGF. De plus, la délétion du domaine N-terminal augmente également la stabilité de TIS11b sans altérer son activité. Nous avons alors généré deux nouvelles protéines thérapeutiques, la protéine ZnC et la protéine ZnC334D pour laquelle la troncation du domaine N-terminal et la substitution de la sérine S334 par un aspartate mimant une phosphorylation ont été combinées. Les nouvelles protéines ont été fusionnées à une étiquette polyarginine R9 leur permettant de traverser les membranes cellulaires (R9-ZnC et R9-ZnCS334D). Nous avons montré que R9-ZnC et R9-ZnCS334D inhibent l'expression de VEGF in vitro dans la lignée de cancer du sein murin 4T1. De plus, R9-ZnCS334D exerce une activité anti-proliférative, anti-migratoire et anti-invasive dans ces cellules. In vivo, l'injection intra-tumorale de R9-ZnCS334D dans des tumeurs 4T1 préétablies inhibe significativement l'expression du VEGF, la croissance et la vascularisation tumorales. De façon remarquable, l'analyse des extraits tumoraux indique que le traitement diminue l'expression de chimiokines clés dans les processus d'angiogenèse, d'inflammation et d'invasion (Fractalkine, MCP-1, NOV, SDF-1, Pentraxin…). Enfin, R9-ZnC et R9-ZnCS334D inhibent l'expression de marqueurs de la transition épithélio-mésenchymateuse, un processus impliqué dans la dissémination métastatique. L'ensemble de ces travaux indique que R9-ZnC et R9-ZnCS334D sont des molécules anti-tumorales multi-cibles, qui inhibent plusieurs étapes clés de la progression tumorale. Cette étude confirme que le ciblage de la stabilité des ARNm est une stratégie prometteuse et novatrice pour le développement de nouvelles thérapies anti-cancéreuses. / One of the innovative aspects of anti-cancer therapies is the possibility of preventing tumor growth by blocking blood supply. Cancer cells induce the formation of their own blood vessels from pre-existing vasculature, a process called angiogenesis. One of the most important proangiogenic factors is vascular endothelial growth factor (VEGF). The success of bevacizumab (a humanized anti-VEGF monoclonal antibody) combined to chemotherapy for the treatment of human metastatic cancers has validated VEGF as an efficient target. However, despite the initial enthusiasm, resistance to these anti-angiogenic treatments resulting from compensatory mechanisms occurs upon time. For this reason, there is a real need for new anti-angiogenic drugs that will target the angiogenic process through distinct mechanisms. In 2010, our laboratory has successfully developed an anti-angiogenic and anti-tumoral therapy based on destabilization of short-lived mRNAs by the zinc finger protein TIS11b. However, the therapeutic protein was highly unstable, thus making it difficult to further characterize the experimental therapy. In this context, the main task of my thesis was the optimization of TIS11b stability and activity followed by the evaluation of the multi-target action of our novel protein on tumor development. In a first part of this work, biochemical and molecular approaches allowed us to demonstrate that phosphorylation of the C-terminal serine S334 in TIS11b protein markedly increases its stability. In addition, deletion of the N-terminal domain of TIS11b highly increases its protein stability without affecting its activity. Therefore, we integrated N-terminal truncation (ZnC) and C-terminal substitution of S334 by an aspartate to mimic a permanent phosphorylation at S334 (ZnCS334D) as a novel TIS11b engineering strategy. Both proteins were fused subsequently to a cell-penetrating peptide polyarginine (R9). In vitro studies revealed that R9-ZnC and R9-ZnCS334D inhibit VEGF expression in the murine breast cancer cells 4T1. In addition, R9-ZnCS334D impaired proliferation, migration, invasion and anchorage-independent growth of 4T1 cells. In vivo, intra-tumoral injection of either protein significantly reduced VEGF expression and tumor vascularization. Strikingly, antibody array analyses of tumor extracts demonstrated a reduced expression of several chemokines such as Fractalkine, MCP-1, NOV, SDF-1 and Pentraxin upon R9-ZnC or R9-ZnCS334D treatment. These factors, which are produced by several cell types within tumor tissue, are key drivers of tumor angiogenesis, tumor-promoting inflammation and invasion. Furthermore, the expression of markers of the epithelial-to-mesenchymal transition was also significantly reduced, suggesting an anti-metastatic effect of R9-ZnC and R9-ZnCS334D. Thus, we provide R9-ZnC and R9-ZnCS334D as potential novel multi-target agents which inhibit key hallmarks of cancer progression. This work supports the emerging link between mRNA stability and cancer and proposes novel concepts for the development of innovative anti-cancer therapies.