When activated by growth factors and mitogens, cellular receptors like EGFR become activated and transmit signals from the cell surface through the MAPK pathway into the nucleus of the cell, in order to elicit a cellular response like growth, apoptosis, proliferation, and survival. Over activation of EGFR even without receptor binding and members of the MAPK pathways like BRAF and MEK1 have been reported in tumorigenesis. We used Gateway Technology to study the recombinant protein expressions from these 3 proto-oncogenes in HEK293T and HeLa cell lines, using pLenti 6.3/V5 as the expression system. BRAF was neither detected in the HeLa nor in the HEK 293T cell lines, by mass spectrometry after transfection. Only in the HeLa cell line was EGFR identified meanwhile MEK1 was identified both in HEK293T and HeLa cell lines. However, the results showed that the proteins identified were endogenous to these cell lines and therefore, no recombinant proteins were expressed using the pLenti 6.3/V5, as the expression system.

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Contexte. Le cancer bronchopulmonaire (CBP) demeure la première cause de mortalité par cancer dans le monde. Malgré l’espoir suscité par le développement des thérapies ciblées, son pronostic demeure sombre, particulièrement dans les cas de CBP à petites cellules (CBP-PC) et de CBP non à petites cellules (CBP-NPC) présentant une activation de l’oncogène KRAS. Matériel et Méthodes. Nous avons mené 3 études successives, visant à (i) radiosensibiliser des modèles de CBP-PC par l’ajout d’un inhibiteur de BCL2, (ii) cibler des modèles de CBP-NPC mutés KRAS par l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF, et (iii) créer un modèle préclinique orthotopique murin de CBP reproduisant la progression tumorale observée en clinique. Résultats. Dans la première étude, l’inhibiteur de BCL2 oblimersen a présenté un effet radiosensibilisant sur des modèles de CBP-PC, in vitro et in vivo. Dans la seconde étude, l’association de l’inhibiteur de mTOR everolimus et de l’inhibiteur de RAF/VEGFR RAF265 a présenté un effet synergique sur des lignées cellulaires de cancers présentant la double mutation de KRAS et de PIK3CA, in vitro et in vivo. Dans la troisième étude, l’injection orthotopique d’une lignée bioluminescente de CBP-NPC chez des souris nude a permis d’établir des tumeurs intra pulmonaires évoluant vers une extension métastatique ganglionnaire et hématogène, et de détecter la présence de cellules tumorales circulantes. Conclusion. L’association d’un inhibiteur de BCL2 à la radiothérapie est une stratégie intéressante dans le CBP-PC, l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF/VEGFR est une stratégie intéressante dans le CBP-NPC présentant une double mutation KRAS-PIK3CA, mais ces données doivent être confirmées sur des modèles orthotopiques afin de gagner en pertinence avant d’envisager un transfert en clinique. / Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Activation of phosphatidylinositol-3-kinase (PI3K)-AKT and Kirsten rat sarcoma viral oncogene homologue (KRAS) can induce cellular immortalization, proliferation, and resistance to anticancer therapeutics such as epidermal growth factor receptor inhibitors or chemotherapy. This study assessed the conséquences of inhibiting these two pathways in tumor cells with activation of KRAS, PI3K-AKT, or both. We investigated whether the combination of a novel RAF/vascular endothelial growth factor receptor inhibitor, RAF265, with a mammalian target of rapamycin (mTOR) inhibitor, RAD001 (everolimus), could lead to enhanced antitumoral effects in vitro and in vivo. To address this question, we used cell lines with different status regarding KRAS, PIK3CA, and BRAF mutations, using immunoblotting to evaluate the inhibitors, and MTT and clonogenic assays for effects on cell viability and proliferation. Subcutaneous xenografts were used to assess the activity of the combination in vivo. RAD001 inhibited mTOR downstream signaling in all cell lines, whereas RAF265 inhibited RAF downstream signaling only in BRAF mutant cells. In vitro, addition of RAF265 to RAD001 led to decreased AKT, S6, and Eukaryotic translation initiation factor 4E binding protein 1 phosphorylation in HCT116 cells. In vitro and in vivo, RAD001 addition enhanced the antitumoral effect of RAF265 in HCT116 and H460 cells (both KRAS mut, PIK3CA mut); in contrast, the combination of RAF265 and RAD001 yielded no additional activity in A549 and MDAMB231 cells. The combination of RAF and mTOR inhibitors is effective for enhancing antitumoral effects in cells with deregulation of both RAS-RAF and PI3K, possibly through the cross-inhibition of 4E binding protein 1 and S6 protein. We then focus on animal models. Preclinical models of NSCLC require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC. Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours. Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.

Neste estudo, geramos ensaios de espectrometria de massa para detecção de 111 mutações nos genes RET, BRAF, NRAS, HRAS, KRAS, PIK3CA e AKT1 e avaliamos inúmeras linhagens celulares e tumores tiroidianos. Mostramos que as mutações dos genes BRAF e RAS refletem prognósticos distintos e que as mutações BRAF são altamente prevalentes em câncer metastático. Mutações dos genes PIK3CA e AKT1, esta última sendo reportada pela primeira vez no câncer de tiróide, são relativamente frequentes neste câncer. Avaliamos ainda a função do microRNA let-7 neste câncer. Mostramos que a ativação do rearranjo RET/PTC3 em células de tiróide PCCl3 reduz a expressão de let-7. Além disso, a transfecção deste microRNA em células TPC-1, que apresentam o rearranjo RET/PTC1, inibe a fosforilação de ERK, o crescimento celular e modula a expressão de genes do ciclo celular e diferenciação. Estes dados contribuem na aplicação de terapias dirigidas a efetores das vias PI3K e MAPK no câncer de tiróide, além de salientar o envolvimento do miRNA let-7 como um gene supressor tumoral nesta doença. / In this study, we designed an assay panel for genotyping 111 mutations by mass spectrometry in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1 and other related genes in many thyroid cancer cell lines and tumors. We show that patients with BRAF and RAS mutations have distinct prognosis and that BRAF mutations are highly prevalent in metastatic thyroid cancers. Mutations of PIK3CA and AKT1, the latter not previously described in this disease, are comparatively frequent in thyroid cancers. In addition, we evaluated the role of let-7 microRNA in this cancer. We show that RET/PTC3 activation in PCCl3 thyroid cells reduces let-7 expression. Moreover, transfection of let-7 in TPC-1 cells, which harbor RET/PTC1 rearrangement, inhibits MAPK activation, reduces cell growth and modulates the expression of cell cycle and differentiation genes. These findings may contribute to the design of therapies directed at MAPK and PI3K effectors and to highlight the function of let-7 as tumor suppressor gene in thyroid cancer.

La v-ATPase est une pompe à protons. Elle permet l’acidification d’organelles, ce qui est indispensable à de nombreux processus cellulaires. Cette enzyme est composée de 14 sous-unités différentes, organisées en deux domaines, le domaine catalytique V1 et le domaine membranaire V0. La sous-unité a du domaine V0 est essentielle au transport des protons. Il en existe 4 isoformes (a1 à a4) et des variants d’épissage (a1-I à a1-IV pour a1) permettant à la v-ATPase d’être adressée vers différents compartiments et donc d’être impliquée dans différents processus. Deux projets visant à étudier cette sous-unité ont été réalisés.En plus de son rôle dans le transport des protons, il a été montré que le domaine V0 de la v-ATPase est impliqué dans des évènements de trafic membranaire, tel que l’exocytose de vésicules de sécrétion. Ce rôle semble nécessiter des interactions avec les protéines SNAREs. J’ai montré, pendant la première partie de ma thèse, que les sous-unités flag-a1-I et flag-a1-IV sont toutes deux adressées aux granules de sécrétion de cellules neurosécrétrices et interagissent avec les protéines SNAREs VAMP2 et syntaxine-1. De façon intéressante la syntaxine-1 semble interagir préférentiellement avec la sous-unité a1-I qui dans les neurones est l’isoforme adressée aux terminaisons nerveuses. Les sous-unités a1-IV ne diffèrent d’a1-I que par l’ajout de 7 acides aminés dans sa moitié N-terminale. Le domaine d’interaction de la sous-unité a avec la syntaxine-1 semble donc être localisé dans cette région.Dans la deuxième partie de ma thèse, j’ai mis en évidence et étudié l’expression de la sous-unité rénale a4 dans des gliomes humains. Ces tumeurs sont les tumeurs cérébrales les plus fréquentes et sont en général associées à un mauvais pronostic. L’OMS distingue, en fonction de paramètres histologiques, les astrocytomes (de grade I à IV), les oligodendrogliomes et les gliomes mixtes (chacun de grade II ou III). Cette classification est controversée, notamment à cause de son manque de reproductibilité, et la prise en compte de marqueurs moléculaires semble s’imposer comme une solution pour la renforcer.J’ai quantifié par RT-PCR quantitative l’expression du gène ATP6V0A4 (codant la sous-unité a4) dans 188 prélèvements de gliomes humains. Nous avons ainsi montré que l’expression de la sous-unité a4 peut être utilisée comme marqueur diagnostique des oligodendrogliomes anaplasiques (35 % l’expriment). Dans un prélèvement, la présence de la codélétion 1p/19q et l’expression de a4, tout deux marqueurs indépendants des oligodendrogliomes, permettra le renforcement du diagnostique oligodendrogliome anaplasique. De plus a4 est fréquemment exprimée par les astrocytomes pilocytiques (70%), où elle est associée à la duplication en tandem de la région chromosomique 7q34 située à proximité directe du gène ATP6V0A4. Enfin une observation prometteuse est que l’expression de a4 pourrait être un marqueur de mauvais pronostic pour les patients atteints d’oligodendrogliome anaplasique ne présentant pas la co-délétion 1p/19q, observation qui devra être confirmée sur une plus grande cohorte de patients. / Vacuolar type H+-ATPase is a proton pump, which acidifies numerous organelles, crucial for many cellular processes. This enzyme is composed of 14 different subunits organized in two domains, a catalytic V1 domain and a V0 membrane domain. The a-subunit of V0 is essential for proton transport. There are 4 isoforms of a (a1 to a4) and splicing variants (a1-I to a1-IV for the a1 subunit). v-ATPases containing different a-subunit isoforms are localized in different compartments allowing v-ATPase to participate in different processes. The a-subunits were studied in this work in two distinct projects.Besides its role in proton pumping, V0 domain of v-ATPase is implicated in organelles trafficking events, like vesicles exocytosis. This role seems to require interactions of V0 with SNARE proteins. During my thesis work, I showed that flag-a1-I and flag-a1-IV are both targeted to secretion granules in PC12 neurosecretory cells. These subunits interact with the SNARE proteins VAMP2 and syntaxin-1. Interestingly, syntaxin-1 seems to preferentially interact with the a1-I subunit, isoform which in neurons is sorted to nerve terminals. The only difference between a1-I and a1-IV subunits is the addition of 7 amino acids in the N-terminal half of a1-IV. So syntaxin-1 probably interacts with a1-I at this location. In a second project, I studied the expression of the renal a4-subunit in human gliomas. These tumors are the most frequent brain tumors and are generally associated with a poor prognosis. Based on histological parameters,WHO distinguishes, astrocytomas (grade I to IV), oligodendrogliomas and oligoastrogliomas (each of grade II or III). This classification suffers of a lack of reproducibility, which could be overcome by the identification of specific molecular markers.In the present work, by real time quantitative PCR, ATP6V0A4 gene (encoding the renal a4) expression was quantified in 188 human glioma biopsies. We established a4 expression as a new marker of grade III oligodendrogliomas (35 % express it), independent of the 1p/19q codeletion, an established marker of oligodendrogliomas. Moreover, a4 is expressed in 70% of pilocytic astrocytomas, in which it is associated with the tandem duplication of 7q34, localized at direct proximity of the ATP6V0A4 gene. Of promising interest is the observation that a4 expression could be considered as a bad prognostic marker for patients with 1p/19q non-deleted oligodendrogliomas, an observation that should be confirmed on larger cohorts of patients.

Neste estudo, geramos ensaios de espectrometria de massa para detecção de 111 mutações nos genes RET, BRAF, NRAS, HRAS, KRAS, PIK3CA e AKT1 e avaliamos inúmeras linhagens celulares e tumores tiroidianos. Mostramos que as mutações dos genes BRAF e RAS refletem prognósticos distintos e que as mutações BRAF são altamente prevalentes em câncer metastático. Mutações dos genes PIK3CA e AKT1, esta última sendo reportada pela primeira vez no câncer de tiróide, são relativamente frequentes neste câncer. Avaliamos ainda a função do microRNA let-7 neste câncer. Mostramos que a ativação do rearranjo RET/PTC3 em células de tiróide PCCl3 reduz a expressão de let-7. Além disso, a transfecção deste microRNA em células TPC-1, que apresentam o rearranjo RET/PTC1, inibe a fosforilação de ERK, o crescimento celular e modula a expressão de genes do ciclo celular e diferenciação. Estes dados contribuem na aplicação de terapias dirigidas a efetores das vias PI3K e MAPK no câncer de tiróide, além de salientar o envolvimento do miRNA let-7 como um gene supressor tumoral nesta doença. / In this study, we designed an assay panel for genotyping 111 mutations by mass spectrometry in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1 and other related genes in many thyroid cancer cell lines and tumors. We show that patients with BRAF and RAS mutations have distinct prognosis and that BRAF mutations are highly prevalent in metastatic thyroid cancers. Mutations of PIK3CA and AKT1, the latter not previously described in this disease, are comparatively frequent in thyroid cancers. In addition, we evaluated the role of let-7 microRNA in this cancer. We show that RET/PTC3 activation in PCCl3 thyroid cells reduces let-7 expression. Moreover, transfection of let-7 in TPC-1 cells, which harbor RET/PTC1 rearrangement, inhibits MAPK activation, reduces cell growth and modulates the expression of cell cycle and differentiation genes. These findings may contribute to the design of therapies directed at MAPK and PI3K effectors and to highlight the function of let-7 as tumor suppressor gene in thyroid cancer.

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Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lukáš Slatinský Supervisor: Assoc. prof. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Interactions of selected anticancer drugs of the MAPK/ERK signaling pathway inhibitors group with the ABC drug transporters ABCB1 (Pgp, P-glycoprotein) and ABCG2 (BCRP, breast cancer resistance protein) are members of a transmembrane efflux ATP dependent transporter family, so called ATP-binding cassettes (ABC). Physiologicaly they are expressed in the cellular membrane and protect body tissues against potentially toxic xenobiotics including drugs. They represent also one of the tumor defense mechanisms when being able to efflux a wide variety of cytotoxic drugs out of the cancer cells leading to treatment failure. BRAF protein plays an important regulatory and signal role in MAPK/ERK pathway affecting cell division, differentiation and secretion. Mutations of BRAF lead to overactivity in MAPK/ERK pathway in many cancer cells and can be therefore targeted by anticancer therapy. Cobimetinib and dabrafenib are relatively new anticancer therapeutics inhibiting the signal pathway mentioned above and they are used in treatment of melanoma carrying the BRAF mutation. The aims of this project were to...