Das Multiple Myelom (MM) ist eine seltene, maligne Störung der Plasmazellen, welche trotz gehöriger Therapiefortschritte in den letzten Jahrzehnten nach wie vor als unheilbare Erkrankung betrachtet werden muss. Obwohl eine sehr große intra- und interindividuelle Heterogenität beim Multiplen Myelom beobachtet werden kann, gibt es verschiedene Mutationen, die mit höherer Frequenz in Myelompatientinnen und -patienten gefunden werden. Eines dieser häufiger betroffenen Proteine ist KRas mit Mutationen in etwa 20% der Fälle. Da die Ras-Proteine und somit auch ihre Isoform KRas zu Beginn der Ras/Raf/Mek/Erk-Signalkaskade stehen und dementsprechend einen großen Einfluss auf die Übermittlung von Wachstums- und Überlebenssignalen in Zellen besitzen, ist eine nähere funktionelle Analyse verschiedener KRas-Mutationen von großer Relevanz. Während für einige Mutationen von KRas bereits funktionelle Analysen existieren, wurden die häufig auftretende Exon 2-Mutation KRasp.G12A, sowie die beiden seltenen Exon 4-Mutationen KRasp.A146T und KRasp.A146V bisher in ihrer funktionellen Rolle im MM noch nicht näher charakterisiert. Um die funktionellen Aspekte dieser genannten Mutationen von KRas näher zu untersuchen, kamen im Rahmen meiner Versuchsreihe Sleeping Beauty Transposon System basierte Expressionsvektoren zur transienten und dauerhaften Proteinexpression in verschiedenen Myelomzelllinien zum Einsatz. Durch Transfektion dieser Plasmide in die KRas-Wildtyp tragenden Zellen mit nachfolgender Transposition in die genomische DNA konnte gezielt die Überexpression der verschiedenen Mutationen realisiert werden. So konnte durch die funktionelle Proteinauslese mittels der Anfertigung von Western Blots gezeigt werden, dass jede der drei getesteten Mutationen zu einer verstärkten Phosphorylierung und damit Aktivierung von KRas-nachgeschalteten Proteinen wie z.B. Erk führt. Zusätzlich wurde für die KRas-Mutationen auch ein aktivierender Effekt auf den PI3K/Akt-Signalweg anhand einer erhöhten Phosphorylierung des Proteins Akt nachgewiesen. Ebenso wie andere bereits besser charakterisierte KRas-Mutationen haben demnach auch die getesteten KRas-Mutationen KRasp.G12A, KRasp.A146T und KRasp.A146V einen positiven Einfluss auf die intrazellulären Überlebenssignale und könnten daher eine elementare Rolle in der Entwicklung des Multiplen Myeloms bei Patientinnen und Patienten spielen. Es gilt daher, die drei in dieser Arbeit untersuchten KRas-Mutationen, zukünftig in die Wirkstoffsuche KRas-spezifischer Therapeutika miteinzubeziehen. / Multiple Myeloma (MM) is a rare, malignant disorder of plasma cells, which despite the progress in therapy over the last decades, must still be considered an incurable disease. Although a very large intra- and interindividual heterogeneity can be observed in multiple myeloma, there are various mutations that are found at higher frequencies in myeloma patients. One of these more common proteins affected by mutations in myeloma patients is KRas, with mutations in about 20% of cases. Since the Ras proteins and thus also their KRas isoform are at the beginning of the Ras/Raf/Mek/Erk signaling cascade and therefore have a major influence on the transmission of survival signals in cells, a closer functional analysis of various KRas mutations is of great relevance. While functional analyses already exist for some KRas mutations, the frequently occurring exon 2 mutation KRasp.G12A and the two rare exon 4 mutations KRasp.A146T and KRasp.A146V have not yet been characterized in their functional role in MM. To further investigate the functional aspects of these KRas mutations, I used protein expression vectors based on the Sleeping Beauty Transposon System for transient and sustained protein expression in different myeloma cell lines. By transfection of these plasmids into KRas wild-type cells the overexpression of the different mutations could be realized. By functional protein readout using Western blots it could be shown that each of the three tested mutations leads to increased phosphorylation and thus activation of KRas downstream proteins such as Erk. In addition, an activating effect on the PI3K/Akt signaling pathway could be demonstrated for the KRas mutations by showing an increased phosphorylation of the Akt protein. As with other KRas mutations that have already been better characterized, the KRas mutations KRasp.G12A, KRasp.A146T and KRasp.A146V have a positive influence on intracellular survival signals and could therefore play a fundamental role in the development of multiple myeloma in patients. It is therefore important to include the three KRas mutations investigated in this work in the drug discovery process for KRas-specific therapeutics in the future.

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El gen K-ras, és molt important clínicament ja que es troba mutat en un 40% dels casos de carcinomes colorectals i en un 90% dels casos de carcinomes de pàncreas, que constitueixen la segona i quarta causa de mort per càncer en països desenvolupats i juga un paper important en el desenvolupament dels sarcomes. Degut a la importància clínica del gen K-ras, un model que millori el seu coneixement en l'àmbit molecular, ajudarà a predir el comportament biològic del tumor abans de decidir el tractament més adequat per un pacient concret. Aquests coneixements, poden portar a una millora substancial des del punt de vista terapèutic. Les mutacions activants de l'oncogen K-ras, s'han localitzat al codó 12 o 13 del mateix. Prèviament, s'ha associat la presència de mutacions al codó 13 amb un fenotip tumoral menys agressiu que quan aquestes es localitzen al codó 12. Concretament, la mutació al codó 13 s'associa amb menor capacitat invasiva local i metastàsica. L'objectiu d'aquesta tesi es trobar una explicació molecular al diferent comportament clínic descrit amb anterioritat en tumors humans segons si la mutació activant es troba al codó 12 o al codó 13 del gen K-ras. També es preten trobar una explicació molecular a la transformació produïda pel gen normal sobrexpressat, que prèviament s'havia descrit que en determinats models era tumorogènic. Aquest estudi s'ha realitzat en un model in vitro (fibroblasts NIH3T3 de ratolí), transfectats establement amb el gen K-ras humà amb les distintes mutacions i en un modelo in vivo, injectant subcutàniament els transfectants en ratolins atímics i estudiant els sarcomes generats.Les conclusions d'aquest treball són que en el nostre model d'estudi, la mutació al codó 12 de K-ras confereix un fenotip més transformant que la mutació al codó 13 o que la sobreexpresió del gen normal. La causa principal d'aquestes diferències en transformació és la major activació de la via de supervivència cel·lular PI3K/AKT als transfectants amb mutació al codó 12 que els confereix major resistència a l'apoptosi, tant en el model in vitro com in vivo. Per últim, la posició del codó mutat en K-ras confereix especifitat histològica als tumors generats que de manera coherent amb el seu origen mesenquimàtic (NIH3T3) són sarcomes. Els tumors generats amb mutació al codó 12 del gen K-ras constitueixen un possible model de fibrosarcoma i els generats amb mutació al codó 13 del gen K-ras constitueixen un possible model d'histioctioma fibrós maligne; dos tipus de sarcomes humans. / K-ras gene is the most frequently mutated ras gene in human tumors. It's mutated in 40% of cases of colorectal carcinomas and 90% of pancreatic adenocarcinomas. These are the second and fourth causes of cancer death in developed countries. K-ras play also an important role in development of sarcomas. A model that improve knowledge of K-ras in a molecular way will help to decide the right treatment to a specific patient.K-ras became oncogenic by single point mutations at codon 12 or 13. Several lines of evidence suggest that the malignant potencial of tumor cells may be influenced not only by the presence or absence of K-ras mutations, but by its molecular nature. Codon 13 mutations have been associated with less aggressive tumoral phenotype than codon 12 mutations in K-ras gene. Codon 13 mutation is associated with less invasive and metastasic capacity.In this thesis, is tested whether K-ras codon 12 mutation would confer upon the cell a more oncogenic phenotype than a K-ras codon 13 mutation and try to find a molecular explanation for these differences. It is also tested the transformation capacity of the K-ras proto-oncogene and molecular explanation of this effect.To this end, NIH3T3 cells (mouse fibroblasts) are transfected with a plasmid containing human K-ras with point mutations at codon 12 or at codon 13 or containing the K-ras proto-oncogene; selected stable transfectants; and evaluated the possible changes in different functions contributing to transformation. It's tested also in vivo model. We subcutaneously injected transfectants in nude mice and generated tumors were compared morphocally, functionally, and molecularly.The conclusion of this work is that K-ras with point mutations at codon 12 confer a more aggressive transforming phenotype, increasing the thresfold of apoptotic induction. In contrast, mutations at codon 13 or the overexpression of K-ras proto-oncogene reduce this thresfold. Increased activation of cellular survive pathway PI3K/AKT confer to K-ras with point mutations at codon 12 resistance to apoptosis, in vitro and in vivo model.And last; the position of mutated codon in K-ras gene confer histologic specificity to generated tumors.Tumors derived from transfectants with K-ras point mutations at codon 12 constitute a possible animal model of human fibrosarcoma. Tumors derived from transfectants with K-ras point mutations at codon 13 constitute a possible animal model of human malignant fibrous histiocytoma.

La technique d'une DPC pour adénocarcinome a évolué ces dernières années tant au niveau sécurité qu'au niveau carcinologique mais cela n'a pas suffit à faire progresser la survie. On peut se demander si la modification de la technique chirurgicale pourrait avoir un impact significatif sur la survie.A) Nous avons recherché, sur 23 pièces de DPC encrées, la présence de la mutation K-ras au niveau de la marge veineuse affirmée R0 en analyse histologique : 13 spécimens (groupe kras+) exprimaient une mutation K-ras au sein de la marge veineuse versus 10 spécimens (groupe kras-) ne l'exprimant pas. Les tumeurs des 2 groupes étaient comparables (taille, envahissement ganglionnaire, engainement périnerveux…). La survie globale à 1 an et 3 ans des groupes kras- versus kras+ étaient de 80% versus 84,6% et 16,7% versus 0% (p=0,03), respectivement. Les médianes de survie des groupes kras- versus kras+ étaient de 24 mois versus 16 mois (p=0,04), respectivement.B) Nous avons comparé, après appariement, 19 patients ayant eu une DPC avec résection « par excès » de la veine porte (groupe VP) avec 19 patients ayant eu une DPC sans résection de la veine porte (groupe contrôle). Les survies médianes et à 3 ans du groupe VP versus groupe contrôle étaient 42 mois versus 22 mois (p=0,04) et 60% versus 31% (p=0,03), respectivement.En conclusion, notre travail a montré qu'au-delà de la marge déterminée par le chirurgien pendant la chirurgie, de celle de l'anatomopathologiste déterminée par l'analyse microscopique, il existait une marge génique. La résection systématique de la veine porte semblait bénéfique car elle permettait sans doute de passer au-delà de cette marge génique. / Pancreticoduodenectomy (PD) for adenocarcinoma was safer during the last decades but did not improve survival. We sought to determine if technical changes during PD could improve survival.A) In a first study, we determine the presence of K-ras mutation in the venous margin of 23 PD's specimens. Thirteen specimens had K-ras mutation (kras+ group) and 10 specimens did not (kras- group). Except K-ras mutation status, tumors of the 2 groups were not different when comparing major histological findings (margin status, lymph node invasion, perineural invasion…). Overall 1- and 3-years survival of patients of kras- group versus kras+ group were 80% versus 84,6% and 16,7% versus 0% (p=0,03), respectively. Median survival of patients of kras- group versus kras+ group were 24 months versus 16 months (p=0,04), respectively.B) In a second study, we compared 19 patients with “excessive” portal vein resection during PD (PV group) with 19 matched patients who underwent PD without venous resection (control group). Median survival of patients of PV group versus control group were 42 months versus 22 months (p=0,04), respectively.In conclusion, we showed that the « genic margin » concept was consistent. Systematic portal vein resection could avoid positive genic margin and might be benefic for patient who underwent PD for resecable adenocarcinoma.

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Normal tissue structure and function are maintained by a dynamic interaction between epithelial cells and the stroma consisting of fibroblasts, adipose, vasculature and resident immune cells, and a multitude of cytokines and growth factors. Stroma was usually studied in the background of the malignant lesion, only in recent years researchers have started considering its role before carcinogenic lesions appear. Recent studies have shown that stromal cells and their products can cause the transformation of adjacent cells through transient signaling during phenomena like adipogenesis and inflammation by secreting various cytokines and chemokines into the matrix which can lead to apoptosis resistance, proliferation, mutations etc. Research in the last few years has demonstrated a functional role for stroma in the initiation and progression of breast, colon and prostate carcinomas. In this study effect of adipogenesis and/or inflammation on prostaglandin biosynthesis is investigated and the effects that these prostaglandins can have on epithelial cells is highlighted. This work demonstrates that normal colonic fibroblasts CCD18Co can produce anti-tumorigenic and pro-tumorigenic prostaglandins during adipogenesis and that this signaling is mediated via COX-2 activation. Although deoxycholic acid (DCA), a secondary bile acid that is responsible for inflammation in the gastro-intestinal tract, induces COX-2 signaling in the fibroblasts the downstream signaling of prostaglandin synthases is suppressed. Adipogenesis also leads to an increased polyamine catabolism. Effects of the prostaglandins were studied on various epithelial colon cancer cell lines. It was seen that 15d-PGJ2 causes growth inhibition and apoptosis in all cell lines tested and it was demonstrated that an activated K-RAS suppressed this phenomena. It was also seen that 15d-PGJ2 treatment could induce MAPK signaling and that an activated K-RAS suppressed JNK activation via AKT and MKK4. In conclusion this work reports that colonic fibroblasts can produce anti-tumorigenic factors like 15d-PGJ2 which may then induce apoptosis in epithelial cancer cells. This would be suppressed by an activated K-RAS and at the same time 15d-PGJ2 mediated MAPK signaling could confer a growth advantage for these cells and thus aid in tumor progression.

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The treatment and/or prevention of cancer by selective down regulation of cancer causing gene (oncogene) transcription would represent a significant advance in the area of anticancer drug design. Non-canonical higher order DNA structures formed in oncogene promoter regions are novel targets for the modulation of oncogene expression. An obvious advantage of selectively targeting oncogene expression would be that general cytotoxicity would be minimized and the negative side effects of current chemotherapy approaches could be minimized or eliminated. To provide a foundation for the design of drugs that target oncogene promoter G-quadruplexes and i-Motifs, the basic understanding is required of the folding of guanine and cytosine rich sequences and how small molecules bind to these structures. The research reported here focuses on higher order DNA structures of two oncogenes, K-ras that is overexpressed in pancreatic cancer, and Bcl-2 that is overexpressed in a number of cancers, and one non-oncogene, HAR1. We have probed the overall structure, stability, and binding of a model drug compounds to G-quadruplex and i-Motif DNA structures in these genes. The overall objectives of this work were: 1) to understand the relationship between oligonucleotide sequence and intramolecular folding topology and stability, and 2) to understand the mechanisms for the selective binding of small molecules to these structures. Biophysical techniques including: microcalorimetry, spectroscopy, analytical ultracentrifugation, gel electrophoresis, and computational methods were used to characterize both the folding and the binding interactions. We have shown that the native K-ras purine and pyrimidine rich sequences form stable G-quadruplexes and i-Motifs. We have also characterized four G-rich sequences found within the reading frame of the human HAR1 gene. This is the first report on the formation of stable G-quadruplex motifs within the RF of any gene. The model drug, TMPyP4, binds to the Bcl-2, K-ras, and HAR1 G-quadruplexes by two different binding modes, end binding and intercalation. The significance of this research is that the results of the K-ras and Bcl-2 studies could lead to the design of drugs that selectively target oncogenes while the HAR1 results could provide new approaches to the treatment of Schizophrenia and Alzheimer’s disease.

L’oncogène K-ras est une petite GTPase de la super famille RAS, fréquemment impliquée dans les cancers, en particulier celui du pancréas, l’un des plus mortels dans les pays occidentaux. Les mutations de l’oncogène K-ras sont considérées comme l’un des événements initiateurs de la cancérogenèse pancréatique et son activité oncogénique est un élément indispensable à la progression tumorale. Cependant, le ciblage thérapeutique de K-ras reste inefficace, à ce jour. Se focaliser sur les cibles précoces de son activité oncogénique semble ainsi être une stratégie alternative intéressante. La mucine membranaire MUC4 est une glycoprotéine de haut poids moléculaire fréquemment dérégulée dans les cancers. Dans le cancer du pancréas, MUC4 est néo-exprimée dès les stades prénéoplasiques et est impliquée dans les propriétés biologiques permettant la progression tumorale et la chimiorésistance. La régulation de MUC4 par K-ras dans la cancérogenèse pancréatique reste à décrypter. En utilisant le modèle murin de cancérogenèse pancréatique Pdx1-Cre; LStopL-K-rasG12D, nous avons mis en évidence que la néo-expression précoce de la mucine Muc4, dans les lésions pancréatiques prénéoplasiques intraépithéliales (PanINs) formées suite à la présence du K-ras muté, est corrélée avec l’activation des voies de signalisation ERK, JNK and NF-κB. In vitro, la transfection de mutants constitutivement activés de K-rasG12V dans les cellules tumorales humaines induit une augmentation de l’expression de MUC4. Cette activation intervient au niveau transcriptionel par le recrutement des facteurs de transcription AP-1 et NF-κB via les voies MAPK, JNK and NF-κB et au niveau post-transcriptionel par un mécanisme impliquant la RalB GTPase. Ensemble, ces résultats démontre que MUC4 est une cible transcriptionelle et post-transcriptionelle de l’activité oncogénique de K-ras dans le cancer du pancréas. Ces résultats ouvrent de nouvelles pistes pour développer des stratégies ciblant les étapes précoces de ce cancer. / K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer, mainly in pancreatic cancer, one of the most deadly cancers in occidental countries. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras clinical targeting remains ineffective until now. Focus on early downstream K-ras signalling may thus appear as an interesting strategy target in this cancer. The membrane-bound mucin MUC4 is a high molecular weight glycoprotein frequently deregulated in cancer. In pancreatic cancer, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-ΚB signalling pathways. In vitro, transfection of constitutively activated K-rasG12V in human pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was shown to be mediated at the transcriptional level by AP-1 and NF-ΚB transcription factors via MAPK, JNK and NF-ΚB pathways and at the post-transcriptional level by a mechanism involving the RalB GTPase. Altogether, these results identifies MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.