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Aquesta tesi té com a principals objectius:1- Identificació de nous substrats de cdk2 en diferents models cel·lulars: cèl·lules HeLa, hepatòcits normals activats a proliferar mitjançant una hepatectomia parcial, limfòcits humans normals activats a proliferar, i la línia limfoide Namalwa.2- Localització intranuclear de proteïnes que participen en el control del cicle cel·lular en dos models de proliferació un normal i l'altre transformat: hepatòcits normals activats a proliferar mitjançant una hepatectomia parcial, i la línia cel·lular transformada d'origen humà HeLa.3- Anàlisi dels mecanismes d'activació de la cdk4 i la cdk2 durant la regeneració hepàtica.

[eng] Analysis of KRAS phosphorylation and KRAS effector domain as targets for cancer therapy Oncogenic mutations of KRAS are found in the most aggressive human tumors, including colorectal cancer (CRC). It has been suggested that oncogenic KRAS phosphorylation at Ser181 modulates its activity and favors cell transformation. Using non-phosphorylatable (S181A), phosphomimetic (S181D) and phospho/dephosphorylatable (S181) oncogenic KRAS mutants, we analyzed the role of this phosphorylation to the maintenance of tumorigenic properties of CRC cells. Surprisingly, oncogenic KRAS phosphomutants did not show great differences in cell growth and signaling activation between them in 2D cell cultures. However, the presence of phospho/dephosphorylatable oncogenic KRAS is required for α-E-catenin expression and for preserving the epithelial organization of CRC cells, and for supporting subcutaneous tumor growth in mice. Interestingly, the expression of other genes differed according to the phosphorylation status of KRAS. Lack of oncogenic KRAS phosphorylation leaded to changes in expression of SERPINE1, PRSS1,2,3 and NEO1 and to a decreased cell invasion capacity, while expression of phosphomimetic oncogenic KRAS resulted in a diminished expression of genes involved in enterocyte differentiation, such as HNF4G. Interestingly, activating or inhibiting PKC in cells expressing the phosphorylatable mutant reproduced the expression of SERPINE1, PRSS1,2,3 and HNF4G observed in the phosphomutant expressing cells. Finally, the analysis, in a public data set of human CRCs, of the gene expression signatures associated with phosphomimetic and non-phosphorylatable oncogenic KRAS suggests that this post-translational modification regulates tumor progression in patients. In conclusion, CRC cells depend on KRAS phosphorylation at Ser181 to maintain their tumorigenic properties. Therefore, specific interference with this modification may have therapeutic benefits. On the other hand, one of the strategies that is widely being followed to inhibit RAS is to search for drugs able to disrupt RAS interactions with other proteins, among them its effectors. For that reason, peptidomimetics have emerged as a promising tool to modulate the biological activity of protein-protein interactions. Accordingly, we studied whether different peptidomimetics against the effector domain of RAS, designed by the Iproteos technology to inhibit RAS, were able to interfere with oncogenic KRAS activity. The cancer model selected for this research was the pancreatic ductal adenocarcinoma, which is a highly metastatic disease with a high mortality rate. Our data have demonstrated that the compound P1.3 negatively affected downstream KRAS signaling in non-transformed cells. Moreover, it efficiently inhibited the interaction of oncogenic KRAS with its effectors. Finally, the compound P1.3 showed high selectivity killing pancreatic cancer cells expressing oncogenic KRAS versus normal cells. Therefore, from the therapeutic point of view, the compound P1.3 has been a promising lead compound. Nevertheless, the impact of this project will be determined once the bioavailability of P1.3 has been assessed along with its pharmacokinetic properties. Consequently, more experiments will be conducted in the near future in order to reach the preclinical phase.

[eng] Colorectal cancer (CRC) is a leading cause of death worldwide and tumour recurrence is a frequent complication that arises from minimal residual disease and shows up after a period of clinical dormancy. Slow-cycling cancer cells (SCCC), also called dormant tumour cells, have been shown to be responsible for tumour relapse due to their enhanced chemoresistance and tumour-initiating capacity. Although the recent efforts invested in the characterization of SCCC, our knowledge about the mechanisms underlying tumour dormancy is still limited. Thanks to the identification, isolation and molecular characterization of SCCC in our laboratory, we identified a set of pluripotency factors overexpressed in these cells, among them, DPPA3. In the present work, we characterized for the first time the role of DPPA3 in the biology of tumour dormancy. DPPA3 is an epigenetic factor essential for early development and predominantly expressed in embryonic stem cells (ESCs) and primordial germ cells (PGCs). Its function is linked to the protection of imprinted loci and transposable elements in the genome from active demethylation processes. Besides maintaining a repressive state in specific loci, DPPA3 is also related to the passive demethylation phenomenon observed in cells at these developmental stages. By the use of genetically modified CRC cell lines we revealed a central role of DPPA3 promoting cell dormancy. We unmasked its capacity controlling the response to hypoxia as a key mechanism to govern cancer cell phenotype. DPPA3 overexpression stimulated the hypoxia program by increasing hypoxia inducible factor 1 subunit alpha (HIF1α) protein levels and enhancing its transcriptional activity. Besides, overexpression of DPPA3 alone was sufficient to induce a G2/M-phase cell cycle arrest and reduce tumour growth. Interestingly, DPPA3 enhanced chemoresistance to CRC standard of care adjuvant chemotherapies. Finally, a cohort of CRC patients with high expression of DPPA3 or enriched in a DPPA3 signature showed a shorter disease-free survival. Altogether, these results pioneer the importance of DPPA3 in cancer and contribute to the understanding of tumour malignancy associated to hypoxia, chemoresistance and dormancy, the unravelling of which is of foremost importance to progress in the battle against the disease, [cat] El càncer colorectal (CRC) presenta una elevada mortalitat arreu del món i la recurrència és una complicació freqüent que sorgeix com a conseqüència de la malaltia mínima residual que es presenta després d'un període de latència clínica. Diversos estudis han descrit la presència de cèl·lules canceroses amb una baixa taxa proliferativa (slow-cycling cancer cells, SCCC) responsables de la recaiguda del pacient a causa de la seva elevada quimioresistència i capacitat regenerativa de tumors. Tot i l'esforç invertit en la caracterització de les SCCC, anomenades també cèl·lules dorments, el nostre coneixement sobre els mecanismes moleculars subjacents a la seva latència és encara limitat. Gràcies a la identificació, aïllament i caracterització molecular de les SCCC feta al nostre laboratori, vam poder identificar un conjunt de factors de pluripotència sobreexpressats en aquestes cèl·lules, entre ells, DPPA3. DPPA3 és un factor epigenètic essencial per al desenvolupament embrionari i expressat en cèl·lules mare embrionàries (ESCs) i cèl·lules germinals primordials (PGCs). La seva funció està relacionada amb la protecció en loci improntats i elements transposables del genoma de processos de desmetilació activa. A part de mantenir un estat repressiu en regions concretes del genoma, DPPA3 també està relacionat amb processos de desmetilació passiva durant el desenvolupament embrionari. Amb l’ús de línies de CRC modificades genèticament per sobreexpressar exògenament o reduir els nivells endògens de DPPA3, vam poder esbrinar el rol que exerceix aquest factor en els programes d’hipòxia i latència tumoral. La sobreexpressió de DPPA3 en línies de CRC va induir el programa d’hipòxia augmentant els nivells de proteïna i activitat transcripcional de HIF1α. A més, també va ser suficient per generar una parada del cicle cel·lular en la fase G2/M in vitro i un retard del creixement tumoral i major quimioresistència a tractaments d’adjuvància estàndards en CRC in vivo. Finalment, vam observar que una elevada expressió de DPPA3 en pacients de CRC està associada amb una major quimioresistència. El conjunt d’aquests resultats contribueixen a la comprensió de la rellevància de DPPA3 en càncer i la malignitat tumoral associada a hipòxia, quimioresistència i latència tumoral