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151 p., E2F-dependent transcriptional network ensures the timely entry of cells into S-phase through the regulation of a vast array of genes involved in DNA replication and cell cycle progression. In this Thesis, we aimed to decipher the role of E2F1 and E2F2 in the metabolism of nucleotides and the implications that E2F activity might have in tumor progression and resistance against chemotherapy. We show that in non-tumoral cells, targeted depletion of E2f1 and E2f2 in mice leads to a downregulation of genes encoding Tk1 and Dck (rate-limiting enzymes of the salvage pathway for nucleotide production), as well as to the activation of DNA damage. Importantly, this phenotype could be rescued with an exogenous supply of nucleotides.In tumor cells, enhanced E2F activity is thought to supply the nucleotides required for their exacerbated DNA replication. We found that the expression of E2F1/E2F2 correlates with malignancy in human prostate cancer and is necessary to maintain steady-state levels of TK1, DCK and TYMS and to prevent genomic instability during S phase. Silencing of E2F1/E2F2 in metastatic prostate cancer (mPCa) cells leads to DNA damage and compromises cellular viability, whereas addition of nucleosides, or the products of TK1 and DCK enzymatic activities improves cellular viability of E2F1/E2F2 knockdown cells, suggesting that the E2F/TK1-DCK-TYMS axis is crucial to preserve DNA integrity in mPCa cells. Furthermore, we show that mPCa cells are resistant to the apoptosis induced by the nucleoside analog 5-fluorouracil (5-FU) through the induction of TK1, DCK and TYMS expression. Remarkably, combined silencing of E2F1/E2F2 prevents 5-FU-induced upregulation of TK1, DCK and TYMS and reverses resistance of mPCa cells to 5-FU, whereas adding nucleosides or the products of TK1 and DCK activities increases the viability of E2F1/E2F2 knockdown cells treated with 5-FU. Interestingly, treatment of mPCa cells with the E2F pathway inhibitor Palbociclib in combination with 5-FU

151 p., E2F-dependent transcriptional network ensures the timely entry of cells into S-phase through the regulation of a vast array of genes involved in DNA replication and cell cycle progression. In this Thesis, we aimed to decipher the role of E2F1 and E2F2 in the metabolism of nucleotides and the implications that E2F activity might have in tumor progression and resistance against chemotherapy. We show that in non-tumoral cells, targeted depletion of E2f1 and E2f2 in mice leads to a downregulation of genes encoding Tk1 and Dck (rate-limiting enzymes of the salvage pathway for nucleotide production), as well as to the activation of DNA damage. Importantly, this phenotype could be rescued with an exogenous supply of nucleotides.In tumor cells, enhanced E2F activity is thought to supply the nucleotides required for their exacerbated DNA replication. We found that the expression of E2F1/E2F2 correlates with malignancy in human prostate cancer and is necessary to maintain steady-state levels of TK1, DCK and TYMS and to prevent genomic instability during S phase. Silencing of E2F1/E2F2 in metastatic prostate cancer (mPCa) cells leads to DNA damage and compromises cellular viability, whereas addition of nucleosides, or the products of TK1 and DCK enzymatic activities improves cellular viability of E2F1/E2F2 knockdown cells, suggesting that the E2F/TK1-DCK-TYMS axis is crucial to preserve DNA integrity in mPCa cells. Furthermore, we show that mPCa cells are resistant to the apoptosis induced by the nucleoside analog 5-fluorouracil (5-FU) through the induction of TK1, DCK and TYMS expression. Remarkably, combined silencing of E2F1/E2F2 prevents 5-FU-induced upregulation of TK1, DCK and TYMS and reverses resistance of mPCa cells to 5-FU, whereas adding nucleosides or the products of TK1 and DCK activities increases the viability of E2F1/E2F2 knockdown cells treated with 5-FU. Interestingly, treatment of mPCa cells with the E2F pathway inhibitor Palbociclib in combination with 5-FU

E2F1/E2F2 expression correlates with malignancy in prostate cancer (PCa), but its functional significance remains unresolved. To define the mechanisms governed by E2F in PCa, we analyzed the contribution of E2F target genes to the control of genome integrity, and the impact of modulating E2F activity on PCa progression. We show that silencing or inhibiting E2F1/E2F2 induces DNA damage during S phase and potentiates 5-FU-induced replication stress and cellular toxicity. Inhibition of E2F downregulates the expression of E2F targets involved in nucleotide biosynthesis (TK1, DCK, TYMS), whose expression is upregulated by 5-FU. However, their enzymatic products failed to rescue DNA damage of E2F1/E2F2 knockdown cells, suggesting additional mechanisms for E2F function. Interestingly, targeting E2F1/E2F2 in PCa cells reduced WEE1 expression and resulted in premature CDK1 activation during S phase. Inhibition of CDK1/CDK2 prevented DNA damage induced by E2F loss, suggesting that E2F1/E2F2 safeguard genome integrity by restraining CDK1/CDK2 activity. Importantly, combined inhibition of E2F and ATR boosted replication stress and dramatically reduced tumorigenic capacity of PCa cells in xenografts. Collectively, inhibition of E2F in combination with drugs targeting nucleotide biosynthesis or DNA repair is a promising strategy to provoke catastrophic levels of replication stress that could be applied to PCa treatment.

Targeted disruption of E2f2 in mice causes T-cell hyperactivation and a disproportionate cell cycle entry upon stimulation. However, E2f2−/− mice do not develop a lymphoproliferative condition. We report that E2f2 plays a Fas-dependent anti-apoptotic function in vitro and in vivo. TCR-stimulated murine E2f2−/− T cells overexpress the proapoptotic genes Fas and FasL and exhibit enhanced apoptosis, which is prevented by treatment with neutralizing anti-FasL antibodies. p53 pathway is activated in TCR-stimulated E2f2−/− lymphocytes, but targeted disruption of p53 in E2f2−/− mice does not abrogate Fas/FasL expression or apoptosis, implying a p53-independent apoptotic mechanism. We show that E2f2 is recruited to Fas and FasL gene promoters to repress their expression. in vivo, E2f2−/− mice are prone to develop immune-mediated liver injury owing to an aberrant lymphoid Fas/FasL activation. Taken together, our results suggest that E2f2-dependent inhibition of Fas/FasL pathway may play a direct role in limiting the development of immune-mediated pathologies

Lipid metabolism rearrangements in nonalcoholic fatty liver disease (NAFLD) contribute to disease progression. NAFLD has emerged as a major risk for hepatocellular carcinoma (HCC), where metabolic reprogramming is a hallmark. Identification of metabolic drivers might reveal therapeutic targets to improve HCC treatment. Here, we investigated the contribution of transcription factors E2F1 and E2F2 to NAFLD-related HCC and their involvement in metabolic rewiring during disease progression. In mice receiving a high-fat diet (HFD) and diethylnitrosamine (DEN) administration, E2f1 and E2f2 expressions were increased in NAFLD-related HCC. In human NAFLD, E2F1 and E2F2 levels were also increased and positively correlated. E2f1-/- and E2f2-/- mice were resistant to DEN-HFD-induced hepatocarcinogenesis and associated lipid accumulation. Administration of DEN-HFD in E2f1-/- and E2f2-/- mice enhanced fatty acid oxidation (FAO) and increased expression of Cpt2, an enzyme essential for FAO, whose downregulation is linked to NAFLD-related hepatocarcinogenesis. These results were recapitulated following E2f2 knockdown in liver, and overexpression of E2f2 elicited opposing effects. E2F2 binding to the Cpt2 promoter was enhanced in DEN-HFD-administered mouse livers compared with controls, implying a direct role for E2F2 in transcriptional repression. In human HCC, E2F1 and E2F2 expressions inversely correlated with CPT2 expression. Collectively, these results indicate that activation of the E2F1-E2F2-CPT2 axis provides a lipid-rich environment required for hepatocarcinogenesis.

Existing therapeutic strategies for breast cancer are limited by tumor recurrence and drug-resistance. Antioxidant plant-derived compounds such as flavonoids reduce adverse outcomes and have been identified as a potential source of antineoplastic agent with less undesirable side effects. Here, we describe the novel regulation of fatty-acid synthase (FASN), the key enzyme in de novo fatty-acid synthesis, whereby Vitis vinifera L. cv Vermentino leaf hydroalcoholic extract lowers its protein stability that is regulated by small ubiquitin-like modifier (SUMO)ylation. The phenolic compounds characterization was performed by liquid chromatography–mass spectrometry (LC–MS), whereas mass spectrometry (LC–MS/MS), Western blotting/co-immunoprecipitation (Co-IP) and RT-PCR, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenicity assays, and FACS analysis were used to measure the expression of targets and tumorigenicity. Vermentino extract exhibits antitumorigenic effects, and we went on to determine that FASN and ubiquitin-conjugating enzyme 9 (UBC9), the sole E2 enzyme required for SUMOylation, were significantly reduced. Moreover, FASN was found SUMOylated in human breast cancer tissues and cell lines, and lack of SUMOylation caused by SUMO2 silencing reduced FASN protein stability. These results suggest that SUMOylation protects FASN against proteasomal degradation and may exert oncogenic activity through alteration of lipid metabolism, whereas Vermentino extract inhibits these effects which supports the additional validation of the therapeutic value of this compound in breast cancer.

[ES] El cáncer es, sin duda, una de las enfermedades que mayores retos presenta en la investigación biomédica. Son muchos los genes involucrados en el cáncer con los que se está investigando, y en muchos casos se ha observado que algunos genes aparecen mutados en una gran parte de los tumores, lo que da a entender que la alteración de estos genes puede ser clave en el desarrollo de gran parte de los tumores, aun cuando no esté directamente ligada al inicio de la formación del tumor. Un claro ejemplo de este grupo es el gen PTEN, el segundo supresor tumoral más mutado en el cáncer, por detrás de p53. Se estima que se pueden encontrar mutaciones somáticas de PTEN en el 30% de los cánceres. La proteína codificada por este gen (PTEN) tiene un papel central en la regulación de una de las rutas de señalización más importantes en el metabolismo, crecimiento, proliferación, supervivencia y migración: la ruta PI3K/AKT. Por ello, además de ser la causante del síndrome de Cowden, la pérdida de función de PTEN es un evento importante en el desarrollo y migración tumoral. Por ello, en este trabajo se plantea la hipótesis de que la sobreexpresión de PTEN pueda prevenir el crecimiento tumoral y metástasis en células portadoras de mutaciones en genes relacionados con el cáncer, concretamente en los genes BRCA1, KRAS, MYC y PIK3CA. Por tanto, nos planteamos como objetivo prevenir el desarrollo del cáncer en las vías de los genes nombrados mediante la sobreexpresión de PTEN. Con este objetivo, se introducirá una copia de PTEN con un sistema de expresión inducible Tet-On en una línea celular inmortalizada, y luego se introducirán las mutaciones en oncogenes y supresores tumorales. Con el fin de imitar las mutaciones más comunes en estos genes, se introducirá una copia extra de los genes comúnmente amplificados (MYC y PIK3CA) en un vector de alta expresión. Se editarán las regiones codificantes de los dos restantes (BRCA1 y KRAS) con el sistema CRISPT/Cas9, de modo que se expresen las, [EN] The cancer is, undoubtedly, one of the most challenging disease being faced by the biomedical research. There are plenty of genes on which the scientific community is working on, and in many cases it has been shown that some of those genes are involved in most types of cancer. This statement leads to think that alterations in those genes have a mayor importance in the development of tumors, even when the mutation itself is not directly bounded to the starting event of the oncogenesis. PTEN gene is a good representative of those genes, as it is the second most frequently mutated tumor suppressor gene in cancer, just behind p53. Nearby in 30% of cancers mutations on PTEN can be found. The protein encoded by this gene (PTEN) plays a key role in the regulation of one of the most important signaling pathway in cell metabolism, growth, proliferation and migration — the PI3K/AKT pathway. In addition to be the direct responsible of the cancer, as it is in the Cowden syndrome, loss of PTEN function is a mayor determinant in the tumor growth. Here we hypothesize that PTEN overexpression might prevent tumor growth and metastasis in cells harboring mutations on genes deeply involved in cancer — BRCA1, KRAS, MYC and PIK3CA. Therefore, we purpose to prevent cancer growth mediated by these genes through the overexpression of PTEN. In order to fulfil that goal, we propose the following pilot project — a copy of PTEN under inducible expression with Tet-On system will be introduced in an immortalized cell line. Then we will introduce mutations in several oncogenes and tumor suppressors. As a means of mimicking the most common mutations in those genes, extra copies of the two commonly amplified genes — MYC and PIK3CA — harbored in high expression vectors will be transfected. Coding regions of the remaining two — BRCA1 and KRAS — will be edited by the CRISPR/Cas9 system, so the most common mutations can be expressed in the cells. In order to test the functional consequences of the

The Golgi organelle duplicates its protein and lipid content to segregate evenly between two daughter cells after mitosis. However, how Golgi biogenesis is regulated during interphase remains largely unknown. Here we show that messenger RNA (mRNA) expression of GOLPH3 and GOLGA2, two genes encoding Golgi proteins, is induced specifically in G1 phase, suggesting a link between cell cycle regulation and Golgi growth. We have examined the role of E2F transcription factors, critical regulators of G1 to S progression of the cell cycle, in the expression of Golgi proteins during interphase. We show that promoter activity for GOLPH3, a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the GOLPH3 promoter region abrogates E2F1-mediated induction of a GOLPH3 luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the GOLPH3 promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of GOLPH3 with small interfering RNA (siRNA) delays the G1 to S transition in synchronized U2OS cells. Taken together, our results reveal a link between cell cycle regulation and Golgi function, and suggest that E2F-mediated regulation of Golgi genes is required for the timely progression of the cell cycle.

[EUS] Gaur egungo gizartean gero eta minbizi kasu gehiago diagnostikatzen dira, eta horien artean % 23a bular-minbiziari dagokio. Gainera, nagusienetarikoa da emakumeen heriotza kausen artean, % 14a alegia. Hori dela eta, azterketa asko egin izan dira bular minbiziaren inguruan eta bi gene izan dira, nagusiki, bular minbiziarekin lotu direnak, BRCA1 eta BRCA2, hain zuzen. Lan honetan bi hauetako bat aztertuko da, BRCA1 genea, gizakiotan 17. kromosoman kokatzen dena (17q21 ), gene honen zenbait mutazio komunak direlako bular-minbizia duten gaixoen artean eta baita familia berekoen artean. Gene honek tumore-supresorea den p220 proteina kondetzen du. Proteina horren ziklo zelularraren kontrolean funtsezko papera betetzen du, izan ere, zelulen zikloko zenbait bidezidor konplexutan parte hartzen du, beste proteina batzuekin elkar eraginez, p53 proteinarekin adibidez. BRCA1 genean mutazioak gertatzean, baina, ziklo zelularraren kontrola inhibitu egiten da eta zelulen gehiegizko proliferazioa gertatzen da, bular zein obario minbizia dakarrena. Gene honetan deskribatutako mutazioen artean bi dira nagusitzen direnak: 2. exonean ematen den 2 nukleotidotako delezioa, 187delAG, eta 19. exonean ematen den nukleotido baten insertzioa, 5382insC. Lan honetan azken mutazio hau landu da, izan ere, handia da mutazio honengatik bular-minbizia garatzeko arriskua (% 67). Lan honetan beraz, mutazioa honen edizioak bular minbizia garatzeko arrisku murrizten inpaktua izango duela proposatzen dugu. Hortaz, kontzeptuen proba moduan hurrengo proiektu pilotua proposatzen da. Mutatutako HCC 1937 lerro zelularra erabiliko dugu, BRCA1 5382insC mutaziorako homozigotoa dena, in vitro modelo esperimental moduan. Edizioa aurrera eramateko CRISPR/Cas9 edizio sistema erabiltzea proposatzen da mutatutako zelulak basati bihurtzeko helburuarekin. Edizioa zuzentzeko, HCC 1937 zelulak 2 plasmido erabilita transfektatuko dira: bat RNA gudaren eta Cas9 nukleasaren ko-espreziorako, eta bestea DNA emailea eraman, [EN] Nowadays, a large amount of cancer cases are diagnosed every year, of which 23% correspond to breast cancer. In addition, breast cancer is the main cause (14%) of death among women and therefore, many studies have been accomplished in it. Those studies have proven that two genes are mainly related to breast cancer, namely BRCA1 and BRCA2. In this project we are going to focus on one of these two genes will be analysed, the BRCA1 gene, which in humans is found in chromosome 17 (17q21), because some of the mutations of this gene are common among breast cancer patients and among the people from the same family. This gene encodes for a tumor-suppressor protein, known as p220. This protein has a key role in the control of cell cycle, as it interacts with other proteins, like p53. Moreover, when mutations in the BRCA1 gene appears cell cycle control is inhibited, and an over-proliferation of cells occurs. This can lead to the developing of a breast or ovarian cancer. Among the mutations described in this gene, two are those that prevail: the 2-nucleotide deletion in exon 2, 187delAG, and the insertion of a nucleotide in exon 19, 5382insC. Because of the high risk of developing cancer in carriers of this latest mutation (67%), BRCA1 5382insC has been addressed in the present work. Here we propose that editing this mutation would have an impact in reducing the risk of developing cancer. Thus, as a proof of principle the following pilot project is proposed. We will use HCC 1937 mutated cell line is proposed, which is a homozygous for BRCA1 5382insC mutation, as an experimental in vitro model. It is proposed to perform the edition using the CISPR/Cas9 edition-system in order to make the mutated cell line become wild-type. To conduct the edition, we will transfect HCC 1937 cells with 2 plasmids: one coexpressing a guide-RNA to the mutant sequence and Cas9 nuclease, and another one providing the donor DNA wild-type for BRCA1 to facilitate the repair of the cleaved DNA by t

[EN] Single gene disorders are the most studied genetic diseases due to its simple inheritance pattern (recessive or dominant). Huntington’s disease (HD) is a single gene disorder which appears between the third and fifth decade of life. It is a progressive neurodegenerative disorder, which leads to 15-20 years of life expectancy after the first symptoms appear. Due to the late onset of the disease, an early diagnosis can be crucial, that is why an easy and cheap method of diagnosis is important. The disease is caused by the number of (CAG)n repeats in the huntingtin (Htt) gene; people with over 36 CAG repeats can develop the disorder. Previous molecular diagnosis had been developed, but were expensive or unsuitable for routine use. In this study, we developed a new, cheap and easy method to detect Huntington´s disease via PCR (polymerase chain reaction). To develop the new method, we tested specific primers in positive and negative control samples to verify the effectiveness. We tested the best concentrations of the reagents and conditions for the PCR. Due to the high concentration of the bases C and G, secondary structures were formed in the DNA chain in the amplicon, we used betaine for the disruption of this secondary structures. To measure the exact number of CAG repeats we made a semilogarithmic regression and used its equation. Results showed that the developed method was effective for the detection of the Huntington’s disease in positive and negative controls. With the developed method, we were able to perform the molecular diagnosis of an unknown individual with chances of developing the disease. Taken together, we have developed a new method of molecular diagnosis for the Huntington´s disease via PCR., [ES] Los trastornos monogénicos son las enfermedades genéticas más estudiadas debido a su simple modelo de herencia (dominante o recesivo). La enfermedad de Huntington (HD) es un trastorno monogénico que aparece entre la tercera y quinta década de vida. Es una enfermedad progresiva y neurodegenerativa, que lleva a 15-20 años de esperanza de vida después de que los primeros síntomas aparezcan. Debido a la tardía expresión de la enfermedad, un diagnóstico temprano es crucial, es por ello que un diagnóstico barato y sencillo es tan 4 importante. La enfermedad depende de las repeticiones de (CAG)n en el gen de la huntingtina (Htt); las personas con más de 36 copias de CAG pueden desarrollar la enfermedad. Anteriormente se han desarrollado diagnósticos moleculares pero eran caros o inadecuados para el uso rutinario. En este estudio hemos desarrollado nuevo, barato y fácil método para detectar la enfermedad de Huntington vía PCR (reacción en cadena de la polimerasa). Para desarrollar este nuevo método, hemos testado unos primers específicos en controles positivos y negativos para verificar la efectividad. Hemos testado las mejores concentraciones de los reactivos y las condiciones de la PCR. Debido a la gran concentración de bases C y G, se formaron estructuras secundarias en la cadena de ADN del amplicón, utilizamos betaína para deshacer esas estructuras secundarias. Para medir el número exacto de repeticiones de CAG, hicimos una regresión semilogarítmica y usamos la ecuación de esta regresión. Los resultados mostraron que el método desarrollado para detectar la enfermedad de Huntington era efectivo, tanto en controles positivos como negativos. Con el método desarrollado, fuimos capaces de hacer el diagnóstico molecular de un individuo con probabilidades de desarrollar la enfermedad. Resumiendo, hemos creado un método nuevo de diagnóstico molecular para detectar la enfermedad de Huntington vía PCR.

Increasing evidence links metabolic signals to cell proliferation, but the molecular wiring that connects the two core machineries remains largely unknown. E2Fs are master regulators of cellular proliferation. We have recently shown that E2F2 activity facilitates the completion of liver regeneration after partial hepatectomy (PH) by regulating the expression of genes required for S-phase entry. Our study also revealed that E2F2 determines the duration of hepatectomy-induced hepatic steatosis. A transcriptomic analysis of normal adult liver identified "lipid metabolism regulation" as a major E2F2 functional target, suggesting that E2F2 has a role in lipid homeostasis. Here we use wild-type (E2F2(+/+)) and E2F2 deficient (E2F2(-/-)) mice to investigate the in vivo role of E2F2 in the composition of liver lipids and fatty acids in two metabolically different contexts: quiescence and 48-h post-PH, when cellular proliferation and anabolic demands are maximal. We show that liver regeneration is accompanied by large triglyceride and protein increases without changes in total phospholipids both in E2F2(+/+) and E2F2(-/-) mice. Remarkably, we found that the phenotype of quiescent liver tissue from E2F2(-/-) mice resembles the phenotype of proliferating E2F2(+/+) liver tissue, characterized by a decreased phosphatidylcholine to phosphatidylethanolamine ratio and a reprogramming of genes involved in generation of choline and ethanolamine derivatives. The diversity of fatty acids in total lipid, triglycerides and phospholipids was essentially preserved on E2F2 loss both in proliferating and non-proliferating liver tissue, although notable exceptions in inflammation-related fatty acids of defined phospholipid classes were detected. Overall, our results indicate that E2F2 activity sustains the hepatic homeostasis of major membrane glycerolipid components while it is dispensable for storage glycerolipid balance.