Trabajo presentado en el III CIBERONC Young Researchers Meeting, celebrado en modalidad virtual el 14 de diciembre de 2020.

Trabajo presentado en el XVIII Congreso de la Sociedad Espanola de Biologia Celular (SEBC), celebrado en Badajoz (Espana) del 15 al 18 de octubre de 2019.

Introduction The homologous, ubiquitously expressed, members (Sos1 and Sos2) of the Sos family of RasGEFs participate in multiple signalling pathways but their specific cellular functions are not clearly defined yet. Material and methods Using a tamoxifen (4OHT)-inducible, conditional Sos1 null mutation, here we generated and analysed wild type (WT), single Sos1-KO, constitutive Sos2-KO and double Sos1/2-DKO primary mouse embryonic fibroblasts (MEFs) in an effort to ascertain the functional specificity or redundancy of Sos1 and Sos2 at the cellular level. Results and discussions Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organisation that were not observed in WT and Sos2 KO counterparts. Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation of cytoplasmic bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Consistent with a mitophagic phenotype, in vivo labelling using specific fluorophores revealed increased levels of mitochondrial oxidative stress in the Sos1-KO and the Sos1/2-DKO cells as compared to Sos2-KO or WT MEFs. Treatment of the MEF cultures with antioxidants such as GSH and NAC corrected the altered perimeter size and proliferative rate of Sos1-KO and Sos1/2-DKO MEFs to levels similar to those of WT and Sos2-KO, but not recover oxidative stress. Furthermore, treatment with the specific mitochondrial superoxide scavenger mitoTEMPO recovered endogenous redox-homeostasis in Sos1-KO and Sos1/2-DKO to normal levels. Analysis of MEFs concomitantly loaded with MitoTracker Green (Δψ-independent) and MitoTracker Red (Δψ-dependent) showed a significantly increased of dysfunctional mitochondria in the Sos1/2-DKO MEFs in comparison to all other genotypes. Finally, Seahorse-based measurements of mitochondrial parameters showed that basal and maximal mitochondrial respiration, spare respiratory capacity, and ATP production, were also significantly decreased in Sos1/2-DKO cells as compared to MEFs of all the other genotypes. Conclusion Our data uncover a direct mechanistic link between Sos RasGEF proteins and the control of mitochondrial oxidative stress and respiration. Dysfunctional mitochondria are a key element in a variety of serious diseases, including cancer, and thus a promising therapeutic target. This work was supported by grants PI16/02137 (FIS) and CIBERONC (ONCG19//2017) from ISCIII; SA043U16 from FEDER-JCyL; FS/35–2017 from FSB and AECC, Spain.

Using a 4-hydroxytamoxifen (4OHT)-inducible, conditional Sos1-null mutation, we analyzed wild-type (WT), single Sos1-KO, Sos2-KO and double Sos1/2 KO primary mouse embryonic fibroblasts (MEF) with an aim at evaluating the functional specificity or redundancy of the Sos1 and Sos2 alleles at the cellular level. The 4OHT-induced Sos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organization that were not observed in the WT and Sos2-KO counterparts. The Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation, in comparison with WT and Sos2-KO MEFs, of cytoplasmic vesicular bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Cellular proliferation and migration were impaired in Sos1-KO and Sos1/2-DKO MEFs in comparison with WT and Sos2-KO MEFs, whereas cell adhesion was only impaired upon depletion of both Sos isoforms. RasGTP formation was practically absent in Sos1/2-DKO MEFs as compared with the other genotypes and extracellular signal-regulated kinase phosphorylation showed only significant reduction after combined Sos1/2 depletion. Consistent with a mitophagic phenotype, in vivo labeling with specific fluorophores uncovered increased levels of oxidative stress (elevated intracellular reactive oxygen species and mitochondrial superoxide and loss of mitochondrial membrane potential) in the Sos1-KO and the Sos1/2-DKO cells as compared with Sos2-KO and WT MEFs. Interestingly, treatment of the MEF cultures with antioxidants corrected the altered phenotypes of Sos1-KO and Sos1/2-DKO MEFs by restoring their altered perimeter size and proliferative rate to levels similar to those of WT and Sos2-KO MEFs.Our data uncover a direct mechanistic link between Sos1 and control of intracellular oxidative stress, and demonstrate functional prevalence of Sos1 over Sos2 with regards to cellular proliferation and viability.

The cyclopentenone prostaglandin A 1 (PGA 1 ) is an inducer of cell death in cancer cells. However, the mechanism that initiates this cytotoxic response remains elusive. Here we report that PGA 1 triggers apoptosis by a process that entails the specific activation of H- and N-Ras isoforms, leading to caspase activation. Cells without H- and N-Ras did not undergo apoptosis upon PGA 1 treatment; in these cells, the cellular demise was rescued by overexpression of either H-Ras or N-Ras. Consistently, the mutant H-Ras-C118S, defective for binding PGA 1 , did not produce cell death. Molecular analysis revealed a key role for the RAF-MEK-ERK signaling pathway in the apoptotic process through the induction of calpain activity and caspase-12 cleavage. We propose that PGA 1 evokes a specific physiological cell death program, through H- and N-Ras, but not K-Ras, activation at endomembranes. Our results highlight a novel mechanism that may be of potential interest for tumor treatment.

Introduction: Chemotherapy with fluoropyrimidine in combination with either oxaliplatin or irinotecan remains the mainstay of treatment for metastatic colorectal cancer (CRC) but their outcomes are mixed, with no clear validated response predictors. It is known that the docking/scaffold protein SPRY2 modulates the EGFR signaling pathway and also regulates cellular apoptosis in response to DNA damage. Moreover, SPRY2 is up-regulated in CRC, especially in the KRAS mutant status. In this study we are going to assess SPRY2 as a key determinant of response to standard chemotherapy in CRC. Methods: We designed a retrospective study of metastatic CRC patients harboring KRAS mutation that have been treated in Institut Català d'Oncologia with fluoropyrimidine and oxaliplatin-based chemotherapy (FOLFOX or CAPOX) in the first or second line during the period of 2009-2013. All of them had paraffin sample of the tumor available (primary tumor or metastatic) and clinical information. For each sample, we macrodissected the tumoral from the non-tumoral tissue and extracted the RNA with the High Pure RNA Micro FFPE Kit. The cDNA was done with random hexamers using the GE Healthcare "Ready-To-Go You-prime First-Strand Beads" kit. Finally, quantitative PCR was performed using SYBR Green technology with the SPRY2 and GAPDH (control) primers for the human sequence. With this information we correlated the levels of SPRY2 expression with clinical parameters like response according RECIST v1.1 criteria and progression free survival. Results: In this pilot study we have analyzed 16 patients. The best radiological response reached was partial response (PR) in 7 patients, stable disease (SD) in 6 patients and progression disease (PD) in 3 patients. The median of progression free survival (mPFS) was 8.5 months (IC95 [2-13]). The median of the expression of SPRY2 (mRNA SPRY2/mRNA GADPH) was 0.07 with a rank of 0.01-0.35. Most of them had levels of expression lower than 0.15 with a mPFS of 8 months but 3 patients had higher levels (2 PR and 1 SD with a mPFS of 13 months). Be mentioned that despite having low values of SPRY2, patients could achieve 38% of PR. The 9 stage IV patients at diagnosis had a median of the expression of SPRY2 of 0.15, higher than the 0.07 of the 7 patients with localized disease at diagnosis (p-value 0.17). Conclusion: As it has been described before, SPRY2 seems to be overexpressed in more aggressive CRC: in the high-grade undifferentiated tumor in the study of Barbachano et al, and in the stage IV in our study. On the other hand, with this preliminary data, we cannot affirm that the level of expression of SPRY2 is directly linked to tumor response to FOLFOX / CAPOX in CRC. The role of SPRY2 in cancer is complex, orchestrating a multilayered regulatory system and mediating a crosstalk among different signaling pathways that remains to be further elucidated.

Patients that receive a T-cell depleted (TCD) hematopoietic stem cell transplantation (SCT) show higher risk of graft failure/rejection and of disease relapse than those that receive unmanipulated grafts. The purpose of the present investigation was to analyze the usefulness of chimaerism quantification in bone marrow (BM), peripheral blood (PB), and leukocyte lineages such as T lymphocytes (CD3+,both CD4+ and CD8+), B lymphocytes (CD19+) and myeloid cells (CD15+), for the early detection of graft failure/rejection episodes and disease relapse after TCD-PBSCT. Two of the ten (2/10) patients included in the study showed stable complete chimaerism (CC). The other 8/10 patients showed decreasing mixed chimaerism (MC) and 7 of them had either graft failure (n = 1)/rejection (n = 3) or disease relapse (n = 3). In two patients relapsed from chronic myeloid leukemia, MC was observed in BM and PB, with higher percentages of autologous cells in BM, as well as in leukocyte lineages, with higher percentages of recipient cells in the myeloid lineage than in lymphocytes. Combined analysis of chimaerism and minimal residual disease allowed early diagnosis of relapse and successful rescue therapy with donor leukocyte infusions (DLI), before the onset of hematological relapse. Chimaerism analysis allowed early diagnosis of incipient graft rejection in 3 patients. These patients showed MC both in BM and PB, with greater percentages of recipient cells in PB. Analysis of leukocyte lineages showed higher percentages of autologous cells in T lymphocytes (mainly CD8+) than in B or myeloid cells. Two of these patients were successfully treated with DLI and recovered normal PB counts and BM cellularity, as well as CC. The graft versus recipient hemopoiesis effect harbored by the donor immunocompetent cells infused seems useful forthe treatment of graft rejection, provided that an early diagnosis is made.

El articulo analiza la restauracion vegetal realizada en la obra de acondicionamiento de la carretera N-320, en el tramo comprendido entre Sacedon y Horche, provincia de Guadalajara, cuyo objetivo era minimizar el dano originado en el paisaje por las obras realizadas, restaurando la cubierta y controlando la erosion. La evaluacion de impacto ambiental proponia medidas correctoras como las plantaciones con especies autoctonas de los taludes de terraplenes y desmontes, de las diferentes estructuras, asi como la restauracion de las plantas de aglomerados y de zahorras. Se describe la serie de vegetacion principal que atraviesa el trazado y como se produjeron las partidas vegetales destinadas a la obra, dando una relacion detallada de las especies introducidas, asi como de sus cantidades. En la seleccion de las especies se siguieron los criterios de seguridad vial establecidos en el proyecto base para conseguir la maxima seguridad vial. En la restauracion destaca la alta diversidad estructural, las diferentes especies no se colocaron agrupadas por especies, sino mezclando las de caracteristicas similares.

Progressive myoclonus epilepsy of the Lafora type or Lafora disease (EPM2; McKusick no. 254780) is an autosomal recessive disorder characterized by epilepsy, myoclonus, progressive neurological deterioration and glycogen-like intracellular inclusion bodies (Lafora bodies). A gene for EPM2 previously has been mapped to chromosome 6q23-q25 using linkage analysis and homozygosity mapping. Here we report the positional cloning of the 6q EPM2 gene. A microdeletion within the EPM2 critical region, present in homozygosis in an affected individual, was found to disrupt a novel gene encoding a putative protein tyrosine phosphatase (PTPase). The gene, denoted EPM2,presents alternative splicing in the 5' and 3' end regions. Mutational analysis revealed that EPM2 patients are homozygous for loss-of-function mutations in EPM2. These findings suggest that Lafora disease results from the mutational inactivation of a PTPase activity that may be important in the control of glycogen metabolism.