Inhibition of PTEN by peroxynitrite activates the phosphoinositide-3-kinase/Akt neuroprotective signaling pathway

El derivado del óxido nítrico, peroxinitrito, activa una cascada de señalización de supervivencia en neuronas. El mecanismo implica la oxidación de la fosfatasa PTEN, que permite incrementar la forma fosforilada (neuroprotectora) de akt.Peroxynitrite is usually considered as a neurotoxic nitric oxidederivative. However, an increasing body of evidence suggests that, at low concentrations, peroxynitrite affords transient cytoprotection, both in vitro and in vivo. Here, we addressed the signaling mechanism responsible for this effect, and found that rat cortical neurons in primary culture acutely exposed to peroxynitrite (0.1 mmol/L) rapidly elicited Akt-Ser473 phosphorylation. Inhibition of phosphoinositide-3-kinase (PI3K)/Akt pathway with wortmannin or Akt small hairpin RNA (shRNA) abolished the ability of peroxynitrite to prevent etoposide-induced apoptotic death. Endogenous peroxynitrite formation by short-term incubation of neurons with glutamate stimulated Akt-Ser473 phosphorylation, whereas Akt shRNA enhanced the vulnerability of neurons against glutamate. We further show that Akt-Ser473 phosphorylation was consequence of the oxidizing, but not the nitrating properties of peroxynitrite. Peroxynitrite failed to nitrate or phosphorylate neurotrophin tyrosine kinase receptors (Trks), and it did not modify the ability of brain-derived neurotrophic factor (BDNF), to phosphorylate its cognate receptor, TrkB; however, peroxynitrite enhanced BDNF-mediated Akt-Ser473 phosphorylation. Finally, we found that peroxynitrite-stimulated Akt-Ser473 phosphorylation was associated with an increased proportion of oxidized phosphoinositide phosphatase, PTEN, in neurons. Moreover, peroxynitrite prevented the increase of apoptotic neuronal death caused by over-expression of PTEN. Thus, peroxynitrite exerts neuroprotection by inhibiting PTEN, hence activating the anti-apoptotic PI3K/Akt pathway in primary neurons

Bex3 dimerization regulates NGF-dependent neuronal survival and differentiation by enhancing trkA gene transcription

The development of the nervous system is a temporally and spatially coordinated process that relies on the proper regulation of the genes involved. Neurotrophins and their receptors are directly responsible for the survival and differentiation of sensory and sympathetic neurons; however, it is not fully understood how genes encoding Trk neurotrophin receptors are regulated. Here, we show that rat Bex3 protein specifically regulates TrkA expression by acting at the trkA gene promoter level. Bex3 dimerization and shuttling to the nucleus regulate the transcription of the trkA promoter under basal conditions and also enhance nerve growth factor (NGF)-mediated trkA promoter activation. Moreover, qChIP assays indicate that Bex3 associates with the trkA promoter within a 150 bp sequence, immediately upstream from the transcription start site, which is sufficient to mediate the effects of Bex3. Consequently, the downregulation of Bex3 using shRNA increases neuronal apoptosis in NGF-dependent sensory neurons deprived of NGF and compromises PC12 cell differentiation in response to NGF. Our results support an important role for Bex3 in the regulation of TrkA expression and in NGF-mediated functions through modulation of the trkA promoter.This work was supported by Ministerio de Ciencia e Innovación Grant BFU2008-00162, by Ministerio de Economía y Competitividad Grant (BFU2011-22898 to J.C.A. and BFU2013-39394-P to P.P.), by a Marie Curie International Reintegration Grant within the European Community 7th Framework Programme, by the Consejería de Educación Grant SA074A08, and by a Consejería de Sanidad de Junta Castilla y León grant to J.C.A. This work was in part supported by the FP7-PAINCAGE integrative project. D.M.-Z. is funded by a grant from the Spanish “Instituto de Saludo Carlos III” PI1202847. L.C. has been the recipient of a Predoctoral Fellowship from the University of Salamanca. J.C.A. was a “Ramón y Cajal” Investigator from the University of Salamanca and a NARSAD 2009 Young Investigator Awardee.Peer Reviewe

Sensitivity of cervical cytology in endometrial cancer detection in a tertiary hospital in Spain

Introduction: Cervical cytology is a well-stablished cervical cancer screening method. However, due to the anatomical continuity of the genital tract, it can also detect signs of endometrial disease. Our aim was to estimate the sensitivity of cervical cytology in endometrial cancer detection and prognosis in a large population over a 30-year period in a large academic tertiary hospital in Spain. Methodology: We performed a search for women diagnosed with endometrial cancer from 1990 to 2020, who were surgically treated and had a previous cervical cytology result. Information Technologies Department databases from Bellvitge University Hospital and the Screenwide case-control study's database were used. Cervical cytology results were classified as abnormal when squamous lesions, glandular atypia or malignant cells were identified. Results: Overall, we evaluated 371 women with endometrial cancer and a documented cervical cytology performed within 3 years previous to surgical treatment. Overall, the sensitivity of cervical cytology for endometrial cancer detection was 25.6%. Several clinico-pathological characteristics, such as non-endometrioid histology and a higher stage, were correlated with higher sensitivity

Inhibition of nuclear PTEN tyrosine phosphorylation enhances glioma radiation sensitivity through attenuated DNA repair

Ionizing radiation (IR) and chemotherapy are standard of care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240FPten knock-in mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy

An Integrated Approach for the Early Detection of Endometrial and Ovarian Cancers (Screenwide Study): Rationale, Study Design and Pilot Study

Screenwide is a case-control study (2017-2021) including women with incident endometrial and ovarian cancers (EC and OC), BRCA1/2 and MMR pathogenic variant carriers, and age-matched controls from three centers in Spain. Participants completed a personal interview on their sociodemographic factors, occupational exposure, medication, lifestyle, and medical history. We collected biological specimens, including blood samples, self-collected vaginal specimens, cervical pap-brush samples, uterine specimens, and, when available, tumor samples. The planned analyses included evaluation of the potential risk factors for EC/OC; evaluation of molecular biomarkers in minimally invasive samples; evaluation of the cost-effectiveness of molecular tests; and the generation of predictive scores to integrate different epidemiologic, clinical, and molecular factors. Overall, 182 EC, 69 OC, 98 BRCA pathogenic variant carriers, 104 MMR pathogenic variant carriers, and 385 controls were enrolled. The overall participation rate was 85.7%. The pilot study using 61 samples from nine EC cases and four controls showed that genetic variants at the variant allele fraction > 5% found in tumors (n = 61 variants across the nine tumors) were detected in paired endometrial aspirates, clinician-collected cervical samples, and vaginal self-samples with detection rates of 90% (55/61), 79% (48/61), and 72% (44/61) by duplex sequencing, respectively. Among the controls, only one somatic mutation was detected in a cervical sample. We enrolled more than 800 women to evaluate new early detection strategies. The preliminary data suggest that our methodological approach could be useful for the early detection of gynecological cancers

Evaluation of somatic mutations in cervicovaginal samples as a non-invasive method for the detection and molecular classification of endometrial cancer

Background The incidence of endometrial cancer is increasing worldwide. While delays in diagnosis reduce survival, case molecular misclassification might be associated with under- and over-treatment. The objective of this study was to evaluate genetic alterations to detect and molecularly classify cases of endometrial cancer using non-invasive samples. Methods Consecutive patients with incident endometrial cancer (N = 139) and controls (N = 107) from a recent Spanish case–control study were included in this analysis. Overall, 339 cervicovaginal samples (out of which 228 were clinician-collected and 111 were self-collected) were analysed using a test based on next-generation sequencing (NGS), which targets 47 genes. Immunohistochemical markers were evaluated in 133 tumour samples. A total of 159 samples were used to train the detection algorithm and 180 samples were used for validation. Findings Overall, 73% (N = 94 out of 129 clinician-collected samples, and N = 66 out of 90 self-collected samples) of endometrial cancer cases had detectable mutations in clinician-collected and self-collected samples, while the specificity was 80% (79/99) for clinician-collected samples and 90% (19/21) for self-collected samples. The molecular classifications obtained using tumour samples and non-invasive gynaecologic samples in our study showed moderate-to-good agreement. The molecular classification of cases of endometrial cancer into four groups using NGS of both clinician-collected and self-collected cervicovaginal samples yielded significant differences in disease-free survival. The cases with mutations in POLE had an excellent prognosis, whereas the cases with TP53 mutations had the poorest clinical outcome, which is consistent with the data on tumour samples. Interpretation This study classified endometrial cancer cases into four molecular groups based on the analysis of cervicovaginal samples that showed significant differences in disease-free survival. The molecular classification of endometrial cancer in non-invasive samples may improve patient care and survival by indicating the early need for aggressive surgery, as well as reducing referrals to highly specialized hospitals in cancers with good prognosis. Validation in independent sets will confirm the potential for molecular classification in non-invasive samples. Funding This study was funded by a competitive grant from Instituto de Salud Carlos III through the projects PI19/01835, PI23/00790, and FI20/00031, CIBERESP CB06/02/0073 and CIBERONC CB16/12/00231, CB16/12/00234 (Co-funded by European Regional Development Fund. ERDF: A way to build Europe). Samples and data were provided by Biobank HUB-ICO-IDIBELL, integrated into the Spanish Biobank Network, and funded by the Instituto de Salud Carlos III (PT20/00171) and by Xarxa de Bancs de Tumors de Catalunya (XBTC) sponsored by Pla Director d’Oncologia de Catalunya. This work was supported in part by the AECC, Grupos estables (GCTRA18014MATI). It also counts with the support of the Secretariat for Universities and Research of the Department of Business and Knowledge of the Generalitat de Catalunya, and grants to support the activities of research groups 2021SGR01354 and 2021SGR1112

A Neutralizing RNA Aptamer against EGFR Causes Selective Apoptotic Cell Death

Nucleic acid aptamers have been developed as high-affinity ligands that may act as antagonists of disease-associated proteins. Aptamers are non immunogenic and characterised by high specificity and low toxicity thus representing a valid alternative to antibodies or soluble ligand receptor traps/decoys to target specific cancer cell surface proteins in clinical diagnosis and therapy. The epidermal growth factor receptor (EGFR) has been implicated in the development of a wide range of human cancers including breast, glioma and lung. The observation that its inhibition can interfere with the growth of such tumors has led to the design of new drugs including monoclonal antibodies and tyrosine kinase inhibitors currently used in clinic. However, some of these molecules can result in toxicity and acquired resistance, hence the need to develop novel kinds of EGFR-targeting drugs with high specificity and low toxicity. Here we generated, by a cell-Systematic Evolution of Ligands by EXponential enrichment (SELEX) approach, a nuclease resistant RNA-aptamer that specifically binds to EGFR with a binding constant of 10 nM. When applied to EGFR-expressing cancer cells the aptamer inhibits EGFR-mediated signal pathways causing selective cell death. Furthermore, at low doses it induces apoptosis even of cells that are resistant to the most frequently used EGFR-inhibitors, such as gefitinib and cetuximab, and inhibits tumor growth in a mouse xenograft model of human non-small-cell lung cancer (NSCLC). Interestingly, combined treatment with cetuximab and the aptamer shows clear synergy in inducing apoptosis in vitro and in vivo. In conclusion, we demonstrate that this neutralizing RNA-aptamer is a promising bio-molecule that can be developed as a more effective alternative to the repertoire of already existing EGFR-inhibitors

Climate Effects on Breeding Phenology of Peregrine and Lanner Falcons in the Mediterranean

We explored the effects of weather on the timing and reproduction of the Mediterranean Peregrine Falcon Falco peregrinus brookei and the Lanner Falcon F. biarmicus feldeggii living on the Mediterranean island of Sicily. We found that the start date of incubation has changed during 1979–2019 and analysed whether incubation timing affected the productivity of both populations and whether the change of incubation date and the quality of breeding sites depended on climatic conditions. Overall spring temperature and rainfall increased on Sicily and the incubation date of the Peregrine and the Lanner Falcon has shifted to be about one week later over the time period 1979 to 2019. Linear mixed modelling showed the influence of winter conditions and random effects (climate sector of island, year of study) on incubation date in both species. The increase in February rainfall has delayed incubation in the Peregrine Falcon, while we could not identify a specific monthly effect delaying incubation in the Lanner Falcon. In both species, the shift in incubation date has resulted in a decrease in productivity (number of fledglings). Weather conditions in late spring predicted the quality of the breeding site of Lanner Falcons but not of Peregrines. The breeding phenology of both falcons shows a common response to weather conditions on Sicily, however the Lanner Falcon seems more sensitive than the Peregrine to the changing climate. Climate effects add to other anthropogenic impacts negatively affecting the future survival of this insular population, which is the largest in Europe

Generation of a knock-in mouse model expressing a regulatable TRP53 protein localized in the mitochondria

Resumen del póster presentado al 27th International Mammalian Genome Conference (IMGC) celebrado en Salamanca (España) del 14 al 18 de septiembre de 2013.TP53 or components of its signaling pathways are inactivated during tumor formation or progression in the majority of human cancers. This suggests that TP53 acts as a block that has to be eliminated for tumor development. Consequently, restoring TP53 function has long been considered an attractive anticancer therapeutic approach. To asses this possibility, we are using a mouse knock-in model that expresses TRP53ERTAM protein, whose function can be reversibly switched on and off in vitro and in vivo. Using this model we have been able to show that transient restoration of TRP53 in a mouse model of chronic myeloid leukemia delays disease onset and prolongs the life of leukemic mice. In recent years, it has been shown that TP53 can induce apoptosis and necrosis acting directly on the mitochondria, independently of its function as a transcription factor. To explore the possible anti tumor activity of mitochondrial TRP53 we have generated a novel knock-in mouse model that expresses a regulatable TRP53 protein fused to a canonical mitochondrial localization signal. This protein, TRP53mitoERTAM, is inactive in the absence of tamoxifen but can be switched to a functional status in the presence of the drug. As expected, in the absence of tamoxifen, these mice behave as Trp53 null mice developing early onset tumors. We will use this model to analyze in vivo the role of mitochondrial TRP53 in tumor suppression and other pathophysiological processes such as ischemic necrosis, and eventually to identify novel therapeutic targets. We will present a progress report of our research using this mouse model.Peer Reviewe