Facultad de Ciencias
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- Protein biomarkers in lung cancer screening: technical considerations and feasibility assessment(Elsevier, 2024) Seijo, L. (Luis); Calle-Arroyo, C. (Carlos) de la; Pineda-Lucena, A. (Antonio); Detterbeck, F. (Frank); Bernasconi-Bisio, F. (Franco); Johansson, M. (Mattias); Montuenga-Badia, L.M. (Luis M.); Orive-Mauleón, D. (Daniel); Hung, J.R. (Rayjean); Valencia, K. (Karmele); Echepare, M. (Mirari); Robbins, H.A. (Hilary); Fernandez-Sanmamed, M. (Miguel)Lung cancer remains the leading cause of cancer-related deaths worldwide, mainly due to late diagnosis and the presence of metastases. Several countries around the world have adopted nation-wide LDCT-based lung cancer screening that will benefit patients, shifting the stage at diagnosis to earlier stages with more therapeutic options. Biomarkers can help to optimize the screening process, as well as refine the TNM stratification of lung cancer patients, providing information regarding prognostics and recommending management strategies. Moreover, novel adjuvant strategies will clearly benefit from previous knowledge of the potential aggressiveness and biological traits of a given early-stage surgically resected tumor. This review focuses on proteins as promising biomarkers in the context of lung cancer screening. Despite great efforts, there are still no successful examples of biomarkers in lung cancer that have reached the clinics to be used in early detection and early management. Thus, the field of biomarkers in early lung cancer remains an evident unmet need. A more specific objective of this review is to present an up-to-date technical assessment of the potential use of protein biomarkers in early lung cancer detection and management. We provide an overview regarding the benefits, challenges, pitfalls and constraints in the development process of protein-based biomarkers. Additionally, we examine how a number of emerging protein analytical technologies may contribute to the optimization of novel robust biomarkers for screening and effective management of lung cancer.
- Matrix metalloproteinase-10 deficiency delays atherosclerosis progression and plaque calcification(Elsevier, 2018) Belzunce, M. (Miriam); Parks, W.C. (William C.); Paramo, J.A. (José Antonio); Andres, V. (Vicente); Purroy, A. (A.); Orbe, J. (Josune); Rodriguez, J.A. (José Antonio); Villa-Bellosta, R. (Ricardo); Roncal, C. (Carmen); Zalba, G. (Guillermo); Meilhac, O. (Olivier)Background and aims: Matrix metalloproteinases (MMPs) have been implicated in atherosclerosis and vascular calcification. Among them, we reported that MMP10 is present in human atheroma, associated with atherosclerosis. However, it remains unclear whether MMP10 is involved in atherogenesis and vascular calcification. Methods: MMP10 was measured in serum from patients with subclinical atherosclerosis and analyzed in carotid endarterectomies by immunostaining. ApoE-deficient mice (Apoe-/-) were crossed to MMP10-deficient (Mmp10-/-) mice and followed up to 20 months. Plaque area and composition were assessed by histology and immunohistochemistry. Inflammatory markers were measured in atherosclerotic plaques by RT-qPCR, and leukocyte subpopulations were analyzed by flow cytometry. In vitro calcification assays were performed in aortic vascular smooth muscle cells (VSMC). Results: MMP10 serum levels were associated with coronary calcification in subjects with subclinical atherosclerosis. Immunostaining revealed MMP10 expression in human atheromas, spatially associated with calcification areas, and complicated plaques released higher amounts of MMP10 than non-diseased segments. Interestingly, vascular MMP10 expression was confined to the atherosclerotic lesion in Apoe-/- mice, and Apoe-/-Mmp10-/- showed a substantial reduction in atherosclerotic lesion size, macrophage content and plaque calcification. Reduced local and systemic inflammatory markers could be demonstrated in Apoe-/-Mmp10-/- by gene expression and flow cytometry analysis. Calcium phosphate deposition and vascular calcification markers were downregulated in VSMC from Apoe-/-Mmp10-/- mice. Conclusions: Delayed plaque progression and altered cellular composition in the absence of MMP10 suggests that MMP10 plays a role in atherosclerosis, favoring inflammation, development and complication of the plaque.
- The involvement of ADAM10 in acantholysis in mucocutaneous pemphigus vulgaris depends on the autoantibody profile of each patient(Oxford, 2020) Ivars-Lleó, M. (Marta); España, A. (Agustín); Alzuguren, P. (Pilar); López-Zabala, M.J. (M. J.); Pelacho, B. (Beatriz); Lasarte, J.J. (Juan José)Background Acantholysis in pemphigus vulgaris (PV) may be triggered by desmoglein (Dsg) and non‐Dsg autoantibodies. The autoantibody profile of each patient results in distinct intracellular signalling patterns. Objectives Based on our previous findings, we aimed to elucidate whether PV acantholysis in a mouse model may be mediated by activation of a disintegrin and metalloproteinase 10 (ADAM10). Methods We used three PV‐IgG fractions from different patients containing high or low levels of anti‐Dsg1 and anti‐Dsg3 antibodies, and the presence or not of anti‐desmocollin (Dsc) antibodies, using a passive transfer mouse model of PV. Results Although all of the PV‐IgG fractions produced suprabasal acantholysis, only those containing anti‐Dsg1/3, but not anti‐Dsc2/3 antibodies, induced ADAM10 activation in a Src‐dependent way, and an increase in the epidermal growth factor (EGF) receptor ligands EGF and betacellulin (BTC). In contrast, the presence of anti‐Dsc2/3 antibodies, in addition to anti‐Dsg1/3, triggered earlier and ADAM10‐independent epidermal detachment, with no increase in EGF and BTC, which was associated with an earlier and more intense acantholysis. Conclusions All PV‐IgG fractions produced suprabasal acantholysis, but our results reveal that depending on the levels of anti‐Dsg antibodies or the presence of non‐Dsg antibodies, such as anti‐Dsc, more severe cell–cell epidermal detachment will occur at different times, and in an ADAM10‐dependent manner or not. Acantholysis in these different groups of patients with PV may be a consequence of the activation of specific intracellular mechanisms downstream of Autoantibodies binding to Dsg or non‐Dsg proteins, and therefore more specific therapeutic approaches in PV should be used.
- PD-1/PD-L1 blockers in NSCLC brain metastases: Challenging paradigms and clinical practice(American Association for Cancer Research, 2020) Gil-Bazo, I. (Ignacio); Lu, B.Y. (Benjamin Y.); Eguren-Santamaría, I. (Iñaki); Goldberg, S. (S.); Kluger, H. (Harriet); Herbst, R.S. (Roy S.); Idoate, M.A. (Miguel Ángel); Corral, J. (Jesús); Schalper, K.A. (Kurt A.); Fernandez-Sanmamed, M. (Miguel)Immune checkpoint inhibitors (ICI) have revolutionized the management of advanced non-small cell lung cancer (NSCLC). However, most pivotal phase III trials systematically excluded patients with active brain metastases, precluding the generalization of the results. Although theoretically restricted from crossing the blood-brain barrier, the novel pharmacokinetic/pharmacodynamic profiles of anti-PD-1/PD-L1 drugs have prompted studies to evaluate their activity in patients with NSCLC with active central nervous system (CNS) involvement. Encouraging results have suggested that ICI could be active in the CNS in selected patients with driver-negative advanced NSCLC with high PD-L1 expression and low CNS disease burden. Single-agent CNS response rates around 30% have been reported. Beyond this particular setting, anti-PD-1/PD-L1 antibodies have been evaluated in patients receiving local therapy for brain metastases (BM), addressing concerns about potential neurologic toxicity risks associated with radiotherapy, more specifically, radionecrosis (RN). Accordingly, a variety of clinical and imaging strategies are being appropriately developed to evaluate tumor response and to rule out pseudoprogression or radionecrosis. Our purpose is to critically summarize the advances regarding the role of systemic anti-PD-1/PD-L1 antibodies for the treatment of NSCLC BM. Data were collected from the PubMed database, reference lists, and abstracts from the latest scientific meetings. Recent reports suggest anti-PD-1/PD-L1 agents are active in a subset of patients with NSCLC with BM showing acceptable toxicity. These advances are expected to change soon the management of these patients but additional research is required to address concerns regarding radionecrosis and the appropriate sequencing of local and systemic therapy combinations.
- Signature-driven repurposing of Midostaurin for combination with MEK1/2 and KRASG12C inhibitors in lung cancer(2023) Gil-Bazo, I. (Ignacio); Drosten, M. (Mathias); Barbacid, M. (Mariano); Kovalski, J. (Joanna); Pineda-Lucena, A. (Antonio); Román, M. (Marta); Jantus-Lewintre, E. (Eloisa); Calabuig-Fariñas, S. (Silvia); Vicent, S. (Silvestre); Ludwig, I.A. (Iziar Amaia); Entrialgo-Cadierno, R. (Rodrigo); Palomino-Echeverría, S. (Sara); Salmon, M. (Marina); Fernandez-Irigoyen, J. (Joaquín); Ruggero, D. (Davide); Lara-Astiaso, D. (David); Santos, A. (Alba); Ponz-Sarvise, M. (Mariano); Macaya, I. (Irati); Feliu, I. (Iker); Rodríguez-Remírez, M. (M.); Guruceaga, E. (Elizabeth); Paz-Ares, L. (Luis); Ferrer, I. (Irene); Narayanan, S. (Shruthi); Ferrero, M. (Macarena); Lecanda, F. (Fernando); Welch, C. (Connor); Lopez, I. (Inés); Lonfgren, S.M. (Shane M.); Santamaria, E. (Enrique); Khatri, P. (Purvesh)Drug combinations are key to circumvent resistance mechanisms compromising response to single anti-cancer targeted therapies. The implementation of combinatorial approaches involving MEK1/2 or KRASG12C inhibitors in the context of KRAS-mutated lung cancers focuses fundamentally on targeting KRAS proximal activators or effectors. However, the antitumor effect is highly determined by compensatory mechanisms arising in defined cell types or tumor subgroups. A potential strategy to find drug combinations targeting a larger fraction of KRAS-mutated lung cancers may capitalize on the common, distal gene expression output elicited by oncogenic KRAS. By integrating a signature-driven drug repurposing approach with a pairwise pharmacological screen, here we show synergistic drug combinations consisting of multi-tyrosine kinase PKC inhibitors together with MEK1/2 or KRASG12C inhibitors. Such combinations elicit a cytotoxic response in both in vitro and in vivo models, which in part involves inhibition of the PKC inhibitor target AURKB. Proteome profiling links dysregulation of MYC expression to the effect of both PKC inhibitor-based drug combinations. Furthermore, MYC overexpression appears as a resistance mechanism to MEK1/2 and KRASG12C inhibitors. Our study provides a rational framework for selecting drugs entering combinatorial strategies and unveils MEK1/2- and KRASG12C-based therapies for lung cancer.
- Stabilization of 14-3-3 protein-protein interactions with Fusicoccin-A decreases alpha-synuclein dependent cell-autonomous death in neuronal and mouse models(Elsevier, 2023) Aymerich, M.S. (María S.); González-Aseguinolaza, G. (Gloria); Perez-Mediavilla, L.A. (Luis Alberto); Vales, A. (África); Fernandez-Irigoyen, J. (Joaquín); Boncristiani, C. (Chiara); Bravo-González, J.J. (Jorge Juan); Basurco, L. (Leyre); Aragón, T. (Tomás); Marcilla, I. (Irene); Arrasate, M. (Montserrat); Luquin, M.R. (María Rosario); Santamaria, E. (Enrique); Vinueza-Gavilanes, R. (Rodrigo)Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14-3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14-3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14-3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.
- Pilot multi-omic analysis of human bile from benign and malignant biliary strictures: A machine-learning approach(MDPI AG, 2020) Latasa, M.U. (María Ujué); Banales, J.M. (Jesús M.); Carrascosa, J. (Juan); González, B. (Belén); Corrales, F.J. (Fernando José); Macias, R.I.R. (Rocío I. R.); Berasain, C. (Carmen); Arechederra, M. (María); Fernández-Barrena, M.G. (Maite G.); Pineda-Lucena, A. (Antonio); Bolado, F. (Federico); Urman, J.M. (Jesús M.); Uriarte, I. (Iker); Avila, M.A. (Matías Antonio); Gil, I. (Iñigo); Alonso, C. (Cristina); Rullán, M. (María); Purroy, A. (A.); Iruarrizaga-Lejarreta, M. (Marta); Sangro, B. (Bruno); Fernandez-Urien, I. (Ignacio); Monte, M.J. (María J.); Herranz, J.M. (José M.); Iraburu-Elizalde, M. (María); Vila, J.J. (Juan J.); Álvarez-Sola, G. (Gloria); Oyón, D. (Daniel); Colyn, L. (Leticia); Romero, M. (Marta); Puchades-Carrasco, L. (Leonor); Cubero, F.J. (Francisco Javier); Zabalza, L. (Lucía); Martinez-Chantar, M.L. (María Luz); Marin, J.J.G (Jose J.G.); Carmona, L. (Lorena)Cholangiocarcinoma (CCA) and pancreatic adenocarcinoma (PDAC) may lead to the development of extrahepatic obstructive cholestasis. However, biliary stenoses can also be caused by benign conditions, and the identification of their etiology still remains a clinical challenge. We performed metabolomic and proteomic analyses of bile from patients with benign (n = 36) and malignant conditions, CCA (n = 36) or PDAC (n = 57), undergoing endoscopic retrograde cholangiopancreatography with the aim of characterizing bile composition in biliopancreatic disease and identifying biomarkers for the differential diagnosis of biliary strictures. Comprehensive analyses of lipids, bile acids and small molecules were carried out using mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (1H-NMR) in all patients. MS analysis of bile proteome was performed in five patients per group. We implemented artificial intelligence tools for the selection of biomarkers and algorithms with predictive capacity. Our machine-learning pipeline included the generation of synthetic data with properties of real data, the selection of potential biomarkers (metabolites or proteins) and their analysis with neural networks (NN). Selected biomarkers were then validated with real data. We identified panels of lipids (n = 10) and proteins (n = 5) that when analyzed with NN algorithms discriminated between patients with and without cancer with an unprecedented accuracy
- NADPH Oxidase Overactivity Underlies Telomere Shortening in Human Atherosclerosis(2020) Montero, L. (Laura); Beloqui, O. (Óscar); Marqués, J. (Javier); Fortuño, A. (Ana); Cortés, A. (Adriana); Orbe, J. (Josune); Pejenaute-Martínez-de-Lizarrondo, Á. (Álvaro); Zalba, G. (Guillermo); Marti-del-Moral, A. (Amelia)Telomere shortening and oxidative stress are involved in the pathogenesis of atherosclerosis. Different studies have shown that phagocytic NADPH oxidase is associated with this disease. This study aimed to investigate the association between phagocytic NADPH oxidase and telomere shortening in human atherosclerosis. To assess this potential association, telomere length and phagocytic NADPH oxidase activity were determined by PCR and chemiluminescence, respectively, in a population of asymptomatic subjects free of overt clinical atherosclerosis. We also measured serum 8-hydroxy-2-deoxyguanosine (8-OHdG) levels (an index of oxidative stress) and carotid intima-media thickness (IMT), a surrogate marker of atherosclerosis. After adjusting them for age and sex, telomere length inversely correlated (p < 0.05) with NADPH oxidase-mediated superoxide production, with 8-OHdG values, and with carotid IMT. Interestingly, the asymptomatic subjects with plaques have a lower telomere length (p < 0.05), and higher values of plasma 8-OHdG and superoxide production (p < 0.05). These data were confirmed in a second population in which patients with coronary artery disease showed lower telomere length and higher 8-OHdG and superoxide production than the asymptomatic subjects. In both studies, NADPH oxidase-dependent superoxide production in phagocytic cells was only due to the specific expression of the Nox2 isoform. In conclusion, these findings suggest that phagocytic NADPH oxidase may be involved in oxidative stress-mediated telomere shortening, and that this axis may be critically involved in human atherosclerosis.
- Microglia and astrocyte activation is region-dependent in the alfa-synuclein mouse model of Parkinson's disease(Wiley, 2023) Vilas, A. (Amaia); González-Aseguinolaza, G. (Gloria); Alonso-Roldán, M.M. (Marta María); Vales, A. (África); Abellanas-Sánchez, M.A. (Miguel Ángel); San-Martín-Uriz, P. (Patxi); Aymerich-Soler, M.S. (María Soledad); Ayerra, L. (Leyre); Basurco, L. (Leyre); Hervas-Stubbs, S. (Sandra); Hernaez, M. (Mikel); Mengual, E. (Elisa); Clavero, P. (P.); Arrasate, M. (Montserrat); Tamayo, I. (Ibon); Conde, E. (Enrique); Luquin, E. (Esther); Vinueza-Gavilanes, R. (Rodrigo)Inflammation is a common feature in neurodegenerative diseases that contributes to neuronal loss. Previously, we demonstrated that the basal inflammatory tone differed between brain regions and, consequently, the reaction generated to a pro-inflammatory stimulus was different. In this study, we assessed the innate immune reaction in the midbrain and in the striatum using an experimental model of Parkinson's disease. An adeno-associated virus serotype 9 expressing the α-synuclein and mCherry genes or the mCherry gene was administered into the substantia nigra. Myeloid cells (CD11b+ ) and astrocytes (ACSA2+ ) were purified from the midbrain and striatum for bulk RNA sequencing. In the parkinsonian midbrain, CD11b+ cells presented a unique anti-inflammatory transcriptomic profile that differed from degenerative microglia signatures described in experimental models for other neurodegenerative conditions. By contrast, striatal CD11b+ cells showed a pro-inflammatory state and were similar to disease-associated microglia. In the midbrain, a prominent increase of infiltrated monocytes/macrophages was observed and, together with microglia, participated actively in the phagocytosis of dopaminergic neuronal bodies. Although striatal microglia presented a phagocytic transcriptomic profile, morphology and cell density was preserved and no active phagocytosis was detected. Interestingly, astrocytes presented a pro-inflammatory fingerprint in the midbrain and a low number of differentially displayed transcripts in the striatum. During α-synuclein-dependent degeneration, microglia and astrocytes experience context-dependent activation states with a different contribution to the inflammatory reaction. Our results point towards the relevance of selecting appropriate cell targets to design neuroprotective strategies aimed to modulate the innate immune system during the active phase of dopaminergic degeneration.
- Set protein is involved in FLT3 membrane trafficking(2023) Marcotegui-Arza, N. (Nerea); Peris-Martínez, I. (Irene); Romero-Murillo, S. (Silvia); Berrozpe-Casado, B. (B.); Arriazu-Ruiz, E. (Elena); Odero, M.D. (Maria Dolores); Marco-Sanz, J. (Javier); Vicente, C. (Carmen)Internal tandem duplication mutations in the FLT3 tyrosine kinase receptor (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia. These mutations cause constitutive activation of FLT3, altering the underlying signaling pathways and retaining FLT3 in the endoplasmic reticulum (ER). However, the mechanism that determines this peculiar localization is not fully understood. Here, we show that SET acts as a scaffold protein for nascent wild-type FLT3, facilitating its transport to the membrane. By contrast, the FLT3-ITD mutation impairs SET/FLT3 binding, leading to its retention in the ER. Of note, the tyrosine kinase inhibitor midostaurin promotes SET/FLT3 binding, increasing FLT3 in the membrane.The in-frame internal tandem duplication (ITD) of the FLT3 gene is an important negative prognostic factor in acute myeloid leukemia (AML). FLT3-ITD is constitutive active and partially retained in the endoplasmic reticulum (ER). Recent reports show that 3'UTRs function as scaffolds that can regulate the localization of plasma membrane proteins by recruiting the HuR-interacting protein SET to the site of translation. Therefore, we hypothesized that SET could mediate the FLT3 membrane location and that the FLT3-ITD mutation could somehow disrupt the model, impairing its membrane translocation. Immunofluorescence and immunoprecipitation assays demonstrated that SET and FLT3 co-localize and interact in FLT3-WT cells but hardly in FLT3-ITD. SET/FLT3 interaction occurs before FLT3 glycosylation. Furthermore, RNA immunoprecipitation in FLT3-WT cells confirmed that this interaction occurs through the binding of HuR to the 3'UTR of FLT3. HuR inhibition and SET nuclear retention reduced FLT3 in the membrane of FLT3-WT cells, indicating that both proteins are involved in FLT3 membrane trafficking. Interestingly, the FLT3 inhibitor midostaurin increases FLT3 in the membrane and SET/FLT3 binding. Therefore, our results show that SET is involved in the transport of FLT3-WT to the membrane; however, SET barely binds FLT3 in FLT3-ITD cells, contributing to its retention in the ER.