ISCIII - Artículoshttp://hdl.handle.net/20.500.12105/21102024-03-29T08:34:57Z2024-03-29T08:34:57ZEfficient generation of human cerebral organoids directly from adherent cultures of pluripotent stem cellsGonzález-Sastre, RosaCoronel Lopez, RaquelBernabeu-Zornoza, AdelaMateos-Martínez, PatriciaRosca, AndreeaLópez-Alonso, VictoriaListe-Noya, Isabelhttp://hdl.handle.net/20.500.12105/190362024-03-22T02:02:57Z2024-02-01T00:00:00ZHuman cerebral organoids (hCOs) offer the possibility of deepening the knowledge of human brain development, as well as the pathologies that affect it. The method developed here describes the efficient generation of hCOs by going directly from two-dimensional (2D) pluripotent stem cell (PSC) cultures to three-dimensional (3D) neuroepithelial tissue, avoiding dissociation and aggregation steps. This has been achieved by subjecting 2D cultures, from the beginning of the neural induction step, to dual-SMAD inhibition in combination with CHIR99021. This is a simple and reproducible protocol in which the hCOs generated develop properly presenting proliferative ventricular zones (VZs) formed by neural precursor and radial glia (RG) that differentiate to give rise to mature neurons and glial cells. The hCOs present additional cell types such as oligodendrocyte precursors, astrocytes, microglia-like cells, and endothelial-like cells. This new approach could help to overcome some of the existing limitations in the field of organoid biotechnology, facilitating its execution in any laboratory setting.
2024-02-01T00:00:00ZBringing to Light the Importance of the miRNA Methylome in Colorectal Cancer Prognosis Through Electrochemical BioplatformsPovedano, EloyRuiz-Valdepeñas Montiel, VíctorSebuyoya, RaveryTorrente-Rodríguez, Rebeca MGarranzo-Asensio, MariaMontero-Calle, Ana MariaPingarrón, José MBarderas Manchado, RodrigoBartosik, MartinCampuzano, Susanahttp://hdl.handle.net/20.500.12105/190342024-03-22T02:02:18Z2024-03-19T00:00:00ZThis work reports the first electrochemical bioplatforms developed for the determination of the total contents of either target miRNA or methylated target miRNA. The bioplatforms are based on the hybridization of the target miRNA with a synthetic biotinylated DNA probe, the capture of the formed DNA/miRNA heterohybrids on the surface of magnetic microcarriers, and their recognition with an antibody selective to these heterohybrids or to the N6-methyladenosine (m6A) epimark. The determination of the total or methylated target miRNA was accomplished by labeling such secondary antibodies with the horseradish peroxidase (HRP) enzyme. In both cases, amperometric transduction was performed on the surface of disposable electrodes after capturing the resulting HRP-tagged magnetic bioconjugates. Because of their increasing relevance in colorectal cancer (CRC) diagnosis and prognosis, miRNA let-7a and m6A methylation were selected. The proposed electrochemical bioplatforms showed attractive analytical and operational characteristics for the determination of the total and m6A-methylated target miRNA in less than 75 min. These bioplatforms, innovative in design and application, were applied to the analysis of total RNA samples extracted from cultured cancer cells with different metastatic profiles and from paired healthy and tumor tissues of patients diagnosed with CRC at different stages. The obtained results demonstrated, for the first time using electrochemical platforms, the potential of interrogating the target miRNA methylation level to discriminate the metastatic capacities of cancer cells and to identify tumor tissues and, in a pioneering way, the potential of the m6A methylation in miRNA let-7a to serve as a prognostic biomarker for CRC.
2024-03-19T00:00:00ZRole of Amyloid Precursor Protein (APP) and Its Derivatives in the Biology and Cell Fate Specification of Neural Stem CellsCoronel Lopez, RaquelBernabeu-Zornoza, AdelaPalmer, CharlotteMuñiz-Moreno, MarZambrano, AlbertoCano, EvaListe-Noya, Isabelhttp://hdl.handle.net/20.500.12105/174522024-02-04T20:52:48Z2018-09-01T00:00:00ZAmyloid precursor protein (APP) is a member of the APP family of proteins, and different enzymatic processing leads to the production of several derivatives that are shown to have distinct biological functions. APP is involved in the pathology of Alzheimer's disease (AD), the most common neurodegenerative disorder causing dementia. Furthermore, it is believed that individuals with Down syndrome (DS) have increased APP expression, due to an extra copy of chromosome 21 (Hsa21), that contains the gene for APP. Nevertheless, the physiological function of APP remains unclear. It is known that APP plays an important role in neural growth and maturation during brain development, possibly by influencing proliferation, cell fate specification and neurogenesis of neural stem cells (NSCs). Proteolytic cleavage of APP occurs mainly via two mutually exclusive pathways, the non-amyloidogenic pathway or the amyloidogenic pathway. Other alternative pathways (η-secretase, δ-secretase and meprin pathways) have also been described for the physiological processing of APP. The different metabolites generated from these pathways, including soluble APPα (sAPPα), soluble APPβ (sAPPβ), β-amyloid (Aβ) peptides and the APP intracellular domain (AICD), have different functions determined by their structural differences, equilibrium and concentration with respect to other fragments derived from APP. This review discusses recent observations regarding possible functions of APP and its proteolytic derivatives in the biology and phenotypic specification of NSCs. This can be important for a better understanding of the pathogenesis and the development of future therapeutic applications for AD and/or DS, diseases in which alterations in neurogenesis have been described.
2018-09-01T00:00:00ZMicromotor-based electrochemical immunoassays for reliable determination of amyloid-β (1-42) in Alzheimer's diagnosed clinical samplesGordón Pidal, José MMoreno-Guzmán, MaríaMontero-Calle, Ana MariaValverde, AlejandroPingarrón, José MCampuzano, SusanaCalero, MiguelBarderas Manchado, RodrigoLópez, Miguel ÁngelEscarpa, Albertohttp://hdl.handle.net/20.500.12105/173882024-02-01T02:01:43Z2024-04-01T00:00:00ZAlzheimer's disease (AD), in addition to being the most common cause of dementia, is very difficult to diagnose, with the 42-amino acid form of Aβ (Aβ-42) being one of the main biomarkers used for this purpose. Despite the enormous efforts made in recent years, the technologies available to determine Aβ-42 in human samples require sophisticated instrumentation, present high complexity, are sample and time-consuming, and are costly, highlighting the urgent need not only to develop new tools to overcome these limitations but to provide an early detection and treatment window for AD, which is a top-challenge. In recent years, micromotor (MM) technology has proven to add a new dimension to clinical biosensing, enabling ultrasensitive detections in short times and microscale environments. To this end, here an electrochemical immunoassay based on polypyrrole (PPy)/nickel (Ni)/platinum nanoparticles (PtNPs) MM is proposed in a pioneering manner for the determination of Aβ-42 in left prefrontal cortex brain tissue, cerebrospinal fluid, and plasma samples from patients with AD. MM combines the high binding capacity of their immunorecognition external layer with self-propulsion through the catalytic generation of oxygen bubbles in the internal layer due to decomposition of hydrogen peroxide as fuel, allowing rapid bio-detection (15 min) of Aβ-42 with excellent selectivity and sensitivity (LOD = 0.06 ng/mL). The application of this disruptive technology to the analysis of just 25 μL of the three types of clinical samples provides values concordant with the clinical values reported, thus confirming the potential of the MM approach to assist in the reliable, simple, fast, and affordable diagnosis of AD by determining Aβ-42.
2024-04-01T00:00:00ZElectroanalytical Immunotool to Determine Matricellular Protein Periostin, a Stromal Biomarker of Prognosis in Colorectal CancerBlázquez‐García, MarinaQuinchia, JenniferRuiz‐Valdepeñas Montiel, VíctorTorrente‐Rodríguez, Rebeca M.Serafín, VerónicaGarranzo-Asensio, MariaGarcía‐Romero, AnaOrozco, JahirBarderas Manchado, RodrigoPingarrón, José M.Campuzano, Susanahttp://hdl.handle.net/20.500.12105/173822024-02-01T02:01:29Z2023-01-01T00:00:00ZTumor-associated stroma biomarkers are emerging as key signaling molecules of metastasis in colorectal cancer (CRC). In this sense, periostin (POSTN), a protein of the extracellular matrix from the stromal compartment, is envisioned as a potential stromal prognostic biomarker, which facilitates the application of pertinent treatments. In this work, we report an easy-to-handle amperometric sandwich-based immunosensing strategy for the determination of POSTN involving commercial magnetic microparticles (MBs), disposable carbon electrodes, and the horseradish peroxidase (HRP)/H2O2/hydroquinone (HQ) electrochemical system. The method allowed a dynamic linear range between 0.47 and 25 ngmL-1 and a limit of detection, LOD, of 0.14 ngmL-1 compatible with clinical demands. The method was applied to the analysis of POSTN in a variety of cancer-related real bio-scenarios including cell extracts and secretomes from CRC cells, and plasma from CRC patients at different stages. The potentials of this firstly developed MBsassisted immunoplatform are justified by distinguishing between metastatic abilities of cultured CRC cells through the analysis of their extracts and secretomes, and by differentiating between healthy controls and CRC patients in just 60 min. Therefore, the developed immunoplatform can be envisaged as a novel and profitable tool for exploring the most uncharacterized but prospective tumor areas.
2023-01-01T00:00:00ZBronchoalveolar cytokine profile differentiates Pulmonary Langerhans cell histiocytosis patients from other smoking-related interstitial lung diseasesBarril, SilviaAcebo, PalomaMillan-Billi, PalomaLuque, AlfonsoSibila, OriolTarín, CarlosTazi, AbdellatifCastillo, DiegoHortelano, Sonsoleshttp://hdl.handle.net/20.500.12105/173772024-01-26T02:01:11Z2023-12-18T00:00:00ZBackground: Pulmonary Langerhans cell histiocytosis (PLCH) is a rare interstitial lung disease (ILD) associated with smoking, whose definitive diagnosis requires the exclusion of other forms of ILD and a compatible surgical lung biopsy. Bronchoalveolar lavage (BAL) is commonly proposed for the diagnosis of ILD, including PLCH, but the diagnostic value of this technique is limited. Here, we have analyzed the levels of a panel of cytokines and chemokines in BAL from PLCH patients, in order to identify a distinct immune profile to discriminate PLCH from other smoking related-ILD (SR-ILD), and comparing the results with idiopathic pulmonary fibrosis (IPF) as another disease in which smoking is considered a risk factor. Methods: BAL samples were collected from thirty-six patients with different ILD, including seven patients with PLCH, sixteen with SR-ILD and thirteen with IPF. Inflammatory profiles were analyzed using the Human Cytokine Membrane Antibody Array. Principal component analysis (PCA) was performed to reduce dimensionality and protein-protein interaction (PPI) network analysis using STRING 11.5 database were conducted. Finally, Random forest (RF) method was used to build a prediction model. Results: We have found significant differences (p < 0.05) on thirty-two cytokines/chemokines when comparing BAL from PLCH patients with at least one of the other ILD. Four main groups of similarly regulated cytokines were established, identifying distinct sets of markers for each cluster. Exploratory analysis using PCA (principal component analysis) showed clustering and separation of patients, with the two first components capturing 69.69% of the total variance. Levels of TARC/CCL17, leptin, oncostatin M (OSM) and IP-10/CXCL10 were associated with lung function parameters, showing positive correlation with FVC. Finally, random forest (RF) algorithm demonstrates that PLCH patients can be differentiated from the other ILDs based solely on inflammatory profile (accuracy 96.25%). Conclusions: Our results show that patients with PLCH exhibit a distinct BAL immune profile to SR-ILD and IPF. PCA analysis and RF model identify a specific immune profile useful for discriminating PLCH.
2023-12-18T00:00:00ZHepatitis E Virus Seroprevalence is Associated with Neurodegenerative Disorders in Older People with Dementia: A Case-Control StudyPerez-Garcia, FelipeVázquez-Morón, SoniaBurgueño-García, IvánMuñoz-Gómez, María JoséZea-Sevilla, María AscensiónCalero, MiguelMartinez, IsidoroRábano, AlbertoResino, Salvadorhttp://hdl.handle.net/20.500.12105/173632024-01-25T02:01:12Z2023-04-12T00:00:00ZIn this case-control study, we evaluated the association between serum antibodies against hepatitis E virus (HEV) and central nervous system (CNS) neurodegenerative disorders (NDs) in older people with dementia. The presence of anti-HEV antibodies was related to a higher adjusted odds ratio (aOR) of having CNS NDs by neuropathological diagnosis (aOR, 2.13; P = .007) and clinical/neuropathological diagnosis (1.84; P = .02). Besides, serum anti-HEV antibodies were directly related to neuropathological injury (higher vascular pathology [aOR, 1.97; P = .006]) and higher probability of Alzheimer-type pathology (1.84; P = .02). In conclusion, the presence of anti-HEV antibodies was related to higher odds of CNS NDs and neuropathological injury in older people.
2023-04-12T00:00:00ZAβ42 Peptide Promotes Proliferation and Gliogenesis in Human Neural Stem CellsBernabeu-Zornoza, AdelaCoronel Lopez, RaquelPalmer, CharlotteCalero, MiguelMartínez-Serrano, ACano, EvaZambrano, AlbertoListe-Noya, Isabelhttp://hdl.handle.net/20.500.12105/173542024-01-24T09:22:02Z2019-06-01T00:00:00ZAmyloid-β 42 [Aβ1-42 (Aβ42)] is one of the main Aβ peptide isoforms found in amyloid plaques of brains with Alzheimer's disease (AD). Although Aβ42 is associated with neurotoxicity, it might mediate several normal physiological processes during embryonic brain development and in the adult brain. However, due to the controversy that exists in the field, relatively little is known about its physiological function. In the present work, we have analyzed the effects of different concentrations of monomeric Aβ42 on cell death, proliferation, and cell fate specification of human neural stem cells (hNSCs), specifically the hNS1 cell line, undergoing differentiation. Our results demonstrate that at higher concentrations (1 μM), Aβ42 increases apoptotic cell death and DNA damage, indicating that prolonged exposure of hNS1 cells to higher concentrations of Aβ42 is neurotoxic. However, at lower concentrations, Aβ42 significantly promotes cell proliferation and glial cell specification of hNS1 cells by increasing the pool of proliferating glial precursors, without affecting neuronal differentiation, in a concentration-dependent manner. At the molecular level, these effects could be mediated, at least in part, by GSK3β, whose expression is increased by treatment with Aβ42 and whose inhibition prevents the glial specification induced by Aβ42. Since the cellular and molecular effects are known to appear decades before the first clinical symptoms, these types of studies are important in discovering the underlying pathophysiological processes involved in the development of AD. This knowledge could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.
2019-06-01T00:00:00ZAdvances in Alzheimer’s Disease Research: Human Cerebral OrganoidsMateos-Martínez, PatriciaGonzález-Sastre, RosaCoronel Lopez, RaquelRosca, AndreeaMartín Benito, SabelaBernabeu-Zornoza, AdelaLópez-Alonso, VictoriaListe-Noya, Isabelhttp://hdl.handle.net/20.500.12105/172252024-01-19T02:00:33Z2023-01-01T00:00:00ZAlzheimer’s disease (AD) is the main neurodegenerative disorder in old age, causing memory impairment and dependency. The histopathology of AD is characterized by the presence of amyloid plaques and neurofibrillary tangles formed by Aβ peptide and hyperphosphorylated Tau, respectively. There is still no cure or effective treatment for AD. This could be due, in part, to the lack of suitable research models since animal models do not recapitulate the full physiological complexity of the human brain. With the development of induced pluripotent stem cells (iPSCs), these limitations could be overcome. Even so, the bi-dimensional (2D) culture models still do not allow to recapitulate all types of brain cells and do not show a three-dimensional (3D) arrangement. Since obtaining 3D cultures called organoids, a new opportunity arises to overcome the limitations of previous models. Human Cerebral Organoids (hCOs) represent a pioneering model, in which part of the complexity of the human brain is present. For this reason, they are fast becoming a very remarkable model for the study of the evolution of the molecular and cellular pathology of AD. This review provides a brief overview of AD research, focusing on the most recent advances achieved through the development of stem cell and cerebral organoid technology
2023-01-01T00:00:00ZEarly and differential autoimmune diseases diagnosis by interrogating specific autoantibody signatures with multiplexed electrochemical bioplatformsArévalo, BeatrizSerafín, VerónicaGarranzo-Asensio, MaríaBarderas Manchado, RodrigoYáñez-Sedeño, PalomaCampuzano, SusanaPingarrón, José M.http://hdl.handle.net/20.500.12105/168702023-12-22T02:01:59Z2023-01-01T00:00:00ZThis work reports the first bioplatform to assist in the early and reliable diagnosis of autoimmune diseases by quadruple determination of autoantibodies (Abs) produced against extractable nuclear antigens (ENAs): La/SSB-Abs, Ro/SSA-Abs, U1snRNP70-Abs and smRNP-Abs. The bioplatform involves indirect immunoassays on the surface of magnetic microcarriers (independent batches for each of the target autoantibodies) and amperometric transduction using the H2O2/hydroquinone (HQ) system on a disposable multiple electrode platform. The magnetic microcarriers were modified with the corresponding antigens using His-tag and carbodiimide/succinimide chemistries and employed for the selective capture of the corresponding autoantibodies. Thereafter, they were enzymatically labelled with a secondary antibody conjugated with horseradish peroxidase (HRP) and magnetically captured on each of the working surfaces of the quadruple platform. The evaluation of the analytical and operational characteristics of the bioplatform for the amperometric determination of standards, performed under optimized experimental conditions, confirmed the bioplatform competitiveness in terms of sensitivity and point-of-care application compared to commercially available ELISA methodologies for the single determination of target Abs. The developed bioplatform was applied to the analysis of serum samples from healthy individuals and from patients with two prevalent autoimmune diseases (systemic lupus erythematosus, SLE, and Sjögren’s syndrome, SS). The obtained results proved the potential of the bioplatform for the differential diagnosis of these two autoimmune diseases through the accurate, simple, and rapid multidetermination of the four target Abs.
2023-01-01T00:00:00ZDual detection system for cancer-associated point mutations assisted by a multiplexed LNA-based amperometric bioplatform coupled with rolling circle amplificationSebuyoya, RaveryValverde, AlejandroMoranova, LudmilaStrmiskova, JohanaHrstka, RomanRuiz-Valdepeñas Montiel, VíctorPingarrón, José M.Barderas Manchado, RodrigoCampuzano, SusanaBartosik, Martinhttp://hdl.handle.net/20.500.12105/168692023-12-22T02:01:50Z2023-01-01T00:00:00ZDNA point mutation in a BRAF proto-oncogene, V600E, is considered an important prognostic and predictive biomarker in various types of cancer, such as melanoma or colorectal cancer. We report here a novel electrochemical (EC) bioplatform for the analysis of BRAF V600E mutation coupled with rolling circle amplification (RCA) and locked nucleic acid (LNA) capture probes. A dual detection system was implemented, whereby two padlock probes complementary to either wild-type (wt) BRAF gene or DNA with V600E mutation (mut) led to amplification of wt or mut variant, respectively. Hybridization with specific LNA capture probes then increased the assay specificity, while EC detection provided rapid measurement times. The bioplatform was applied to analyze BRAF V600E mutation of cancer cells and tumor tissues from patients with melanoma or colorectal cancer. This is the first RCA-based EC bioplatform for BRAF analysis in a dual format without using PCR or sophisticated instrumentation.
2023-01-01T00:00:00ZBenefits of FAIMS to Improve the Proteome Coverage of Deteriorated and/or Cross-Linked TMT 10-Plex FFPE Tissue and Plasma-Derived Exosomes SamplesMontero-Calle, Ana MariaGarranzo-Asensio, MariaRejas-González, RaquelFeliu, JaimeMendiola, MartaPeláez-García, AlbertoBarderas Manchado, Rodrigohttp://hdl.handle.net/20.500.12105/168682023-12-22T02:01:38Z2023-10-24T00:00:00ZThe proteome characterization of complex, deteriorated, or cross-linked protein mixtures as paired clinical FFPE or exosome samples isolated from low plasma volumes (250 µL) might be a challenge. In this work, we aimed at investigating the benefits of FAIMS technology coupled to the Orbitrap Exploris 480 mass spectrometer for the TMT quantitative proteomics analyses of these complex samples in comparison to the analysis of protein extracts from cells, frozen tissue, and exosomes isolated from large volume plasma samples (3 mL). TMT experiments were performed using a two-hour gradient LC-MS/MS with or without FAIMS and two compensation voltages (CV = -45 and CV = -60). In the TMT experiments of cells, frozen tissue, or exosomes isolated from large plasma volumes (3 mL) with FAIMS, a limited increase in the number of identified and quantified proteins accompanied by a decrease in the number of peptides identified and quantified was observed. However, we demonstrated here a noticeable improvement (>100%) in the number of peptide and protein identifications and quantifications for the plasma exosomes isolated from low plasma volumes (250 µL) and FFPE tissue samples in TMT experiments with FAIMS in comparison to the LC-MS/MS analysis without FAIMS. Our results highlight the potential of mass spectrometry analyses with FAIMS to increase the depth into the proteome of complex samples derived from deteriorated, cross-linked samples and/or those where the material was scarce, such as FFPE and plasma-derived exosomes from low plasma volumes (250 µL), which might aid in the characterization of their proteome and proteoforms and in the identification of dysregulated proteins that could be used as biomarkers.
2023-10-24T00:00:00ZModeling of Respiratory Diseases Evolving with Fibrosis from Organoids Derived from Human Pluripotent Stem CellsChamorro-Herrero, IreneZambrano, Albertohttp://hdl.handle.net/20.500.12105/168602023-12-22T02:01:18Z2023-02-23T00:00:00ZRespiratory disease is one of the leading causes of morbidity and mortality worldwide. There is no cure for most diseases, which are treated symptomatically. Hence, new strategies are required to deepen the understanding of the disease and development of therapeutic strategies. The advent of stem cell and organoid technology has enabled the development of human pluripotent stem cell lines and adequate differentiation protocols for developing both airways and lung organoids in different formats. These novel human-pluripotent-stem-cell-derived organoids have enabled relatively accurate disease modeling. Idiopathic pulmonary fibrosis is a fatal and debilitating disease that exhibits prototypical fibrotic features that may be, to some extent, extrapolated to other conditions. Thus, respiratory diseases such as cystic fibrosis, chronic obstructive pulmonary disease, or the one caused by SARS-CoV-2 may reflect some fibrotic aspects reminiscent of those present in idiopathic pulmonary fibrosis. Modeling of fibrosis of the airways and the lung is a real challenge due to the large number of epithelial cells involved and interaction with other cell types of mesenchymal origin. This review will focus on the status of respiratory disease modeling from human-pluripotent-stem-cell-derived organoids, which are being used to model several representative respiratory diseases, such as idiopathic pulmonary fibrosis, cystic fibrosis, chronic obstructive pulmonary disease, and COVID-19.
2023-02-23T00:00:00ZAltered Clock Gene Expression in Female APP/PS1 Mice and Aquaporin-Dependent Amyloid Accumulation in the RetinaCarrero, LauraAntequera, DesireéAlcalde, IgnacioMegías, DiegoOrdoñez-Gutierrez, LaraGutierrez, CristinaMerayo-Lloves, JesúsWandosell, FranciscoMunicio, CristinaCarro, Evahttp://hdl.handle.net/20.500.12105/168452023-12-20T02:01:59Z2023-10-27T00:00:00ZAlzheimer's disease (AD), the most prevalent form of dementia, is a neurodegenerative disorder characterized by different pathological symptomatology, including disrupted circadian rhythm. The regulation of circadian rhythm depends on the light information that is projected from the retina to the suprachiasmatic nucleus in the hypothalamus. Studies of AD patients and AD transgenic mice have revealed AD retinal pathology, including amyloid-β (Aβ) accumulation that can directly interfere with the regulation of the circadian cycle. Although the cause of AD pathology is poorly understood, one of the main risk factors for AD is female gender. Here, we found that female APP/PS1 mice at 6- and 12-months old display severe circadian rhythm disturbances and retinal pathological hallmarks, including Aβ deposits in retinal layers. Since brain Aβ transport is facilitated by aquaporin (AQP)4, the expression of AQPs were also explored in APP/PS1 retina to investigate a potential correlation between retinal Aβ deposits and AQPs expression. Important reductions in AQP1, AQP4, and AQP5 were detected in the retinal tissue of these transgenic mice, mainly at 6-months of age. Taken together, our findings suggest that abnormal transport of Aβ, mediated by impaired AQPs expression, contributes to the retinal degeneration in the early stages of AD.
2023-10-27T00:00:00ZSchnurri-3 drives tumor growth and invasion in cancer cells expressing interleukin-13 receptor alpha 2Bartolomé, Rubén AMartín-Regalado, ÁngelaPintado-Berninches, LauraRobles, JavierRamírez-González, María ABoukich, IssamSánchez-Gómez, PilarBalyasnikova, Irina VCasal, J Ignaciohttp://hdl.handle.net/20.500.12105/167502023-12-06T02:01:35Z2023-11-14T00:00:00ZInterleukin 13 receptor alpha 2 (IL13Rα2) is a relevant therapeutic target in glioblastoma (GBM) and other tumors associated with tumor growth and invasion. In a previous study, we demonstrated that protein tyrosine phosphatase 1B (PTP1B) is a key mediator of the IL-13/IL13Rα2 signaling pathway. PTP1B regulates cancer cell invasion through Src activation. However, PTP1B/Src downstream signaling mechanisms that modulate the invasion process remain unclear. In the present research, we have characterized the PTP1B interactome and the PTP1B-associated phosphoproteome after IL-13 treatment, in different cellular contexts, using proteomic strategies. PTP1B was associated with proteins involved in signal transduction, vesicle transport, and with multiple proteins from the NF-κB signaling pathway, including Tenascin-C (TNC). PTP1B participated with NF-κB in TNC-mediated proliferation and invasion. Analysis of the phosphorylation patterns obtained after PTP1B activation with IL-13 showed increased phosphorylation of the transcription factor Schnurri-3 (SHN3), a reported competitor of NF-κB. SHN3 silencing caused a potent inhibition in cell invasion and proliferation, associated with a down-regulation of the Wnt/β-catenin pathway, an extensive decline of MMP9 expression and the subsequent inhibition of tumor growth and metastasis in mouse models. Regarding clinical value, high expression of SHN3 was associated with poor survival in GBM, showing a significant correlation with the classical and mesenchymal subtypes. In CRC, SHN3 expression showed a preferential association with the mesenchymal subtypes CMS4 and CRIS-B. Moreover, SHN3 expression strongly correlated with IL13Rα2 and MMP9-associated poor prognosis in different cancers. In conclusion, we have uncovered the participation of SNH3 in the IL-13/IL13Rα2/PTP1B pathway to promote tumor growth and invasion. These findings support a potential therapeutic value for SHN3.
2023-11-14T00:00:00ZFunctional Proteomics Characterization of the Role of SPRYD7 in Colorectal Cancer Progression and MetastasisMontero-Calle, Ana MariaJiménez de Ocaña, SofíaBenavente-Naranjo, RuthRejas-González, RaquelBartolomé, Rubén AMartínez-Useros, JavierSanz, RodrigoDziaková, JanaFernández-Aceñero, María JesúsMendiola, MartaCasal, José IgnacioPeláez-García, AlbertoBarderas Manchado, Rodrigohttp://hdl.handle.net/20.500.12105/167482023-12-06T02:01:44Z2023-10-31T00:00:00ZSPRY domain-containing protein 7 (SPRYD7) is a barely known protein identified via spatial proteomics as being upregulated in highly metastatic-to-liver KM12SM colorectal cancer (CRC) cells in comparison to its isogenic poorly metastatic KM12C CRC cells. Here, we aimed to analyze SPRYD7's role in CRC via functional proteomics. Through immunohistochemistry, the overexpression of SPRYD7 was observed to be associated with the poor survival of CRC patients and with an aggressive and metastatic phenotype. Stable SPRYD7 overexpression was performed in KM12C and SW480 poorly metastatic CRC cells and in their isogenic highly metastatic-to-liver-KM12SM-and-to-lymph-nodes SW620 CRC cells, respectively. Upon upregulation of SPRYD7, in vitro and in vivo functional assays confirmed a key role of SPRYD7 in the invasion and migration of CRC cells and in liver homing and tumor growth. Additionally, transient siRNA SPRYD7 silencing allowed us to confirm in vitro functional results. Furthermore, SPRYD7 was observed as an inductor of angiogenesis. In addition, the dysregulated SPRYD7-associated proteome and SPRYD7 interactors were elucidated via 10-plex TMT quantitative proteins, immunoproteomics, and bioinformatics. After WB validation, the biological pathways associated with the stable overexpression of SPRYD7 were visualized. In conclusion, it was demonstrated here that SPRYD7 is a novel protein associated with CRC progression and metastasis. Thus, SPRYD7 and its interactors might be of relevance in identifying novel therapeutic targets for advanced CRC.
2023-10-31T00:00:00Z