Genoma mitocondrial, mitocondriopatías y cáncer

Tesis doctoral inédita, leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 11 de marzo de 2016La mitocondria resulta clave para numerosas funciones celulares como la obtención de energía y la señalización celular. Este orgánulo alberga su propio genoma que contiene la información únicamente para 37 genes cuya función es esencial para el sistema OXPHOS. Cambios en la secuencia del ADNmt pueden producir desde enfermedades severas (mutaciones) a no tener manifestación alguna (polimorfismos silentes). El sistema de cíbridos transmitocondriales representa un modelo de estudio ampliamente utilizado ya que permite aislar el efecto producido por diferentes variantes de ADNmt. La neuropatía óptica hereditaria de Leber (LHON) está considerada como la patología mitocondrial más común. En ella, 3 mutaciones (m.3460G>A, m.11778G>A y m.14484T>C), todas ellas afectando al Complejo I, explican el 95% de los casos. A pesar de ser una enfermedad considerablemente estudiada existen preguntas sin resolver. En esta tesis describimos el primer caso de un paciente portador de 3 mutaciones LHON en su molecula de ADNmt (m.11778G>A, m.14484T>C y m.11253T>C). El estudio molecular sobre cíbridos demostró la ausencia de sinergia entre estas mutaciones, presentando todos los cíbridos de pacientes LHON una afectación moderada y sugiriendo un mecanismo de acción común. La función mitocondrial, además de ser clave en las patologías mitocondriales clásicas, ha sido implicada en otras como el cáncer. En este sentido, el efecto Warburg (glucolisis aerobia) ha sido descrito como uno de los sellos característicos de las células tumorales. Por otro lado, se han encontrado mutaciones en el ADNmt en la mayoría de tumores, aunque su papel es aún controvertido. Para estudiar el proceso de reprogramación metabólica se estudió la función mitocondrial en una serie de 4 líneas celulares epiteliales, MCA3D, PB, A5 y CarC con un potencial tumorigénico creciente. Los resultados mostraron un aumento de la tasa glucolítica a medida que aumenta la tumorigenicidad de las líneas celulares, pero sin una afectación de la función mitocondrial. Estos resultados apoyan la teoría de que la mitocondria no tiene por qué estar afectada necesariamente en las células tumorales, pero sí el metabolismo energético. Por otro lado, sumándose a esta tendencia natural de acumulación de mutaciones en tumores, la línea parental 143B de osteosarcoma humano (sobre la que se realizaron los cíbridos LHON) posee una mutación en MT-COI en el ADNmt. Curiosamente, la eliminación del ADNmt o la sustitución de este ADNmt por una molécula sin mutaciones o con la mutación severa m.8363G>A eliminaban su tumorigenicidad, sugiriendo que la presencia de mutaciones moderadas eran necesarias para mantener el fenotipo tumoral. Esta hipótesis que fue confirmada mediante la utilización de cíbridos portadores de las 3 mutaciones primarias LHON y sus controles de haplogrupo. Los principales mecanismos alterados que explica estas diferencias en función de ADNmt fueron una mayor producción de ROS y un aumento de resistencia a apoptosis.Mitochondria play a key role in many cellular functions such as energy production and cell signaling. This organelle has its own genome containing exclusively the information for 37 genes whose function is essential for the OXPHOS system. Changes in mtDNA sequence produce from severe diseases to silent polymorphisms. The transmitochondrial cybrid system represents a widely used study-model to isolate the effect of different variants of mtDNA. The Leber's hereditary optic neuropathy is considered the most common mitochondrial pathology in which three mutations (m.3460G> A, m.11778G> A and m.14484T> C), all of them affecting complex I, account for 95% of the cases. Despite being a significantly studied disease some questions remain unresolved. In this thesis we describe the first case of a patient with 3 LHON mutations (m.11778G> A, m.14484T> C and m.11253T> C). The molecular study on cybrids demonstrated the absence of synergy for these mutations. All cybrids harboring LHON mutations showed a similar moderate impairment, suggesting a common mechanism of action. Mitochondrial function, besides being a key factor in classical mitochondrial pathologies, has been implicated in other pathologies like cancer. In this aspect, the Warburg effect has been described as one of the hallmarks of tumor cells. Furthermore, mutations in mtDNA for most tumors have been found, although its role is still controversial. To study the metabolic reprogramming process we studied the mitochondrial function in a series of 4 epithelial cell lines, MCA3D, PB, A5 and CarC with increasing tumorigenic potential. The results showed an increase in glycolytic function with increasing tumorigenicity of the cell lines, but no affectation of mitochondrial function was detected. These results support the theory that mitochondria are not necessarily affected in tumor cells, but the energy metabolism is. Furthermore, adding to this natural tendency of accumulation of mutations in tumors, the osteosarcoma 143B parental line (on which the LHON cybrids were performed) possesses a mutation in MT-COI on mtDNA. Interestingly, mtDNA removal or replacement by a wild type mtDNA or with the severe mutation m.8363G>A hampered the tumorigenicity, suggesting that the presence of moderate mutations were necessary to maintain the tumorigenic phenotype. This hypothesis was confirmed using cybrids carrying the 3 primary LHON mutations and haplogroup controls. The main altered mechanism explaining these differences depending on mtDNA were a higher ROS production and an increased apoptosis resistance

A look at ancestral knowledge for an intercultural education in the compartir el recuerdo district educational institution and the Cundinamarca college in Bogotá Colombia, period 2021-2022

Esta investigación pretende hacer un reconocimiento y dignificación a nuestros pueblos indígenas en cuanto a su identidad, cosmovisión, idioma, espiritualidad y elementos culturales. Se hace necesario crear un proyecto pedagógico para que en la escuela se dé a conocer la educación propia de las diferentes etnias indígenas que habitan el territorio colombiano. La estrategia metodológica utilizada es un estudio etnográfico en el cual se tendrá en cuenta la visión holística de la integración de saberes indígenas. El grupo focal está conformado por estudiantes, docentes y etnoeducadores. El objetivo es identificar las estrategias pedagógicas interculturales desarrolladas en las instituciones educativas El Recuerdo y el Colegio Cundinamarca en Bogotá Colombia y, que permitan la sistematización de contenidos educativos desde la perspectiva de los saberes y narrativas indígenas para que sean integrados en el currículo escolar, es decir en los Proyectos Educativos Institucionales PEI en los colegios públicos de Bogotá. Posteriormente se plantean las conclusiones que permiten determinar que sí es posible construir una educación propia indígena en contextos urbanos. Finalmente se presenta una propuesta pedagógica que fortalece las actitudes y habilidades de todos los miembros de la comunidad educativa como agentes transformadores.This research aims to recognize and dignify our indigenous peoples in terms of their identity, worldview, language, spirituality, cultural elements, social organization, and mythology, among others. It is necessary to create a pedagogical project so that the education of the different indigenous ethnic groups that inhabit the Colombian territory is made known in the school. The approaches of Catherine Walsh, Aníbal Quijano, Denisse Najmanovich and Sousa Santos are especially considered. The methodological strategy used is an ethnographic study in which the holistic vision of the integration of indigenous knowledge will be taken into account. The focus group is made up of students, teachers, teaching directors, ethno-educators or facilitators. The objective is to identify the intercultural pedagogical strategies developed in the educational institutions Compartir el Recuerdo and Colegio Cundinamarca in Bogotá Colombia and, that allow the systematization of educational contents from the perspective of indigenous knowledge and narratives so that they are integrated into the school curriculum, that is, in the PEI Institutional Educational Projects in public schools in Bogotá. Subsequently, the conclusions that allow us to determine that it is possible to build an indigenous education in urban contexts are presented. Finally, a pedagogical proposal is presented that strengthens the attitudes and skills of all members of the educational community as transforming agents

Clinical and molecular parameters associated to pneumonitis development in non-small-cell lung cancer patients receiving chemoimmunotherapy from NADIM trial

Background Pneumonitis (Pn) is one of the main immune-related adverse effects, having a special importance in lung cancer, since they share affected tissue. Despite its clinical relevance, Pn development remains an unpredictable treatment adverse effect, whose mechanisms are mainly unknown, being even more obscure when it is associated to chemoimmunotherapy.Methods In order to identify parameters associated to treatment related Pn, we analyzed clinical variables and molecular parameters from 46 patients with potentially resectable stage IIIA non-small-cell lung cancer treated with neoadjuvant chemoimmunotherapy included in the NADIM clinical trial (NCT03081689). Pn was defined as clinical or radiographic evidence of lung inflammation without alternative diagnoses, from treatment initiation to 180 days. Results Among 46 patients, 12 developed Pn (26.1%). Sex, age, smoking status, packs-year, histological subtype, clinical or pathological response, progression free survival, overall survival and number of nivolumab cycles, were not associated to Pn development. Regarding molecular parameters at diagnosis, Pn evelopment was not associated to programmed death ligand 1, TPS, T cell receptor repertoire parameters, or tumor mutational burden. However, patients who developed Pn had statistically significant lower blood median levels of platelet to monocyte ratio (p=0.012) and teratocarcinoma derived growth factor 1 (p=0.013; area under the curve (AUC) 0.801), but higher median percentages of natural killers (NKs) (p=0.019; AUC 0.786), monocytes (p=0.017; AUC 0.791), MSP (p=0.006; AUC 0.838), PARN (p=0.017; AUC 0.790), and E-Cadherin (p=0.022; AUC 0.788). In addition, the immune scenario of Pn after neoadjuvant treatment involves: high levels of neutrophils and NK cells, but low levels of B and T cells in peripheral blood; increased clonality of intratumoral T cells; and elevated plasma levels of several growth factors (EGF, HGF, VEGF, ANG-1, PDGF, NGF, and NT4) and inflammatory cytokines (MIF, CCL16, neutrophil gelatinase-associated lipocalin, BMP-4, and u-PAR). Conclusions Although statistically underpowered, our results shed light on the possible mechanisms behind Pn development, involving innate and adaptative immunity, and open the possibility to predict patients at high risk. If confirmed, this may allow the personalization of both, the surveillance strategy and the therapeutic approaches to manage Pn in patients receiving chemoimmunotherapy

The role of metabolism in tumor immune evasion: Novel approaches to improve immunotherapy

The tumor microenvironment exhibits altered metabolic properties as a consequence of the needs of tumor cells, the natural selection of the most adapted clones, and the selfish relationship with other cell types. Beyond its role in supporting uncontrolled tumor growth, through energy and building materials obtention, metabolism is a key element controlling tumor immune evasion. Immunotherapy has revolutionized the treatment of cancer, being the first line of treatment for multiple types of malignancies. However, many patients either do not benefit from immunotherapy or eventually relapse. In this review we overview the immunoediting process with a focus on the metabolism-related elements that are responsible for increased immune evasion, either through reduced immunogenicity or increased resistance of tumor cells to the apoptotic action of immune cells. Finally, we describe the main molecules to modulate these immune evasion processes through the control of the metabolic microenvironment as well as their clinical developmental statusWork in the authors’ laboratories was supported by “Instituto de Salud Carlos III” (ISCIII) PI19/01652 grant, Ministry of Science and Innovation RTC2017-6502-1 INmunoSIGHT and European Union’s Horizon 2020 research and innovation programme, CLARIFY 875160 grant, to M.P. A.C.-B. received a Spanish Lung Cancer Group (SLCG) grant and is supported by a ISCIII-“Sara Borrell” contract CD19/00170. M.C. is supported by PEJD-2019-PRE/BMD-17006 contract granted to A.C.-B. R.L.-B. was supported by PEJ16/MED/AI-1972 and PEJD-2018-PRE/SAL-8641 from European Social Fund and Comunidad de Madrid, both granted to M.

Evaluation of mitochondrial function and metabolic reprogramming during tumor progression in a cell model of skin carcinogenesis

Metabolic reprogramming from mitochondrial aerobic respiration to aerobic glycolysis is a hallmark of cancer. However, whether it is caused by a dysfunction in the oxidative phosphorylation pathway is still under debate. In this work, we have analyzed the bioenergetic cellular (BEC) index and the relative cell ability to grow in the presence of either galactose or glucose as sources of sugar (Gal/Glu index) of a system formed by four epidermal cell lines with increasing tumorigenic potentials, ranging from nontumorigenic to highly malignant. We find that the BEC index gradually decreases whereas the Gal/Glu index increases with tumorigenicity, indicating that a progressive metabolic adaptation to aerobic glycolysis occurs in tumor cells associated with malignancy. Interestingly, this metabolic adaptation does not appear to be caused by damaged respiration, since the expression and activity of components of the respiratory chain complexes were unchanged in the cell lines. Moreover, the corresponding mitochondrial ATP synthetic abilities of the cell lines were found similar. The production of reactive oxygen species was also measured. A shift in ROS generation was found when compared nontumorigenic with tumorigenic cell lines, the latter exhibiting about threefold higher ROS levels than nontumorigenic cells. This result indicates that oxidative stress is an early event during tumor progression. © 2013 Elsevier Masson SAS. All rights reserved.This work was supported by grants of the Center for Biomedical Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III (grants PI 07/0167 and PI 10/0703 to RG), Comunidad Autónoma de Madrid (grant number GEN-0269 to RG and grant S2010/BMD-2359, SkinModel-CM, to MQ) and the Spanish Ministry of Science and Innovation (grant SAF2010-19152 to MQ).Peer Reviewe

Dynamic circulating tumor DNA quantificaton for the individualization of non-small-cell lung cancer patients treatment

Background: Liquid biopsy has evolved from being a promising line to becoming a validated approach for biomarker testing. However, its utility for individualization of therapy has been scarcely reported. In this study, we show how monitoring levels of EGFR mutation in plasma can be useful for the individualization of treatment. Results: Longitudinal EGFR mutation levels in plasma always correlated with tumor response ascertained by RECIST criteria. Moreover, decreasing EGFR mutation levels were detected in all patients benefiting from locoregional radiotherapy, whereas the opposite occurred when a patient progressed soon after radiotherapy treatment. Similarly, increasing EGFR mutation levels anticipated disease progression after TKI dose reduction, discontinuation of treatment, or reduced bioavailability due to drug interactions. In addition, EGFR mutation levels were useful to monitor treatment outcome of new therapies and constituted a decisive factor when the clinical situation of the patient did not correlate with responses ascertained by radiologist. Finally, our results indicate that cancer associated body fluids (pleural, pericardial or cerebrospinal fluid) are certainly a suitable source for biomarker testing that can extend EGFR mutation detection to biofluids other than blood. Materials and Methods: A total of 180 serial plasma samples from 18 non-smallcell lung cancer patients who carried an activating EGFR mutation were investigated by digital PCR. Conclusions: Monitoring levels of EGFR mutation in plasma allows resolving doubts that frequently arise in daily clinical practice and constitutes a major step towards achieving personalized medicineThis study was supported by Carlos III Institute of Health, Spanish Ministry of Science and Innovation, and European Regional Development Fund (grant number: PI16/01818 and PIE14/00064), A Romero is supported by Joan Rodés fellowship (grant number: JR14/00017) and CP pre-doctoral studies are supported by Jose Luís Castaño Foundatio

Concordance between circulating tumor cells and clinical status during follow-up in anaplastic lymphoma kinase (ALK) non-small-cell lung cancer patients

Background: The identification of anaplastic lymphoma kinase (ALK) rearrangements is found in approximately 5% of non-small-cell lung cancers (NSCLCs). However, the development of liquid biopsies as a diagnostic tool is less developed in these cases. This study investigates the use of CTCs during treatment, together with an extended follow-up to correlate with clinical evolution. Patients and Methods: A total of 13 patients out of a cohort of 212 patients with lung adenocarcinoma, presented ALK rearrangements (6%) confirmed by tumor biopsy. A total of 60 serial blood samples were collected from these patients who were prospectively enrolled in the study. Results: All patients had a positive CTC count at baseline (mean = 3). The median follow-up was 9 months (range 1-17 months). Three patients underwent surgery and their CTC counts decreased after the procedure but still remained detectable. After radiotherapy, 3 cases showed an average decrease of 5 CTCs. A total of 6 patients were treated with ALK inhibitors and a partial response was observed in 3 of them, who also presented decreased CTC counts. The other 3 patients presented primary resistance, and their CTC counts were higher than those obtained prior to progression. Conclusion: We believe that the use of CTCs for dynamic monitoring of NSCLC with ALK rearrangement and to detect disease persistence or recurrence may be a reliable technique. CTC counts may also have potential use to monitor the efficacy of ALK inhibitors, facilitating detection of resistance to treatmentThis study was supported by Carlos III Institute of Health, Spanish Ministry of Science and Innovation, and European Regional Development Fund (grant number: PI16/01818 and PIE14/00064), D. Pérez-Callejo is supported by SEOM-Río-Hortega contract, A Romero is supported by Joan Rodés fellowship (grant number: JR14/00017) and M Sánchez-Beato is supported by Miguel Servet contract (CP11/00018 and CPII16/00024

Pretreatment Tissue TCR Repertoire Evenness Is Associated with Complete Pathologic Response in Patients with NSCLC Receiving Neoadjuvant Chemoimmunotherapy

Neoadjuvant Chemoimmunotherapy; Lung cancer; T-cell receptorQuimioimmunoteràpia neoadjuvant; Càncer de pulmó; Receptor de cèl·lules TQuimioinmunoterapia neoadyuvante; Cáncer de pulmón; Receptor de células TPurpose: Characterization of the T-cell receptor (TCR) repertoire may be a promising source for predictive biomarkers of pathologic response to immunotherapy in locally advanced non–small cell lung cancer (NSCLC). Experimental Design: In this study, next-generation TCR sequencing was performed in peripheral blood and tissue samples of 40 patients with NSCLC, before and after neoadjuvant chemoimmunotherapy (NADIM clinical trial, NCT03081689), considering their complete pathologic response (CPR) or non-CPR. Beyond TCR metrics, tissue clones were ranked by their frequency and spatiotemporal evolution of top 1% clones was determined. Results: We have found a positive association between an uneven TCR repertoire in tissue samples at diagnosis and CPR at surgery. Moreover, TCR most frequently ranked clones (top 1%) present in diagnostic biopsies occupied greater frequency in the total clonal space of CPR patients, achieving an AUC ROC to identify CPR patients of 0.967 (95% confidence interval, 0.897–1.000; P = 0.001), and improving the results of PD-L1 tumor proportion score (TPS; AUC = 0.767; P = 0.026) or tumor mutational burden (TMB; AUC = 0.550; P = 0.687). Furthermore, tumors with high pretreatment top 1% clonal space showed similar immune cell populations but a higher immune reactive gene expression profile. Finally, the selective expansion of pretreatment tissue top 1% clones in peripheral blood of CPR patients suggests also a peripheral immunosurveillance, which could explain the high survival rate of these patients. Conclusions: We have identified two parameters derived from TCR repertoire analysis that could outperform PD-L1 TPS and TMB as predictive biomarkers of CPR after neoadjuvant chemoimmunotherapy, and unraveled possible mechanisms of CPR involving enhanced tumor immunogenicity and peripheral immunosurveillance

Blood biomarkers associated to complete pathologicalresponse on NSCLC patients treated with neoadjuvantchemoimmunotherapy included in NADIM clinical trial

Background:Immunotherapy is being tested in early-stage non-small cell lungcancer (NSCLC), and achieving higher rates of complete pathological responses(CPR) as compared to standard of care. Early identification of CPR patients hasvital clinical implications. In this study, we focused on basal peripheral immunecells and their treatment-related changes to find biomarkers associated toCPR.Methods:Blood from 29 stage IIIA NSCLC patients participating in the NADIMtrial (NCT03081689) was collected at diagnosis and post neoadjuvant treatment.More than 400 parameters of peripheral blood mononuclear cells (PBMCs) phe-notype and plasma soluble factors were analyzed.Results:Neoadjuvant chemoimmunotherapy altered more than 150 immuneparameters. At diagnosis, 11 biomarkers associated to CPR were described, withan area under the ROC curve>0.70 andp-value<.05. CPR patients had sig-nificantly higher levels of CD4+PD-1+cells, NKG2D, and CD56 expression onT CD56 cells, intensity of CD25 expression on CD4+CD25hi+cells and CD69expression on intermediate monocytes; but lower levels of CD3+CD56–CTLA-4+cells, CD14++CD16+CTLA-4+cells, CTLA-4 expression on T CD56 cells andlower levels of b-NGF, NT-3, and VEGF-D in plasma compared to non-CPR. Posttreatment, CPR patients had significantly higher levels of CD19 expression on Bcells, BCMA, 4-1BB, MCSF, and PARC and lower levels of MPIF-1 and Flt-3L inplasma compared to non-CPR.Conclusions:Patients achieving CPR seem to have a distinctive peripheralblood immune status at diagnosis, even showing different immune response totreatment.TheseresultsreinforcethedifferentbiologybehindCPRandnon-CPRresponses