Re-definition of non-small cell lung cancer transcriptional subtypes using integrative bioinformatics approaches

[eng] Recent technological advances and the utilization of high- throughput DNA and RNA sequencing analyses have increased our understanding of cancer diseases, including non-small cell lung cancer. For instance, genomic characterization has allowed the identification of some gene alterations which can be targeted with specific drug compounds. However, detection of genomic alterations does not fully recapitulate the heterogeneity of the disease and it sometimes fail to predict the subset of patients that most benefit from chemo- or immunotherapy. In this context, transcriptional profiling has emerged as a promising tool for patient selection and treatment guidance. Comprehensive evaluation of the expression level of pathways and genes involved in tumor progression and immune response has demonstrated to predict clinical benefit potentially better than single agents such as PD-L1 or tumor mutational burden in the case of immunotherapy regimens. This study aimed to establish a robust classification of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tumors, respectively, based on the transcriptional profiling of 50 landmark molecular pathways. This work also aimed to characterize the subtypes at different levels and to identify potential specific vulnerabilities and drug candidates. Thus, in this thesis we present a new tumor classification framework based on the expression profiling of 50 signaling pathways, which is more robust than the use of single gene expression levels, prone to multiple sources of variability. Also, this approach allows for the integration of different gene expression datasets, significantly increasing the sample size and the amount of molecular heterogeneity that can be considered. In fact, to our knowledge, no previous LUAD or LUSC classification has been derived from such a large sample size. Moreover, we have provided a way for drug prioritization, based on the molecular characteristics of each subtype and the integration of huge cancer cell lines pharmacogenomics projects. In the end, this work could lay the foundation for improving patient stratification beyond genomics and single biomarkers and pave the way for more personalized treatment avenues in non-small cell lung cancer.[cat] Els avenços tecnològics recents i la utilització d'anàlisis de seqüenciació d'ADN i ARN d'alt rendiment han augmentat la nostra comprensió de les malalties del càncer, inclòs el càncer de pulmó de cèl·lula no petita. Per exemple, la caracterització genòmica ha permès identificar algunes alteracions genètiques que poden ser tractades amb fàrmacs específics. No obstant això, la detecció d'alteracions genòmiques no recapitula del tot l'heterogeneïtat de la malaltia i a vegades no aconsegueix predir el subconjunt de pacients que més es beneficiarien del tractament amb quimioteràpia o immunoteràpia. En aquest context, l’anàlisi dels perfils transcripcionals ha sorgit com una eina prometedora per a la selecció del pacient i l'orientació del tractament. Per exemple, l'avaluació del nivell d'expressió de vies i gens implicats en la progressió tumoral i la resposta immunitària ha demostrat predir el benefici clínic de millor manera que marcadors individuals com el PD-L1 o la càrrega mutacional del tumor en el cas de la resposta a immunoteràpia. Aquest estudi tenia com a objectiu establir una classificació robusta dels tumors d'adenocarcinoma pulmonar i carcinoma de cèl·lules escamoses pulmonars, respectivament. Aquest treball també tenia com a objectiu caracteritzar els subtipus a diferents nivells i identificar possibles vulnerabilitats específiques. Així, en aquesta tesi presentem un nou marc de classificació tumoral basat en el perfil d'expressió de 50 vies de senyalització, que és més robust que l'ús de nivells d'expressió de gens individuals, propens a múltiples fonts de variabilitat. A més, aquest enfocament permet la integració de diferents conjunts de dades d'expressió gènica, augmentant significativament la mida de la mostra i la quantitat d'heterogeneïtat molecular que es pot considerar. De fet, segons el nostre coneixement, no s'ha obtingut cap classificació prèvia d’ adenocarcinoma pulmonar o carcinoma de cèl·lules escamoses pulmonars a partir d'una quantitat de mostra tan gran. D'altra banda, hem donat una aproximació per a la priorització de fàrmacs, basada en les característiques moleculars de cada subtipus i la integració de grans projectes de farmacogenòmica de línies cel·lulars de càncer. Al final, aquest treball podria establir les bases per millorar l'estratificació del pacient més enllà de la genòmica i els biomarcadors individuals i aplanar el camí per a opcions de tractament més personalitzades en càncer de pulmó de cèl·lula no petita

G6PD overexpression protects from oxidative stress and age-related hearing loss

Aging of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative damage in macromolecules, which contributes to cellular malfunction, compromises cell viability, and, ultimately, leads to functional decline. Cellular detoxification relies in part on the production of NADPH, which is an important cofactor for major cellular antioxidant systems. NADPH is produced principally by the housekeeping enzyme glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the rate-limiting step in the pentose phosphate pathway. We show here that G6PD transgenic mice (G6PD-Tg), which show enhanced constitutive G6PD activity and NADPH production along life, have lower auditory thresholds than wild-type mice during aging, together with preserved inner hair cell (IHC) and outer hair cell (OHC), OHC innervation, and a conserved number of synapses per IHC. Gene expression of antioxidant enzymes was higher in 3-month-old G6PD-Tg mice than in wild-type counterparts, whereas the levels of pro-apoptotic proteins were lower. Consequently, nitration of proteins, mitochondrial damage, and TUNEL apoptotic cells were all lower in 9-month-old G6PD-Tg than in wild-type counterparts. Unexpectedly, G6PD overexpression triggered low-grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophage infiltration. Our results lead us to propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the production of ROS and cellular detoxification power along aging and thus prevents hearing loss progression.Secretaría de Estado de Investigación, Desarrollo e Innovación, Grant/Award Number: MINECO/FEDER SAF2017-86107-R; Comunidad de Madrid, Grant/Award Number: FEDER/CM-B2017/BMD-368

Integrative transcriptome analysis of malignant pleural mesothelioma reveals a clinically relevant immune-based classification

Background: Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasia affecting the lung mesothelium. Immune checkpoint inhibitors (ICI) in MPM have not been extremely successful, likely due to poor identification of suitable candidate patients for the therapy. We aimed to identify cellular immune fractions associated with clinical outcome and classify patients with MPM based on their immune contexture. For each defined group, we sought for molecular specificities that could help further define our MPM classification at the genomic and transcriptomic level, as well as identify differential therapeutic strategies based on transcriptional signatures predictive of drug response. Methods: The abundance of 20 immune cell fractions in 516 MPM samples from 7 gene expression datasets was inferred using gene set variation analysis. Identification of clinically relevant fractions was performed with Cox proportional-hazards models adjusted for age, stage, sex, and tumor histology. Immune-based groups were defined based on the identified fractions. Results: T-helper 2 (TH2) and cytotoxic T (TC) cells were found to be consistently associated with overall survival. Three immune clusters (IG) were subsequently defined based on TH2 and TC immune infiltration levels: IG1 (54.5%) was characterized by high TH2 and low TC levels, IG2 (37%) had either low or high levels of both fractions, and IG3 (8.5%) was defined by low TH2 and high TC levels. IG1 and IG3 groups were associated with worse and better overall survival, respectively. While no differential genomic alterations were identified among immune groups, at the transcriptional level, IG1 samples showed upregulation of proliferation signatures, while IG3 samples presented upregulation of immune and inflammation-related pathways. Finally, the integration of gene expression with functional signatures of drug response showed that IG3 patients might be more likely to respond to ICI. Conclusions: This study identifies a novel immune-based signature with potential clinical relevance based on TH2 and TC levels, unveiling a fraction of patients with MPM with better prognosis and who might benefit from immune-based therapies. Molecular specificities of the different groups might be used to tailor specific potential therapies in the future

G6PD overexpression protects from oxidative stress and ameliorates ARHL progression

Trabajo presentado en el 56th Inner Ear Biology Workshop, celebrado en Padua (Italia) del 7 al 10 de septiembre de 2019.Ageing of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative-derived damage in macromolecules, which contribute to cellular malfunction, compromise cell viability and, finally, causes functional decline. The cellular detoxification power partially relies in NADPH production, which serves as cofactor for the activity of major cellular antioxidant enzymes. NADPH is mainly produced by glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the rate-limiting step in the pentose phosphate pathway. We show here that the transgenic mouse G6PD-Tg, which shows enhanced NADPH production along life, maintains lower auditory thresholds than wild type mice during ageing. G6PD overexpression preserves irreplaceable cochlear cell populations, thus G6PD-Tg mice exhibit higher number of inner and outer hair cells (OHC), more widespread OHC innervation and higher number of synapses per IHC than wild type mice. Transcripts for antioxidant enzymes and pro-apoptotic proteins levels were increased and reduced respectively in 3-month-old G6PD-Tg. Accordingly, tyrosine modification by nitration in proteins and mitochondrial damage was reduced in 9-month-old G6PD-Tg compared with wild type mice. As well, as lesser TUNEL positive apoptotic cells were detected in whole mount preparations in G6PD-Tg mice. Interestingly, G6PD overexpression turned out to trigger an inflammatory response effectively resolved without cellular damage or macrophage infiltration in the cochlea. In conclusion, we propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the generation of ROS and the cell detoxification power along ageing and, therefore to prevent hearing loss progression. Acknowledgements. This work was supported by FP7-2013-TARGEAR and FEDER/SAF 2014-AGEAR and 2017-HEARCOD

G6PD overexpression protects from Oxidative Stress and Retard Age¿related Hearing Loss Porgression

Resumen del trabajo presentado en el 43rd Annual MidWinter Meeting-ARO, celebrado en San José, California (Estados Unidos) del 25 al 29 de enero de 2020.Ageing of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative-derived damage in macromolecules, which contribute to cellular malfunction, compromise cell viability and, finally, causes functional decline. The cellular detoxification power partially relies in NADPH production, which is a cofactor for major cellular antioxidant enzymes. NADPH is mainly produced by glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the rate-limiting step in the pentose phosphate pathway. We show here that the transgenic mouse G6PD-Tg, which shows enhanced NADPH production along life, maintains lower auditory thresholds than wild type mice during ageing. G6PD overexpression preserved inner (IHC) and outer hair cells (OHC), OHC innervation and number of synapses per IHC. Transcripts for antioxidant enzymes were increased whereas levels of pro-apoptotic proteins were reduced in 3-month-old G6PD-Tg. Consequently, nitration of proteins, mitochondrial damage and TUNEL+ apoptotic cells were reduced in 9-month-old G6PDTg compared to wild type mice. Unexpectedly, G6PD overexpression triggered low grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophages infiltration. In conclusion, we propose here that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the generation of ROS and the cell detoxification power along ageing and, therefore to prevent hearing loss progression.This work has been founded by Spanish MINECO/ FEDER SAF2017-86107-R to IVN. JMBM was supported by contracts from MINECO/FEDER SAF2014-AGEAR and MINECO/FEDER SAF2017-86107-R

Gene Expression Profiling as a Potential Tool for Precision Oncology in Non-Small Cell Lung Cancer

Recent technological advances and the application of high-throughput mutation and transcriptome analyses have improved our understanding of cancer diseases, including non-small cell lung cancer. For instance, genomic profiling has allowed the identification of mutational events which can be treated with specific agents. However, detection of DNA alterations does not fully recapitulate the complexity of the disease and it does not allow selection of patients that benefit from chemo- or immunotherapy. In this context, transcriptional profiling has emerged as a promising tool for patient stratification and treatment guidance. For instance, transcriptional profiling has proven to be especially useful in the context of acquired resistance to targeted therapies and patients lacking targetable genomic alterations. Moreover, the comprehensive characterization of the expression level of the different pathways and genes involved in tumor progression is likely to better predict clinical benefit from different treatments than single biomarkers such as PD-L1 or tumor mutational burden in the case of immunotherapy. However, intrinsic technical and analytical limitations have hindered the use of these expression signatures in the clinical setting. In this review, we will focus on the data reported on molecular classification of non-small cell lung cancer and discuss the potential of transcriptional profiling as a predictor of survival and as a patient stratification tool to further personalize treatments

DUAL NOX4/NOX1 INHIBITION BY SETANAXIB HALTS CHOLANGIOCARCINOMA PROGRESSION BY IMPAIRING CANCER ASSOCIATED FIBROBLAST ACTIVATION

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