Design of bioinformatic tools for integrative analysis of microRNA-mRNA interactome applied to digestive cancers

[spa] En esta tesis se han desarrollado e implementado distintas herramientas bioinformáticas que permiten el estudio de las interacciones miRNA-mRNA en contextos celulares específicos. oncretamente se ha creado un paquete de R (miRComb) que calcula las interacciones miRNA-mRNA partiendo de expresión de miRNAs y mRNAs, y predicciones bloinformáticas de bases de datos preexistentes. Las interacciones miRNA-mRNA finales son aquellas que muestran una correlación negativa y han estado predichas por al meno una base de datos. Como valor añadido, el paquete miRComb realiza un resumen en pdf con los resultados básicos del análisis (número de interacciones, número de mRNAs target por miRNA, análisis funcional, etc.), que permite comparar los datos de distintos estudios. Hemos aplicado esta metodología en el contexto de cánceres digestivos. En un primer estudio hemos utilizado datos públicos de 5 cánceres digestivos (colon, recto, esófago, stómago e hígado) y hemos determinado las interacciones miRNA-mRNA comunes entre ellos y específicas de cada uno. En un segundo estudio, hemos utilizado la misma metodología para analizar datos de IRNA-mRNA en biopsias de pacientes del Hospital Clínic de Barcelona con cáncer de páncreas. En este estudio hemos descrito interacciones miRNA-mRNA en el contexto de cáncer pancreático y hemos podido validar dos de ellas a nivel experimental. En resumen, podemos concluir que el paquete miRComb es una herramienta útil para el estudio del interactoma de miRNA-mRNA, y que ha servido para establecer hipótesis biológicas que luego se han podido comprobar en el laboratori

Novel Circulating miRNA Signatures for Early Detection of Pancreatic Neoplasia

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) presents the lowest survival rate of all cancers because only 6% of patients reach five-year survival. Alterations in the expression of several microRNAs (miRNAs) occur in the tumor of PDAC and in preneoplastic lesions as the called intraductal papillary mucinous neoplasm (IPMN). Here, we aimed at identifying which miRNAs are significantly altered in liquid biopsies from patients with PDAC and IPMN to find new noninvasive biomarkers for early detection of PDAC. METHODS: We analyzed by real-time quantitative reverse transcription-PCR (qRT-PCR) the expression of 17 circulating miRNAs, previously found to be significantly overexpressed in tissue pancreatic neoplasms, in a set of 182 plasma samples (94 PDAC, 19 IPMN, 18 chronic pancreatitis, and 51 disease-free controls). Then, we analyzed CA19.9 levels in the same plasma set, and we assessed the diagnostic values of differentially expressed miRNAs, CA19.9, and all possible combinations. RESULTS: Of note, 16, 14, and 9 miRNAs were significantly increased in PDAC, IPMN, and chronic pancreatitis, respectively, compared with control plasmas. miR-21-5p, miR-33a-3p, miR-320a, and miR-93-5p showed the highest discriminating capacity for pancreatic neoplasia (PDAC or IPMN) with an area under the receiver operating characteristic curve (AUC) of 0.86, 0.85, 0.85, and 0.80, respectively. 2-miRNA combinations improved these performances reaching AUC = 0.90 for "miR-33a-3p+miR-320a." Addition of CA19.9 increased the diagnostic potential of miRNA signatures even further achieving an AUC of 0.95 (93% sensitivity and 85% specificity) for the combination of "miR-33a-3p+miR-320a+CA19.9." CONCLUSIONS: Novel signatures combining miRNAs and CA19.9 could be used as noninvasive biomarkers for early detection of PDAC

Carm1-arginine methylation of the transcription factor C/EBPα regulates transdifferentiation velocity

Developmental biology; Gene regulation; Transcription factorBiologia del desenvolupament; Regulació gènica; Factor de transcripcióBiología del desarrollo; Regulación génica; Factor de transcripciónHere, we describe how the speed of C/EBPα-induced B cell to macrophage transdifferentiation (BMT) can be regulated, using both mouse and human models. The identification of a mutant of C/EBPα (C/EBPαR35A) that greatly accelerates BMT helped to illuminate the mechanism. Thus, incoming C/EBPα binds to PU.1, an obligate partner expressed in B cells, leading to the release of PU.1 from B cell enhancers, chromatin closing and silencing of the B cell program. Released PU.1 redistributes to macrophage enhancers newly occupied by C/EBPα, causing chromatin opening and activation of macrophage genes. All these steps are accelerated by C/EBPαR35A, initiated by its increased affinity for PU.1. Wild-type C/EBPα is methylated by Carm1 at arginine 35 and the enzyme’s perturbations modulate BMT velocity as predicted from the observations with the mutant. Increasing the proportion of unmethylated C/EBPα in granulocyte/macrophage progenitors by inhibiting Carm1 biases the cell’s differentiation toward macrophages, suggesting that cell fate decision velocity and lineage directionality are closely linked processes.TG was supported by the Center for Genomic Regulation, Barcelona, the Spanish Ministry of Economy, Industry and Competitiveness, (Plan Estatal PID2019-109354GB-100), AGAUR (SGR 006713) and the 4D-Genome European Research Council Synergy grant. KSZ was supported by the NIH grant R01GM36477. We have used ChatGPT to improve parts of the text

CNApp, a tool for the quantification of copy number alterations and integrative analysis revealing clinical implications.

Somatic copy number alterations (CNAs) are a hallmark of cancer, but their role in tumorigenesis and clinical relevance remain largely unclear. Here, we developed CNApp, a web-based tool that allows a comprehensive exploration of CNAs by using purity-corrected segmented data from multiple genomic platforms. CNApp generates genome-wide profiles, computes CNA scores for broad, focal and global CNA burdens, and uses machine learning-based predictions to classify samples. We applied CNApp to the TCGA pan-cancer dataset of 10,635 genomes showing that CNAs classify cancer types according to their tissue-of-origin, and that each cancer type shows specific ranges of broad and focal CNA scores. Moreover, CNApp reproduces recurrent CNAs in hepatocellular carcinoma and predicts colon cancer molecular subtypes and microsatellite instability based on broad CNA scores and discrete genomic imbalances. In summary, CNApp facilitates CNA-driven research by providing a unique framework to identify relevant clinical implications. CNApp is hosted at https://tools.idibaps.org/CNApp/

The trophectoderm acts as a niche for the inner cell mass through C/EBPα-regulated IL-6 signaling

IL-6 has been shown to be required for somatic cell reprogramming into induced pluripotent stem cells (iPSCs). However, how Il6 expression is regulated and whether it plays a role during embryo development remains unknown. Here, we describe that IL-6 is necessary for C/EBPα-enhanced reprogramming of B cells into iPSCs but not for B cell to macrophage transdifferentiation. C/EBPα overexpression activates both Il6 and Il6ra genes in B cells and in PSCs. In embryo development, Cebpa is enriched in the trophectoderm of blastocysts together with Il6, while Il6ra is mostly expressed in the inner cell mass (ICM). In addition, Il6 expression in blastocysts requires Cebpa. Blastocysts secrete IL-6 and neutralization of the cytokine delays the morula to blastocyst transition. The observed requirement of C/EBPα-regulated IL-6 signaling for pluripotency during somatic cell reprogramming thus recapitulates a physiologic mechanism in which the trophectoderm acts as niche for the ICM through the secretion of IL-6.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved

Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells.

Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs

Unique genomic profile of fibrolamellar hepatocellular carcinoma

BACKGROUND & AIMS: Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary hepatic cancer that develops in children and young adults without cirrhosis. Little is known about its pathogenesis, and it can be treated only with surgery. We performed an integrative genomic analysis of a large series of patients with FLC to identify associated genetic factors. METHODS: By using 78 clinically annotated FLC samples, we performed whole-transcriptome (n = 58), single-nucleotide polymorphism array (n = 41), and next-generation sequencing (n = 48) analyses; we also assessed the prevalence of the DNAJB1-PRKACA fusion transcript associated with this cancer (n = 73). We performed class discovery using non-negative matrix factorization, and functional annotation using gene-set enrichment analyses, nearest template prediction, ingenuity pathway analyses, and immunohistochemistry. The genomic identification of significant targets in a cancer algorithm was used to identify chromosomal aberrations, MuTect and VarScan2 were used to identify somatic mutations, and the random survival forest was used to determine patient prognoses. Findings were validated in an independent cohort. RESULTS: Unsupervised gene expression clustering showed 3 robust molecular classes of tumors: the proliferation class (51% of samples) had altered expression of genes that regulate proliferation and mammalian target of rapamycin signaling activation; the inflammation class (26% of samples) had altered expression of genes that regulate inflammation and cytokine enriched production; and the unannotated class (23% of samples) had a gene expression signature that was not associated previously with liver tumors. Expression of genes that regulate neuroendocrine function, as well as histologic markers of cholangiocytes and hepatocytes, were detected in all 3 classes. FLCs had few copy number variations; the most frequent were focal amplification at 8q24.3 (in 12.5% of samples), and deletions at 19p13 (in 28% of samples) and 22q13.32 (in 25% of samples). The DNAJB1-PRKACA fusion transcript was detected in 79% of samples. FLC samples also contained mutations in cancer-related genes such as BRCA2 (in 4.2% of samples), which are uncommon in liver neoplasms. However, FLCs did not contain mutations most commonly detected in liver cancers. We identified an 8-gene signature that predicted survival of patients with FLC. CONCLUSIONS: In a genomic analysis of 78 FLC samples, we identified 3 classes based on gene expression profiles. FLCs contain mutations and chromosomal aberrations not previously associated with liver cancer, and almost 80% contain the DNAJB1-PRKACA fusion transcript. By using this information, we identified a gene signature that is associated with patient survival time

Colon capsule endoscopy versus CT colonography in FIT-positive colorectal cancer screening subjects: a prospective randomised trial-the VICOCA study

Background: Colon capsule endoscopy (CCE) and CT colonography (CTC) are minimally invasive techniques for colorectal cancer (CRC) screening. Our objective is to compare CCE and CTC for the identification of patients with colorectal neoplasia among participants in a CRC screening programme with positive faecal immunochemical test (FIT). Primary outcome was to compare the performance of CCE and CTC in detecting patients with neoplastic lesions. Methods: The VICOCA study is a prospective, single-centre, randomised trial conducted from March 2014 to May 2016; 662 individuals were invited and 349 were randomised to CCE or CTC before colonoscopy. Endoscopists were blinded to the results of CCE and CTC. Results: Three hundred forty-nine individuals were included: 173 in the CCE group and 176 in the CTC group. Two hundred ninety individuals agreed to participate: 147 in the CCE group and 143 in the CTC group. In the intention-toscreen analysis, sensitivity, specificity and positive and negative predictive values for the identification of individuals with colorectal neoplasia were 98.1%, 76.6%, 93.7% and 92.0% in the CCE group and 64.9%, 95.7%, 96.8% and 57.7% in the CTC group. In terms of detecting significant neoplastic lesions, the sensitivity of CCE and CTC was 96.1% and 79.3%, respectively. Detection rate for advanced colorectal neoplasm was higher in the CCE group than in the CTC group (100% and 93.1%, respectively; RR = 1.07; p = 0.08). Both CCE and CTC identified all patients with cancer. CCE detected more patients with any lesion than CTC (98.6% and 81.0%, respectively; RR = 1.22; p = 0.002). Conclusion: Although both techniques seem to be similar in detecting patients with advanced colorectal neoplasms, CCE is more sensitive for the detection of any neoplastic lesion

Integrated Analysis of Germline and Tumor DNA Identifies New Candidate Genes Involved in Familial Colorectal Cancer

Colorectal cancer (CRC) shows aggregation in some families but no alterations in the known hereditary CRC genes. We aimed to identify new candidate genes which are potentially involved in germline predisposition to familial CRC. An integrated analysis of germline and tumor whole-exome sequencing data was performed in 18 unrelated CRC families. Deleterious single nucleotide variants (SNV), short insertions and deletions (indels), copy number variants (CNVs) and loss of heterozygosity (LOH) were assessed as candidates for first germline or second somatic hits. Candidate tumor suppressor genes were selected when alterations were detected in both germline and somatic DNA, fulfilling Knudson's two-hit hypothesis. Somatic mutational profiling and signature analysis were also performed. A series of germline-somatic variant pairs were detected. In all cases, the first hit was presented as a rare SNV/indel, whereas the second hit was either a different SNV (3 genes) or LOH affecting the same gene (141 genes). BRCA2, BLM, ERCC2, RECQL, REV3L and RIF1 were among the most promising candidate genes for germline CRC predisposition. The identification of new candidate genes involved in familial CRC could be achieved by our integrated analysis. Further functional studies and replication in additional cohorts are required to confirm the selected candidates

A pan-cancer clinical platform to predict immunotherapy outcomes and prioritize immuno-oncology combinations in early-phase trials

Immunooncology; Predictive biomarkers; Tumor microenvironmentInmunooncología; Biomarcadores predictivos; Microambiente tumoralImmunooncologia; Biomarcadors predictius; Microambient tumoralBackground Immunotherapy is effective, but current biomarkers for patient selection have proven modest sensitivity. Here, we developed VIGex, an optimized gene signature based on the expression level of 12 genes involved in immune response with RNA sequencing. Methods We implemented VIGex using the nCounter platform (Nanostring) on a large clinical cohort encompassing 909 tumor samples across 45 tumor types. VIGex was developed as a continuous variable, with cutoffs selected to detect three main categories (hot, intermediate-cold and cold) based on the different inflammatory status of the tumor microenvironment. Findings Hot tumors had the highest VIGex scores and exhibited an increased abundance of tumor-infiltrating lymphocytes as compared with the intermediate-cold and cold. VIGex scores varied depending on tumor origin and anatomic site of metastases, with liver metastases showing an immunosuppressive tumor microenvironment. The predictive power of VIGex-Hot was observed in a cohort of 98 refractory solid tumor from patients treated in early-phase immunotherapy trials and its clinical performance was confirmed through an extensive metanalysis across 13 clinically annotated gene expression datasets from 877 patients treated with immunotherapy agents. Last, we generated a pan-cancer biomarker platform that integrates VIGex categories with the expression levels of immunotherapy targets under development in early-phase clinical trials. Conclusions Our results support the clinical utility of VIGex as a tool to aid clinicians for patient selection and personalized immunotherapy interventions.A.H.C. would like to acknowledge fellowship funding from the Spanish Society of Medical Oncology (SEOM), CRIS Contra el Cancer and Hold'em For Life Oncology Fellowship. This research has been funded by the Comprehensive Program of Cancer Immunotherapy & Immunology II (CAIMI-II) supported by the BBVA Foundation (grant 53/2021) and the 2020–2021 Division of Medical Oncology and Hematology (DMOH) Fellowship award at Princess Margaret Cancer Centre. VHIO would like to acknowledge the Cellex Foundation for providing research facilities and equipment and the CERCA Programme from the Generalitat de Catalunya for their support of this research. Authors from VHIO acknowledge the State Agency for Research (Agencia Estatal de Investigación) for providing financial support as a Center of Excellence Severo Ochoa (CEX2020-001024-S/AEI/10.13039/501100011033). A.V. was the recipient of a project award from the FAECC (AVP/18/AECC/3219) and received funding from the Advanced Molecular Diagnostic (DIAMAV) program from the FERO Foundation. Graphical abstract was created with BioRender.com. Diagram in Figure 3B was created with SankeyMATIC (sankeymatic.com)