MicroRNA-4732-3p Is Dysregulated in Breast Cancer Patients with Cardiotoxicity, and Its Therapeutic Delivery Protects the Heart from Doxorubicin-Induced Oxidative Stress in Rats.

Anthracycline-induced cardiotoxicity is the most severe collateral effect of chemotherapy originated by an excess of oxidative stress in cardiomyocytes that leads to cardiac dysfunction. We assessed clinical data from patients with breast cancer receiving anthracyclines and searched for discriminating microRNAs between patients that developed cardiotoxicity (cases) and those that did not (controls), using RNA sequencing and regression analysis. Serum levels of 25 microRNAs were differentially expressed in cases versus controls within the first year after anthracycline treatment, as assessed by three different regression models (elastic net, Robinson and Smyth exact negative binomial test and random forest). MiR-4732-3p was the only microRNA identified in all regression models and was downregulated in patients that experienced cardiotoxicity. MiR-4732-3p was also present in neonatal rat cardiomyocytes and cardiac fibroblasts and was modulated by anthracycline treatment. A miR-4732-3p mimic was cardioprotective in cardiac and fibroblast cultures, following doxorubicin challenge, in terms of cell viability and ROS levels. Notably, administration of the miR-4732-3p mimic in doxorubicin-treated rats preserved cardiac function, normalized weight loss, induced angiogenesis, and decreased apoptosis, interstitial fibrosis and cardiac myofibroblasts. At the molecular level, miR-4732-3p regulated genes of TGFß and Hippo signaling pathways. Overall, the results indicate that miR-4732-3p is a novel biomarker of cardiotoxicity that has therapeutic potential against anthracycline-induced heart damage.

Corrigendum to "Plasmatic Membrane Expression of Adhesion Molecules in Human Cardiac Progenitor/Stem Cells Might Explain Their Superior Cell Engraftment after Cell Transplantation".

[This corrects the article DOI: 10.1155/2020/8872009.].

Intratumoral immunosuppression profiles in 11q-deleted neuroblastomas provide new potential therapeutic targets.

High-risk neuroblastoma (NB) patients with 11q deletion frequently undergo late but consecutive relapse cycles with fatal outcome. To date, no actionable targets to improve current multimodal treatment have been identified. We analyzed immune microenvironment and genetic profiles of high-risk NB correlating with 11q immune status. We show in two independent cohorts that 11q-deleted NB exhibits various immune inhibitory mechanisms, including increased CD4+ resting T cells and M2 macrophages, higher expression of programmed death-ligand 1, interleukin-10, transforming growth factor-beta-1, and indoleamine 2,3-dioxygenase 1 (P < 0.05), and also higher chromosomal breakages (P ≤ 0.02) and hemizygosity of immunosuppressive miRNAs than MYCN-amplified and other 11q-nondeleted high-risk NB. We also analyzed benefits of maintenance treatment in 83 high-risk stage M NB patients focusing on 11q status, either with standard anti-GD2 immunotherapy (n = 50) or previous retinoic acid-based therapy alone (n = 33). Immunotherapy associated with higher EFS (50 vs. 30, P = 0.028) and OS (72 vs. 52, P = 0.047) at 3 years in the overall population. Despite benefits from standard anti-GD2 immunotherapy in high-risk NB patients, those with 11q deletion still face poor outcome. This NB subgroup displays intratumoral immune suppression profiles, revealing a potential therapeutic strategy with combination immunotherapy to circumvent this immune checkpoint blockade.

Plasmatic Membrane Expression of Adhesion Molecules in Human Cardiac Progenitor/Stem Cells Might Explain Their Superior Cell Engraftment after Cell Transplantation.

Human bone marrow mesenchymal stem cells (BM-MSCs) and cardiac progenitor/stem cells (CPCs) have been extensively studied as a potential therapeutic treatment for myocardial infarction (MI). Previous reports suggest that lower doses of CPCs are needed to improve cardiac function relative to their bone marrow counterparts. Here, we confirmed this observations and investigated the surface protein expression profile that might explain this effect. Myocardial infarction was performed in nude rats by permanent ligation of the left coronary artery. Cardiac function and infarct size before and after cell transplantation were evaluated by echocardiography and morphometry, respectively. The CPC and BM-MSC receptome were analyzed by proteomic analysis of biotin-labeled surface proteins. Rats transplanted with CPCs showed a greater improvement in cardiac function after MI than those transplanted with BM-MSCs, and this was associated with a smaller infarct size. Analysis of the receptome of CPCs and BM-MSCs showed that gene ontology biological processes and KEGG pathways associated with adhesion mechanisms were upregulated in CPCs compared with BM-MSCs. Moreover, the membrane protein interactome in CPCs showed a strong relationship with biological processes related to cell adhesion whereas the BM-MSCs interactome was more related to immune regulation processes. We conclude that the stronger capacity of CPCs over BM-MSCs to engraft in the infarcted area is likely linked to a more pronounced cell adhesion expression program.

Cancer testis antigens in myelodysplastic syndromes revisited: a targeted RNA-seq approach.

Cancer-Testis antigens (CTA) are named after the tissues where they are mainly expressed: in germinal and in cancer cells, a process that mimics many gametogenesis features. Mapping accurately the CTA gene expression signature in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) is a prerequisite for downstream immune target-discovery projects. In this study, we take advantage of the use of azacitidine to treat high-risk MDS and CMML to draw the CTAs landscape, before and after treatment, using an ad hoc targeted RNA sequencing (RNA-seq) design for this group of low transcript genes. In 19 patients, 196 CTAs were detected at baseline. Azacitidine did not change the number of CTAs expressed, but it significantly increased or decreased expression in nine and five CTAs, respectively. TFDP3 and DDX53, emerged as the main candidates for immunotherapeutic targeting, as they showed three main features: i) a significant derepression on day +28 of cycle one in those patients who achieved complete remission with hypomethylating treatment (FC = 6, p = .008; FC = 2.1, p = .008, respectively), ii) similar dynamics at the protein level to what was observed at the RNA layer, and iii) to elicit significant specific cytotoxic immune responses detected by TFDP3 and DDX53 HLA-A*0201 tetramers. Our study addresses the unmet landscape of CTAs expression in MDS and CMML and revealed a previously unrecognized TFDP3 and DDX53 reactivation, detectable in plasma and able to elicit a specific immune response after one cycle of azacitidine.

Immunosuppressive profiles in liquid biopsy at diagnosis predict response to neoadjuvant chemotherapy in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterised by high pathological complete response to neoadjuvant chemotherapy (NAC). However, refractory and poor NAC responders still face very poor outcome, emphasising the urgent need for tools that facilitate identification of these patients, so that surgery or alternatives to NAC are considered early in the treatment protocol. MATERIALS AND METHODS: We combined metabolomics, exosome circulating miRNAs and flow cytometry experimental approaches in TNBC patients at diagnosis with immunohistochemistry in needle biopsy tumours to generate NAC-response predictive models. We also co-cultured and studied crosstalk between isolated patient-derived early myeloid-derived suppressor cells (eMDSCs) and TNBC cancer cell lines. RESULTS: Blood-derived liquid biopsy biomarkers display a novel immunosuppressive profile of tryptophan-derived metabolites and eMDSC levels that significantly predict NAC response. Notably, indoleamine 2,3-dioxygenase 1 (IDO1) expression in tumour cells inversely correlated with circulating tryptophan levels but directly correlated with the level of eMDSCs. In addition, a set of circulating exosome miRNAs that target pathways of immune maturation also predicted poor NAC response prior to chemotherapy. Interestingly, expression of IDO1 increased when TNBC cell lines were co-cultured with patient-derived eMDSCs and this, in turn, promoted proliferation of eMDSCs. CONCLUSION: Our findings demonstrate that the suppressive pathways of the immune system play a key role in modulating the NAC response in TNBC. We identify a crosstalk mechanism between tumour cells and eMDSCs that exacerbates immunosuppression. These results provide a potential new tool to identify poor NAC responders for alternative strategies of treatment, including early surgical resection of the tumour, and to explore in them alternative immune therapies.

RNA Sequencing Analysis for the Identification of a PCM1/PDGFRB Fusion Gene Responsive to Imatinib.

The platelet-derived growth factor receptor ß (PDGFRB) gene translocations lead to a spectrum of chronic myeloid neoplasms, frequently associated with eosinophilia. Clinical heterogeneity is associated with a molecular one. Here, we report a novel case of a patient harboring a t(5;8)(q33;p22) translocation, resulting in the PCM1/PDGFRB fusion. Conventional cytogenetics and RNA sequencing were performed to identify the chromosomes and the genes involved in the rearrangement, respectively. This study shows that the combination of different strategies is pivotal to fine-tune the diagnosis and the clinical management of the patient. After 1 year of treatment with imatinib, the patient achieves hematological and molecular remission. We present an attractive strategy to identify novel and/or cryptic fusions, which will be relevant for clinicians dealing with the diagnosis of the patients with myelodysplastic syndrome/myeloproliferative diseases with atypical manifestations.

Clinical Utility of a Next-Generation Sequencing Panel for Acute Myeloid Leukemia Diagnostics.

Next-generation sequencing (NGS) has redefined the genetic landscape of acute myeloid leukemia (AML), providing new molecular markers for diagnostic and prognostic classifications. However, its application in the clinical setting is still challenging. We hypothesized that a 19-gene AML-targeted NGS panel could be a valid approach to obtain clinically relevant information. Thus, we assessed the ability of this panel to classify AML patients according to diagnostic and prognostic indexes in a cohort of 162 patients. The assay yielded a median read depth >2000×, with 88% of on-target reads and a mean uniformity >93% without significant global strand bias. The method was sensitive and specific, with a valid performance at the clinical variant allele frequency cutoff of 3% for point mutations and 5% for insertions or deletions (INDELs). Three-hundred thirty-nine variants were found (36% INDELs and 64% single nucleotide variants). Concordance between NGS and other conventional techniques was 100%, but the NGS approach was able to identify more clinically relevant mutations. Finally, all patients could be classified into one of the 2016 World Health Organization diagnostic categories and virtually all into the recently proposed prognostic indexes (2017 European LeukemiaNet and Genomic classification). To sum up, we validate a reliable and reproducible method for AML diagnosis and demonstrate that small, well-designed NGS panels are sufficient to guide clinical decisions according to the current standards.

The MELAS mutation m.3243A>G promotes reactivation of fetal cardiac genes and an epithelial-mesenchymal transition-like program via dysregulation of miRNAs.

The pathomechanisms underlying oxidative phosphorylation (OXPHOS) diseases are not well-understood, but they involve maladaptive changes in mitochondria-nucleus communication. Many studies on the mitochondria-nucleus cross-talk triggered by mitochondrial dysfunction have focused on the role played by regulatory proteins, while the participation of miRNAs remains poorly explored. MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) is mostly caused by mutation m.3243A>G in mitochondrial tRNALeu(UUR) gene. Adverse cardiac and neurological events are the commonest causes of early death in m.3243A>G patients. Notably, the incidence of major clinical features associated with this mutation has been correlated to the level of m.3243A>G mutant mitochondrial DNA (heteroplasmy) in skeletal muscle. In this work, we used a transmitochondrial cybrid model of MELAS (100% m.3243A>G mutant mitochondrial DNA) to investigate the participation of miRNAs in the mitochondria-nucleus cross-talk associated with OXPHOS dysfunction. High-throughput analysis of small-RNA-Seq data indicated that expression of 246 miRNAs was significantly altered in MELAS cybrids. Validation of selected miRNAs, including miR-4775 and miR-218-5p, in patient muscle samples revealed miRNAs whose expression declined with high levels of mutant heteroplasmy. We show that miR-218-5p and miR-4775 are direct regulators of fetal cardiac genes such as NODAL, RHOA, ISL1 and RXRB, which are up-regulated in MELAS cybrids and in patient muscle samples with heteroplasmy above 60%. Our data clearly indicate that TGF-ß superfamily signaling and an epithelial-mesenchymal transition-like program are activated in MELAS cybrids, and suggest that down-regulation of miRNAs regulating fetal cardiac genes is a risk marker of heart failure in patients with OXPHOS diseases.

Integrated gene set analysis for microRNA studies.

MOTIVATION: Functional interpretation of miRNA expression data is currently done in a three step procedure: select differentially expressed miRNAs, find their target genes, and carry out gene set overrepresentation analysis Nevertheless, major limitations of this approach have already been described at the gene level, while some newer arise in the miRNA scenario.Here, we propose an enhanced methodology that builds on the well-established gene set analysis paradigm. Evidence for differential expression at the miRNA level is transferred to a gene differential inhibition score which is easily interpretable in terms of gene sets or pathways. Such transferred indexes account for the additive effect of several miRNAs targeting the same gene, and also incorporate cancellation effects between cases and controls. Together, these two desirable characteristics allow for more accurate modeling of regulatory processes. RESULTS: We analyze high-throughput sequencing data from 20 different cancer types and provide exhaustive reports of gene and Gene Ontology-term deregulation by miRNA action. AVAILABILITY AND IMPLEMENTATION: The proposed methodology was implemented in the Bioconductor library mdgsa http://bioconductor.org/packages/mdgsa For the purpose of reproducibility all of the scripts are available at https://github.com/dmontaner-papers/gsa4mirna CONTACT: : david.montaner@gmail.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Regulation of salt tolerance by Torulaspora delbrueckii calcineurin target Crz1p.

Recently, the academic interest in the yeast Torulaspora delbrueckii has increased notably due to its high resistance to several types of stress, including salt and osmotic imbalance. However, the molecular mechanisms underlying these unusual properties are poorly understood. In Saccharomyces cerevisiae, the high-salt response is mediated by calcineurin, a conserved Ca(2+)/calmodulin-modulated protein phosphatase that regulates the transcriptional factor Crz1p. Here, we cloned the T. delbrueckii TdCRZ1 gene, which encodes a putative zinc finger transcription factor homologue to Crz1p. Consistent with this, overexpression of TdCRZ1 enhanced the salt tolerance of S. cerevisiae wild-type cells and suppressed the sensitivity phenotype of cnb1Delta and crz1Delta mutants to monovalent and divalent cations. However, T. delbrueckii cells lacking TdCrz1p showed phenotypes distinct from those previously observed in S. cerevisiae crz1Delta mutants. Quite remarkably, Tdcrz1-null cells were insensitive to high Na(+) and were more Li(+) tolerant than wild-type cells. Clearly, TdCrz1p was not required for the salt-induced transcriptional activation of the TdENA1 gene, encoding a putative P-type ATPase homologue to the main S. cerevisiae Na(+) pump ENA1. Furthermore, T. delbrueckii cells were insensitive to the immunosuppressive agents FK506 and cyclosporine A, both in the presence and in the absence of NaCl. Signaling through the calcineurin/Crz1 pathway appeared to be essential only on high-Ca(2+)/Mn(2+) media. Hence, T. delbrueckii and S. cerevisiae differ in the regulatory circuits and mechanisms that drive the adaptive response to salt stress.

Heterologous expression of type I antifreeze peptide GS-5 in baker's yeast increases freeze tolerance and provides enhanced gas production in frozen dough.

The demand for frozen-dough products has increased notably in the baking industry. Nowadays, no appropriate industrial baker's yeast with optimal gassing capacity in frozen dough is, however, available, and it is unlikely that classical breeding programs could provide significant improvements of this trait. Antifreeze proteins, found in diverse organisms, display the ability to inhibit the growth of ice, allowing them to survive at temperatures below 0 degrees C. In this study a recombinant antifreeze peptide GS-5 was expressed from the polar fish grubby sculpin (Myoxocephalus aenaeus) in laboratory and industrial baker's yeast strains of Saccharomyces cerevisiae. Production of the recombinant protein increased freezing tolerance in both strains tested. Furthermore, expression of the GS-5 encoding gene enhanced notably the gassing rate and total gas production in frozen and frozen sweet doughs. These effects are unlikely to be due to reduced osmotic damage during freezing/thawing, because recombinant cells showed growth behavior similar to that of the parent under hypermosmotic stress conditions.