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Introduction The aim of this study was to investigate endogenous retrovirus (ERV) expression and type I interferon (IFN) activation in human pleural mesothelioma (PM) and their association with clinical outcome. Methods The expression of ERV was determined from PM cohorts and mesothelial precursor RNA sequencing data. The expression of ERV was confirmed by quantitative polymerase chain reaction (qPCR). Methylation of genomic DNA was assessed by quantitative methylation-specific PCR. DNA demethylation was induced in cells by demethylating agent 5-Aza-2’-deoxycytidine (5-Aza-CdR) treatment. To block type I IFN signaling, the cells were treated with ruxolitinib or MAVS silencing. The expression of IFN-stimulated genes (ISGs) was determined by qPCR and Western blot. Circulating ERVs were detected by qPCR. Results Long terminal repeats (LTRs) represent the most abundant transposable elements up-regulated in PM. Within the LTR, ERVmap_1248 and LTR7Y, which are specifically enriched in PM, were further analyzed. The 5-Aza-CdR treatment increased the levels of ERVmap_1248 expression and induced ERVmap_1248 promoter demethylation in mesothelial cells. In addition, ERVmap_1248 promoter was more demethylated in the mesothelioma tissue compared with nontumor tissue. The 5-Aza-CdR treatment of the mesothelial cells also increased the levels of ISGs. Basal ISG expression was higher in the mesothelioma cells compared with the mesothelial cells, and it was significantly decreased by ruxolitinib treatment or MAVS silencing. Furthermore, ISG expression was higher in the tumor tissue with high expression levels of ERVmap_1248. High expression of ERVmap_1248 was associated with longer overall survival and BAP1 mutations. ERVmap_1248 and LTR7Y can be detected in the PM plasma. Conclusions We provide clues for patient stratification especially for immunotherapy where best clinical responses are associated with an activated basal immune response.

We previously observed increased levels of adenosine-deaminase-acting-on-dsRNA (Adar)-dependent RNA editing during mesothelioma development in mice exposed to asbestos. The aim of this study was to characterize and assess the role of ADAR-dependent RNA editing in mesothelioma. We found that tumors and mesothelioma primary cultures have higher ADAR-mediated RNA editing compared to mesothelial cells. Unsupervised clustering of editing in different genomic regions revealed heterogeneity between tumor samples as well as mesothelioma primary cultures. ADAR2 expression levels are higher in BRCA1-associated protein 1 wild-type tumors, with corresponding changes in RNA editing in transcripts and 3'UTR. ADAR2 knockdown and rescue models indicated a role in cell proliferation, altered cell cycle, increased sensitivity to antifolate treatment, and type-1 interferon signaling upregulation, leading to changes in the microenvironment in vivo. Our data indicate that RNA editing contributes to mesothelioma heterogeneity and highlights an important role of ADAR2 not only in growth regulation in mesothelioma but also in chemotherapy response, in addition to regulating inflammatory response downstream of sensing nucleic acid structures.

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies.

Mesothelioma is an aggressive cancer associated with asbestos exposure. RNA-binding motif protein 8a (RBM8A) mRNA editing increases in mouse tissues upon asbestos exposure. The aim of this study was to further characterize the role of RBM8A in mesothelioma and the consequences of its mRNA editing. RBM8A protein expression was higher in mesothelioma compared to mesothelial cells. Silencing RBM8A changed splicing patterns in mesothelial and mesothelioma cells but drastically reduced viability only in mesothelioma cells. In the tissues of asbestos-exposed mice, editing of Rbm8a mRNA was associated with increased protein immunoreactivity, with no change in mRNA levels. Increased adenosine deaminase acting on dsRNA (ADAR)-dependent editing of Alu elements in the RBM8A 3'UTR was observed in mesothelioma cells compared to mesothelial cells. Editing stabilized protein expression. The unedited RBM8A 3'UTR had a stronger interaction with Musashi (MSI) compared to the edited form. The silencing of MSI2 in mesothelioma or overexpression of Adar2 in mesothelial cells resulted in increased RBM8A protein levels. Therefore, ADAR-dependent editing contributes to maintaining elevated RBM8A protein levels in mesothelioma by counteracting MSI2-driven downregulation. A wider implication of this mechanism for the translational control of protein expression is suggested by the editing of similarly structured Alu elements in several other transcripts.

Early events in an experimental model of mesothelioma development include increased levels of editing in double-stranded RNA (dsRNA). We hypothesised that expression of endogenous retroviruses (ERV) contributes to dsRNA formation and type-I interferon signaling. ERV and interferon stimulated genes (ISGs) expression were significantly higher in tumor compared to non-tumor samples. 12 tumor specific ERV ("MesoERV1-12") were identified and verified by qPCR in mouse tissues. "MesoERV1-12" expression was lower in mouse embryonic fibroblasts (MEF) compared to mesothelioma cells. "MesoERV1-12" levels were significantly increased by demethylating agent 5-Aza-2'-deoxycytidine treatment and were accompanied by increased levels of dsRNA and ISGs. Basal ISGs expression was higher in mesothelioma cells compared to MEF and was significantly decreased by JAK inhibitor Ruxolitinib, by blocking Ifnar1 and by silencing Mavs. "MesoERV7" promoter was demethylated in asbestos-exposed compared to sham mice tissue as well as in mesothelioma cells and MEF upon 5-Aza-CdR treatment. These observations uncover novel aspects of asbestos-induced mesothelioma whereby ERV expression increases due to promoter demethylation and is paralleled by increased levels of dsRNA and activation of type-I IFN signaling. These features are important for early diagnosis and therapy.

Calretinin (CALB2) is a diagnostic and prognostic marker in malignant pleural mesothelioma (MPM). We previously reported that calretinin expression is regulated at the mRNA level. The presence of a medium-sized (573 nucleotide) 3′ untranslated region (3′UTR) predicted to contain binding sites for miR-30a/b/c/d/e and miR-9 as well as an adenine/uridine-rich element (ARE) in all three transcripts arising from the CALB2 gene, suggests that calretinin expression is regulated via posttranscriptional mechanisms. Our aim was to investigate the role of the CALB2-3′UTR in the posttranscriptional regulation of calretinin expression in MPM. CALB2-3′UTR was inserted downstream of the luciferase reporter gene using pmiRGLO vector and reporter expression was determined after transfection into MPM cells. Targeted mutagenesis was used to generate variants harboring mutated miR-30 family and ARE binding sites. Electrophoretic mobility shift assay was used to test for the presence of ARE binding proteins. CALB2-3′UTR significantly decreased luciferase activity in MPM cells. Analysis of mutation in the ARE site revealed a further destabilization of the reporter and human antigen R (HuR) binding to the ARE sequence was detected. The mutation of two miR-30 binding sites abolished CALB2-3′UTR destabilization effect; a transient delivery of miR-30e-5p mimics or anti-miR into MPM cells resulted in a significant decrease/increase of the luciferase reporter expression and calretinin protein, respectively. Moreover, overexpression of CALB2-3′UTR quenched the effect of miR-30e-5p mimics on calretinin protein levels, possibly by sequestering the mimics, thereby suggesting a competitive endogenous RNA network. Finally, by data mining we observed that expression of miR-30e-5p was negatively correlated with the calretinin expression in a cohort of MPM patient samples. Our data show the role of (1) adenine-uridine (AU)-binding proteins in calretinin stabilization and (2) miR-30e-5p in the posttrans

Calretinin (CALB2) is a diagnostic and prognostic marker in malignant pleural mesothelioma (MPM). We previously reported that calretinin expression is regulated at the mRNA level. The presence of a medium-sized (573 nucleotide) 3′ untranslated region (3′UTR) predicted to contain binding sites for miR-30a/b/c/d/e and miR-9 as well as an adenine/uridine-rich element (ARE) in all three transcripts arising from the CALB2 gene, suggests that calretinin expression is regulated via posttranscriptional mechanisms. Our aim was to investigate the role of the CALB2-3′UTR in the posttranscriptional regulation of calretinin expression in MPM. CALB2-3′UTR was inserted downstream of the luciferase reporter gene using pmiRGLO vector and reporter expression was determined after transfection into MPM cells. Targeted mutagenesis was used to generate variants harboring mutated miR-30 family and ARE binding sites. Electrophoretic mobility shift assay was used to test for the presence of ARE binding proteins. CALB2-3′UTR significantly decreased luciferase activity in MPM cells. Analysis of mutation in the ARE site revealed a further destabilization of the reporter and human antigen R (HuR) binding to the ARE sequence was detected. The mutation of two miR-30 binding sites abolished CALB2-3′UTR destabilization effect; a transient delivery of miR-30e-5p mimics or anti-miR into MPM cells resulted in a significant decrease/increase of the luciferase reporter expression and calretinin protein, respectively. Moreover, overexpression of CALB2-3′UTR quenched the effect of miR-30e-5p mimics on calretinin protein levels, possibly by sequestering the mimics, thereby suggesting a competitive endogenous RNA network. Finally, by data mining we observed that expression of miR-30e-5p was negatively correlated with the calretinin expression in a cohort of MPM patient samples. Our data show the role of (1) adenine-uridine (AU)-binding proteins in calretinin stabilization and (2) miR-30e-5p in the posttrans