Identification of redox-sensitive transcription factors as markers of malignant pleural mesothelioma.

Although asbestos has been banned in most countries around the world, malignant pleural mesothelioma (MPM) is a current problem. MPM is an aggressive tumor with a poor prognosis, so it is crucial to identify new markers in the preventive field. Asbestos exposure induces oxidative stress and its carcinogenesis has been linked to a strong oxidative damage, event counteracted by antioxidant systems at the pulmonary level. The present study has been focused on some redox-sensitive transcription factors that regulate cellular antioxidant defense and are overexpressed in many tumors, such as Nrf2 (Nuclear factor erythroid 2-related factor 2), Ref-1 (Redox effector factor 1), and FOXM1 (Forkhead box protein M1). The research was performed in human mesothelial and MPM cells. Our results have clearly demonstrated an overexpression of Nrf2, Ref-1, and FOXM1 in mesothelioma towards mesothelium, and a consequent activation of downstream genes controlled by these factors, which in turn regulates antioxidant defense. This event is mediated by oxidative free radicals produced when mesothelial cells are exposed to asbestos fibers. We observed an increased expression of Nrf2, Ref-1, and FOXM1 towards untreated cells, confirming asbestos as the mediator of oxidative stress evoked at the mesothelium level. These factors can therefore be considered predictive biomarkers of MPM and potential pharmacological targets in the treatment of this aggressive cancer

Micro-RNA-215 and -375 regulate thymidylate synthase protein expression in pleural mesothelioma and mediate epithelial to mesenchymal transition

The standard front-line treatment for pleural mesothelioma (PM) is pemetrexed-based chemotherapy, whose major target is thymidylate synthase (TS). In several cancer models, miR-215 and miR-375 have been shown to target TS, while information on these miRNAs in PM are still limited although suggest their role in epithelial to mesenchymal transition. Seventy-one consecutive PM tissues (4 biphasic, 7 sarcomatoid, and 60 epithelioid types) and 16 commercial and patient-derived PM cell lines were screened for TS, miR-215, and miR-375 expression. REN and 570B cells were selected for miR-215 and miR-375 transient transfections to test TS modulation. ZEB1 protein expression in tumor samples was also tested. Moreover, genetic profile was investigated by means of BAP1 and p53 immunohistochemistry. Expression of both miR-215 and miR-375 was significantly higher in epithelioid histotype. Furthermore, inverse correlation between TS protein and both miR-215 and miR-375 expression was found. Efficiently transfected REN and 570B cell lines overexpressing miR-215 and miR-375 showed decreased TS protein levels. Epithelioid PM with a mesenchymal component highlighted by reticulin stain showed significantly higher TS and ZEB1 protein and lower miRNA expression. A better survival was recorded for BAP1 lost/TS low cases. Our data indicate that miR-215 and miR-375 are involved in TS regulation as well as in epithelial-to-mesenchymal transition in PM. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00428-022-03321-8

Circulating microRNAs found dysregulated in ex-exposed asbestos workers and pleural mesothelioma patients as potential new biomarkers

Malignant pleural mesothelioma (MPM), a fatal cancer, is an occupational disease mostly affecting workers ex-exposed to asbestos fibers. The asbestos, a cancerogenic mineral of different chemical composition, was widely employed in western Countries in industrial manufactures of different types. MPM may arise after a long latency period, up to five decades. MPM is resistant to conventional chemo- and radio-therapies. Altogether, these data indicate that the identification of new and specific markers are of a paramount importance for an early diagnosis and treatment of MPM. In recent years, microRNAs expression was found dysregulated in patients, both in cancer cells and sera, affected by tumors of different histotypes, including MPM. Cell and circulanting microRNAs, found to be dysregulated in this neoplasia, were proposed as new biomarkers. It has been reported that circulating microRNAs are stable in biological fluids and could be employed as potential MPM biomarkers. In this investigation, circulating microRNAs (miR) from serum samples of MPM patients and workers ex-exposed to asbestos fibers (WEA) and healthy subjects (HS) were comparatively analyzed by microarray and RT-qPCR technologies. Our results allowed (i) to select MiR-3665, an endogenous stable microRNA, as the internal control to quantify in our analyses circulating miRNAs; to detect (ii) miR-197-3p, miR-1281 and miR 32-3p up-regulated in MPM compared to HS; (iii) miR-197-3p and miR-32-3p up-regulated in MPM compared to WEA; (iv) miR-1281 up-regulated in both MPM and WEA compared to HS. In conclusion, three circulating up-regulated microRNAs, i.e. miR-197-3p, miR-1281 and miR-32-3p are proposed as potential new MPM biomarker

New DNA methylation signals for malignant pleural mesothelioma risk assessment

SIMPLE SUMMARY: Our study investigated DNA methylation differences in easily accessible white blood cells (WBCs) between malignant pleural mesothelioma (MPM) cases and asbestos-exposed cancer-free controls. A multiple regression model highlighted that the methylation level of two single CpGs (cg03546163 in FKBP5 and cg06633438 in MLLT1) are independent MPM markers. The epigenetic changes at the FKBP5 and MLLT1 genes were robustly associated with MPM in asbestos-exposed subjects. Interaction analyses showed that MPM cases and cancer-free controls showed DNAm differences which may be linked to asbestos exposure. ABSTRACT: Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasm. Patients are usually diagnosed when current treatments have limited benefits, highlighting the need for noninvasive tests aimed at an MPM risk assessment tool that might improve life expectancy. Three hundred asbestos-exposed subjects (163 MPM cases and 137 cancer-free controls), from the same geographical region in Italy, were recruited. The evaluation of asbestos exposure was conducted considering the frequency, the duration and the intensity of occupational, environmental and domestic exposure. A genome-wide methylation array was performed to identify novel blood DNA methylation (DNAm) markers of MPM. Multiple regression analyses adjusting for potential confounding factors and interaction between asbestos exposure and DNAm on the MPM odds ratio were applied. Epigenome-wide analysis (EWAS) revealed 12 single-CpGs associated with the disease. Two of these showed high statistical power (99%) and effect size (>0.05) after false discovery rate (FDR) multiple comparison corrections: (i) cg03546163 in FKBP5, significantly hypomethylated in cases (Mean Difference in beta values (MD) = −0.09, 95% CI = −0.12|−0.06, p = 1.2 × 10(−7)), and (ii) cg06633438 in MLLT1, statistically hypermethylated in cases (MD = 0.07, 95% CI = 0.04|0.10, p = 1.0 × 10(−6)). Based on the interaction analysis, asbestos exposure and epigenetic profile together may improve MPM risk assessment. Above-median asbestos exposure and hypomethylation of cg03546163 in FKBP5 (OR = 20.84, 95% CI = 8.71|53.96, p = 5.5 × 10(−11)) and hypermethylation of cg06633438 in MLLT1 (OR = 11.71, 95% CI = 4.97|29.64, p = 5.9 × 10(−8)) genes compared to below-median asbestos exposure and hyper/hypomethylation of single-CpG DNAm, respectively. Receiver Operation Characteristics (ROC) for Case-Control Discrimination showed a significant increase in MPM discrimination when DNAm information was added in the model (baseline model, BM: asbestos exposure, age, gender and white blood cells); area under the curve, AUC = 0.75; BM + cg03546163 at FKBP5. AUC = 0.89, 2.1 × 10(−7); BM + cg06633438 at MLLT1. AUC = 0.89, 6.3 × 10(−8). Validation and replication procedures, considering independent sample size and a different DNAm analysis technique, confirmed the observed associations. Our results suggest the potential application of DNAm profiles in blood to develop noninvasive tests for MPM risk assessment in asbestos-exposed subjects

Dna methylation of fkbp5 as predictor of overall survival in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is an aggressive tumor with median survival of 12 months and limited effective treatments. The scope of this study was to study the relationship between blood DNA methylation (DNAm) and overall survival (OS) aiming at a noninvasive prognostic test. We investigated a cohort of 159 incident asbestos exposed MPM cases enrolled in an Italian area with high incidence of mesothelioma. Considering 12 months as a cut-off for OS, epigenome-wide association study (EWAS) revealed statistically significant (p value = 7.7 7 10 129 ) OS-related differential methylation of a single-CpG (cg03546163), located in the 5\u2032 UTR region of the FKBP5 gene. This is an independent marker of prognosis in MPM patients with a better performance than traditional inflammation-based scores such as lymphocyte-to-monocyte ratio (LMR). Cases with DNAm < 0.45 at the cg03546163 had significantly poor survival compared with those showing DNAm 65 0.45 (mean: 243 versus 534 days; p value< 0.001). Epigenetic changes at the FKBP5 gene were robustly associated with OS in MPM cases. Our results showed that blood DNA methylation levels could be promising and dynamic prognostic biomarkers in MPM

Evaluation of the preclinical efficacy of lurbinectedin in malignant pleural mesothelioma

Background: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer generally diagnosed at an advanced stage and characterized by a poor prognosis. The absence of alterations in druggable kinases, together with an immune-suppressive tumor microenvironment, limits the use of molecular targeted therapies, making the treatment of MPM particularly challenging. Here we investigated the in vitro susceptibility of MPM to lurbinectedin (PM01183), a marine-derived drug that recently received accelerated approval by the FDA for the treatment of patients with metastatic small cell lung cancer with disease progression on or after platinum-based chemotherapy. Methods: A panel of primary MPM cultures, resembling the three major MPM histological subtypes (epithelioid, sarcomatoid, and biphasic), was characterized in terms of BAP1 status and histological markers. Subsequently, we explored the effects of lurbinectedin at nanomolar concentration on cell cycle, cell viability, DNA damage, genotoxic stress response, and proliferation. Results: Stabilized MPM cultures exhibited high sensitivity to lurbinectedin independently from the BAP1 mutational status and histological classification. Specifically, we observed that lurbinectedin rapidly promoted a cell cycle arrest in the S-phase and the activation of the DNA damage response, two conditions that invariably resulted in an irreversible DNA fragmentation, together with strong apoptotic cell death. Moreover, the analysis of long-term treatment indicated that lurbinectedin severely impacts MPM transforming abilities in vitro. Conclusion: Overall, our data provide evidence that lurbinectedin exerts a potent antitumoral activity on primary MPM cells, independently from both the histological subtype and BAP1 alteration, suggesting its potential activity in the treatment of MPM patients