Mapping new non-genetic dependencies in malignant pleural mesothelioma

Malignant Pleural Mesothelioma (MPM) is a very aggressive cancer whose incidence is growing worldwide. MPM escapes the classical models of carcinogenesis and lacks a distinctive genetic fingerprint, keeping obscure the molecular events that lead to tumorigenesis. This severely impacts on the limited therapeutic options and on the lack of specific biomarkers, concurring to make MPM one of the deadliest cancers. Here we combined a functional genome-wide loss of function CRISPR/Cas9 screening with patients’ transcriptomic and clinical data, to identify genes essential for MPM progression. Besides, we explored the role of non-coding RNAs to MPM progression by analysing gene expression profiles and clinical data from the MESO-TCGA dataset. We identified TRIM28 and the lncRNA LINC00941 as new vulnerabilities of MPM, associated with disease aggressiveness and bad outcome of patients. TRIM28 is a multi-domain protein involved in many processes, including transcription regulation. We showed that TRIM28 silencing impairs MPM cells’ growth and clonogenicity by blocking cells in mitosis. RNA-seq profiling showed that TRIM28 loss abolished the expression of major mitotic players. Our data suggest that TRIM28 is part of the B-MYB/FOXM1-MuvB complex that specifically drives the activation of mitotic genes, keeping the time of mitosis. In parallel, we found LINC00941 as strongly associated with reduced survival probability in MPM patients. LINC00941 KD profoundly reduced MPM cells’ growth, migration and invasion. This is accompanied by changes in morphology, cytoskeleton organization and cell-cell adhesion properties. RNA-seq profiling showed that LINC00941 KD impacts crucial functions of MPM, including HIF1α signalling. Collectively these data provided new insights into MPM biology and demonstrated that the integration of functional screening with patients’ clinical data is a powerful tool to highlight new non-genetic cancer dependencies that associate to a bad outcome in vivo, paving the way to new MPM-oriented targeted strategies and prognostic tools to improve patients risk-based stratification.

Role of non-coding RNA alterations in melanoma

Cutaneous melanoma (CM) is a potentially lethal form of skin cancer and its most important histopathologic factor for staging is Breslow thickness (BT). Its correct determination is fundamental for pathologists. A deeper understanding of the molecular processes guiding CM pathogenesis could improve diagnosis, treatment and prognosis. MicroRNAs (miRNAs) play a key role in CM biology. The firs aim was to investigate miRNA expression in reference to BT assessment. We found that the combined miRNA expression of miR-21-5p and miR-146a-5p above or below 1.5 was significantly associated with overall survival and successfully identified all superficially spreading melanoma (SSM) patients with relapsing suggesting that the combined assessment of these miRNAs expression could aid in SSM staging. Secondly, we focus on multiple primary melanoma (MPM) patients, which develop multiple primary melanomas in their lifetime, and represent a model of high-risk CM occurrence. We explored the miRNome of single CM and MPM: CM and MPM present several dysregulated miRNAs, including key miRNAs involved in epithelial-mesenchymal transition. A different miRNA profile was observed between 1st and 2nd melanoma from the same patient. MiRNA target analysis revealed a more differentiated and less invasive status of MPMs compared to CMs. This characterization of the miRNA regulatory network of MPMs highlights molecular features differentiating this subtype from CM. Recently, NGS experiments revealed the existence of miRNA variants (isomiRs) with different length and sequence. We identified a shorter 3’isoform as tenfold over-represented compared to the canonical form of miR-125a-5p. Target analysis revealed that miRNA shortening could change the pattern of target gene regulation. Finally, we study miRNA and isomiR dysregulation in benign nevi (BN) and CM and in CM and melanoma metastasis. The reported non-random dysregulation of specific isomiRs contributes to the understanding of the complex melanoma pathogenesis and serves as the basis for further functional studies.