New deep-water cnidarian sites in the southern Adriatic Sea

Recent ROV (Remotely Operated Vehicle) exploration and bottom sampling in the southern Adriatic Sea (Apulian and Montenegrin margins) resulted in the discovery of cnidarian-rich deep-sea habitats in the depth range of ca. 400-700 m. In particular, ROV inspection of Montenegrin canyons reveals the existence of megabenthic communities dominated by a variety of cnidarians, including scleractinians (Madrepora oculata, Lophelia pertusa, Dendrophyllia cornigera),antipatharians (Leiopathes glaberrima) and gorgonians (Callogorgia verticillata) as major habitat forming taxa, often in association with sponges and, subordinately, serpulids. All such cnidarians are new records for the south-eastern side of the Adriatic Sea. Our investigation indicates that an almost continuous belt of patchy cold water coral sites occurs along the entire south-western margin (Apulian),basically connecting the Adriatic populations with those inhabiting the Ionian margin (Santa Maria di Leuca coral province)

A Network of MicroRNAs and mRNAs Involved in Melanosome Maturation and Trafficking Defines the Lower Response of Pigmentable Melanoma Cells to Targeted Therapy

Simple Summary Selective inhibitors of mutant BRAFV600E (BRAFi) have revolutionized the treatment of metastatic melanoma patients and represent a powerful example of the efficacy of targeted therapy. However, one of the main limitations of BRAFi is that treated cells put in place several adaptive response mechanisms, which initially confer drug tolerance and later provide a gateway for the insurgence of genetically acquired resistance mechanisms. We previously discovered that pigmentation is one of these adaptive response mechanisms. Upon BRAFi treatment, those cells that increase their pigmentation level are more resistant to BRAFi than those that do not. Here, we demonstrate that pigmentation limits BRAFi activity through an increase in the number of intracellular mature melanosomes. We also show that this increase derives from increased maturation and/or trafficking. In addition, we identify the miRNAs and mRNAs that are involved in these biological processes. Finally, we provide the rationale for testing a new combinatorial therapeutic strategy that aims at increasing BRAFi efficacy by blocking the adaptive responses that they elicit. This strategy is based on the combined use of BRAFi with inhibitors of pigmentation, specifically inhibitors of melanosome maturation and/or trafficking. Background: The ability to increase their degree of pigmentation is an adaptive response that confers pigmentable melanoma cells higher resistance to BRAF inhibitors (BRAFi) compared to non-pigmentable melanoma cells. Methods: Here, we compared the miRNome and the transcriptome profile of pigmentable 501Mel and SK-Mel-5 melanoma cells vs. non-pigmentable A375 melanoma cells, following treatment with the BRAFi vemurafenib (vem). In depth bioinformatic analyses (clusterProfiler, WGCNA and SWIMmeR) allowed us to identify the miRNAs, mRNAs and biological processes (BPs) that specifically characterize the response of pigmentable melanoma cells to the drug. Such BPs were studied using appropriate assays in vitro and in vivo (xenograft in zebrafish embryos). Results: Upon vem treatment, miR-192-5p, miR-211-5p, miR-374a-5p, miR-486-5p, miR-582-5p, miR-1260a and miR-7977, as well as GPR143, OCA2, RAB27A, RAB32 and TYRP1 mRNAs, are differentially expressed only in pigmentable cells. These miRNAs and mRNAs belong to BPs related to pigmentation, specifically melanosome maturation and trafficking. In fact, an increase in the number of intracellular melanosomes-due to increased maturation and/or trafficking-confers resistance to vem. Conclusion: We demonstrated that the ability of pigmentable cells to increase the number of intracellular melanosomes fully accounts for their higher resistance to vem compared to non-pigmentable cells. In addition, we identified a network of miRNAs and mRNAs that are involved in melanosome maturation and/or trafficking. Finally, we provide the rationale for testing BRAFi in combination with inhibitors of these biological processes, so that pigmentable melanoma cells can be turned into more sensitive non-pigmentable cells

po 220 ran a novel and promising gene for malignant pleural mesothelioma

Introduction RAN is a member of RAS superfamily of GTPases involved in a varied range of cellular processes. Although it is widely demonstrated RAN is overexpressed in many human tumours having an essential role in malignant cell survival and cancer progression, little is known about its role in Malignant Pleural Mesothelioma (MPM). Previous studies showed the RAN gene is upregulated in mesothelioma tissues and cell lines, so it might be involved in carcinogenesis of MPM. We aimed to explore the functional role of RAN in MPM cell lines and its likely use as co-target in mesothelioma treatment. Material and methods The role of RAN in MPM tumorigenesis was investigated through RNA interference, on a panel of one mesothelial cell line (Met-5A) and four MPM cell lines (Mero-14, Mero-25, Istmes-2 and NCI-H28). After monitoring gene knockdown, at both the mRNA and protein levels, a phenotypic study was performed through Caspase-3/7, Sulforhodamine B, Wound-Healing and Colony Formation assays. Flow cytometry was employed to monitor cell cycle. To validate data from siRNA experiments, two different siRNA were independently used to target RAN. The gene was also knocked-out using a lentiviral CRISPR/Cas9 system in Mero-14. Cas9 endonuclease and gRNA were transduced by two different lentiviral transfer vectors.The doxycycline-regulated Cas9 induction was followed by DNA, RNA and proteins extraction to confirm the occurrence of gene disruption. TIDE analysis was carried out to monitor targeted mutations triggered by the genome editing. Results and discussions The siRNA-mediated knockdown was confirmed at both the mRNA and protein level in all cell lines. The silencing caused a statistically significant decrease of proliferation rate and clonogenicity in Mero-14, Mero-25 and Istmes-2.The migration ability was affected in Met-5A and Istmes-2. An increase in apoptosis was observed in all cell lines, being statistically significant only in the malignant ones. Flow cytometry analysis showed an increase of cells in G0/G1 phase and a decrease of cells in S phase, being significant in Mero-14 cell line only. RAN knock-out has been confirmed at both the mRNA and protein level, whereas the TIDE analysis is still ongoing. Conclusion This study showed that MFAP5 is a novel myoepithelial cell marker that appears to be up-regulated in duct epithelium in DCIS and IC-NST during tumourogenesis and that its cytoplasmic expression in invasive tumours seems to have apoor prognostic role manifested by its association with poor prognostic parameters such as high grade, late stage,lymph node invasion and increased MVD

Antitumoral effects of attenuated Listeria monocytogenes in a genetically engineered mouse model of melanoma

Attenuated Listeria monocytogenes (Lmat-LLO) represents a valuable anticancer vaccine and drug delivery platform. Here we show that in vitro Lmat-LLO causes ROS production and, in turn, apoptotic killing of a wide variety of melanoma cells, irrespectively of their stage, mutational status, sensitivity to BRAF inhibitors or degree of stemness. We also show that, when administered in the therapeutic setting to Braf/Pten genetically engineered mice, Lmat-LLO causes a strong decrease in the size and volume of primary melanoma tumors, as well as a reduction of the metastatic burden. At the molecular level, we confirm that the anti-melanoma activity exerted in vivo by Lmat-LLO depends also on its ability to potentiate the immune response of the organism against the infected tumor. Our data pave the way to the preclinical testing of listeria-based immunotherapeutic strategies against metastatic melanoma, using a genetically engineered mouse rather than xenograft models

The landscape of BRAF transcript and protein variants in human cancer

Background: The BRAF protein kinase is widely studied as a cancer driver and therapeutic target. However, the regulation of its expression is not completely understood. Results: Taking advantage of the RNA-seq data of more than 4800 patients belonging to 9 different cancer types, we show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1, and BRAF-X2) that differ in the last part of their coding sequences, as well as in the length (BRAF-ref: 76 nt; BRAF-X1 and BRAF-X2: up to 7 kb) and in the sequence of their 3'UTRs. The expression levels of BRAF-ref and BRAF-X1/X2 are inversely correlated, while the most prevalent among the three isoforms varies from cancer type to cancer type. In melanoma cells, the X1 isoform is expressed at the highest level in both therapy-naïve cells and cells with acquired resistance to vemurafenib driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. In addition to the BRAF-ref protein, the BRAF-X1 protein (the full length as well as the Δ[3-10] variant) is also translated. The expression levels of the BRAF-ref and BRAF-X1 proteins are similar, and together they account for BRAF functional activities. In contrast, the endogenous BRAF-X2 protein is hard to detect because the C-terminal domain is selectively recognized by the ubiquitin-proteasome pathway and targeted for degradation. Conclusions: By shedding light on the repertoire of BRAF mRNA and protein variants, and on the complex regulation of their expression, our work paves the way to a deeper understanding of a crucially important player in human cancer and to a more informed development of new therapeutic strategies

Circulating microRNAs in metastatic colorectal cancer (mCRC) patients (pts) treated with regorafenib

Abstract not availabl

Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types

Most human pre-mRNAs are spliced into linear molecules that retain the exon order defined by the genomic sequence. By deep sequencing of RNA from a variety of normal and malignant human cells, we found RNA transcripts from many human genes in which the exons were arranged in a non-canonical order. Statistical estimates and biochemical assays provided strong evidence that a substantial fraction of the spliced transcripts from hundreds of genes are circular RNAs. Our results suggest that a non-canonical mode of RNA splicing, resulting in a circular RNA isoform, is a general feature of the gene expression program in human cells