Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer.

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.

Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila.

N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.

CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells.

BACKGROUND: Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The capacity to entrap heterogeneous molecular cargoes and target specific cell populations through EV functionalization promises advancements in biomedical applications. However, the efficiency of the obtained EVs, the contribution of cell-exposed receptors to EV interactions, and the predictability of functional cargo release with potential sharing of high molecular weight recombinant mRNAs are crucial for advancing heterologous EVs in targeted therapy applications. METHODS: In this work, we selected the popular EV marker CD81 as a transmembrane guide for fusion proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. We performed high-content imaging analyses to track EV-cell interactions, including isogenic breast cancer cells with manipulated HER2 expression. We validated the functional cargo delivery of recombinant EVs carrying doxorubicin upon EV-donor cell treatment. Then, we performed an in vivo study using JIMT-1 cells commonly used as HER2-refractory, trastuzumab-resistant model to detect a more than 2000 nt length recombinant mRNA in engrafted tumors. RESULTS: Fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs according to their expression levels in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used to track EV-cell interactions. In time-frames where the global EV distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 exposure in isogenic cells remarkably affected the tropism of heterologous EVs, demonstrating the specificity of antiHER2 EVs representing about 20% of secreted bulk vesicles. The specific interaction strongly correlated with improved cell-killing activity of doxorubicin-EVs in MDA-MB-231 ectopically expressing HER2 and reduced toxicity in SK-BR-3 with a knocked-out HER2 receptor, overcoming the effects of the free drug. Interestingly, the fusion protein-corresponding transcripts present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, improving our sensitivity in detecting penetrant cargoes in tissue biopsies. CONCLUSIONS: This study highlights the quantitative aspects underlying the creation of a platform for secreted heterologous EVs and shows the limits of single receptor-ligand interactions behind EV-cell engagement mechanisms, which now become the pivotal step to predict functional tropism and design new generations of EV-based nanovehicles.

Zebrafish Melanoma-Derived Interstitial EVs Are Carriers of ncRNAs That Induce Inflammation.

Extracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carrier of bioactive molecules is boosted by cells that actively secrete them in biological fluids or in the intercellular space (interstitial EVs, iEVs). Here we have optimised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are around 140 nm in diameter, as determined by nanoparticle tracking and transmission electron microscopy (TEM) analysis. Western blot analysis shows enrichment for CD63 and Alix in the iEV fraction, but not in melanoma cell lysates. Super resolution and confocal microscopy reveal that purified zebrafish iEVs are green fluorescent protein positive (GFP+), indicating that they integrate the oncogene GFP-HRASV12G used to induce melanoma in this model within their vesicular membrane or luminal content. Analysis of RNA-Seq data found 118 non-coding (nc)RNAs differentially distributed between zebrafish melanoma and their iEVs, with only 17 of them being selectively enriched in iEVs. Among these, the RNA components of RNAses P and MRP, which process ribosomal RNA precursors, mitochondrial RNAs, and some mRNAs, were enriched in zebrafish and human melanoma EVs, but not in iEVs extracted from brain tumours. We found that melanoma iEVs induce an inflammatory response when injected in larvae, with increased expression of interferon responsive genes, and this effect is reproduced by MRP- or P-RNAs injected into circulation. This suggests that zebrafish melanoma iEVs are a source of MRP- and P-RNAs that can trigger inflammation in cells of the innate immune system.

Investigation of the link between PROP taste perception and vegetables consumption using FAOSTAT data.

In this work we investigated, in populations located in Central Asia, the relationship between PROP taste perception and vegetables liking and consumption using FAOSTAT dataset. Collected data were analysed using distance matrices, Mantel test and Pearson correlation. Populations showing similar ability in tasting PROP bitterness are more similar as respect to vegetable consumption (r = 0.63, p-value = .05). Moreover, a significant negative correlation was found between the percentage of Non Taster (NT) in different countries and the percentage of vegetable consumption (r = -0.87, p-value = .02), while a significant positive correlation emerged between the percentage of Super Taster (ST) and the percentage of vegetable liking (r = 0.87, p-value = .02). In our work we showed that differences in bitter perception among populations contributes to differences in vegetable liking and vegetable consumption. More in detail, populations with higher percentage of ST consume more vegetables than population where the majority of individuals are NT.

Dp71 Expression in Human Glioblastoma.

BACKGROUND: Dp71 is the most abundant dystrophin (DMD) gene product in the nervous system. Mutation in the Dp71 coding region is associated with cognitive disturbances in Duchenne muscular dystrophy (DMD) patients, but the function of dystrophin Dp71 in tumor progression remains to be established. This study investigated Dp71 expression in glioblastoma, the most common and aggressive primary tumor of the central nervous system (CNS). METHODS: Dp71 expression was analyzed by immunofluorescence, immunohistochemistry, RT-PCR, and immunoblotting in glioblastoma cell lines and cells isolated from human glioblastoma multiforme (GBM) bioptic specimens. RESULTS: Dp71 isoform was expressed in normal human astrocytes (NHA) cell lines and decreased in glioblastoma cell lines and cells isolated from human glioblastoma multiforme bioptic specimens. Moreover, Dp71 was localized in the nucleus in normal cells, while it was localized into the cytoplasm of glioblastoma cells organized in clusters. We have shown, by double labeling, that Dp71 colocalizes with lamin B in normal astrocytes cells, confirming the roles of Dp71 and lamin B in maintaining nuclear architecture. Finally, we demonstrated that decreased Dp71 protein in cells isolated from human bioptic specimens was inversely correlated with the Ki-67 tumor proliferative index. CONCLUSION: A decreased Dp71 expression is associated with cancer proliferation and poor prognosis in glioblastoma.

The DNA-repair protein APE1 participates with hnRNPA2B1 to motif-enriched and prognostic miRNA secretion.

The base excision repair (BER) Apurinic/apyrimidinic endonuclease 1 (APE1) enzyme is endowed with several non-repair activities including miRNAs processing. APE1 is overexpressed in many cancers but its causal role in the tumorigenic processes is largely unknown. We recently described that APE1 can be actively secreted by mammalian cells through exosomes. However, APE1 role in EVs or exosomes is still unknown, especially regarding a putative regulatory function on vesicular small non-coding RNAs. Through dedicated transcriptomic analysis on cellular and vesicular small RNAs of different APE1-depleted cancer cell lines, we found that miRNAs loading into EVs is a regulated process, dependent on APE1, distinctly conveying RNA subsets into vesicles. We identified APE1-dependent secreted miRNAs characterized by enriched sequence motifs and possible binding sites for APE1. In 33 out of 34 APE1-dependent-miRNA precursors, we surprisingly found EXO-motifs and proved that APE1 cooperates with hnRNPA2B1 for the EV-sorting of a subset of miRNAs, including miR-1246, through direct binding to GGAG stretches. Using TCGA-datasets, we showed that these miRNAs identify a signature with high prognostic significance in cancer. In summary, we provided evidence that the ubiquitous DNA-repair enzyme APE1 is part of the EV protein cargo with a novel post-transcriptional role for this ubiquitous DNA-repair enzyme that could explain its role in cancer progression. These findings could open new translational perspectives in cancer biology.

Integrating extracellular vesicle and circulating cell-free DNA analysis using a single plasma aliquot improves the detection of HER2 positivity in breast cancer patients.

Multi-analyte liquid biopsies represent an emerging opportunity for non-invasive cancer assessment. We developed ONCE (ONe Aliquot for Circulating Elements), an approach for the isolation of extracellular vesicles (EV) and cell-free DNA (cfDNA) from a single aliquot of blood. We assessed ONCE performance to classify HER2-positive early-stage breast cancer (BrCa) patients by combining EV-associated RNA (EV-RNA) and cfDNA signals on n=64 healthy donors (HD) and non-metastatic BrCa patients. Specifically, we isolated EV-enriched samples by a charge-based (CB) method and investigated EV-RNA and cfDNA by next-generation sequencing (NGS) and by digital droplet PCR (ddPCR). Sequencing of cfDNA and EV-RNA from HER2- and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi-analyte liquid biopsy prediction performance was comparable to tissue-based sequencing results from TCGA. Also, imaging flow cytometry analysis revealed HER2 protein on the surface of EV isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EV as companion analyte to cfDNA. This data confirms the relevance of combining cfDNA and EV-RNA for HER2 cancer assessment and supports the ONCE as a valuable tool for multi-analytes liquid biopsies' clinical implementation.

Non-Syndromic Autosomal Dominant Hearing Loss: The First Italian Family Carrying a Mutation in the NCOA3 Gene.

Hearing loss (HL) is the most frequent sensory disorder, affecting about 1-3 per 1000 live births, with more than half of the cases attributable to genetic causes. Despite the fact that many HL causative genes have already been identified, current genetic tests fail to provide a diagnosis for about 40% of the patients, suggesting that other causes still need to be discovered. Here, we describe a four-generation Italian family affected by autosomal dominant non-syndromic hearing loss (ADNSHL), in which exome sequencing revealed a likely pathogenic variant in NCOA3 (NM_181659.3, c.2909G>C, p.(Gly970Ala)), a gene recently described as a novel candidate for ADNSHL in a Brazilian family. A comparison between the two families highlighted a series of similarities: both the identified variants are missense, localized in exon 15 of the NCOA3 gene and lead to a similar clinical phenotype, with non-syndromic, sensorineural, bilateral, moderate to profound hearing loss, with a variable age of onset. Our findings (i.e., the identification of the second family reported globally with HL caused by a variant in NCOA3) further support the involvement of NCOA3 in the etiopathogenesis of ADNSHL, which should, thus, be considered as a new gene for autosomal dominant non-syndromic hearing loss.

Unveiling mutational dynamics in non-small cell lung cancer patients by quantitative EGFR profiling in vesicular RNA.

The mutational status of the epidermal growth factor receptor (EGFR) guides the stratification of non-small cell lung cancer (NSCLC) patients for treatment with tyrosine kinase inhibitors (TKIs). A liquid biopsy test on cell-free DNA is recommended as a clinical decision-supporting tool, although it has limited sensitivity. Here, we comparatively investigated the extracellular vesicle (EV)-RNA as an independent source for multidimensional and longitudinal EGFR profiling in a cohort of 27 NSCLC patients. We introduced and validated a new rapid, highly specific EV-RNA test with wild-type (WT) and mutant-sensitive probes (E746-A750del, L858R, and T790M). We included a cohort of 20 NSCLC patients with EGFR WT tumor tissues and systematically performed molecular EV-RNA and circulating tumor DNA analyses with clinical data statistics and biophysical profiles of EVs. At the single-patient level, we detected variegated tumor heterogeneity dynamics supported by combinations of driver EGFR mutations. EV-RNA-based mutation analysis showed an unprecedented sensitivity of over 90%. The resistance-associated mutation T790M frequently pre-existed at baseline with a gained EV-transcript copy number at progression, while the general mutational burden was mostly decreasing during the intermediate follow-up. The biophysical profile of EVs and the quantitative assessment of T790M revealed an association with tumor size determined by the sum of the longest diameters in target lesions. Vesicular RNA provides a validated tool suitable for use in clinical practice to investigate the dynamics of common driver EGFR mutations in NSCLC patients receiving TKIs.

Inhibition of mitochondrial translation suppresses glioblastoma stem cell growth.

Glioblastoma stem cells (GSCs) resist current glioblastoma (GBM) therapies. GSCs rely highly on oxidative phosphorylation (OXPHOS), whose function requires mitochondrial translation. Here we explore the therapeutic potential of targeting mitochondrial translation and report the results of high-content screening with putative blockers of mitochondrial ribosomes. We identify the bacterial antibiotic quinupristin/dalfopristin (Q/D) as an effective suppressor of GSC growth. Q/D also decreases the clonogenicity of GSCs in vitro, consequently dysregulating the cell cycle and inducing apoptosis. Cryoelectron microscopy (cryo-EM) reveals that Q/D binds to the large mitoribosomal subunit, inhibiting mitochondrial protein synthesis and functionally dysregulating OXPHOS complexes. These data suggest that targeting mitochondrial translation could be explored to therapeutically suppress GSC growth in GBM and that Q/D could potentially be repurposed for cancer treatment.

RNA packaging into extracellular vesicles: An orchestra of RNA-binding proteins?

Extracellular vesicles (EVs) are heterogeneous membranous particles released from the cells through different biogenetic and secretory mechanisms. We now conceive EVs as shuttles mediating cellular communication, carrying a variety of molecules resulting from intracellular homeostatic mechanisms. The RNA is a widely detected cargo and, impressively, a recognized functional intermediate that elects EVs as modulators of cancer cell phenotypes, determinants of disease spreading, cell surrogates in regenerative medicine, and a source for non-invasive molecular diagnostics. The mechanistic elucidation of the intracellular events responsible for the engagement of RNA into EVs will significantly improve the comprehension and possibly the prediction of EV "quality" in association with cell physiology. Interestingly, the application of multidisciplinary approaches, including biochemical as well as cell-based and computational strategies, is increasingly revealing an active RNA-packaging process implicating RNA-binding proteins (RBPs) in the sorting of coding and non-coding RNAs. In this review, we provide a comprehensive view of RBPs recently emerging as part of the EV biology, considering the scenarios where: (i) individual RBPs were detected in EVs along with their RNA substrates, (ii) RBPs were detected in EVs with inferred RNA targets, and (iii) EV-transcripts were found to harbour sequence motifs mirroring the activity of RBPs. Proteins so far identified are members of the hnRNP family (hnRNPA2B1, hnRNPC1, hnRNPG, hnRNPH1, hnRNPK, and hnRNPQ), as well as YBX1, HuR, AGO2, IGF2BP1, MEX3C, ANXA2, ALIX, NCL, FUS, TDP-43, MVP, LIN28, SRP9/14, QKI, and TERT. We describe the RBPs based on protein domain features, current knowledge on the association with human diseases, recognition of RNA consensus motifs, and the need to clarify the functional significance in different cellular contexts. We also summarize data on previously identified RBP inhibitor small molecules that could also be introduced in EV research as potential modulators of vesicular RNA sorting.

Rapid Nickel-based Isolation of Extracellular Vesicles from Different Biological Fluids.

Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers. We recently demonstrated that nickel-based isolation (NBI) is a valuable method for fast, efficient, and easy recovery of heterogeneous EVs from biological fluids. NBI exploits nickel cations to capture negatively charged vesicles. Then, a mix of balanced chelating agents elutes EVs while preserving their integrity and stability in solution. Here, we describe steps and quality controls to functionalize a matrix of agarose beads, obtain an efficient elution of EVs, and extract nucleic acids carried by them. We demonstrate the versatility of NBI method in isolating EVs from media of primary mouse astrocytes, from human blood, urine, and saliva processed in parallel, as well as outer membrane vesicles (OMVs) from cultured Gram-negative bacteria.

Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood.

BACKGROUND: Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice. METHODS: We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility. FINDINGS: From plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 1010/ml) and large EV (~560 nm of mean diameter; ~5 × 108/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies. INTERPRETATION: We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. FUND: Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur).

Frequent NRG1 fusions in Caucasian pulmonary mucinous adenocarcinoma predicted by Phospho-ErbB3 expression.

NRG1 fusions were recently reported as a new molecular feature of Invasive Mucinous Adenocarcinoma (IMA) of the lung. The NRG1 chimeric ligand acts as a strong inductor of phosphorylation and tyrosine kinase activity of the ErbB2/ErbB3 heterodimer, thus enhancing the PI3K-AKT/MAPK pathways. The NRG1 fusions were widely investigated in Asian IMA cohorts, whereas just anecdotal information are available about the occurrence of NRG1 fusions in IMA Caucasian population. Here we firstly explored a large Caucasian cohort of 51 IMAs and 34 non-IMA cases for the occurrence of NRG1 rearrangements by fluorescent in situ hybridization (FISH) and RNA target sequencing. FISH results were correlated to the immunohistochemical expression of phosphorylated-ErbB3 (pErbB3) receptor and the mutational status of KRAS, EGFR and ALK genes. The NRG1 rearrangements were detected in 31% IMAs and 3% non-IMAs and the CD74-NRG1 fusion transcript variant was characterized in 4 NRG1-positive IMAs. Moreover, pErbB3 expression was found to be strictly associated to the mucinous pattern (p = 0.012, Chi-square test) and all IMA cases showing aberrant expression of pErbB3 demonstrated NRG1 rearrangements. No significant correlation between NRG1 rearrangements and EGFR, KRAS or ALK mutations respectively, was observed. We report for the first time that NRG1 fusions are driver alterations clearly associated with mucinous lung adenocarcinoma subtype of Caucasian patients and not exclusive of Asiatic population. pErbB3 immunostaining may represent a strong predictor of NRG1 fusions, pointing out the detection of pErbB3 by IHC as a rapid and effective pre-screening method to select the NRG1-positive patients.