Biological relevance and therapeutic potential of G-quadruplex structures in the human noncoding transcriptome.

Noncoding RNAs are functional transcripts that are not translated into proteins. They represent the largest portion of the human transcriptome and have been shown to regulate gene expression networks in both physiological and pathological cell conditions. Research in this field has made remarkable progress in the comprehension of how aberrations in noncoding RNA drive relevant disease-associated phenotypes; however, the biological role and mechanism of action of several noncoding RNAs still need full understanding. Besides fulfilling its function through sequence-based mechanisms, RNA can form complex secondary and tertiary structures which allow non-canonical interactions with proteins and/or other nucleic acids. In this context, the presence of G-quadruplexes in microRNAs and long noncoding RNAs is increasingly being reported. This evidence suggests a role for RNA G-quadruplexes in controlling microRNA biogenesis and mediating noncoding RNA interaction with biological partners, thus ultimately regulating gene expression. Here, we review the state of the art of G-quadruplexes in the noncoding transcriptome, with their structural and functional characterization. In light of the existence and further possible development of G-quadruplex binders that modulate G-quadruplex conformation and protein interactions, we also discuss the therapeutic potential of G-quadruplexes as targets to interfere with disease-associated noncoding RNAs.

Potential of the Stromal Matricellular Protein Periostin as a Biomarker to Improve Risk Assessment in Prostate Cancer.

Prostate cancer (PCa) ranges from indolent to aggressive tumors that may rapidly progress and metastasize. The switch to aggressive PCa is fostered by reactive stroma infiltrating tumor foci. Therefore, reactive stroma-based biomarkers may potentially improve the early detection of aggressive PCa, ameliorating disease classification. Gene expression profiles of PCa reactive fibroblasts highlighted the up-regulation of genes related to stroma deposition, including periostin and sparc. Here, the potential of periostin as a stromal biomarker has been investigated on PCa prostatectomies by immunohistochemistry. Moreover, circulating levels of periostin and sparc have been assessed in a low-risk PCa patient cohort enrolled in active surveillance (AS) by ELISA. We found that periostin is mainly expressed in the peritumoral stroma of prostatectomies, and its stromal expression correlates with PCa grade and aggressive disease features, such as the cribriform growth. Moreover, stromal periostin staining is associated with a shorter biochemical recurrence-free survival of PCa patients. Interestingly, the integration of periostin and sparc circulating levels into a model based on standard clinico-pathological variables improves its performance in predicting disease reclassification of AS patients. In this study, we provide the first evidence that circulating molecular biomarkers of PCa stroma may refine risk assessment and predict the reclassification of AS patients.

microRNAs as players and signals in the metastatic cascade: Implications for the development of novel anti-metastatic therapies.

microRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. Increasing evidence emerging from human tumor preclinical models clearly indicates that specific miRNAs, collectively termed "metastamirs," play a functional role in different steps of the metastatic cascade, by exerting either pro- or anti-metastatic functions, and behave as signaling mediators to enable tumor cell to colonize a specific organ. miRNAs also actively participate in the proficient interaction of cancer cells with tumor microenvironment, either at the primary or at the metastatic site. Circulating miRNAs, released by multiple cell types, following binding to proteins or encapsulation in extracellular vesicles, play a main role in this cross-talk by acting as transferrable messages. The documented involvement of specific miRNAs in the dissemination process has aroused interest in the development of miRNA-based strategies for the treatment of metastasis. Preclinical research carried out in tumor experimental models, using both miRNA replacement and miRNA inhibitory approaches, is encouraging towards translating miRNA-based strategies into human cancer therapy, based on the observed therapeutic activity in the absence of main toxicity. However, to accelerate their adoption in the clinic, further improvements in terms of efficacy and targeted delivery to the tumor are still necessary.

Dissecting the role of microRNAs in prostate cancer metastasis: implications for the design of novel therapeutic approaches.

Metastatic prostate cancer is a lethal disease that remains incurable despite the recent approval of new drugs, thus making the development of alternative treatment approaches urgently needed. A more precise understanding of the molecular mechanisms underlying prostate cancer dissemination could lead to the identification of novel therapeutic targets for the design of efficient anti-metastatic strategies. MicroRNA (miRNAs) are endogenous, small non-coding RNA molecules acting as key regulators of gene expression at post-transcriptional level. It has been clearly established that altered miRNA expression is a common hallmark of cancer. In addition, emerging evidence suggests their direct involvement in the metastatic cascade. In this review, we present a comprehensive overview of the data generated in experimental tumor models indicating that specific miRNAs may impinge on the different stages of prostate cancer metastasis, including (i) the regulation of epithelial-to-mesenchymal transition and cell migration/invasion, (ii) the interplay between cancer cells and the surrounding stroma, (iii) the control of angiogenesis, (iv) the regulation of anoikis, and (v) the colonization of distant organs. Moreover, we show preliminary evidence of the clinical relevance of some of these miRNAs, in terms of association with tumor aggressiveness/dissemination and clinical outcome, as emerged from translation studies carried out in prostate cancer patient cohorts. We also discuss the potential and the current limitations of manipulating metastasis-related miRNAs, by mimicking or inhibiting them, as a strategy for the development of novel therapeutic approaches for the advanced disease.

Complexity in the tumour microenvironment: Cancer associated fibroblast gene expression patterns identify both common and unique features of tumour-stroma crosstalk across cancer types.

Cancer is a complex disease, driven by the accumulation of several somatic aberrations but fostered by a two-way interaction between tumour cells and the surrounding microenvironment. Cancer associated fibroblasts (CAFs) represent one of the major players in tumour-stroma crosstalk. Recent in vitro and in vivo studies, often conducted by employing high throughput approaches, have started unravelling the key pathways involved in their functional effects. This review focus on open challenges in the study of CAF properties and function, highlighting at the same time the existence of common mechanisms as well as peculiarities in different cancer types (breast, prostate and lung cancer). Although still limited by current experimental models, which are unable to deal with the full level of complexity of the tumour microenvironment, a better understanding of these mechanisms may enable the identification of new biomarkers and therapeutic targets, to improve current strategies for cancer diagnosis and treatment.

PKC-alpha modulation by miR-483-3p in platinum-resistant ovarian carcinoma cells.

The occurrence of drug resistance limits the efficacy of platinum compounds in the cure of ovarian carcinoma. Since microRNAs (miRNAs) may contribute to this phenomenon by regulating different aspects of tumor cell response, the aim of this study was to exploit the analysis of expression of miRNAs in platinum sensitive/resistant cells in an attempt to identify potential regulators of drug response. MiR-483-3p, which may participate in apoptosis and cell proliferation regulation, was found up-regulated in 4 platinum resistant variants, particularly in the IGROV-1/Pt1 subline, versus parental cells. Transfection of a synthetic precursor of miR-483-3p in IGROV-1 parental cells elicited a marked up-regulation of the miRNA levels. Growth-inhibition and colony-forming assays indicated that miR-483-3p over-expression reduced cell growth and conferred mild levels of cisplatin resistance in IGROV-1 cells, by interference with their proliferative potential. Predicted targets of miR-483-3p included PRKCA (encoding PKC-alpha), previously reported to be associated to platinum-resistance in ovarian carcinoma. We found that miR-483-3p directly targeted PRKCA in IGROV-1 cells. In keeping with this finding, cisplatin sensitivity of IGROV-1 cells decreased upon molecular/pharmacological inhibition of PKC-alpha. Overall, our results suggest that overexpression of miR-483-3p by ovarian carcinoma platinum-resistant cells may interfere with their proliferation, thus protecting them from DNA damage induced by platinum compounds and ultimately representing a drug-resistance mechanism. The impairment of cell growth may account for low levels of drug resistance that could be relevant in the clinical setting.

Mesenchymal to amoeboid transition is associated with stem-like features of melanoma cells.

BACKGROUND: Cellular plasticity confers cancer cells the ability to adapt to microenvironmental changes, a fundamental requirement for tumour progression and metastasis. The epithelial to mesenchymal transition (EMT) is a transcriptional programme associated with increased cell motility and stemness. Besides EMT, the mesenchymal to amoeboid transition (MAT) has been described during tumour progression but to date, little is known about its transcriptional control and involvement in stemness. The aim of this manuscript is to investigate (i) the transcriptional profile associated with the MAT programme and (ii) to study whether MAT acquisition in melanoma cancer cells correlates with clonogenic potential to promote tumour growth. RESULTS: By using a multidisciplinary approach, we identified four different treatments able to induce MAT in melanoma cells: EphA2 overexpression, Rac1 functional inhibition using its RacN17 dominant negative mutant, stimulation with Ilomastat or treatment with the RhoA activator Calpeptin. First, gene expression profiling identified the transcriptional pathways associated with MAT, independently of the stimulus that induces the MAT programme. Notably, gene sets associated with the repression of mesenchymal traits, decrease in the secretion of extracellular matrix components as well as increase of cellular stemness positively correlate with MAT. Second, the link between MAT and stemness has been investigated in vitro by analysing stemness markers and clonogenic potential of melanoma cells undergoing MAT. Finally, the link between MAT inducing treatments and tumour initiating capability has been validated in vivo. CONCLUSION: Taken together, our results demonstrate that MAT programme in melanoma is characterised by increased stemness and clonogenic features of cancer cells, thus sustaining tumour progression. Furthermore, these data suggest that stemness is not an exclusive feature of cells undergoing EMT, but more generally is associated with an increase in cellular plasticity of cancer cells.

Cleavage and polyadenylation machinery as a novel targetable vulnerability for human cancer.

The role of alternative polyadenylation of mRNA in sustaining aggressive features of tumors is quite well established, as it is responsible for the 3'UTR shortening of oncogenes and subsequent relief from miRNA-mediated repression observed in cancer cells. However, the information regarding the vulnerability of cancer cells to the inhibition of cleavage and polyadenylation (CPA) machinery is very scattered. Only few recent reports show the antitumor activity of pharmacological inhibitors of CPSF3, one among CPA factors. More in general, the fact that deregulated CPA can be seen as a new hallmark of cancer and as a potential reservoir of novel therapeutic targets has never been formalized. Here, to extend our view on the potential of CPA inhibition (CPAi) approaches as anticancer therapies, we systematically tested the fitness of about one thousand cell lines of different cancer types upon depletion of all known CPA factors by interrogating genome-scale CRISPR and RNAi dependency maps of the DepMap project. Our analysis confirmed core and accessory CPA factors as novel vulnerabilities for human cancer, thus highlighting the potential of CPAi as anticancer therapy. Among all, CPSF1 appeared as a promising actionable candidate for drug development, as it showed low dependency scores pancancer and particularly in highly proliferating cells. In a personalized medicine perspective, the observed differential vulnerability of cancer cell lines to selected CPA factors may be used to build up signatures to predict response of individual human tumors to CPAi approaches.

Linc00941 fuels ribogenesis and protein synthesis by supporting robust cMYC translation in malignant pleural mesothelioma.

Malignant pleural mesothelioma is a rare and lethal cancer caused by exposure to asbestos. The highly inflammatory environment caused by fibers accumulation forces cells to undergo profound adaptation to gain survival advantages. Prioritizing the synthesis of essential transcripts is an efficient mechanism coordinated by multiple molecules, including long non-coding RNAs. Enhancing the knowledge about these mechanisms is an essential weapon in combating mesothelioma. Linc00941 correlates to bad prognosis in various cancers, but it is reported to partake in distinct and apparently irreconcilable processes. In this work, we report that linc00941 supports the survival and aggressiveness of mesothelioma cells by influencing protein synthesis and ribosome biogenesis. Linc00941 binds to the translation initiation factor eIF4G, promoting the selective protein synthesis of cMYC, which, in turn, enhances the expression of key genes involved in translation. We analyzed a retrospective cohort of 97 mesothelioma patients' samples from our institution, revealing that linc00941 expression strongly correlates with reduced survival probability. This discovery clarifies linc00941's role in mesothelioma and proposes a unified mechanism of action for this lncRNA involving the selective translation of essential oncogenes, reconciling the discrepancies about its function.

The pancancer overexpressed NFYC Antisense 1 controls cell cycle mitotic progression through in cis and in trans modes of action.

Antisense RNAs (asRNAs) represent an underappreciated yet crucial layer of gene expression regulation. Generally thought to modulate their sense genes in cis through sequence complementarity or their act of transcription, asRNAs can also regulate different molecular targets in trans, in the nucleus or in the cytoplasm. Here, we performed an in-depth molecular characterization of NFYC Antisense 1 (NFYC-AS1), the asRNA transcribed head-to-head to NFYC subunit of the proliferation-associated NF-Y transcription factor. Our results show that NFYC-AS1 is a prevalently nuclear asRNA peaking early in the cell cycle. Comparative genomics suggests a narrow phylogenetic distribution, with a probable origin in the common ancestor of mammalian lineages. NFYC-AS1 is overexpressed pancancer, preferentially in association with RB1 mutations. Knockdown of NFYC-AS1 by antisense oligonucleotides impairs cell growth in lung squamous cell carcinoma and small cell lung cancer cells, a phenotype recapitulated by CRISPR/Cas9-deletion of its transcription start site. Surprisingly, expression of the sense gene is affected only when endogenous transcription of NFYC-AS1 is manipulated. This suggests that regulation of cell proliferation is at least in part independent of the in cis transcription-mediated effect on NFYC and is possibly exerted by RNA-dependent in trans effects converging on the regulation of G2/M cell cycle phase genes. Accordingly, NFYC-AS1-depleted cells are stuck in mitosis, indicating defects in mitotic progression. Overall, NFYC-AS1 emerged as a cell cycle-regulating asRNA with dual action, holding therapeutic potential in different cancer types, including the very aggressive RB1-mutated tumors.

Repositioning of antiarrhythmics for prostate cancer treatment: a novel strategy to reprogram cancer-associated fibroblasts towards a tumor-suppressive phenotype.

BACKGROUND: Cancer-associated fibroblasts (CAFs) play a significant role in fueling prostate cancer (PCa) progression by interacting with tumor cells. A previous gene expression analysis revealed that CAFs up-regulate genes coding for voltage-gated cation channels, as compared to normal prostate fibroblasts (NPFs). In this study, we explored the impact of antiarrhythmic drugs, known cation channel inhibitors, on the activated state of CAFs and their interaction with PCa cells. METHODS: The effect of antiarrhythmic treatment on CAF activated phenotype was assessed in terms of cell morphology and fibroblast activation markers. CAF contractility and migration were evaluated by 3D gel collagen contraction and scratch assays, respectively. The ability of antiarrhythmics to impair CAF-PCa cell interplay was investigated in CAF-PCa cell co-cultures by assessing tumor cell growth and expression of epithelial-to-mesenchymal transition (EMT) markers. The effect on in vivo tumor growth was assessed by subcutaneously injecting PCa cells in SCID mice and intratumorally administering the medium of antiarrhythmic-treated CAFs or in co-injection experiments, where antiarrhythmic-treated CAFs were co-injected with PCa cells. RESULTS: Activated fibroblasts show increased membrane conductance for potassium, sodium and calcium, consistently with the mRNA and protein content analysis. Antiarrhythmics modulate the expression of fibroblast activation markers. Although to a variable extent, these drugs also reduce CAF motility and hinder their ability to remodel the extracellular matrix, for example by reducing MMP-2 release. Furthermore, conditioned medium and co-culture experiments showed that antiarrhythmics can, at least in part, reverse the protumor effects exerted by CAFs on PCa cell growth and plasticity, both in androgen-sensitive and castration-resistant cell lines. Consistently, the transcriptome of antiarrhythmic-treated CAFs resembles that of tumor-suppressive NPFs. In vivo experiments confirmed that the conditioned medium or the direct coinjection of antiarrhythmic-treated CAFs reduced the tumor growth rate of PCa xenografts. CONCLUSIONS: Collectively, such data suggest a new therapeutic strategy for PCa based on the repositioning of antiarrhythmic drugs with the aim of normalizing CAF phenotype and creating a less permissive tumor microenvironment.

Study of lncRNAs in Pediatric Neurological Diseases: Methods, Analysis of the State-of-Art and Possible Therapeutic Implications.

Long non-coding RNAs (lncRNAs) have emerged as crucial regulators in various cellular processes, and their roles in pediatric neurological diseases are increasingly being explored. This review provides an overview of lncRNA implications in the central nervous system, both in its physiological state and when a pathological condition is present. We describe the role of lncRNAs in neural development, highlighting their significance in processes such as neural stem cell proliferation, differentiation, and synaptogenesis. Dysregulation of specific lncRNAs is associated with multiple pediatric neurological diseases, such as neurodevelopmental or neurodegenerative disorders and brain tumors. The collected evidence indicates that there is a need for further research to uncover the full spectrum of lncRNA involvement in pediatric neurological diseases and brain tumors. While challenges exist, ongoing advancements in technology and our understanding of lncRNA biology offer hope for future breakthroughs in the field of pediatric neurology, leveraging lncRNAs as potential therapeutic targets and biomarkers.

Computational analysis of five neurodegenerative diseases reveals shared and specific genetic loci.

Neurodegenerative diseases (ND) are heterogeneous disorders of the central nervous system that share a chronic and selective process of neuronal cell death. A computational approach to investigate shared genetic and specific loci was applied to 5 different ND: Amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Multiple sclerosis (MS), and Lewy body dementia (LBD). The datasets were analyzed separately, and then we compared the obtained results. For this purpose, we applied a genetic correlation analysis to genome-wide association datasets and revealed different genetic correlations with several human traits and diseases. In addition, a clumping analysis was carried out to identify SNPs genetically associated with each disease. We found 27 SNPs in AD, 6 SNPs in ALS, 10 SNPs in PD, 17 SNPs in MS, and 3 SNPs in LBD. Most of them are located in non-coding regions, with the exception of 5 SNPs on which a protein structure and stability prediction was performed to verify their impact on disease. Furthermore, an analysis of the differentially expressed miRNAs of the 5 examined pathologies was performed to reveal regulatory mechanisms that could involve genes associated with selected SNPs. In conclusion, the results obtained constitute an important step toward the discovery of diagnostic biomarkers and a better understanding of the diseases.

Navigating the Multiverse of Antisense RNAs: The Transcription- and RNA-Dependent Dimension.

Evidence accumulated over the past decades shows that the number of identified antisense transcripts is continuously increasing, promoting them from transcriptional noise to real genes with specific functions. Indeed, recent studies have begun to unravel the complexity of the antisense RNA (asRNA) world, starting from the multidimensional mechanisms that they can exert in physiological and pathological conditions. In this review, we discuss the multiverse of the molecular functions of asRNAs, describing their action through transcription-dependent and RNA-dependent mechanisms. Then, we report the workflow and methodologies to study and functionally characterize single asRNA candidates.

MIR205HG/LEADR Long Noncoding RNA Binds to Primed Proximal Regulatory Regions in Prostate Basal Cells Through a Triplex- and Alu-Mediated Mechanism.

Aside serving as host gene for miR-205, MIR205HG transcribes for a chromatin-associated long noncoding RNA (lncRNA) able to restrain the differentiation of prostate basal cells, thus being reannotated as LEADR (Long Epithelial Alu-interacting Differentiation-related RNA). We previously showed the presence of Alu sequences in the promoters of genes modulated upon MIR205HG/LEADR manipulation. Notably, an Alu element also spans the first and second exons of MIR205HG/LEADR, suggesting its possible involvement in target selection/binding. Here, we performed ChIRP-seq to map MIR205HG/LEADR chromatin occupancy at genome-wide level in prostate basal cells. Our results confirmed preferential binding to regions proximal to gene transcription start site (TSS). Moreover, enrichment of triplex-forming sequences was found upstream of MIR205HG/LEADR-bound genes, peaking at -1,500/-500 bp from TSS. Triplexes formed with one or two putative DNA binding sites within MIR205HG/LEADR sequence, located just upstream of the Alu element. Notably, triplex-forming regions of bound genes were themselves enriched in Alu elements. These data suggest, from one side, that triplex formation may be the prevalent mechanism by which MIR205HG/LEADR selects and physically interacts with target DNA, from the other that direct or protein-mediated Alu (RNA)/Alu (DNA) interaction may represent a further functional requirement. We also found that triplex-forming regions were enriched in specific histone modifications, including H3K4me1 in the absence of H3K27ac, H3K4me3 and H3K27me3, indicating that in prostate basal cells MIR205HG/LEADR may preferentially bind to primed proximal regulatory elements. This may underscore the need for basal cells to keep MIR205HG/LEADR target genes repressed but, at the same time, responsive to differentiation cues.

miR-550a-3p is a prognostic biomarker and exerts tumor-suppressive functions by targeting HSP90AA1 in diffuse malignant peritoneal mesothelioma.

Diffuse malignant peritoneal mesothelioma (DMPM) is a rare and rapidly lethal tumor, poorly responsive to conventional treatments. In this regards, the identification of molecular alterations underlying DMPM onset and progression might be exploited to develop novel therapeutic strategies. Here, we focused on miR-550a-3p, which we found downregulated in 45 DMPM clinical samples compared to normal tissues and whose expression levels were associated with patient outcome. Through a gain-of-function approach using miRNA mimics in 3 DMPM cell lines, we demonstrated the tumor-suppressive role of miR-550a-3p. Specifically, miRNA ectopic expression impaired cell proliferation and invasiveness, enhanced the apoptotic response, and reduced the growth of DMPM xenografts in mice. Antiproliferative and proapoptotic effects were also observed in prostate and ovarian cancer cell lines following miR-550a-3p ectopic expression. miR-550a-3p effects were mediated, at least in part, by the direct inhibition of HSP90AA1 and the consequent downregulation of its target proteins, the levels of which were rescued upon disruption of miRNA-HSP90AA1 mRNA pairing, partially abrogating miR-550a-3p-induced cellular effects. Our results show that miR-550a-3p reconstitution affects several tumor traits, thus suggesting this approach as a potential novel therapeutic strategy for DMPM.

Noncoding RNAs in the Interplay between Tumor Cells and Cancer-Associated Fibroblasts: Signals to Catch and Targets to Hit.

Cancer development and progression are not solely cell-autonomous and genetically driven processes. Dynamic interaction of cancer cells with the surrounding microenvironment, intended as the chemical/physical conditions as well as the mixture of non-neoplastic cells of the tumor niche, drive epigenetic changes that are pivotal for the acquisition of malignant traits. Cancer-associated fibroblasts (CAF), namely fibroblasts that, corrupted by cancer cells, acquire a myofibroblast-like reactive phenotype, are able to sustain tumor features by the secretion of soluble paracrine signals and the delivery extracellular vesicles. In such diabolic liaison, a major role has been ascribed to noncoding RNAs. Defined as RNAs that are functional though not being translated into proteins, noncoding RNAs predominantly act as regulators of gene expression at both the transcriptional and post-transcriptional levels. In this review, we summarize the current knowledge of microRNAs and long noncoding RNAs that act intracellularly in either CAFs or cancer cells to sustain tumor-stroma interplay. We also report on the major role of extracellular noncoding RNAs that are bidirectionally transferred between either cell type. Upon presenting a comprehensive view of the existing literature, we provide our critical opinion regarding the possible clinical utility of tumor-stroma related noncoding RNAs as therapeutic target/tools or prognostic/predictive biomarkers.

Prediction of Grade Reclassification of Prostate Cancer Patients on Active Surveillance through the Combination of a Three-miRNA Signature and Selected Clinical Variables.

Active surveillance (AS) has evolved as a strategy alternative to radical treatments for very low risk and low-risk prostate cancer (PCa). However, current criteria for selecting AS patients are still suboptimal. Here, we performed an unprecedented analysis of the circulating miRNome to investigate whether specific miRNAs associated with disease reclassification can provide risk refinement to standard clinicopathological features for improving patient selection. The global miRNA expression profiles were assessed in plasma samples prospectively collected at baseline from 386 patients on AS included in three independent mono-institutional cohorts (training, testing and validation sets). A three-miRNA signature (miR-511-5p, miR-598-3p and miR-199a-5p) was found to predict reclassification in all patient cohorts (training set: AUC 0.74, 95% CI 0.60-0.87, testing set: AUC 0.65, 95% CI 0.51-0.80, validation set: AUC 0.68, 95% CI 0.56-0.80). Importantly, the addition of the three-miRNA signature improved the performance of the clinical model including clinicopathological variables only (AUC 0.70, 95% CI 0.61-0.78 vs. 0.76, 95% CI 0.68-0.84). Overall, we trained, tested and validated a three-miRNA signature which, combined with selected clinicopathological variables, may represent a promising biomarker to improve on currently available clinicopathological risk stratification tools for a better selection of truly indolent PCa patients suitable for AS.

miR-1227 Targets SEC23A to Regulate the Shedding of Large Extracellular Vesicles.

Cancer cells shed a heterogenous mixture of extracellular vesicles (EVs), differing in both size and composition, which likely influence physiological processes in different manners. However, how cells differentially control the shedding of these EV populations is poorly understood. Here, we show that miR-1227, which is enriched in prostate cancer EVs, compared to the cell of origin, but not in EVs derived from prostate benign epithelial cells, induces the shedding of large EVs (such as large oncosomes), while inhibiting the shedding of small EVs (such as exosomes). RNA sequencing from cells stably expressing miR-1227, a modified RISCTRAP assay that stabilizes and purifies mRNA-miR-1227 complexes for RNA sequencing, and in silico target prediction tools were used to identify miR-1227 targets that may mediate this alteration in EV shedding. The COPII vesicle protein SEC23A emerged and was validated by qPCR, WBlot, and luciferase assays as a direct target of miR-1227. The inhibition of SEC23A was sufficient to induce the shedding of large EVs. These results identify a novel mechanism of EV shedding, by which the inhibition of SEC23A by miR-1227 induces a shift in EV shedding, favoring the shedding of large EV over small EV.

Targeting the telosome: therapeutic implications.

Since telomere integrity is required to guarantee the unlimited replicative potential of cancer cells, telomerase, the enzyme responsible for telomere length maintenance in most human tumors, and lately also telomeres themselves have become extremely attractive targets for new anticancer interventions. At the current status of knowledge, it is still not possible to define the best therapeutic target between telomerase and telomeres. It is noteworthy that interfering with telomeres, through direct targeting of telomeric DNA or proteins involved in the telosome complex, could negatively affect the proliferative potential not only of tumors expressing telomerase activity but also of those that maintain their telomeres through alternative lengthening or still unknown mechanisms. This review presents the different therapeutic approaches proposed thus far and developed in preclinical tumor models and discusses the perspectives for their use in the clinical setting.