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Soft-tissue sarcomas (STS) are a rare and heterogeneous group of tumors that arise from connective tissue and can occur anywhere in the body. Among the plethora of over 50 different STS types, liposarcoma (LPS) is one of the most common. The subtypes of STS are characterized by distinct differences in tumor biology that drive responses to pharmacologic therapy and disparate oncologic outcomes. Small non-coding RNAs (sncRNA) are a heterogeneous class of regulatory RNAs involved in the regulation of gene expression by targeting mRNAs. Among the several types of sncRNAs, miRNAs and tRNA-derived ncRNAs are the most studied in the context of tumor biology, and we are learning more about the role of these molecules as important regulators of STS tumorigenesis and differentiation. However, challenges remain in translating these findings and no biomarkers or therapeutic approaches targeting sncRNAs have been developed for clinical use. In this review, we summarize the current landscape of sncRNAs in the context of STS with an emphasis on LPS, including the role of sncRNAs in the tumorigenesis and differentiation of these rare malignancies and their potential as novel biomarkers and therapeutic targets. Finally, we provide an appraisal of published studies and outline future directions to study sncRNAs in STS, including tRNA-derived ncRNAs.

Nanostring microRNAs data

Supplementary Data from Pan-Cancer Analysis of Canonical and Modified miRNAs Enhances the Resolution of the Functional miRNAome in Cancer

Supplementary Figure from Pan-Cancer Analysis of Canonical and Modified miRNAs Enhances the Resolution of the Functional miRNAome in Cancer

Supplementary Figure 1: Relapse-free survival (RFS) analysis for WWOX in KM plotter. Supplementary Figure 2: Relapse-free survival (RFS) analysis for the WWOX gene in TCGA dataset. Supplementary Figure 3: Boxplots for each comparison of the expression of the specific gene between WWOX+ (>75th percentile) and WWOX- (

Epitranscriptomic studies of miRNAs have added a new layer of complexity to the cancer field. Although there is fast-growing interest in adenosine-to-inosine (A-to-I) miRNA editing and alternative cleavage that shifts miRNA isoforms, simultaneous evaluation of both modifications in cancer is still missing. Here, we concurrently profiled multiple miRNA modification types, including A-to-I miRNA editing and shifted miRNA isoforms, in >13,000 adult and pediatric tumor samples across 38 distinct cancer cohorts from The Cancer Genome Atlas and The Therapeutically Applicable Research to Generate Effective Treatments data sets. The differences between canonical miRNAs and the wider miRNAome in terms of expression, clustering, dysregulation, and prognostic standpoint were investigated. The combination of canonical miRNAs and modified miRNAs boosted the quality of clustering results, outlining unique clinicopathologic features among cohorts. Certain modified miRNAs showed opposite expression from their canonical counterparts in cancer, potentially impacting their targets and function. Finally, a shifted and edited miRNA isoform was experimentally validated to directly bind and suppress a unique target. These findings outline the importance of going beyond the well-established paradigm of one mature miRNA per miRNA arm to elucidate novel mechanisms related to cancer progression.Significance:Modified miRNAs may act as cancer biomarkers and function as allies or antagonists of their canonical counterparts in gene regulation, suggesting the concurrent consideration of canonical and modified miRNAs can boost patient stratification.

TCGA TNBC samples

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Triple-negative breast cancer (TNBC) is a heterogeneous, highly aggressive, and difficult to treat tumor type. The tumor suppressor WWOX spans FRA16D, a common fragile site that is commonly altered in breast cancer. Despite recent progress, the role of WWOX in TNBC metastasis is unknown. Here we report that WWOX inactivation correlates with advanced stages of TNBC and that its levels are frequently altered in TNBC cells. Ectopic restoration of WWOX in WWOX-negative TNBC cells inhibited metastasis while its depletion in WWOX-positive TNBC cells promoted metastasis. WWOX was a negative regulator of c-MYC, which regulated miR-146a expression and consequently fibronectin levels, contributing to an epithelial status of the cell. Treatment of TNBC cells with anti-miR-146a rescued the WWOX antimetastatic phenotype. Moreover, overexpression of MYC in WWOX-expressing TNBC cells overrode WWOX effects on miR-146a and fibronectin levels. Altogether, our data uncover an essential role for WWOX in antagonizing TNBC progression and highlight its potential use as a biomarker for metastasis.Significance:These findings highlight the mechanism by which the tumor suppressor WWOX regulates metastasis of triple-negative breast cancer.See related commentary by Sharma, p. 1746

Novel therapeutic strategies are needed for paediatric patients affected by Acute Myeloid Leukaemia (AML), particularly for those at high-risk for relapse. MicroRNAs (miRs) have been extensively studied as biomarkers in cancer and haematological disorders, and their expression has been correlated to the presence of recurrent molecular abnormalities, expression of oncogenes, as well as to prognosis/clinical outcome. In the present study, expression signatures of different miRs related both to presence of myeloid/lymphoid or mixed-lineage leukaemia 1 and Fms like tyrosine kinase 3 internal tandem duplications rearrangements and to the clinical outcome of paediatric patients with AML were identified. Notably, miR-221-3p and miR-222-3p resulted as a possible relapse-risk related miR. Thus, miR-221-3p and miR-222-3p expression modulation was investigated by using a Bromodomain‑containing protein 4 (BRD4) inhibitor (JQ1) and a natural compound that acts as histone acetyl transferase inhibitor (curcumin), alone or in association, in order to decrease acetylation of histone tails and potentiate the effect of BRD4 inhibition. JQ1 modulates miR-221-3p and miR-222-3p expression in AML with a synergic effect when associated with curcumin. Moreover, changes were observed in the expression of CDKN1B, a known target of miR-221-3p and miR-222-3p, increase in apoptosis and downregulation of miR-221-3p and miR-222-3p expression in CD34+ AML primary cells. Altogether, these findings suggested that several miRs expression signatures at diagnosis may be used for risk stratification and as relapse prediction biomarkers in paediatric AML outlining that epigenetic drugs, could represent a novel therapeutic strategy for high-risk paediatric patients with AML. For these epigenetic drugs, additional research for enhancing activity, bioavailability and safety is needed.

Chronic lymphocytic leukemia (CLL) is a quiescent B-cell malignancy that depends on transcriptional dysregulation for survival. The histone deacetylases are transcriptional regulators whose role within the regulatory chromatin and consequence on the CLL transcriptome is poorly characterized. Here, we profiled and integrated the genome wide occupancy of HDAC1, BRD4, H3K27Ac and H3K9Ac signals with chromatin accessibility, Pol2 occupancy and target expression signatures in CLL cells. We identified that when HDAC1 was recruited within super-enhancers marked by acetylated H3K27 and BRD4, it functioned as a transcriptional activator that drove the de novo expression of select genes to facilitate survival and progression in CLL. Targeting HDACs reduced BRD4 and Pol2 engagement to downregulate the transcript and proteins levels of specific oncogenic driver genes in CLL such as BLK, a key mediator of the B-cell receptor pathway, core transcription factors such as PAX5 and IKZF3 and the anti-apoptotic gene, BCL2. Concurrently, HDAC1, when recruited in the absence of super-enhancers repressed target gene expression. HDAC inhibition reversed silencing of a defined set of protein coding and noncoding RNA genes. We focused on a specific set of microRNA genes and show that their upregulation was inversely correlated with the expression of CLL specific survival, transcription factor and signaling genes. Our findings identify that the transcriptional activator and repressor functions of HDACs cooperate within the same tumor to establish the transcriptional dependencies essential for survival in CLL.

Gliomas are the most prevalent type of brain tumors and one of the leading causes of cancer-related death in the adolescent and young adult population (AYA). Two-thirds of glioma AYA patients are affected by low-grade gliomas (LGGs), but there are no specific treatments. Therefore, a percentage of LGG patients experience tumor relapse and malignant progression to high-grade glioma which leads to fatal outcomes. In part, malignant progression is potentiated by the immunosuppressive stromal component of the tumor microenvironment (TME) underscored by M2-macrophages and a paucity of cytotoxic T cells. As a result, first-line immunotherapies have failed to improve outcomes for patients with progressive high-grade gliomas. Here, we report the efficacy of an in vivo approach that demonstrates the potential for a novel cell-mediated innate immunotherapy designed to abrogate immunosuppressive mechanisms within the glioma TME and enhance the recruitment of activated effector T cells. A single dose of engineered bone marrow-derived myeloid cells that release Interleukin-2 (GEMys-IL2) was used systemically to treat mice with LGG tumors systemically. Our results demonstrate that GEMys-IL2 efficiently crossed the blood brain barrier (BBB), infiltrated the glioma microenvironment, and reprogrammed the infiltrating immune cell composition and transcriptome. In addition, GEMys-IL2 impaired tumor progression and extended survival in a LGG immunocompetent mouse model. In conclusion, we demonstrated that GEMys-IL2 have a therapeutic effect in vivo, thus supporting its potential application as a novel immunotherapy that warrants further investigation.

Epitranscriptomic studies of miRNAs have added a new layer of complexity to the cancer field. Although there is fast-growing interest in adenosine-to-inosine (A-to-I) miRNA editing and alternative cleavage that shifts miRNA isoforms, simultaneous evaluation of both modifications in cancer is still missing. Here, we concurrently profiled multiple miRNA modification types, including A-to-I miRNA editing and shifted miRNA isoforms, in >13,000 adult and pediatric tumor samples across 38 distinct cancer cohorts from The Cancer Genome Atlas and The Therapeutically Applicable Research to Generate Effective Treatments data sets. The differences between canonical miRNAs and the wider miRNAome in terms of expression, clustering, dysregulation, and prognostic standpoint were investigated. The combination of canonical miRNAs and modified miRNAs boosted the quality of clustering results, outlining unique clinicopathologic features among cohorts. Certain modified miRNAs showed opposite expression from their canonical counterparts in cancer, potentially impacting their targets and function. Finally, a shifted and edited miRNA isoform was experimentally validated to directly bind and suppress a unique target. These findings outline the importance of going beyond the well-established paradigm of one mature miRNA per miRNA arm to elucidate novel mechanisms related to cancer progression. Significance: Modified miRNAs may act as cancer biomarkers and function as allies or antagonists of their canonical counterparts in gene regulation, suggesting the concurrent consideration of canonical and modified miRNAs can boost patient stratification.

Lung cancer is the second most commonly diagnosed cancer and the leading cause of cancer deaths worldwide. Among its subtypes, lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) are the most common, accounting for more than 85% of lung cancer diagnoses. Despite the incredible efforts and recent advances in lung cancer treatments, patients affected by this condition still have a poor prognosis. Therefore, novel diagnostic biomarkers are needed. Recently, a class of transposable elements called human endogenous retroviruses (HERVs) has been found to be implicated in cancer development and later employed as novel biomarkers for several tumor types. In this study, we first ever characterized the expression of HERVs at genomic locus-specific resolution in both LUAD and LUSC cohorts available in The Cancer Genome Atlas (TCGA). Precisely, (i) we profiled the expression of HERVs in TCGA-LUAD and TCGA-LUSC cohorts; (ii) we identified the dysregulated HERVs in both lung cancer subtypes; (iii) we evaluated the impact of the dysregulated HERVs on signaling pathways using neural network-based predictions; and (iv) we assessed their association with overall survival (OS) and relapse-free survival (RFS). In conclusion, we believe this study may help elucidate another layer of dysregulation that occurs in lung cancer involving HERVs, paving the way for identifying novel lung cancer biomarkers.

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor (TKR) frequently mutated in lung cancer. Despite initially favorable clinical responses of non-small cell lung cancer (NSCLC) patients harboring an EGFR mutation to treatment with tyrosine kinase inhibitors (TKIs), rapid resistance occurs mainly because of genetic alterations, including amplification of the hepatocyte growth factor receptor (MET). RNA post-transcriptional modifications that contribute to aberrant expression of MET in cancer are under-investigated. Among them, adenosine to inosine (A-to-I) RNA editing regulates RNA sequence, function, degradation, and structure. RNA editing has been observed in microRNAs (miRNAs), a class of small non-coding RNAs involved in post-transcriptional gene regulation. High throughput interrogation of the human genome allows for the identification of miRNA editing deregulation in cancer. However, the biological impact of edited miRNAs on lung cancer progression and drug resistance remains largely unknown. A reduction of A-to-I editing in position 5 of miR-411-5p has been identified in several cancers, including NSCLC. In this study, we determined that edited miR-411-5p negatively affects the ERK1/2 pathway, directly targets MET, and promotes EGFR TKI response in NSCLC.

EVs have emerged as an important component in tumour initiation, progression and metastasis. Although notable progresses have been made, the detection of EV cargoes remain significantly challenging for researchers to practically use; faster and more convenient methods are required to validate the EV cargoes, especially as biomarkers. Here we show, the possibility of examining embedded EVs as substrates to be used for detecting DNA amplification through ultrasensitive in situ hybridization (ISH). This methodology allows the visualization of DNA targets in a more direct manner, without time consuming optimization steps or particular expertise. Additionally, formalin-fixed paraffin-embedded (FFPE) blocks of EVs allows long-term preservation of samples, permitting future studies. We report here: (i) the successful isolation of EVs from liposarcoma tissues; (ii) the EV embedding in FFPE blocks (iii) the successful selective, specific ultrasensitive ISH examination of EVs derived from tissues, cell line, and sera; (iv) and the detection of MDM2 DNA amplification in EVs from liposarcoma tissues, cell lines and sera. Ultrasensitive ISH on EVs would enable cargo study while the application of ISH to serum EVs, could represent a possible novel methodology for diagnostic confirmation. Modification of probes may enable researchers to detect targets and specific DNA alterations directly in tumour EVs, thereby facilitating detection, diagnosis, and improved understanding of tumour biology relevant to many cancer types.

The repository provides the rawread counts expression data (miRNA-Seq and RNA-Seq) and the source code developed for each analysis performedin this study.

MLL is an aggressive subtype of leukemia with a poor prognosis that mostly affects pediatric patients. MLL-rearranged fusion proteins (MLLr) induce aberrant target gene expression resulting in leukemogenesis. MLL and its fusions are tethered to chromatin by LEDGF/p75, a transcriptional co-activator that specifically recognizes H3K36me2/3. LEDGF/p75 is ubiquitously expressed and associated with regulation of gene expression, autoimmune responses, and HIV replication. LEDGF/p75 was proven to be essential for leukemogenesis in MLL. Apart from MLL, LEDGF/p75 has been linked to lung, breast, and prostate cancer. Intriguingly, LEDGF/p75 interacts with Med-1, which co-localizes with BRD4. Both are known as co-activators of super-enhancers. Here, we describe LEDGF/p75-dependent chemoresistance of MLLr cell lines. Investigation of the underlying mechanism revealed a role of LEDGF/p75 in the cell cycle and in survival pathways and showed that LEDGF/p75 protects against apoptosis during chemotherapy. Remarkably, LEDGF/p75 levels also affected expression of BRD4 and Med1. Altogether, our data suggest a role of LEDGF/p75 in cancer survival, stem cell renewal, and activation of nuclear super enhancers. ispartof: Cancer Gene Therapy vol:29 issue:2 pages:133-140 ispartof: location:England status: published

Pancreatic ductal adenocarcinoma (PDAC) is a relatively uncommon malignancy; however, its incidence is rising worldwide, and it is expected to become the second-leading cause of cancer-related death by 2030. From a histological point of view, PDAC is characterized by a prominent desmoplastic reaction that compresses blood vessels, limiting oxygen and nutrient availability in the tumor microenvironment. Despite that, PDAC cells are capable to adapt dynamically to these stress conditions, adopting different strategies that includes the strict regulation of the autophagic flux. Thus, studying these adaptive mechanisms is crucial to understand PDAC progression and to establish new therapeutic modalities to tackle it. Here, we explore the role of the tumor suppressor miR-15a in the regulation of its putative target Fra-2, a transcription factor, commonly activated by cell stress. By employing IPA on PDAC tumors from TCGA dataset, we found that both miR-15a and its target Fra-2 are predicted to regulate the IGF-1 signaling pathway. In an independent cohort of 44 PDAC samples, miR-15a levels inversely correlated with both Fra-2 and IGF1R expression; conversely, Fra-2 significantly correlated with IGF1R. By chromatin immunoprecipitation and luciferase assay, we assessed that miR-15a directly targeted Fra-2 and IGF1R that, in turn, is transcriptionally regulated by Fra-2 activity. Then, we investigated the role of miR-15a/Fra-2 regulation of IGF1R in the response to starvation-induced cell stress. In starved PDAC cell lines, Fra-2 transcriptional activity triggered IGF1R promoter, causing IGF1R overexpression in control cells but not in miR-15a-overexpressing cells. Consistently, the IGF1 release after starvation induced phosphorylation of IGF1R and activation of the downstream mTOR pathway in control cells but not in miR-15a-overexpressing cells. Therefore, TEM and western blot analysis demonstrated that activation of mTOR via IGF1 release reduced the autophagic flux of PDAC control cell lines compared to miR-15a overexpressing cells under starvation. To assess our results in vivo, we injected PDAC cells wild type or Fra-2 knockout into the flank of nude mice. At tumor onset, mice were randomly divided in two groups and fed with control or hypoproteic diet for three weeks. Hypoproteic diet did not interfere with the growth of wild-type tumors, by contrast, significantly impaired Fra-2KO tumors growth rate. Tumor analysis revealed that hypoproteic diet potently induced IGF1R overexpression and mTOR pathway activation in wild-type tumors but not in Fra-2KO tumors. Our findings demonstrate that IGF1R expression is regulated by miR-15a directly and indirectly via Fra-2 in PDAC. This novel miR-15a/Fra-2/IGF1R axis, triggered by starvation, regulates the autophagic flux and growth of PDAC cells in stress condition, and could be targeted by specific small inhibitors. Citation Format: Gian Luca Rampioni Vinciguerra, Marina Capece, Luca Reggiani Bonetti, Paolo Magistri, Federica Calore, Giovanni Nigita, Rosario Distefano, Roberto Ballarin, Fabrizio Di Benedetto, Andrea Vecchione, Barbara Belletti, Gustavo Baldassarre, Francesca Lovat, Carlo M. Croce. The novel miR-15a/Fra-2/IGF1R axis drives response to starvation-induced cell stress in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4823.

Lung cancer is the leading cause of cancer-related mortality in both men and women worldwide. Despite the administration of platinum compounds represents the main treatment, the majority of tumors remains intrinsically resistant. In lung cancer, miR-301a exerts a main role by modulating the activity of transcription factors like NF-kB and Stat3. Here, we dissected the contribution of miR-301a in the regulation of its new identified target Fos-related antigen-2 (Fra-2), a transcription factor that belongs to the Fos/AP-1 family. By microarray analysis, we discovered that Fra-2 overexpression increased glioma pathogenesis-related protein 1 (GLIPR1) levels, a mediator of cisplatin resistance in lung cancer. Its mechanism of regulation is still unclear. We demonstrated that GLIPR1 is also a miR-301a target. In vitro, modulation of miR-301a reduced Fra-2 and GLIPR1 levels; conversely, Fra-2 overexpression significantly increased GLIPR1 and miR-301a. Then, by chromatin immunoprecipitation assay, we confirmed that Fra-2 interacts with the promoter regions of both GLIPR1 and MIR301A genes, supporting that miR-301a/Fra-2/GLIPR1 axis could be autoregulated by feedback loop in which Fra-2 promotes the transcription of MIR301A gene. Investigating the effect of this new axis on the response to cisplatin, we observed that both Fra-2/GLIPR1 overexpression and miR-301a silencing induced cisplatin resistance in lung cancer cells. By contrast, silencing of GLIPR1 and combined administration of low doses of Fos/AP-1 inhibitor restored the cisplatin sensitivity in Fra-2 overexpressing cells. In the lung adenocarcinoma samples from the TCGA dataset, miR-301a overexpression inversely correlated with Fra-2 and GLIPR1 expression. Consistently, the overexpression of miR-301a identified a fraction of tumors with low expression of Fra-2 and GLIPR1 in an internal cohort of lung cancer samples. Altogether, our findings identify the miR-301a/Fra-2/GLIPR1 axis that contributes to cisplatin resistance in lung cancer cells and could serve as biomarker to stratify patients who may benefit from cisplatin administration, alone or in combination with Fos/AP-1 inhibitors. Citation Format: Francesca Lovat, Gian Luca Rampioni Vinciguerra, Marina Capece, Rosario Distefano, Giovanni Nigita, Andrea Vecchione, Carlo M. Croce. Role of the miR-301a/Fra-2/GLIPR1 axis in lung cancer cisplatin resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3903.

Our earlier studies had shown that overexpression of the JmjC domain histone demethylase KDM2B renders mouse embryo fibroblasts (MEFs) resistant to oxidative stress due to the role of KDM2B in the regulation of antioxidant mechanisms. Here we present evidence that the knockdown of KDM2B in basal-like breast cancer cell lines results in a decrease of Glutathione (GSH) levels, a secondary increase of intracellular ROS levels and in enhanced sensitivity to deubiquitinase inhibitors. The expression of the Glutamate-Cystine antiporter, the Glutamate-Cysteine Ligase GCLC/GCLM and the Glutathione Peroxidase GPX4, all of which regulate GSH abundance was not affected. To address the mechanism of the GSH regulation we carried out RNA-Seq, quantitative proteomics and metabolomics analyses in shKDM2B- and empty vector-transduced MDA-MB-231 cells. The results showed that the KD of KDM2B causes major shifts in metabolism and that one of the metabolic pathways whose activity depends on KDM2B is the SGOC pathway, which has a major role in GSH biosynthesis. Experiments in cultured cells confirmed the importance of KDM2B in the regulation of this pathway and they also showed that the inhibition of the pathway via the KD of KDM2B is partly responsible for the shKDM2B-induced inhibition of cell proliferation in culture and in xenograft experiments in NSG mice. More important, the transcriptomic signature of the SGOC pathway correlates with the expression of KDM2B in basal like mammary adenocarcinomas in the TCGA database. The genes encoding the majority of the enzymes in the SGOC pathway are known to be regulated by MYC and ATF4 and our data show that both MYC and ATF4 are under the regulatory control of KDM2B. ATAC-Seq and ChIP-Seq experiments in shKDM2B and control MDA-MB-231 cells also showed that KDM2B binds the promoter region of not only MYC and ATF4, but also of the genes encoding the SGOC enzymes and that it regulates chromatin accessibility and the abundance of H3K4me3/H3K27Ac active histone marks in these promoters. Overall, our data indicate that KDM2B regulates the SGOC pathway by targeting MYC and ATF4 and by making the promoters of the genes encoding the SGOC enzymes accessible to these regulators. Overall, our data provide new evidence on SGOC regulation, and identify novel KDM2B-dependent metabolic vulnerabilities in basal like breast cancer. Citation Format: Evangelia Chavdoula, Vollter Anastas, Allesandro La Ferlita, Julian Aldana, Giuseppe Carota, Mariarita Spampinato, Sameer Parashar, Ilaria Cosentini, Burak Soysal, Giovanni Nigita, Michael Freitas, Philip Tsichlis. KDM2B regulates Serine-Glycine-One Carbon (SGOC) metabolism by targeting the SGOC enzyme genes via a combination of direct and indirect epigenetic mechanisms. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6038.

Significance 13q14.3 deletion is the most common genetic lesion identified in CLLs. This study shows that microdeletions affecting the miR-15a/16-1 cluster are more frequent than expected in all CLL cohorts and are prevalent in patients carrying a trisomy 12. Copy-number variation analysis and an experimental FISH analysis revealed that ∼34% of samples carry previously unidentified microdeletions of miR-15a/16-1. These data may have clinical relevance for the successful stratification of patients for treatment., Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and is characterized by chromosomal aberrations including 13q, 11q, and 17p deletions and a trisomy of chromosome 12 (T12). 13q deletions are often associated with 11q and 17p deletions in aggressive cases. Conversely, T12 CLLs show a variable prognosis, and association with 13q deletions is uncommon. The miR-15a/16-1 cluster is the functional target of 13q deletions, leading to BCL2 overexpression. Chromosomal aberrations in CLL are associated with prognosis, and their identification is carried out by fluorescence in situ hybridization (FISH). Since standard FISH only detects large deletions, we investigated the presence of undetected microdeletions targeting miR-15a/16-1 in CLL cases. We found that ∼34% of CLL samples show an unreported loss of the miR-15a/16-1 locus regardless of their cytogenetic profile. Interestingly, 15 out of 39 (∼39%) of all CLLs with T12, carry microdeletions of miR-15a/16-1, indicating that, in patients with T12, miR-15a/16-1 are mostly inactivated by microdeletions. In addition, ∼40% of CLL cases bearing T12, 17p−, and 11q− showed unidentified microdeletions of miR-15a/16-1, suggesting that miR-15a/16-1 loss cooperates with such chromosomal alterations in CLL. These data may have clinical relevance for the successful stratification of patients for treatment.

Despite the development of targeted therapeutics, immunotherapy, and strategies for early detection, lung cancer carries a high mortality. Further, significant racial disparities in outcomes exist for which the molecular drivers have yet to be fully elucidated. The growing field of Epitranscriptomics has introduced a new layer of complexity to the molecular pathogenesis of cancer. RNA modifications can occur in coding and non-coding RNAs, such as miRNAs, possibly altering their gene regulatory function. The potential role for such modifications as clinically informative biomarkers remains largely unknown. Here, we concurrently profiled canonical miRNAs, shifted isomiRs (templated and non-templated), and miRNAs with single-point modification events (RNA and DNA) in White American (W) and Black or African American (B/AA) lung adenocarcinoma (LUAD) patients. We found that while most deregulated miRNA isoforms were similar in W and B/AA LUAD tissues compared to normal adjacent tissues, there was a subgroup of isoforms with deregulation according to race. We specifically investigated an edited miRNA, miR-151a-3p with an A-to-I editing event at position 3, to determine how its altered expression may be associated with activation of divergent biological pathways between W and B/AA LUAD patients. Finally, we identified distinct race-specific miRNA isoforms that correlated with prognosis for both Ws and B/AAs. Our results suggested that concurrently profiling canonical and non-canonical miRNAs may have potential as a strategy for identifying additional distinct biological pathways and biomarkers in lung cancer.

Novel therapeutic strategies are needed for paediatric patients affected by Acute Myeloid Leukaemia (AML), particularly for those at high-risk for relapse. MicroRNAs (miRs) have been extensively studied as biomarkers in cancer and haematological disorders, and their expression has been correlated to the presence of recurrent molecular abnormalities, expression of oncogenes, as well as to prognosis/clinical outcome. In the present study, expression signatures of different miRs related both to presence of myeloid/lymphoid or mixed-lineage leukaemia 1 and Fms like tyrosine kinase 3 internal tandem duplications rearrangements and to the clinical outcome of paediatric patients with AML were identified. Notably, miR-221-3p and miR-222-3p resulted as a possible relapse-risk related miR. Thus, miR-221-3p and miR-222-3p expression modulation was investigated by using a Bromodomain‑containing protein 4 (BRD4) inhibitor (JQ1) and a natural compound that acts as histone acetyl transferase inhibitor (curcumin), alone or in association, in order to decrease acetylation of histone tails and potentiate the effect of BRD4 inhibition. JQ1 modulates miR-221-3p and miR-222-3p expression in AML with a synergic effect when associated with curcumin. Moreover, changes were observed in the expression of

Lung and breast cancer are the two most common causes of malignant pleural effusion (MPE). MPE diagnosis plays a crucial role in determining staging and therapeutic interventions in these cancers. However, our understanding of the pathogenesis and progression of MPE at the molecular level is limited. Extracellular Vesicles (EVs) and their contents, including microRNAs (miRNAs), can be isolated from all bodily fluids, including pleural fluid. This study aims to compare EV-miRNA patterns of expression in MPE caused by breast (BA-MPE) and lung (LA-MPE) adenocarcinomas compared to the control group of heart-failure-induced effusions (HF-PE). We conducted an analysis of 24 pleural fluid samples (8 LA-MPE, 8 BA-MPE, and 8 HF-PE). Using NanoString technology, we profiled miRNAs within EVs isolated from 12 cases. Bioinformatic analysis demonstrated differential expression of miR-1246 in the MPE group vs. HF-PE group and miR-150-5p and miR-1246 in the BA-MPE vs. LA-MPE group, respectively. This difference was demonstrated and validated in an independent cohort using real-time PCR (RT-PCR). miRNA-1246 demonstrated 4-fold increased expression (OR: 3.87, 95% CI: 0.43, 35) in the MPE vs. HF-PE group, resulting in an area under the curve of 0.80 (95% CI: 0.60, 0.99). The highest accuracy for differentiating MPE vs. HF-PE was seen with a combination of miRNAs compared to each miRNA alone. Consistent with prior studies, this study demonstrates dysregulation of specific EV-based miRNAs in breast and lung cancer; pleural fluid provides direct access for the analysis of these EV-miRNAs as biomarkers and potential targets and may provide insight into the underlying pathogenesis of tumor progression. These findings should be explored in large prospective studies.

INTRODUCTION Gliomas are the most prevalent type of brain tumors in the adolescent and young adult population (AYA) and one of the leading causes of cancer- related death. Two-third of glioma AYA patients are affected by low-grade gliomas (LGGs), but yet there are no specific treatments. Therefore, a significant percentage of LGG patients experience tumor recurrence and malignant progression to high-grade glioma (HGG) and the immune microenvironment plays a critical role in mediating mechanisms of tumor progression and treatment resistance. Specifically, tumor associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) have been shown to restrict cytotoxic effector trafficking and activation. To that end, first-line immunotherapies have limited efficacy in improving outcomes for HGGs. HYPOTHESIS We investigated the preclinical efficacy of a novel immunotherapeutic approach for the treatment of LGGs in AYA mice to modulate and reprogram the immunosuppressive tumor microenvironment (TME) and enhance the trafficking of activated effector T cells. METHODS Immunocompetent RCAS n/tv-a LGG bearing animals were treated at day 28 with a single retroorbital dose of bone-marrow derived myeloid cells, engineered for the release of Interlekin-2 (GEMys-IL2). The treatment was well tolerated and no sign of toxicity was noted. RESULTS Our results demonstrated that GEMys-IL2 efficiently crossed the BBB, infiltrate the glioma microenvironment, and activate infiltrating immune cells while altering the TME underscored by an increased CD8+ T cell population. Of note, the RCAS/t-va immunocompetent LGG to HGG progression mouse model treated with a single dose of GEMys-IL2 demonstrated a transient arrest of tumor burden within 14 days post treatment, which was associated with a significant advantage in overall survival. CONCLUSIONS These results highlight the pre-clinical efficacy and anti-tumoral therapeutic activity of GEMYys-IL2 on CD8+ T cell trafficking and activation to the TME in low-grade glioma in vivo, thus supporting its potential as a novel cell-mediated innate immunotherapy.

Low grade gliomas (LGG) account for about two-thirds of all glioma diagnoses in adolescents and young adults (AYA) and malignant progression of these patients leads to dismal outcomes. Recent studies have shown the importance of the dynamic tumor microenvironment in high-grade gliomas (HGG), yet its role is still poorly understood in low-grade glioma malignant progression. Here, we investigated the heterogeneity of the immune microenvironment using a platelet-derived growth factor (PDGF)-driven RCAS (replication-competent ASLV long terminal repeat with a splice acceptor) glioma model that recapitulates the malignant progression of low to high-grade glioma in humans and also provides a model system to characterize immune cell trafficking and evolution. To illuminate changes in the immune cell landscape during tumor progression, we performed single-cell RNA sequencing on immune cells isolated from animals bearing no tumor (NT), LGG and HGG, with a particular focus on the myeloid cell compartment, which is known to mediate glioma immunosuppression. LGGs demonstrated significantly increased infiltrating T cells, CD4 T cells, CD8 T cells, B cells, and natural killer cells in the tumor microenvironment, whereas HGGs significantly abrogated this infiltration. Our study identified two distinct macrophage clusters in the tumor microenvironment; one cluster appeared to be bone marrow-derived while another was defined by overexpression of Trem2, a marker of tumor associated macrophages. Our data demonstrates that these two distinct macrophage clusters show an immune-activated phenotype (Stat1, Tnf, Cxcl9 and Cxcl10) in LGG which evolves to an immunosuppressive state (Lgals3, Apoc1 and Id2) in HGG that restricts T cell recruitment and activation. We identified CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Interestingly, these results were mirrored by our analysis of the TCGA dataset, which demonstrated a statistically significant association between CD74 overexpression and decreased overall survival in AYA patients with grade II gliomas. Targeting immunosuppressive myeloid cells and intra-tumoral macrophages within this therapeutic window may ameliorate mechanisms associated with immunosuppression before and during malignant progression.

MicroRNAs (miRNAs) are regulatory small non-coding RNAs that function as translational repressors. MiRNAs are involved in most cellular processes, and their expression and function are presided by several factors. Amongst, miRNA editing is an epitranscriptional modification that alters the original nucleotide sequence of selected miRNAs, possibly influencing their biogenesis and target-binding ability. A-to-I and C-to-U RNA editing are recognized as the canonical types, with the A-to-I type being the predominant one. Albeit some bioinformatics resources have been implemented to collect RNA editing data, it still lacks a comprehensive resource explicitly dedicated to miRNA editing. Here, we present MiREDiBase, a manually curated catalog of editing events in miRNAs. The current version includes 3,059 unique validated and putative editing sites from 626 pre-miRNAs in humans and three primates. Editing events in mature human miRNAs are supplied with miRNA-target predictions and enrichment analysis, while minimum free energy structures are inferred for edited pre-miRNAs. MiREDiBase represents a valuable tool for cell biology and biomedical research and will be continuously updated and expanded at https://ncrnaome.osumc.edu/miredibase.

MicroRNAs (miRNAs or miRs) are the most characterized class of non-coding RNAs and are engaged in many cellular processes, including cell differentiation, development, and homeostasis. MicroRNA dysregulation was observed in several diseases, cancer included. Epitranscriptomics is a branch of epigenomics that embraces all RNA modifications occurring after DNA transcription and RNA synthesis and involving coding and non-coding RNAs. The development of new high-throughput technologies, especially deep RNA sequencing, has facilitated the discovery of miRNA isoforms (named isomiRs) resulting from RNA modifications mediated by enzymes, such as deaminases and exonucleases, and differing from the canonical ones in length, sequence, or both. In this review, we summarize the distinct classes of isomiRs, their regulation and biogenesis, and the active role of these newly discovered molecules in cancer and other diseases.

MiRNA Epitranscriptomics has placed a new layer of complexity in the cancer field. Despite the fast-growing interest in miRNA editing and shifted miRNA isoforms, a simultaneous study of both modifications in cancer is still missing. Here, we concurrently profiled multiple miRNA modifications, including A-to-I RNA editing and shifted miRNA isoforms, in >13K adult and pediatric tumor samples across 38 distinct cancer cohorts from The Cancer Genome Atlas and The Therapeutically Applicable Research to Generate Effective Treatments datasets. We investigated the differences among canonical miRNAs and the wider miRNAome in terms of expression, clustering, dysregulation, and prognostic standpoint. The combination of canonical miRNAs/miRNA isoforms boosted the quality of clustering results, outlining unique cohorts’ clinical-pathological features. We described modified miRNAs showing opposite dysregulation with respect to their canonical counterparts in cancer, potentially impacting their targetome and function. The abundance of expressed miRNA isoforms directly impacted the activation/deactivation of critical carcinogenesis pathways. Finally, we experimentally validated unique targeting for a shifted and edited miRNA isoform. Our findings outlined once more the importance of going beyond the well-established paradigm of one-mature-miRNA per miRNA arm to elucidate novel mechanisms related to cancer progression.

The epitranscriptome encompasses all post-transcriptional modifications that occur on RNAs. These modifications can alter the function and regulation of their RNA targets, which, if dysregulated, result in various diseases and cancers. As with other RNAs, miRNAs are highly modified by epitranscriptomic modifications such as m6A methylation, 2′-O-methylation, m5C methylation, m7G methylation, polyuridine, and A-to-I editing. miRNAs are a class of small non-coding RNAs that regulates gene expression at the post-transcriptional level. miRNAs have gathered high clinical interest due to their role in disease, development, and cancer progression. Epitranscriptomic modifications alter the targeting, regulation, and biogenesis of miRNAs, increasing the complexity of miRNA regulation. In addition, emerging studies have revealed crosstalk between these modifications. In this review, we will summarize the epitranscriptomic modifications—focusing on those relevant to miRNAs—examine the recent crosstalk between these modifications, and give a perspective on how this crosstalk expands the complexity of miRNA biology.

Myeloid cells and macrophages have been shown to promote immunosuppression in high-grade gliomas (HGG), however their roles in malignant progression of low-grade glioma (LGG) are poorly understood. Here, we investigated the heterogeneity of the immune microenvironment during glioma progression using a murine model that recapitulates the malignant progression of low to high-grade glioma. To that end, we performed single-cell RNA sequencing on CD45+ immune cells isolated from animals bearing no tumor (NT), LGG, and HGG. We observed an increased infiltration of CD4+ T cells, CD8+ T cells, B cells, and natural killer cells in the tumor microenvironment of LGG, whereas this infiltration was abrogated in HGG. Our study identified two distinct macrophage clusters across all 3 samples, with signatures of bone marrow derived and resident macrophages, respectively. These macrophages showed an immune-activated phenotype (Stat1, Tnf, Cxcl9 and Cxcl10) in LGG, but then evolved to a more immunosuppressive state (Lgals3, Apoc1 and Id2) in HGG, restricting T cell recruitment and activation. In addition, we identified CD74 and macrophage migration inhibition factor (MIF) as potential targets for both these distinct macrophage populations, based on their increased expression in LGG and HGG compared to NT. Targeting these factors during the LGG therapeutic window may inhibit myeloid cells and intra-tumoral macrophages and attenuate their immunosuppressive properties and impair malignant progression.

MicroRNAs (miRNAs) have been extensively reported to be associated with hematological malignancies. The loss of miR-15a/16–1 at chromosome 13q14 is a hallmark of most of human chronic lymphocytic leukemia (CLL). Deletion of murine miR-15a/16–1 and miR-15b/16–2 has been demonstrated to promote B cell malignancies. Here, we evaluate the biological role of miR-15/16 clusters, crossbreeding miR-15a/16–1 and miR-15b/16–2 knockout mice. Unexpectedly, the complete deletion of both clusters promoted myeloproliferative disorders in the majority of the mice by the age of 5 months with a penetrance of 70%. These mice showed a significant enlargement of spleen and abnormal swelling of lymph nodes. Flow cytometry characterization demonstrated an expanded CD11b/Gr-1 double-positive myeloid population both in spleen and in bone marrow. The transplantation of splenocytes harvested from double-KO mice into wild-type recipient mice resulted in the development of myeloproliferative disorders, as observed in the donors. In vivo, miR-15/16 cluster deletion up-regulated the expression of Cyclin D1, Cyclin D2, and Bcl-2. Taken together, our findings identify a driver oncogenic role for miR-15/16 cluster deletion in different leukocytic cell lineages.

Despite advances that have improved the treatment of chronic myeloid leukemia (CML) patients in chronic phase, the mechanisms of the transition from chronic phase CML to blast crisis (BC) are not fully understood. Considering the key role of miR-15/16 loci in the pathogenesis of myeloid and lymphocytic leukemia, here we aimed to correlate the expression of miR-15a/16 and miR-15b/16 to progression of CML from chronic phase to BC. We analyzed the expression of the two miR-15/16 clusters in 17 CML patients in chronic phase and 22 patients in BC and in 11 paired chronic phase and BC CML patients. BC CMLs show a significant reduction of the expression of miR-15a/-15b/16 compared to CMLs in chronic phase. Moreover, BC CMLs showed an overexpression of miR-15/16 direct targets such as Bmi-1, ROR1, and Bcl-2 compared to CMLs in chronic phase. This study highlights the loss of both miR-15/16 clusters as a potential oncogenic driver in the transition from chronic phase to BC in CML patients.

Simple Summary Adenosine to inosine (A-to-I) editing is a type of RNA editing where individual adenosines are enzymatically converted into inosines. A-to-I RNA editing plays an important role in cancer biology. Several studies have demonstrated that A-to-I editing of microRNAs (miRNAs) very often affect miRNA function as oncosuppressors or oncogenes, hence showing clinical relevance. Hence, A-to-I miRNA editing has been suggested as a potential diagnostic and prognostic tool in the monitoring of cancer patients. Nevertheless, the process of identifying and characterizing miRNA editing events in tumor samples still presents several challenges. In this review, we outline molecular aspects linked to miRNA A-to-I editing and retrace methods and approaches dedicated to detection of editing sites and functional characterization of edited miRNAs in cancer. Abstract Adenosine to inosine (A-to-I) editing consists of an RNA modification where single adenosines along the RNA sequence are converted into inosines. Such a biochemical transformation is catalyzed by enzymes belonging to the family of adenosine deaminases acting on RNA (ADARs) and occurs either co- or post-transcriptionally. The employment of powerful, high-throughput detection methods has recently revealed that A-to-I editing widely occurs in non-coding RNAs, including microRNAs (miRNAs). MiRNAs are a class of small regulatory non-coding RNAs (ncRNAs) acting as translation inhibitors, known to exert relevant roles in controlling cell cycle, proliferation, and cancer development. Indeed, a growing number of recent researches have evidenced the importance of miRNA editing in cancer biology by exploiting various detection and validation methods. Herein, we briefly overview early and currently available A-to-I miRNA editing detection and validation methods and discuss the significance of A-to-I miRNA editing in human cancer.

Cancer-associated cachexia is a heterogeneous, multifactorial syndrome characterized by systemic inflammation, unintentional weight loss, and profound alteration in body composition. The main feature of cancer cachexia is represented by the loss of skeletal muscle tissue, which may or may not be accompanied by significant adipose tissue wasting. Such phenotypic alteration occurs as the result of concomitant increased myofibril breakdown and reduced muscle protein synthesis, actively contributing to fatigue, worsening of quality of life, and refractoriness to chemotherapy. According to the classical view, this condition is primarily triggered by interactions between specific tumor-induced pro-inflammatory cytokines and their cognate receptors expressed on the myocyte membrane. This causes a shift in gene expression of muscle cells, eventually leading to a pronounced catabolic condition and cell death. More recent studies, however, have shown the involvement of regulatory non-coding RNAs in the outbreak of cancer cachexia. In particular, the role exerted by microRNAs is being widely addressed, and several mechanistic studies are in progress. In this review, we discuss the most recent findings concerning the role of microRNAs in triggering or exacerbating muscle wasting in cancer cachexia, while mentioning about possible roles played by long non-coding RNAs and ADAR-mediated miRNA modifications.

Lung cancer is the leading cause of cancer mortality worldwide. Increased understanding of the molecular mechanisms of the disease has led to the development of novel therapies and improving outcomes. Recently, extracellular vesicles (EVs) have emerged as vehicles for the transfer of genetic information between tumors and their microenvironment and have been implicated in lung cancer initiation, progression, and response to therapy. However, the mechanisms that drive the biogenesis and selective packaging of EVs remain poorly understood. Rab family guanosine triphosphates (GTPases) and their regulators are important membrane trafficking organizers. In this study, we investigated the role of select Rab GTPases on the regulation of EV release. We found that microRNAs target Rab GTPases to regulate EV release from lung cancer cell lines. In particular, Rab32 is a target of miR-124a, and siRNA and miRNA mediated inhibition of Rab32 leads to impaired EV secretion. The downstream implications for microRNA-based regulation of EV release are currently under investigation.

HDAC1 is a key regulator of gene expression in cancer. We identified a critical role for HDAC1 in establishing the transcriptional dependencies essential for survival in chronic lymphocytic leukemia (CLL) by profiling HDAC1 with BRD4, H3K27Ac superenhancers, H4K9Ac, chromatin accessibility signatures, Pol2 measurements and expression signatures to generate a regulatory chromatin landscape. Superenhancers marked by high levels of acetylation and BRD4 paradoxically also recruited the highest levels of HDAC1. HDAC inhibition poisoned transcription at these loci to selectively disrupt B-cell transcription factors and B-cell receptor signaling. HDAC1 was also recruited genome-wide at promoters without superenhancers to repress expression; HDAC inhibition induces these genes which include key microRNA networks that reciprocally downregulate CLL specific survival and driver genes. Our work provides a compelling rationale for profiling HDAC1 across cancers to characterize its role in driving the transcriptional dysregulation that is a hallmark of most cancers and develop epigenetic therapeutic strategies.SignificanceOur work definitively establishes the composition of the regulatory chromatin that enables HDAC1 to function as an activator and repressor at distinct target genes within the same tumor to drive transcriptional dysregulation and allow the expression of B cell specific signaling and survival networks that are critical for survival.

In the last two decades, RNA post-transcriptional modifications, including RNA editing, have been the subject of increasing interest among the scientific community. The efforts of the Human Genome Project combined with the development of new sequencing technologies and dedicated bioinformatic approaches created to detect and profile RNA transcripts have served to further our understanding of RNA editing. Investigators have determined that non-coding RNA (ncRNA) A-to-I editing is often deregulated in cancer. This discovery has led to an increased number of published studies in the field. However, the eventual clinical application for these findings remains a work in progress. In this review, we provide an overview of the ncRNA editing phenomenon in cancer. We discuss the bioinformatic strategies for RNA editing detection as well as the potential roles for ncRNA A to I editing in tumor immunity and as clinical biomarkers.

Double knockout of the two miR-15/16 loci in mouse resulted in the development of acute myeloid leukemia (AML). This result suggested that, at least, a fraction of human AMLs could be due to a similar mechanism. We analyzed the role of the two miR-15/16 clusters in 93 myelodysplastic syndrome (MDS) patients divided in three subgroups: patients with MDS, patients with MDS before transforming into AML (MDS-T), and patients with AML evolving from MDS (MDS-AML). Then, we tested 139 AML cases and 14 different AML cell lines by assessing microRNA (miRNA) expression, target protein expression, genetic loss, and silencing. MDS-T and MDS-AML patients show a reduction of the expression of miR-15a/-15b/-16 compared to MDS patients. Each miRNA can significantly predict MDS and MDS-T groups. Then, 79% of primary AMLs show a reduced expression of miR-15a and/or miR-15b. The expression of miR-15a/-15b/-16 significantly stratified AML patients in two prognostic classes. Furthermore, 40% of AML cell lines showed a combined loss of the expression of miR-15a/-15b and overexpression of their direct/indirect targets. As potential mechanisms involved in the silencing of the two miR-15/16 loci, we identified a genetic loss of miR-15a and miR-15b and silencing of these two loci by methylation. We identified a potential driver oncogenic role in the loss of expression of both miR-15/16 clusters in the progression of MDS into AML and in AML pathogenesis. The stratification of AML patients, based on miR-15/16 expression, can lead to targeted and combination therapies for the treatment of this incurable disease.

MicroRNAs (miRNAs) are small noncoding RNAs that act as endogenous regulatory molecules targeting specific mRNAs for translational repression. Studies of breast cancer genomics indicate that breast cancer subtypes are distinguished and regulated by specific sets of miRNAs which affect activities such as tumor initiation, progression, and even drug response. Polo-like Kinase 1 (PLK1) is widely considered to be a proto-oncogene due to its increased expression in multiple tumor types, as well as its crucial role in regulating mitosis. Pharmacological inhibition of PLK1 can reduce tumor volume and induce tumor cell death in solid and hematologic malignancies. This prompted us to investigate how PLK1 inhibition with the target-specific inhibitor NMS-P937 would impact breast cancer cells, and how miRNAs may influence the overall response of these cells to this inhibition. We found that miR-183-5p targets PLK1 gene, effectively reducing its protein expression. Such miRNA-driven regulation of PLK1 expression sensitizes breast cancer cells to NMS-P937, resulting in synergistically increased apoptosis. We also show that the miRNA-regulated reduction of PLK1 influences the expression of apoptosis-related key proteins and possibly inducing further indirect PLK1 downmodulation through a DNMT1-p53 axis. These results suggest a potential biologically significant link between the expression of miR-183-5p and the efficacy of PLK1-specific inhibitors in breast cancer cells. Our work further elucidates how miR-183-5p regulates PLK1 gene while also enhancing NMS-P937 effect in breast cancer. Future studies assessing the role of miR-183-5p as a novel biomarker for anti-PLK1 chemotherapy agents are warranted.

Background Pancreatic and peri-pancreatic neoplasms encompass a variety of histotypes characterized by a heterogeneous prognostic impact. miRNAs are considered efficient candidate biomarkers due to their high stability in tissues and body fluids. We applied Nanostring profiling of circulating exosomal miRNAs to distinct pancreatic lesions in order to establish a source for biomarker development. Methods A series of 140 plasma samples obtained from patients affected by pancreatic ductal adenocarcinoma (PDAC, n = 58), pancreatic neuroendocrine tumors (PanNET, n = 42), intraductal papillary mucinous neoplasms (IPMN, n = 20), and ampulla of Vater carcinomas (AVC, n = 20) were analyzed. Comprehensive miRNA profiling was performed on plasma-derived exosomes. Relevant miRNAs were validated by qRT-PCR and in situ hybridization (ISH). Results Lesion specific miRNAs were identified through multiple disease comparisons. Selected miRNAs were validated in the plasma by qRT-PCR and at tissue level by ISH. We leveraged the presence of clinical subtypes with each disease cohort to identify miRNAs that are differentially enriched in aggressive phenotypes. Conclusions This study shows that pancreatic lesions are characterized by specific exosomal-miRNA signatures. We also provide the basis for further explorations in order to better understand the relevance of these signatures in pancreatic neoplasms.

Tumor progression and metastasis are the major causes of death among cancer associated mortality. Metastatic cells acquire features of migration and invasion and usually undergo epithelia-mesenchymal transition (EMT). Acquirement of these various hallmarks rely on different cellular pathways, including TGF-β and Wnt signaling. Recently, we reported that WW domain-containing oxidoreductase (WWOX) acts as a tumor suppressor and has anti-metastatic activities involving regulation of several key microRNAs (miRNAs) in triple-negative breast cancer (TNBC). Here, we report that WWOX restoration in highly metastatic MDA-MB435S cancer cells alters mRNA expression profiles; further, WWOX interacts with various proteins to exert its tumor suppressor function. Careful alignment and analysis of gene and miRNA expression in these cells revealed profound changes in cellular pathways mediating adhesion, invasion and motility. We further demonstrate that WWOX, through regulation of miR-146a levels, regulates SMAD3, which is a member of the TGF-β signaling pathway. Moreover, proteomic analysis of WWOX partners revealed regulation of the Wnt-signaling activation through physical interaction with Disheveled. Altogether, these findings underscore a significant role for WWOX in antagonizing metastasis, further highlighting its role and therapeutic potential in suppressing tumor progression.

// Farzaneh Moshiri 1, 2 , Alessandro Salvi 3 , Laura Gramantieri 4 , Angelo Sangiovanni 5 , Paola Guerriero 1 , Giuseppina De Petro 3 , Cristian Bassi 1 , Laura Lupini 1 , Arash Sattari 2 , Douglas Cheung 2 , Dario Veneziano 2 , Giovanni Nigita 2 , Ram C. Shankaraiah 1 , Nazario Portolani 6 , Paolo Carcoforo 1 , Francesca Fornari 4, 7 , Luigi Bolondi 4, 7 , Antonio Frassoldati 8 , Silvia Sabbioni 9 , Massimo Colombo 5 , Carlo M. Croce 2 and Massimo Negrini 1 1 Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy 2 Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA 3 Department of Molecular and Translational Medicine, Division of Biology and Genetics, University of Brescia, Brescia, Italy 4 Center for Applied Biomedical Research, St. Orsola-Malpighi University Hospital, Bologna, Italy 5 Gastroenterology and Hepatology Division, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milano, Milan, Italy 6 Department of Medical and Surgical Sciences, Surgical Clinic, University of Brescia, Brescia, Italy 7 Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy 8 Oncology Division, University Hospital of Ferrara, Cona (FE), Italy 9 Department of Life Sciences and Biotechnology, University of Ferrara, Ferrara, Italy Correspondence to: Massimo Negrini, email: ngm@unife.it Carlo M. Croce, email: Carlo.Croce@osumc.edu Keywords: hepatocellular carcinoma; cirrhosis; circulating microRNA; diagnostic biomarkers Received: March 09, 2017 Accepted: November 05, 2017 Epub: February 27, 2018 Published: March 16, 2018 ABSTRACT Hepatocellular carcinoma (HCC) is the most common liver cancer and second leading cause of cancer related death worldwide. Most HCCs occur in a damaged cirrhotic background and it may be difficult to discriminate between regenerative nodules and early HCCs. No dependable molecular biomarker exists for the early detection of HCC. MicroRNAs (miRNAs) have attracted attention as potential blood-based biomarkers. To identify circulating miRNAs with diagnostic potential in HCC, we performed preliminary RNAseq studies on plasma samples from a small set of HCC patients, cirrhotic patients and healthy controls. Then, out of the identified miRNAs, we investigated miR-101-3p, miR-106b-3p, miR-1246 and miR-411-5p in plasma of independent HCC patients’ cohorts. The use of droplet digital PCR (ddPCR) confirmed the aberrant levels of these miRNAs. The diagnostic performances of each miRNA and their combinations were measured using Receiver Operating Characteristic (ROC) curve analyses: a classifier consisting of miR-101-3p, miR-1246 and miR-106b-3p produced the best diagnostic precision in plasma of HCC vs. cirrhotic patients (AUC = 0.99). A similar performance was found when the levels of miRNAs of HCC patients were compared to healthy controls (AUC = 1.00). We extended the analyses of the same miRNAs to serum samples. In serum of HCC vs. cirrhotic patients, the combination of miR-101-3p and miR-106b-3p exhibited the best diagnostic accuracy with an AUC = 0.96. Thus, circulating miR-101-3p, miR-106b-3p and miR-1246, either individually or in combination, exhibit a considerable potential value as diagnostic biomarkers of HCC.

Introduction: Extracellular vesicles (EVs) are membrane vesicles containing proteins and nucleic acids that aresecreted by all cells and circulate in the blood. In the tumor microenvironment their contents havebeen shown to carry important pro-carcinogenic molecules. Soft tissue sarcoma of theretroperitoneum of which the most common types are well differentiated/dedifferentiatedliposarcoma (WD/DDLPS) are marked by recurrence rates of >50% even after complete resection.A molecular hallmark of RLS WD/DDLPS are high levels of MDM2 DNA, a finding observed innearly all WD/DDLPS tumors. Indeed, FISH assessment of MDM2 in resected tissues is currentlythe definitive diagnostic methodology for WD/DDLPS. Prognosis in WD/DDLPS is enhanced byearly detection of recurrent lesions. However, current scanning technologies cannot resolverecurrent liposarcoma vs postoperative scarring in many patients (97% sensitivity/51%specificity); the need for easily retrieved and analyzed circulating factors that are diagnostic andprognostic is urgent. Methods: A cohort of 11 WD/DDLPS serum samples were collected at the time of surgery and during followup visits (6-12 months after surgery). Serum from normal healthy people (N=15) were used as acontrol. EVs were isolated from the serum through precipitation technology (ExoQuick) andultracentrifugation. The EVs-DNA was then isolated and the level of MDM2 was measured by Q-PCR. The level of MDM2 DNA in the WD/DDLPS tissues was also measured by Q-PCR andcompared with Normal Adjacent tissues. Results: MDM2 DNA was detected in EVs of the serum of RLS WD/DDLPS patients at significantly higherlevels than normal controls (geometric mean MDM2 DNA number of molecules value=74,434 vs10,146, p=0.0025). This increase was concordant with the level of MDM2 DNA as measured inWD/DDLPS tissues compared to normal adjacent tissues (p= 0.0009). Moreover, serumWD/DDLPS EV MDM2 was dramatically decreased in RLS WD/DDLPS patients after tumorresection (geometric mean MDM2 DNA number of molecules value pre-op=74,434 vs post-op=13,879, p=0.0048), and appears to correlate with CT-scan evidence of recurrent or persistentdisease after surgery. Importantly, the level of MDM2 DNA in EV's post-operatively decreasedreaching a level similar to the level of MDM2 in the controls (difference between post- surgeryand controls: p=0.47). Conclusion: In conclusion RLS WD/DDLPS EVMDM2 may potentially serve as an efficacious RLSWD/DDLPS first-ever prognostic and therapy predictive biomarker for a lethal disease currentlylacking such determinants. Citation Format: Lucia Casadei, Patricia Sarchet, Luciano Cascione, Giovanni Nigita, Fernanda Costas Casal de Faria, Carlo Maria Croce, Valerie Grignol, Raphael E. Pollock. Extracellular vesicle - MDM2 as liquid biopsy biomarker for disease identification in retroperitoneal liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 474.

In the last two decades noncoding RNAs have been the recipients of increasing scientific interest. In particular, miRNAs, short (~22 nts) noncoding transcripts, have been thoroughly investigated since their essential role in posttranscriptional gene expression regulation had been established in the early 2000s. With the advent and the advancements of high-throughput sequencing technologies in recent years, long noncoding RNAs have also started to emerge as important actors in cellular functions and processes. Such transcripts, on average longer than 200 nt, whose functions have yet to be fully characterized, have recently been identified as regulatory elements of the RNAi pathway, harboring several miRNA response elements, uncovering the phenomena of competing endogenous RNAs (ceRNAs), or "sponge RNAs." The present chapter aims to provide a brief update on the actual biomedical relevance of ceRNAs, together with a summary of resources, tools, and practical examples of their application to aid researchers in the discovery and further elucidation of lncRNA-miRNA interactions.