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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.

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.

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.

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.

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.

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.

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.

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.

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.

Germ cell tumors (GCT) are the most common malignancies in males between 15 and 35 years of age. Despite the high cure rate, achieved through chemotherapy and/or surgery, the molecular basis of GCT etiology is still largely obscure. Here, we show a positive correlation between MAPK15 (ERK8; ERK7) expression and specific GCT subtypes, with the highest levels found in the aggressive embryonal carcinomas (EC). Indeed, in corresponding cellular models for EC, MAPK15 enhanced tumorigenicity in vivo and promoted cell proliferation in vitro, supporting a role for this kinase in human GCT. At molecular level, we demonstrated that endogenous MAPK15 is necessary to sustain cell cycle progression of EC cells, by limiting p53 activation and preventing the triggering of p53-dependent mechanisms resulting in cell cycle arrest. To understand MAPK15-dependent mechanisms impinging on p53 activation, we demonstrate that this kinase efficiently protects cells from DNA damage. Moreover, we show that the ability of MAPK15 to control the autophagic process is necessary for basal management of DNA damage and for tumor formation controlled by the kinase. In conclusion, our findings suggest that MAPK15 overexpression may contribute to the malignant transformation of germ cells by controlling a "stress support" autophagic pathway, able to prevent DNA damage and the consequent activation of the p53 tumor suppressor. Moreover, in light of these results, MAPK15-specific inhibitors might represent new tools to enhance the therapeutic index of cytotoxic therapy in GCT treatment, and to increase the sensitivity to DNA-damaging drugs in other chemotherapy-resistant human tumors.

MicroRNAs (miRs), a class of regulatory single-stranded small non-coding RNAs, are frequently deregulated in cancer and have been proposed as diagnostic and prognostic biomarkers in this fatal disease. Recent improvements in high-throughput sequencing (HTS) technologies have allowed the discovery of post-transcriptional modification phenomena involving miRs (e.g., isomiRs, A-to-I editing sites) able to alter the miR canonical sequence and thus their function. However, albeit earlier studies have proposed the exploitation of modified miRs as potential cancer biomarkers, their comprehensive and concurrent profiling, in cancer, still lacks. In light of this, we have applied the miRge2 pipeline (PubmedID:30153801) to more than 12K samples across 38 different cancer types from TCGA and TARGET, aiming at profiling the expression of all detectable canonical and modified miR isoforms in cancer. We identified over 29K unique miR isoforms (21 novel ones) expressed in at least one tumor type, whereby about 85% contains at least one modification (e.g., SNPs, RNA editing). According to our results, taking into account both canonical and modified miR isoforms throughout the analysis leads to an increase of about 20-fold in the identification of potential diagnostic and prognostic miRs with respect to conventional methods, which exclusively consider canonical miRs (miRBase v22). Then, leveraging on an independent cohort of 38 colorectal cancers and 21 adjacent healthy tissues (EGAS00001001127), we found that ~600 significantly deregulated miR isoforms were in common with the ones derived from the TCGA COAD dataset. Focusing on two miR isoforms, the canonical miR-151a-3p and its isoform (miR-151a-3p_-2|-2) with two left-shifted residues in both 5' and 3' end, which we found to be up-regulated in both cohorts, we performed a target prediction coupled with a miR/protein anti-correlation analysis. Interestingly, we found that the two miRs have a distinct set of targets. Finally, employing an Ingenuity(R) Pathway Analysis on their predicted targets, we observed that miR-151a-3p_-2|-2, in contrast to the canonical counterpart, is significantly involved in oncogenic pathways, such as PAK and CXCR4 signaling. This suggests that the final tumor phenotype could be substantially affected by modified miR isoforms. Here, we present one of the most in-depth and extensive miRNome profiling in cancer performed to date, revealing one magnitude of novel potential diagnostic and prognostic cancer biomarkers and enlightening the contribution of miR isoforms to the wider miRNome in cancer pathogenesis. Citation Format: Rosario Distefano, Luisa Tomasello, Gioacchino P. Marceca, Marina Bagnoli, Alessandro Lagana', Mario Acunzo, Chi Song, Pierluigi Gasparini, Giovanni Nigita, Carlo M. Croce. Concurrent profiling of canonical and modified miRNAomes from TCGA and TARGET cohorts leads to enhanced resolution in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2543.

RNA editing in microRNAs has been recently proposed as a novel biomarker in cancer. Here, we investigated RNA editing by leveraging small-RNA sequencing data from 87 NSCLC (Non-Small Cell Lung Cancer) samples paired with normal lung tissues from The Cancer Genome Atlas (TCGA) combined with 26 plasma-derived exosome samples from an independent cohort. Using both the editing levels and microRNA editing expression, we detected deregulated microRNA editing events between NSCLC tumor and normal tissues. Interestingly, and for the first time, we also detected editing sites in the microRNA cargo of circulating exosomes, providing the potential to non-invasively discriminate between normal and tumor samples. Of note, miR-411-5p edited in position 5 was significantly dysregulated in tissues as well as in exosomes of NSCLC patients, suggesting a potential targetome shift relevant to lung cancer biology.

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

The World Health Organization has recently introduced molecular prognostic-diagnostic biomarkers in the classification of Central Nervous System (CNS) tumors. In order to characterize subclasses of tumors that cannot find a precise location in the current classification, and, or cannot be tested because of scant material, it is important to find new molecular biomarkers in tissue and, or biological fluid samples. In this study, we identified serum microRNAs that could serve as biomarkers for the diagnosis and prognosis of patients with tumors of glial origin. We retrospectively analyzed microRNA expression in the serum extracellular vesicles of patients with tumors of glial origin. Extracellular vesicles RNA was analyzed by Nanostring. qRT-PCR confirmed 6 overexpressed microRNAs: hsa-miR-4443, hsa-miR-422a, hsa-miR-494-3p, hsa-miR-502-5p, hsa-miR-520f-3p, and hsa-miR-549a. Hsa-miR-4443 was the only microRNA that showed significant differences in most comparisons. In situ hybridization (ISH), confirmed that our signature was mostly expressed in cancer cells. Importantly, hsa-miR-549a and hsa-miR-502-5p expression predicted prognosis in patients with tumors of glial origin. Although more studies are needed, we demonstrated that serum vesicles microRNA profiles are promising diagnostic and prognostic molecular biomarkers that will find an actual application in the clinical practice of CNS tumors., Highlights • We identified a microRNA signature in the serum extracellular vesicles of patients with glial tumors. • The signature was differentially expressed among tumors and controls, showing potentials in the diagnosis of Gliomas. • Hsa-miR-549a and hsa-miR-502-5p expression predicted prognosis in patients with tumors of glial origin. Research in context To find targeted therapies and patients sensitive to specific treatments, we need to better classify different groups of brain tumors identifying novel molecular biomarkers. The most common brain tumors are Gliomas. In this article, we identified a group of very short genes (microRNAs) in the extra-cellular vesicles of the serum of 8 normal and of 28 patients with gliomas. The expression of such genes differs among the different groups of tumors and normal patients, and can help in the diagnosis of gliomas. Interestingly, two microRNAs could predict the outcome of the disease and might find future clinical applications.

Drug resistance imposes severe limitations to the efficacy of targeted therapy in BRAF-mutated metastatic melanoma. Although this issue has been mitigated by the development of combination therapies with BRAF plus MEK inhibitors, drug resistance inevitably occurs with time and results in clinical recurrences and untreatable disease. Hence, there is strong need of developing new combination therapies and non-invasive diagnostics for the early identification of drug-resistant patients. We report here that the development of drug resistance to BRAFi is dominated by a dynamic deregulation of a large population of miRNAs, leading to the alteration of cell intrinsic proliferation and survival pathways, as well as of proinflammatory and proangiogenic cues, where a prominent role is played by the miR-199b-5p/VEGF axis. Significant alterations of miRNA expression levels are detectable in tumor biopsies and plasma from patients after disease recurrence. Targeting these alterations blunts the development of drug resistance.

Mutated protein-coding genes drive the molecular pathogenesis of many diseases, including cancer. Specifically, mutated KRAS is a documented driver for malignant transformation, occurring early during the pathogenesis of cancers such as lung and pancreatic adenocarcinomas. Therapeutically, the indiscriminate targeting of wild-type and point-mutated transcripts represents an important limitation. Here, we leveraged on the design of miRNA-like artificial molecules (amiRNAs) to specifically target point-mutated genes, such as KRAS, without affecting their wild-type counterparts. Compared with an siRNA-like approach, the requirement of perfect complementarity of the microRNA seed region to a given target sequence in the microRNA/target model has proven to be a more efficient strategy, accomplishing the selective targeting of point-mutated KRAS in vitro and in vivo.

Background: We previously identified miR-135b as a possible biomarker for gemcitabine sensitivity in metastatic breast cancer patients. Here we aimed to better understand the role of this microRNA in breast cancer cells in order to identify new clinically relevant approaches to revert gemcitabine resistance. Methods: miR-135b KO MDA-MB-231 breast cancer cells were generated through CRISPR-Cas9 technology and tested for gemcitabine sensitivity by cell survival and apoptosis activation assays. Gene expression profile was performed by GeneChip® Human Transcriptome Array 2.0 (HTA 2.0 - Affymetrix). The One-Way between-subject ANOVA algorithm was used to calculate statistical significance of pairwise comparisons. The validation of different gene expression was assessed by qRT-PCR. The mesenchymal phenotype of miR-135b KO cells was tested by wound-healing and migration assay. mTOR pathway has been tested by Western blot analysis. Results: miR-135b KO MDA-MB-231 showed increased resistance to gemcitabine than WT cells. Gene expression profiling of miR-135b WT and KO cells revealed the upregulation of several genes, including miR-21 and gene involved in the epithelial-to-mesenchymal transition (EMT) in the absence of the microRNA. Functional enrichment analysis indicated the deregulation of multiple tumor-associated pathways, including focal adhesion, senescence and autophagy. Accordingly, miR-135b KO cells showed higher migrating properties and expression of mesenchymal genes. Moreover, miR-135b KO cells showed deregulation of the mTOR-signaling pathway upon starvation or treatment with gemcitabine. Based on these observations, we assessed whether inhibition of mTOR activation using a clinically approved inhibitor (everolimus) could enhance gemcitabine sensitivity in the absence of miR-135b. Our data indicate that blocking mTOR pathway could represent a valid approach to revert resistance to gemcitabine even in cancer cells with low levels of miR-135b. Conclusions: We identified several deregulated genes and pathways in the absence of miR-135b in breast cancer cells, in particular related to the EMT and mTOR pathway. These results confirm the role of miR-135b in breast cancer and pave the way for new studies for the combination of gemcitabine and mTOR inhibitors in a specific subset of metastatic breast cancer patients that could largely benefit from it. Citation Format: Anna Tessari, Meghan Pawlikowski, Kareesma Parbhoo, Olivia Willetts, Marianna Hernandez, Giovanni Nigita, Ashley Braddom, Joseph J. Mills, Dario Palmieri, Carlo M. Croce. miR-135b mediates gemcitabine sensitivity in breast cancer cells by modulating epithelial-to-mesenchymal transition and mTOR-signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 473.

RNA editing is a finely tuned, dynamic mechanism for post-transcriptional gene regulation that has been thoroughly investigated in the last decade. In this work we analyse the variations in microRNA seed region editing activity during a time course of a hypoxia-exposed breast cancer cell line. By leveraging on recent innovative bioinformatics approaches applied to a more informative experimental context, we have analysed small RNA-seq (sRNA-seq) data obtained from a hypoxia-treated breast cancer cell line over time, focusing specifically on miRNA post-transcriptional modifications occurring in the miRNA seed region (MSR), with particular attention to A-to-I editing events. To estimate the impact of editing on miRNA function during hypoxia progression, we decided to investigate A-to-G miRNA modifications, as these are expected to be the result of A-to-I editing. Specifically, we decided to consider all A-to-I editing events occurring in MSRs (2-8 nt seed region). Subsequently, in order to compare the target sets of the wild type (WT) and edited (ED) versions of each miRNA, we performed binding site prediction on gene 3’-UTRs by multiple prediction tool consensus. Furthermore, our target prediction results were validated experimentally. Finally, employing gene microarray data associated to the sRNA-seq data, we performed differential gene expression analysis followed by functional enrichment, focusing on hypoxia-related pathways, such as VEGF and PI3K/Akt, to evaluate how the miRNA editing phenomenon could affect these pathways during the hypoxic time course. We identified a total of 29 statistically significant canonical and non-canonical editing sites in 20 different miRNAs, with 8 sites previously known. The average modification levels of the majority of MSRs generally increased with time of exposure to hypoxia. We found that microRNA editing events in the seed region are not depended on microRNA expression. Additionally, the evidence that emerges from our functional data shows, indeed, how the editing of certain miRNAs is significantly associated with the cellular response to hypoxic stimulus, unprecedentedly providing insights on the meaning of the target set shift derived from these modifications. This reveals that microRNA editing acts under the influence of environmentally induced stimuli. Our results, thus, show a microRNA editing activity trend in favour of cellular pathways closely associated to hypoxia, providing novel insights on the functional role of miRNA seed region editing. This provides a foundation for further elucidation of the miRNA editing phenomenon, giving a direction to future innovative investigation of its biological importance and potential involvement in physiological and pathological cellular changes. Citation Format: Mario Acunzo, Giovanni Nigita, Giulia Romano, Dario Veneziano, Alessandro Lagana’, Dorothee Wernicke, Alfredo Ferro, Carlo Croce. miRNA editing in seed region is in synergy with cellular changes in hypoxic conditions. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-166.

540 Background: Collecting duct carcinoma (CDC) of the kidney is a rare tumor, originating from renal medulla, characterized by younger age at diagnosis and poor prognosis. Since targeted therapies are currently not available for CDC, we aimed to identify the most deregulated pathways in this type of cancer to gain insights into new molecular targets. Methods: Total RNA was extracted from FFPE samples of primary CDC (n=9), clear cell carcinoma (CCC, n=7) and healthy normal (n=7) adjacent renal tissues (23 total samples). Gene expression profile was performed by GeneChip Human Transcriptome Array 2.0 (HTA 2.0 -Affymetrix). The One-Way between-subject ANOVA algorithm was used to calculate statistical significances of pairwise comparisons. Transcripts with a linear fold change (f.c.) of 2 (p-value

Introduction JMML is a rare leukemia characterized by aberrant myeloid proliferation and hypersensitivity to GM-CSF. Mutually exclusive mutations in PTPN11, N/K-RAS, CBL, or NF1 are found in ~90% of patients. These mutations cause the disease at least in part by activating STAT5 through phosphorylation and promoting cell growth. MicroRNAs (miRs) are small (~22 nucleotides) noncoding RNAs, regulating gene expression and often deregulated in leukemia. We investigated whether miRs are deregulated in JMML and whether miRs target critical proteins involved in the disease pathogenesis. Patients and Methods MiRs expression profile of 40 bone marrow (BM) samples from untreated JMML patients and 8 BM samples from healthy controls was performed using the Ncounter Human v2 miRNA Expression Assay (nanostring). MiR150-5p and STAT5b mRNA expression were assessed using quantitative (q)RT-PCR. Selected miRs and target expression were measured in BM and spleen from a JMML (PTPN11) murine model (D61Y mutation). We overexpressed miR-150-5p in AML cell lines (K562, OCI-AML-3, KG1a) using a miR-150-5p precursor plasmid and STAT5b mRNA was assessed by qRT-PCR. STAT5 and phospho-STAT5 protein levels were assessed by Western Blot in miR-150-5p transfected AML cell lines, human JMML and mice BM cells. STAT5b 3'UTR sequence was cloned in a Firefly/Renilla Luc construct and co-transfected with miR-150-5p miRNA mimic into 293T cell line. Results We found that 25 miRs were differentially expressed in whole BM cells from JMML patients respect to healthy controls (Table 1). MiR-150-5p was the most downregulated miR in JMML. We focused on miR-150-5p, since it has been described to be downregulated in AML cases and is predicted to target STAT5b, a critical gene in JMML biology. We validated that miR-150-5p was down-regulated in JMML cases respect to controls performing qRT-PCR on 38 BM samples from JMML patients. Likewise, miR-150-5p was downregulated in BM and spleen samples from PTPN11 mutated mice respect to controls (0.35 and 0.27 Average Fold Change decrease respectively). STAT5 protein, a predicted target for miR-150-5p, was highly expressed in the JMML patients and mice BM samples respect to their controls (4.3 and 1.3 Average Fold Change increase respectively). MiR-150-5p overexpression in K562, OCI-AML-3 and KG1a cell lines led to decrease of STAT5 protein levels and phosphorilation at 48 hours. Direct interaction of STAT5b 3'UTR with miR-150-5p was demonstrated by luciferase assay (~50% Luc activity inhibition, P Discussion We showed that miR-150-5p is downregulated in JMML samples and in JMML animal models. Functionally, miR-150-5p directly inhibits the translation of STAT5b mRNA resulting also in a decrease of phosphorylation of STAT5 total protein. These findings identify an alternative mechanism that supports STAT5 deregulation in JMML and that could be therapeutically targeted. Table 1. Deregulated miRNAs in JMML Gene Accession # P value Fold-Change (Log2) miR-150-5p MIMAT0000451 0,001 -2,382 let-7g-5p MIMAT0000414 0,002 -1,660 miR-1260a MIMAT0005911 0,009 -1,592 let-7a-5p MIMAT0000062 0,010 -1,574 miR-4454 MIMAT0018976 0,021 -1,399 miR-148a-3p MIMAT0000243 0,030 -1,211 miR-146b-5p MIMAT0002809 0,009 -1,084 miR-342-3p MIMAT0000753 0,010 -1,075 let-7f-5p MIMAT0000067 0,021 -1,022 miR-26a-5p MIMAT0000082 0,034 -1,008 let-7d-5p MIMAT0000065 0,038 -1,005 miR-30b-5p MIMAT0000420 0,019 -0,972 miR-29b-3p MIMAT0000100 0,044 -0,956 miR-29a-3p MIMAT0000086 0,024 -0,761 miR-338-3p MIMAT0000763 0,042 0,654 miR-23a-3p MIMAT0000078 0,040 0,689 miR-222-3p MIMAT0000279 0,018 0,756 miR-548ai MIMAT0018989 0,009 0,993 miR-494 MIMAT0002816 0,023 1,021 miR-320e MIMAT0015072 0,007 1,175 miR-224-5p MIMAT0000281 0,024 1,227 miR-4508 MIMAT0019045 0,016 1,369 miR-575 MIMAT0003240 0,014 1,429 miR-3195 MIMAT0015079 0,014 1,432 miR-630 MIMAT0003299 0,001 2,272 Figure 1. miR-150-5p Relative Expression for each subset of JMML patients with different mutational profiles and Healthy Controls Figure 1. miR-150-5p Relative Expression for each subset of JMML patients with different mutational profiles and Healthy Controls Disclosures No relevant conflicts of interest to declare.

Background: MicroRNAs are a class of small non-coding RNAs that modulate gene expression at post-transcriptional level, resulting crucial in many physiological and pathological processes. Their involvement in cancer initiation and tumor progression has been clearly established, as well as their linkage with chemoresistance. Gemcitabine is a nucleoside analog used in the treatment of metastatic breast cancer (MBC), characterized by a favorable safety profile. Only a small percentage of pts show a strong and prolonged response to this drug, thus suggesting the need of predictive biomarkers. Here, we aimed to identify microRNAs whose expression could be related to gemcitabine sensitivity in MBC. Methods: 24 MBC pts were treated with gemcitabine as single-agent therapy from 1999 to 2011 at Fondazione IRCCS Istituto Nazionale Tumori (Milan). They were selected and classified by the response to the treatment (11 gemcitabine sensitive, stable or partial response after >24 weeks of treatment, and 13 gemcitabine resistant, progressive disease within 16 weeks from the first drug administration). MicroRNA expression profiling was performed by Nanostring nCounter Technology using RNA from FFPE metastatic lesions. Biological normalization was executed to correct for differences in sample abundances. Each sample was normalized to the geometric mean of the top 100 most highly expressed microRNAs. Student's t test was used to calculate statistical significances of pairwise comparisons. The nCounter analysis was validated on a wider population of 50 MBC pts (17 sensitive and 33 resistant) by qRT-PCR. The correlation between microRNA expression levels and gemcitabine half maximal inhibitory concentration (IC50) was evaluated using different BC cell lines. Finally, gemcitabine sensitivity was assessed on these cell lines by cell survival assays following exogenous modulation of microRNA expression levels. Results: Median age at diagnosis was 47 yrs (range 21-74 yrs). Hormone receptors were positive in thirty-one pts (62%), while Her2 in 15 cases (30%). Soft tissues were the most frequent site of metastasis (84%), followed by bone (64%), liver (36%), lung (34%) and CNS (16%). Sixteen pts (32%) had local recurrences. Median number of chemotherapies and endocrine therapies received prior to gemcitabine was 3 (range 1-6) and 2 (range 1-5) respectively. From the nCounter analysis, a subset of 20 microRNAs was significantly deregulated (p Conclusions: We identified miR-135b, miR-146b and miR-155 as potentially predictive for gemcitabine sensitivity in MBC pts. In particular, miR-135b appears to be causally involved in BC cells response to gemcitabine. These results pave the way to a better understanding of the molecular mechanisms underlying gemcitabine resistance in BC, and may have a clinical impact to identify those patients expected to obtain strong and prolonged benefits from this well-tolerated treatment. Citation Format: Anna Tessari, Dario Palmieri, Giovanni Nigita, Dario Veneziano, Sara Cresta, Biagio Paolini, Maria Silvia Cona, Taylor Vargo, Erika Reese, Tyler Sheetz, Vincenzo Coppola, Filippo De Braud, Carlo M. Croce. Role of miR-135b in gemcitabine sensitivity for metastatic breast cancer patients. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C17.