microRNA Cluster Research Articles

Abstract 1097: The miR-17-92 microRNA cluster plays a crucial role in osteosarcoma progression

Abstract Osteosarcoma is the most common primary bone malignancy that affects adolescents. Around 30% of patients with localized osteosarcoma and 70% of patients with metastasis will experience treatment failure within 5 years of diagnosis. The complex biology of osteosarcoma and astounding genetic heterogeneity has made it challenging to identify effective new gene targets and therapeutic agents. Our studies found an overall overexpression of a microRNA cluster, miR-17-92 microRNAs in human primary osteosarcoma compared to normal bone. We learned that upregulation of this miR-17-92 cluster simultaneously silences a suite of key tumor suppressors. Using data from a novel spontaneous osteosarcoma mouse model and genetic screen, we discovered miR-17-92 targets, such as PTEN, PTRPD and SRGAP2, which are potential tumor suppressor genes. Specifically blocking miR-17-92 function in osteosarcoma cells reduced their migration and ability to grow larger tumors in immunodeficient mice compared to the controls. Also, knockdown of miR-17-92 cluster microRNAs led to increase in the levels of PTPRD and SRGAP2 in cells as well as tumors; further suggesting that miR-17-92 is targeting these genes. We also performed gain-of-function of miR-17-92 studies in a poorly aggressive osteosarcoma cell line and found that overexpression of miR-17-92 leads to ability to grow in an anchorage independent manner and form tumors in immunodeficient mice, both features that are lacking in the parental line. Ongoing RNA sequencing studies on miR-17-92 target transcripts in osteosarcoma cells, and functional analyses of miR-17-92 deletion mutants, will be presented. In an attempt to target miR-17-92 miRNA expression, we have tested small molecules. Our data suggests that triptolide, a diterpenoid epoxide, inhibits MYC expression and downregulates miR-17-92 miRNAs resulting in upregulation of several tumor suppressor driver proteins including PTEN, PTPRD, and SRGAP2. Together, our data suggests that upregulation of miR-17-92 miRNAs contributes to osteosarcoma progression and triptolide inhibits miR-17-92 expression. These data have implications for how sarcomas develop in general and suggest a new way to treat cancer by targeting microRNAs using small molecules. Citation Format: Jyotika Varshney, Nicholas J. Slipek, John Osborne, Adrian Chang, Anne E. Sarver, Ingrid Cornax, Gerry M. O’ Sullivan, Subbaya Subramanian, David A. Largaespada. The miR-17-92 microRNA cluster plays a crucial role in osteosarcoma progression. [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 1097.

Abstract 970: Circulating microRNA signature in group 4 medulloblastoma patients

Abstract Medulloblastoma is the most common malignant brain tumor in childhood. Four main clinically and molecularly distinct MB subtypes have been identified: WNT, SHH, Group 3, and Group 4. Among them, Group 4 MB (G4MB) appears to have the most skewed age ratio, is predominant in males and shows an intermediate prognosis. In spite of being the most frequent (35%), it is also the least understood of all the subgroups. Diagnosis is based on patients’ clinical symptoms and neuro-imaging techniques with high risk of false positive and/or late tumor discovery. The early detection of the disease is essential to reduce both cancer mortality and long time therapy related toxicity. To date, there is no reliable marker for G4MB that could facilitate the early diagnosis and allow the evaluation of the response to radio and chemotherapy treatment. Therefore, the discovery of new biomarkers as a specific signature of G4MB patients, potentially useful as diagnostic factor, would be extraordinarily worthwhile. MicroRNAs are single-stranded non coding RNA molecules of 19-24 nucleotides in length involved in the post-transcriptional regulation of gene expression, whose expression is strongly deregulated in several types of tumors. Recent studies have shown that microRNAs are released into the bloodstream and are stably detectable thanks to their association with extracellular vescicle, or complex with RNA-binding protein that protect them from RNAse degradation. The minimally invasive liquid biopsy flanked to the reliability and specificity of microRNAs in cancer detection indicates circulating microRNAs not only as promising biomarkers to get early diagnosis, but also to follow treatment response and detect recurrence. Here we report a high-throughput screening of microRNAs in G4MB plasma samples before surgery and during follow-up. Two different approaches (DeepSeq and Low Denstity Array) were used to detect the differentially expressed circulating microRNAs in 4 patients and in 4 age- and sex- matched healthy donors. Baseline microRNA expression profiling allowed us to disclose a signature of 34 miRNAs able to identify patients versus healthy children. Moreover, selected microRNAs were validated by RT-qPCR technology on a wider cohort of G4MB patients (n = 8) and healthy donors (n = 8). Specifically, we identified two important oncogenic microRNA clusters (miR-17/92 and miR-106b/25) significantly upregulated in G4MB plasma patients. Therapy reduced the expression of these microRNAs up to levels of healthy donors. In conclusion we identified circulating microRNA signature in G4MB. Citation Format: Evelina Miele, Vincenzo Alfano, Zein Mersini Besharat, Giuseppina Catanzaro, Angela Mastronuzzi, Andrea Carai, Agnese Po, Luana Abballe, Antonella Cacchione, Franco Locatelli, Elisabetta Ferretti. Circulating microRNA signature in group 4 medulloblastoma patients. [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 970.

Loss of MYC and E-box3 binding contributes to defective MYC-mediated transcriptional suppression of human MC-let-7a-1~let-7d in glioblastoma.

Previously, we reported that MYC oncoprotein down-regulates the transcription of human MC-let-7a-1~let-7d microRNA cluster in hepatocarcinoma (HCC). Surprisingly, in silico analysis indicated that let-7 miRNA expression levels are not reduced in glioblastoma (GBM). Here we investigated the molecular basis of this differential expression. Using human GBM U87 and U251 cells, we first demonstrated that forced over-expression of MYC indeed could not down-regulate the expression of human MC-let-7a-1~let-7d microRNA cluster in GBM. Furthermore, analysis of MC-let-7a-1~let-7d promoter in GBM indicated that MYC failed to inhibit the promoter activity. Pearson's correlation and Linear Regression analysis using the expression data from GSE55092 (HCC) and GSE4290 (GBM) demonstrated a converse relationship of MC-let-7a-1~let-7d and MYC only in HCC but not in GBM. To understand the underlying mechanisms, we examined whether MYC could bind to the non-canonical E-box 3 located in the promoter of MC-let-7a-1~let-7d. Results from both chromatin immune-precipitation (ChIP) and super-shift assays clearly demonstrated the loss of MYC and E-box 3 binding in GBM, suggesting for the first time that a defective MYC and E-box3 binding in GBM is responsible for the differential MYC mediated transcriptional inhibition of MC-let-7a-1~let-7d and potentially other tumor suppressors. MYC and let-7 are key oncoprotein and tumor suppressor, respectively. Understanding the molecular mechanisms of their regulations will provide new insight and have important implications in the therapeutics of GBM as well as other cancers.

Regulation of Nuclear Hormone Receptors by MYCN-Driven miRNAs Impacts Neural Differentiation and Survival in Neuroblastoma Patients

MYCN amplification and MYC signaling are associated with high-risk neuroblastoma with poor prognosis. Treating these tumors remains challenging, although therapeutic approaches stimulating differentiation have generated considerable interest. We have previously shown that the MYCN-regulated miR-17∼92 cluster inhibits neuroblastoma differentiation by repressing estrogen receptor alpha. Here, wedemonstrate that this microRNA (miRNA) cluster selectively targets several members of the nuclear hormone receptor (NHR) superfamily, and we present a unique NHR signature associated with the survival of neuroblastoma patients. We found that suppressing glucocorticoid receptor (GR) expression in MYCN-driven patient and mouse tumors was associated with an undifferentiated phenotype and decreased survival. Importantly, MYCN inhibition and subsequent reactivation of GR signaling promotes neural differentiation and reduces tumor burden. Our findings reveal a key role for the miR-17∼92-regulated NHRs in neuroblastoma biology, thereby providing a potential differentiation approach for treating neuroblastoma patients.

Pervasive microRNA Duplication in Chelicerates: Insights from the Embryonic microRNA Repertoire of the Spider Parasteatoda tepidariorum.

MicroRNAs are small (∼22 nt) noncoding RNAs that repress translation and therefore regulate the production of proteins from specific target mRNAs. microRNAs have been found to function in diverse aspects of gene regulation within animal development and many other processes. Among invertebrates, both conserved and novel, lineage specific, microRNAs have been extensively studied predominantly in holometabolous insects such as Drosophila melanogaster . However little is known about microRNA repertoires in other arthropod lineages such as the chelicerates. To understand the evolution of microRNAs in this poorly sampled subphylum, we characterized the microRNA repertoire expressed during embryogenesis of the common house spider Parasteatoda tepidariorum . We identified a total of 148 microRNAs in P. tepidariorum representing 66 families. Approximately half of these microRNA families are conserved in other metazoans, while the remainder are specific to this spider. Of the 35 conserved microRNAs families 15 had at least two copies in the P. tepidariorum genome. A BLAST-based approach revealed a similar pattern of duplication in other spiders and a scorpion, but not among other chelicerates and arthropods, with the exception of a horseshoe crab. Among the duplicated microRNAs we found examples of lineage-specific tandem duplications, and the duplication of entire microRNA clusters in three spiders, a scorpion, and in a horseshoe crab. Furthermore, we found that paralogs of many P. tepidariorum microRNA families exhibit arm switching, which suggests that duplication was often followed by sub- or neofunctionalization. Our work shows that understanding the evolution of microRNAs in the chelicerates has great potential to provide insights into the process of microRNA duplication and divergence and the evolution of animal development.

PNR-12GENOME-WIDE PROFILING OF EMBRYONAL TUMORS WITH MULTILAYERED ROSETTES (ETMR)

PNR-12. GENOME-WIDE PROFILING OF EMBRYONAL TUMORS WITH MULTILAYERED ROSETTES (ETMR) Sander Lambo1, Jens Bunt2, Christine Schmidt1, Stefan M. Pfister1, Linda J. Richards2, Lukas Chavez1, Andrey Korshunov3, and Marcel Kool1; Division of Pediatric Neurooncology, DKFZ, Heidelberg, Germany; Cortical Development and Axon Guidance, Queensland Brain Institute, Brisbane, Australia; Clinical Cooperation Unit Neuropathology, DKFZ, Heidelberg, Germany ETMRs are rare pediatric brain tumors with a dismal prognosis and largely unknown mechanisms driving the tumors. Characteristic to ETMR tumors are high expression of LIN28A/B and fusion of a Chr19 microRNA cluster (C19MC) to TTYH1 coupled with amplification of the cluster. Our goal is to understand the mechanisms that play a role in tumorigenesis and possibly identify new treatment strategies. We generated genomic data from 23 ETMRs, including three that lack the C19MC amplification, to understand their genome, transcriptome and epigenome. Whole genome sequencing identified no other recurrent genetic events than the fusion and C19MC amplification, frequentlyaccompanied byother complex rearrangements. Interestingly, gene and miRNA expression profiles of ETMRs w/o C19MC amplification wereverysimilar.Analysisof whole genome bisulfite sequencing data supported this similarity, demonstrating large overlapping regions of strong hypomethylation between tumors. Additionally, we generated genome-wide chromatin maps for the major (ENCODE) histone marks. The resulting enhancer landscape, combined with gene and miRNA expression, revealed high similarity with neural tube cells in the forebrain lineage. miRNA target prediction algorithms predicted that NFI transcription factors, highly downregulated in ETMRs, are targeted by multiple miRNAs in C19MC, suggesting a role for NFI genes in ETMR tumorigenesis. Inhibition of NFI protein function in the mouse brain indeed resulted in formation of ectopic rosette-like structures in the forebrain and an increased size of the ventricular zone, but the mice died before tumors could eventually develop. Together our data provides many novel insights into (epi)genetic aberrations and transcriptional networks driving ETMRs. Neuro-Oncology 18:iii7–iii15, 2016. doi:10.1093/neuonc/now067.10 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Small Cell Lung Cancer Screen of Oncology Drugs, Investigational Agents, and Gene and microRNA Expression.

Small cell lung carcinoma (SCLC) is an aggressive, recalcitrant cancer, often metastatic at diagnosis and unresponsive to chemotherapy upon recurrence, thus it is challenging to treat. Sixty-three human SCLC lines and three NSCLC lines were screened for response to 103 US Food and Drug Administration-approved oncology agents and 423 investigational agents. The investigational agents library was a diverse set of small molecules that included multiple compounds targeting the same molecular entity. The compounds were screened in triplicate at nine concentrations with a 96-hour exposure time using an ATP Lite endpoint. Gene expression was assessed by exon array, and microRNA expression was derived by direct digital detection. Activity across the SCLC lines was associated with molecular characteristics using pair-wise Pearson correlations. Results are presented for inhibitors of targets: BCL2, PARP1, mTOR, IGF1R, KSP/Eg5, PLK-1, AURK, and FGFR1. A relational map identified compounds with similar patterns of response. Unsupervised microRNA clustering resulted in three distinct SCLC subgroups. Associating drug response with micro-RNA expression indicated that lines most sensitive to etoposide and topotecan expressed high miR-200c-3p and low miR-140-5p and miR-9-5p. The BCL-2/BCL-XL inhibitors produced similar response patterns. Sensitivity to ABT-737 correlated with higher ASCL1 and BCL2. Several classes of compounds targeting nuclear proteins regulating mitosis produced a response pattern distinct from the etoposide response pattern. Agents targeting nuclear kinases appear to be effective in SCLC lines. Confirmation of SCLC line findings in xenografts is needed. The drug and compound response, gene expression, and microRNA expression data are publicly available at http://sclccelllines.cancer.gov.

Differential MicroRNA Expression Profile in Myxomatous Mitral Valve Prolapse and Fibroelastic Deficiency Valves.

Myxomatous mitral valve prolapse (MMVP) and fibroelastic deficiency (FED) are two common variants of degenerative mitral valve disease (DMVD), which is a leading cause of mitral regurgitation worldwide. While pathohistological studies have revealed differences in extracellular matrix content in MMVP and FED, the molecular mechanisms underlying these two disease entities remain to be elucidated. By using surgically removed valvular specimens from MMVP and FED patients that were categorized on the basis of echocardiographic, clinical and operative findings, a cluster of microRNAs that expressed differentially were identified. The expressions of has-miR-500, -3174, -17, -1193, -646, -1273e, -4298, -203, -505, and -939 showed significant differences between MMVP and FED after applying Bonferroni correction (p < 0.002174). The possible involvement of microRNAs in the pathogenesis of DMVD were further suggested by the presences of in silico predicted target sites on a number of genes reported to be involved in extracellular matrix homeostasis and marker genes for cellular composition of mitral valves, including decorin (DCN), aggrecan (ACAN), fibromodulin (FMOD), α actin 2 (ACTA2), extracellular matrix protein 2 (ECM2), desmin (DES), endothelial cell specific molecule 1 (ESM1), and platelet/ endothelial cell adhesion molecule 1 (PECAM1), as well as inverse correlations of selected microRNA and mRNA expression in MMVP and FED groups. Our results provide evidence that distinct molecular mechanisms underlie MMVP and FED. Moreover, the microRNAs identified may be targets for the future development of diagnostic biomarkers and therapeutics.

MiR-23~27~24 clusters control effector T cell differentiation and function

Abstract Coordinated repression of gene expression by evolutionarily conserved microRNA (miRNA) clusters and paralogs ensures that miRNAs efficiently exert their biological impact. Combining both loss- and gain-of-function genetic approaches, here we show that the miR-23~27~24 clusters regulate multiple aspects of T cell biology, particularly Th2 immunity. Low expression of this miRNA family confers proper effector T cell function at both physiological and pathological settings. Further studies in T cells with exaggerated regulation by individual members of the miR-23~27~24 clusters revealed miR-24 and miR-27 collaboratively limit Th2 responses through targeting IL-4 and GATA3 in both direct and indirect manners. Intriguingly, while overexpression of the entire miR-23 cluster also negatively impacts other Th lineages, enforced expression of miR-24, in contrast to miR-23 and miR-27, actually promotes the differentiation of Th1, Th17 and iTreg cells, implying that under certain conditions, miRNA families can fine tune the biological effects of their regulation by having individual members antagonize rather than cooperate with each other. Together, our results identify a miRNA family with important immunological roles and suggest tight regulation of miR-23~27~24 clusters in T cells is required to maintain optimal effector function and to prevent aberrant immune responses.

Global, cancer-specific microRNA cluster hypomethylation was functionally associated with the development of non-B non-C hepatocellular carcinoma.

BackgroundWhile hepatitis B and C viral infection have been suppressed, non-B non-C hepatocellular carcinoma (NBNC-HCC) is considered to be rising in incidence terms in some developed countries where prevalence of those viral infections among HCC patients had been very high (such as Japan, Korea, and Italy). To elucidate critical molecular changes in NBNC-HCC, we integrated three large datasets relating to comprehensive array-based analysis of genome-wide DNA methylation (N = 43 pairs) and mRNA/miRNA expression (N = 15, and 24 pairs, respectively) via statistical modeling.ResultsHierarchical clustering of DNA methylation in miRNA coding regions clearly distinguished NBNC-HCC tissue samples from relevant background tissues, revealing a remarkable tumor-specific hypomethylation cluster. In addition, miRNA clusters were extremely hypomethylated in tumor samples (median methylation change for non-clustered miRNAs: -2.3%, clustered miRNAs: -24.6%). The proportion of CpGs hypomethylated in more than 90% of the samples was 55.9% of all CpGs within miRNA clusters, and the peak methylation level was drastically shifted from 84% to 39%. Following statistical adjustment, the difference in methylation levels within miRNA coding regions was positively associated with their expression change. Receiver operating characteristic (ROC) analysis revealed a great discriminatory ability in respect to cluster-miRNA methylation. Moreover, miRNA methylation change was negatively correlated with corresponding target gene expression amongst conserved and highly matched miRNA sites.ConclusionsWe observed a drastic negative shift of methylation levels in miRNA cluster regions. Changes in methylation status of miRNAs were more indicative of target gene expression and pathological diagnosis than respective miRNA expression changes, suggesting the importance of genome-wide miRNA methylation for tumor development. Our study dynamically summarized global miRNA hypomethylation and its genome-wide scale consequence in NBNC-HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0514-6) contains supplementary material, which is available to authorized users.

The mirn23a microRNA cluster antagonizes B cell development.

Ablation of microRNA synthesis by deletion of the microRNA-processing enzyme Dicer has demonstrated that microRNAs are necessary for normal hematopoietic differentiation and function. However, it is still unclear which specific microRNAs are required for hematopoiesis and at what developmental stages they are necessary. This is especially true for immune cell development. We previously observed that overexpression of the products of the mirn23a gene (microRNA-23a, -24-2, and 27a) in hematopoietic progenitors increased myelopoiesis with a reciprocal decrease in B lymphopoiesis, both in vivo and in vitro. In this study, we generated a microRNA-23a, -24-2, and 27a germline knockout mouse to determine whether microRNA-23a, -24-2, and 27a expression was essential for immune cell development. Characterization of hematopoiesis in microRNA-23a, -24-2, and 27a-/- mice revealed a significant increase in B lymphocytes in both the bone marrow and the spleen, with a concomitant decrease in myeloid cells (monocytes/granulocytes). Analysis of the bone marrow progenitor populations revealed a significant increase in common lymphoid progenitors and a significant decrease in both bone marrow common myeloid progenitors and granulocyte monocyte progenitors. Gene-expression analysis of primary hematopoietic progenitors and multipotent erythroid myeloid lymphoid cells showed that microRNA-23a, -24-2, and 27a regulates essential B cell gene-expression networks. Overexpression of microRNA-24-2 target Tribbles homolog 3 can recapitulate the microRNA-23a, -24-2, and 27a-/- phenotype in vitro, suggesting that increased B cell development in microRNA-23a, -24-2, and 27a null mice can be partially explained by a Tribbles homolog 3-dependent mechanism. Data from microRNA-23a, -24-2, and 27a-/- mice support a critical role for this microRNA cluster in regulating immune cell populations through repression of B lymphopoiesis.

MicroRNA-17~92 inhibits colorectal cancer progression by targeting angiogenesis

The miR-17~92 microRNA (miRNA) cluster host gene is upregulated in a broad spectrum of human cancers including colorectal cancer (CRC). Previous studies have shown that miR-17~92 promotes tumorigenesis and cancer angiogenesis in some tumor models. However, its role in the initiation and progression of CRC remains unknown. In this study, we found that transgenic mice overexpressing miR-17~92 specifically in epithelial cells of the small and large intestines exhibited decreased tumor size and tumor angiogenesis in azoxymethane and dextran sulfate sodium salt (AOM-DSS)-induced CRC model as compared to their littermates control. Further study showed that miR-17~92 inhibited the progression of CRC via suppressing tumor angiogenesis through targeting multiple tumor angiogenesis-inducing genes, TGFBR2, HIF1α, and VEGFA in vivo and in vitro. Collectively, we demonstrated that miR-17~92 suppressed tumor progression by inhibiting tumor angiogenesis in a genetically engineered mouse model, indicating the presence of cellular context-dependent pro- and anti-cancer effects of miR-17~92.

Dysregulation of a family of short noncoding RNAs, tsRNAs, in human cancer

Chronic lymphocytic leukemia (CLL) is the most common human leukemia, and transgenic mouse studies indicate that activation of the T-cell leukemia/lymphoma 1 (TCL1) oncogene is a contributing event in the pathogenesis of the aggressive form of this disease. While studying the regulation of TCL1 expression, we identified the microRNA cluster miR-4521/3676 and discovered that these two microRNAs are associated with tRNA sequences and that this region can produce two small RNAs, members of a recently identified class of small noncoding RNAs, tRNA-derived small RNAs (tsRNAs). We further proved that miR-3676 and miR-4521 are tsRNAs using Northern blot analysis. We found that, like ts-3676, ts-4521 is down-regulated and mutated in CLL. Analysis of lung cancer samples revealed that both ts-3676 and ts-4521 are down-regulated and mutated in patient tumor samples. Because tsRNAs are similar in nature to piRNAs [P-element-induced wimpy testis (Piwi)-interacting small RNAs], we investigated whether ts-3676 and ts-4521 can interact with Piwi proteins and found these two tsRNAs in complexes containing Piwi-like protein 2 (PIWIL2). To determine whether other tsRNAs are involved in cancer, we generated a custom microarray chip containing 120 tsRNAs 16 bp or more in size. Microarray hybridization experiments revealed tsRNA signatures in CLL and lung cancer, indicating that, like microRNAs, tsRNAs may have an oncogenic and/or tumor-suppressor function in hematopoietic malignancies and solid tumors. Thus, our results show that tsRNAs are dysregulated in human cancer.

MiR-17-92 cluster components analysis in Burkitt lymphoma: overexpression of miR-17 is associated with poor prognosis.

Burkitt lymphoma (BL) is an aggressive B cell lymphoma characterized by the reciprocal translocation of the c-Myc gene with immunoglobulin genes. Recently, MYC has been shown to maintain the neoplastic state via the miR-17-92 microRNA cluster that suppresses chromatin regulatory genes and the apoptosis regulator Bim. However, the expression and prognostic impact of miR-17-92 members in pediatric BL (pBL) are unknown. Therefore, we investigated miR-17, miR-19a, miR-19b, miR-20, and miR-92a expression and prognostic impact in a series of 41 pBL samples. In addition, Bim protein expression was evaluated and compared to miR-17, miR-19a, miR-19b, miR-20, and miR-92a levels and patient outcomes. The expression of miR-17-92 members was evaluated by qPCR and Bim protein by immunohistochemistry. Log-rank test was employed to assess prognostic impact. We found that upregulated expression of miR-17 and miR-20a correlates with lack of pro-apoptotic Bim expression. Patients bearing tumors with upregulated miR-17 displayed decreased overall survival (OS), and multivariate analysis revealed that miR-17 was a significant predictor of shortened OS. Using hairpin inhibitors, we showed that inhibition of miR-17 resulted in enhanced Bim expression in a BL cell line overexpressing the miR-17-92 cluster. Our results describe for the first time miR-17, miR-19a, miR-19b, miR-20a, and miR-92a expression profiles in pBL. The prognostic impact of miR-17 should be validated in a larger series, and may provide new therapeutic avenues in the era of anti-miRNA therapy research. Additional functional studies are further required to understand the specific role of miR-17-92 cluster members in BL.

Abstract A15: MiR-377 targets E2F3 and alters the NF-κB signaling pathway through MAP3K7 in malignant melanoma

Abstract Background: The incidence of cutaneous malignant melanoma continues to rise, and once the disease metastasizes it is almost inevitably fatal. We were the first to report that a large micro-RNA (miRNA) cluster on human chromosome 14q32, implicated in many types of cancers, is significantly down-regulated in melanoma, and have been studying this ‘tumor-suppressor miRNA cluster’ for several years now. MiR-377, one of the miRNAs located within this cluster, was studied in this work. Methods: qRT-pCR was used to quantify miR-377 levels in melanoma cell lines and samples. Melanoma cell lines ectopically expressing miR-377 were generated by stable transfection and mRNA expression was assessed using mRNA arrays. Potential targets of miR-377 were identified through luciferase reporter assays. Cellular proliferation, migration and soft-agar colony formation were monitored in control and miR-377-expressing cells using cell biology techniques and protein expression was assessed by western blot. Results: miR-377 is expressed in normal melanocytes but not in melanoma cell lines or samples. Its ectopic stable expression in melanoma cell lines decreased their proliferative and migratory capacity and their colony-forming capability. mRNA arrays of melanoma cells over-expressing miR-377 pointed to several down-regulated mRNAs that have putative binding sites for miR-377 in their 3'UTR, of which both E2F3 and MAP3K7 were found to be direct targets of miR-377. E2F3, a potent transcriptional inducer of cell-cycle progression, was found to be elevated in melanoma cell lines, but decreased following ectopic expression of miR-377. Ectopic miR-377 also led to a decrease in the activity of a reporter plasmid containing three E2F DNA-binding sites linked to a luciferase cDNA sequence, demonstrating that miR-377 down-regulates E2F3-induced transcription. MAP3K7, a serine/threonine kinase along the MAPK signaling pathway, was over-expressed in melanoma but decreased following ectopic expression of miR-377. MAP3K7 is known to be involved in the activation of NF-κB. MiR-377 over-expression led to decreased activity of a reporter plasmid containing two NF-κB DNA-binding sites and to decreased output along the NF-κB signaling pathway. Conclusion Our results suggest that miR-377 is an important negative regulator of E2F and of the MAP3K7/NF-κB signaling pathway in melanoma cells. The NF-κB signaling has been implicated in the acquisition of resistance to B-Raf inhibition. It is tempting to speculate that silencing of miR-377 in melanoma promotes the tumorigenic and metastatic potential of the cells through activation of these pathways. Indeed we found in preliminary results that overexpression of miR-377 in melanoma cells resistance to PLX-4032 (Zelboraf), re-sensitized the cells to the drug. Citation Format: Liron Zehavi, Adi Layani, Jasmine Jacob-Hirsch, Yechezkel Sidi, Raya Leibowitz-Amit, Dror Avni. MiR-377 targets E2F3 and alters the NF-κB signaling pathway through MAP3K7 in malignant melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A15.

Abstract B43: MYCN-amplified neuroblastoma maintain an undifferentiated phenotype through interference with estrogen and NGF signaling

Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. It is a highly heterogenic cancer of the sympathetic nervous system and features clinical phenotypes ranging from spontaneous differentiation and regression to aggressive growth and metastasis. Amplification of the MYCN gene is found in approximately 20-30% of all neuroblastoma cases and is linked to an undifferentiated phenotype and a poor prognosis. MYCN is a member of the MYC network of transcription factors, which are key players in the regulation of various fundamental cellular processes, e.g., cell growth, apoptosis and differentiation. We have previously demonstrated that miR-18a from the MYCN-regulated miR-17~92 microRNA cluster interferes with neuronal differentiation through repression of estrogen receptor alpha (ERα) levels. This study aims to analyze the effect of ERα overexpression on the morphology and functional behavior of MYCN-amplified neuroblastoma cells. Real time PCR, Western Blot and immunofluorescence analysis were performed to analyze mRNA levels and the amount, activation and localization of proteins. The morphological phenotype after activation of estrogen and/or NGF signaling was studied using bright field microscopy. Anchorage independent growth, WST-1 cell viability and transwell migration and invasion assays were conducted to study the effect of ERα overexpression and activation of NGF signaling on the functional phenotype of NB cells. Here we show that overexpression of ERα or interference with miR-18a is sufficient to increase mRNA and protein levels of the nerve growth factor (NGF) receptors TrkA and p75NTR in the MYCN-amplified neuroblastoma cell line SK-N-BE(2). Both NGF receptors negatively correlate with MYCN-amplification and are strongly linked to an improved prognosis in neuroblastoma patients. Importantly, neuronal differentiation was enhanced upon activation of NGF signaling in neuroblastoma cells with increased ERα levels, suggesting a crosstalk between estrogen and NGF signaling. Furthermore, overexpression of ERα resulted in a robust interference with processes linked to tumorigenesis, including cell survival, proliferation, migration, and anchorage independent growth. Together our results suggest a new mechanism by which MYCN-amplified neuroblastoma maintain an undifferentiated phenotype: MYCN-induced miR-18a targets ERα and thereby interferes with activation of NGF signaling and induction of differentiation. Conversely, reintroduction of ERα potentiates NGF signaling, facilitates neuronal differentiation and inhibits processes linked to tumorigenesis. Our data propose that restauration of ERα expression and activity, e.g., by interference with miR-18a, could be a promising strategy for the development of new therapies for neuroblastoma. Citation Format: Johanna Dzieran, Ulrica K. Westermark, Marie Arsenian Henriksson. MYCN-amplified neuroblastoma maintain an undifferentiated phenotype through interference with estrogen and NGF signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B43.

The multifactorial nature of microRNAs in vascular remodelling.

Vascular remodelling is a multifactorial process that involves both adaptive and maladaptive changes of the vessel wall through, among others, cell proliferation and migration, but also apoptosis and necrosis of the various cell types in the vessel wall. Vascular remodelling can be beneficial, e.g. during neovascularization after ischaemia, as well as pathological, e.g. during atherosclerosis and aneurysm formation. In recent years, it has become clear that microRNAs are able to target many genes that are involved in vascular remodelling processes and either can promote or inhibit structural changes of the vessel wall. Since many different processes of vascular remodelling are regulated by similar mechanisms and factors, both positive and negative vascular remodelling can be affected by the same microRNAs. A large number of microRNAs has been linked to various aspects of vascular remodelling and indeed, several of these microRNAs regulate multiple vascular remodelling processes, including both the adaptive processes angiogenesis and arteriogenesis as well as maladaptive processes of atherosclerosis, restenosis and aneurysm formation. Here, we discuss the multifactorial role of microRNAs and microRNA clusters that were reported to play a role in multiple forms of vascular remodelling and are clearly linked to cardiovascular disease (CVD). The microRNAs reviewed are miR-126, miR-155 and the microRNA gene clusters 17-92, 23/24/27, 143/145 and 14q32. Understanding the contribution of these microRNAs to the entire spectrum of vascular remodelling processes is important, especially as these microRNAs may have great potential as therapeutic targets for treatment of various CVDs.

MiR-137 Modulates a Tumor Suppressor Network-Inducing Senescence in Pancreatic Cancer Cells.

Activating K-Ras mutations occurs frequently in pancreatic cancers and is implicated in their development. Cancer-initiating events, such as oncogenic Ras activation, lead to the induction of cellular senescence, a tumor suppressor response. During senescence, the decreased levels of KDM4A lysine demethylase contribute to p53 activation, however, the mechanism by which KDM4A is downregulated is unknown. We show that miR-137 targets KDM4A mRNA during Ras-induced senescence and activates both p53 and retinoblastoma (pRb) tumor suppressor pathways. Restoring the KDM4A expression contributed to bypass of miR-137-induced senescence and inhibition of endogenous miR-137 with an miRNA sponge-compromised Ras-induced senescence. miR-137 levels are significantly reduced in human pancreatic tumors, consistent with previous studies revealing a defective senescence response in this cancer type. Restoration of miR-137 expression inhibited proliferation and promoted senescence of pancreatic cancer cells. These results suggest that modulating levels of miR-137 may be important for triggering tumor suppressor networks in pancreatic cancer.

MiR-23∼27∼24 clusters control effector T cell differentiation and function

Coordinated repression of gene expression by evolutionarily conserved microRNA (miRNA) clusters and paralogs ensures that miRNAs efficiently exert their biological impact. Combining both loss- and gain-of-function genetic approaches, we show that the miR-23∼27∼24 clusters regulate multiple aspects of T cell biology, particularly helper T (Th) 2 immunity. Low expression of this miRNA family confers proper effector T cell function at both physiological and pathological settings. Further studies in T cells with exaggerated regulation by individual members of the miR-23∼27∼24 clusters revealed that miR-24 and miR-27 collaboratively limit Th2 responses through targeting IL-4 and GATA3 in both direct and indirect manners. Intriguingly, although overexpression of the entire miR-23 cluster also negatively impacts other Th lineages, enforced expression of miR-24, in contrast to miR-23 and miR-27, actually promotes the differentiation of Th1, Th17, and induced regulatory T cells, implying that under certain conditions, miRNA families can fine tune the biological effects of their regulation by having individual members antagonize rather than cooperate with each other. Together, our results identify a miRNA family with important immunological roles and suggest that tight regulation of miR-23∼27∼24 clusters in T cells is required to maintain optimal effector function and to prevent aberrant immune responses.

HDAC3-Dependent Epigenetic Pathway Controls Lung Alveolar Epithelial Cell Remodeling and Spreading via miR-17-92 and TGF-β Signaling Regulation

The terminal stages of pulmonary development, called sacculation and alveologenesis, involve both differentiation of distal lung endoderm progenitors and extensive cellular remodeling of the resultant epithelial lineages. These processes are coupled with dramatic expansion of distal airspace and surface area. Despite the importance of these late developmental processes and their relation to neonatal respiratory diseases, little is understood about the molecular and cellular pathways critical for their successful completion. We show that a histone deacetylase 3 (Hdac3)-mediated epigenetic pathway is critical for the proper remodeling and expansion of the distal lung saccules into primitive alveoli. Loss of Hdac3 in the developing lung epithelium leads to a reduction of alveolar type 1 cell spreading and a disruption of lung sacculation. Hdac3 represses miR-17-92 expression, a microRNA cluster that regulates transforming growth factor β (TGF-β) signaling. De-repression of miR-17-92 in Hdac3-deficient lung epithelium results in decreased TGF-β signaling activity. Importantly, inhibition of TGF-β signaling and overexpression of miR-17-92 can phenocopy the defects observed in Hdac3 null lungs. Conversely, loss of miR-17-92 expression rescues many of the defects caused by loss of Hdac3 in the lung. These studies reveal an intricate epigenetic pathway where Hdac3 is required to repress miR-17-92 expression to allow for proper TGF-β signaling during lung sacculation.