Alessia Baccarini

microRNA-181a has a critical role in ovarian cancer progression through the regulation of the epithelial-mesenchymal transition.

Ovarian cancer is a leading cause of cancer deaths among women. Effective targets to treat advanced epithelial ovarian cancer (EOC) and biomarkers to predict treatment response are still lacking because of the complexity of pathways involved in ovarian cancer progression. Here we show that miR-181a promotes TGF-β-mediated epithelial-to-mesenchymal transition via repression of its functional target, Smad7. miR-181a and phosphorylated Smad2 are enriched in recurrent compared with matched-primary ovarian tumours and their expression is associated with shorter time to recurrence and poor outcome in patients with EOC. Furthermore, ectopic expression of miR-181a results in increased cellular survival, migration, invasion, drug resistance and in vivo tumour burden and dissemination. In contrast, miR-181a inhibition via decoy vector suppression and Smad7 re-expression results in significant reversion of these phenotypes. Combined, our findings highlight an unappreciated role for miR-181a, Smad7, and the TGF-β signalling pathway in high-grade serous ovarian cancer.

TLR Signals Induce Phagosomal MHC-I Delivery from the Endosomal Recycling Compartment to Allow Cross-Presentation

Adaptation of the endoplasmic reticulum (ER) pathway for MHC class I (MHC-I) presentation in dendritic cells enables cross-presentation of peptides derived from phagocytosed microbes, infected cells, or tumor cells to CD8 T cells. How these peptides intersect with MHC-I molecules remains poorly understood. Here, we show that MHC-I selectively accumulate within phagosomes carrying microbial components, which engage Toll-like receptor (TLR) signaling. Although cross-presentation requires Sec22b-mediated phagosomal recruitment of the peptide loading complex from the ER-Golgi intermediate compartment (ERGIC), this step is independent of TLR signaling and does not deliver MHC-I. Instead, MHC-I are recruited from an endosomal recycling compartment (ERC), which is marked by Rab11a, VAMP3/cellubrevin, and VAMP8/endobrevin and holds large reserves of MHC-I. While Rab11a activity stocks ERC stores with MHC-I, MyD88-dependent TLR signals drive IκB-kinase (IKK)2-mediated phosphorylation of phagosome-associated SNAP23. Phospho-SNAP23 stabilizes SNARE complexes orchestrating ERC-phagosome fusion, enrichment of phagosomes with ERC-derived MHC-I, and subsequent cross-presentation during infection.

Correcting miR-15a/16 genetic defect in New Zealand Black mouse model of CLL enhances drug sensitivity

Alterations in the human 13q14 genomic region containing microRNAs mir-15a and mir-16-1 are present in most human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black murine model to be associated with a point mutation in the primary mir-15a/16-1 region, which correlated with a decrease in mature miR-16 and miR-15a levels. In this study, addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G1 in non–New Zealand Black B cell and New Zealand Black–derived malignant B-1 cell lines. However, the New Zealand Black line had significantly greater G1 accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments showed a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G1/S transition, in the New Zealand Black cell line following miR-15a/16 addition. These microRNAs were shown to directly target the cyclin D1 3′ untranslated region using a green fluorescent protein lentiviral expression system. miR-16 was also shown to augment apoptosis induction by nutlin, a mouse double minute 2 (MDM2) antagonist, and genistein, a tyrosine kinase inhibitor, when added to a B-1 cell line derived from multiple in vivo passages of malignant B-1 cells from New Zealand Black mice with CLL. miR-16 synergized with nutlin and genistein to induce apoptosis. Our data support a role for the mir-15a/16-1 cluster in cell cycle regulation and suggest that these mature microRNAs in both the New Zealand Black model and human CLL may be targets for therapeutic efficacy in this disease. [Mol Cancer Ther 2009;8(9):2684–92]

The miR-126-VEGFR2 axis controls the innate response to pathogen-associated nucleic acids

miR-126 is a microRNA expressed predominately by endothelial cells and controls angiogenesis. We found miR-126 was required for the innate response to pathogen-associated nucleic acids and that miR-126-deficient mice had greater susceptibility to infection with pseudotyped HIV. Profiling of miRNA indicated that miR-126 had high and specific expression by plasmacytoid dendritic cells (pDCs). Moreover, miR-126 controlled the survival and function of pDCs and regulated the expression of genes encoding molecules involved in the innate response, including Tlr7, Tlr9 and Nfkb1, as well as Kdr, which encodes the growth factor receptor VEGFR2. Deletion of Kdr in DCs resulted in reduced production of type I interferon, which supports the proposal of a role for VEGFR2 in miR-126 regulation of pDCs. Our studies identify the miR-126–VEGFR2 axis as an important regulator of the innate response that operates through multiscale control of pDCs.

Myeloid Dysregulation in a Human Induced Pluripotent Stem Cell Model of PTPN11-Associated Juvenile Myelomonocytic Leukemia

Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML). Germline PTPN11 defects cause Noonan syndrome (NS), and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs) harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and upregulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were upregulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations, but not those without PTPN11 defects. Reducing miR-223s function in NS/JMML hiPSCs normalized myelogenesis. MicroRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover microRNA dysregulation, establishing a genotype-phenotype association for JMML and providing therapeutic targets.

Quiescent Tissue Stem Cells Evade Immune Surveillance

Summary Stem cells are critical for the maintenance of many tissues, but whether their integrity is maintained in the face of immunity is unclear. Here we found that cycling epithelial stem cells, including Lgr5+ intestinal stem cells, as well as ovary and mammary stem cells, were eliminated by activated T cells, but quiescent stem cells in the hair follicle and muscle were resistant to T cell killing. Immune evasion was an intrinsic property of the quiescent stem cells resulting from systemic downregulation of the antigen presentation machinery, including MHC class I and TAP proteins, and is mediated by the transactivator NLRC5. This process was reversed upon stem cell entry into the cell cycle. These studies identify a link between stem cell quiescence, antigen presentation, and immune evasion. As cancer‐initiating cells can derive from stem cells, these findings may help explain how the earliest cancer cells evade immune surveillance. Graphical Abstract Figure. No Caption available. HighlightsLgr5+ intestinal, ovary, and mammary stem cells are subject to T cell clearanceHair follicle stem cells evade detection and killing by innate and adaptive immunityHair follicle stem cells downregulate Nlrc5 and MHC class I in their quiescent stateExpression of Nlrc5 upregulates MHC class I on hair follicle stem cells &NA; Agudo et al. find that cycling tissue stem cells are subject to immune clearance, but quiescent stem cells downregulate the antigen presentation machinery and evade immune surveillance.

70. Knocking Down the Circular RNA ciRS-E2 Blocks Cancer Cell Proliferation Demonstrating Circular RNAs as a New Therapeutic Target

Circular RNAs (circRNA) are a newly discovered class of abundant and well conserved non-coding RNAs (Memczak et al. and Hansen et al. Nature 2013). They are comprised of RNA sequences in which the 3’ end of a downstream exon has been backspliced to the 5’ end of an upstream exon, causing the formation of a continuous RNA loop. Thousand of different circRNAs have been identified so far in different cell types, including cancer cells, and their expression levels often reaches or exceeds many coding RNAs in the cell. A small number of circRNAs have been shown to control the activity of specific microRNAs by acting as molecular sponges, however the functions of the vast majority of circRNAs are still not known. Because the sequence of the 3’-5’ exon junction of a circRNA is unique in the transcriptome, we hypothesized that targeting this region may be used to generate RNAi vectors that can knockdown circRNAs without impacting their host gene.Using a deep-sequencing approach that we call Capture-seq and a novel mapping algorithm, we detected an abundant circRNA expressed in highly proliferative cells, which we dub circular E2 (ciRS-E2). ciRS-E2 is comprised of exon 2 of a coding gene which has circularized as a result of the 3’ end of exon 2 backsplicing to the 5’ end of the exon. Using a PCR that was specific for the backspliced junction, we found ciRS-E2 to be highly expressed in a number of cancers, including leukemia, melanoma, and ovarian.We generated two different shRNAs to target the unique 3’-5’ exon junction of ciRS-E2 and designed them to have different seed regions so that potential off-target effects could be accounted for. The shRNAs were cloned into lentiviral vectors encoding GFP (LV.GFP), and introduced into acute myelocytic leukemia or ovarian adenocarcinoma cells. More than 95% of cells were transduced. For comparison, the cells were transduced with two different control LV.GFP vectors encoding scrambled sequences. Impressively, both ciRS-E2 shRNAs knocked down ciRS-E2 by more than 80%, and there was no change in the expression of ciRS-E2s host genes mRNA or protein. Strikingly, within 7 days of transduction there was a major loss in cells transduced with either ciRS-E2 shRNA, which did not happen with control cells. By 10 days, there were less than 20% GFP+ cells, indicating that ciRS-E2 is important for cancer cell fitness. Further analysis revealed that this was due to impaired cell proliferation as a result of the cells arresting in G0/G1. Transcriptomics analysis by RNA-seq indicated that loss of ciRS-E2 resulted in a significant decrease (P 2-fold) in the expression of a number of key oncogenes including Myc and Max.This study is one of the first to assign an important cellular function to a circRNA, and the first to identify a circRNA that controls cancer cell fitness. Importantly, we show that RNAi vectors can be generated to efficiently and specifically knock down a circRNA without impacting their host gene. circRNAs represent a potential new therapeutic target, and our work suggests that knockdown of ciRS-E2 may be used to control cancer cell proliferation.

MicroRNA activity profile in the ovarian cancer cell line OVCAR3 identifies a proapoptotic effect of miR-23a

License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php Advances in Genomics and Genetics 2015:5 355–364 Advances in Genomics and Genetics Dovepress

Abstract B47: microRNA-181a plays a critical role in ovarian cancer progression through the regulation of epithelial-mesenchymal transition

Ovarian cancer is the most lethal gynecological cancer primarily due to advanced stage of disease at diagnosis. Effective therapeutic targets and prognostic biomarkers are still lacking due to insufficient knowledge of the pathways that regulate ovarian cancer progression. Here, we identify miR-181a as a mediator of disease dissemination through the induction of EMT and direct activation of the TGF-β signaling pathway via repression of its functional target, Smad7. High expression of miR-181a and phospho-Smad2 were associated with poor patient outcome and were enriched in recurrent compared to matched-primary tumors. Ectopic expression of miR-181a resulted in increased cellular survival, migration, drug resistance, and in vivo tumor burden, and dissemination. Conversely, targeting this miRNA using a decoy vector resulted in significant decreases in cell survival, migration, and MET in ovarian cancer cell lines. Combined, our findings identify miR-181a as a novel modulator of ovarian cancer dissemination through the induction of EMT and highlight its role as a potential biomarker and therapeutic target for aggressive late-stage ovarian cancer. Citation Format: Aditya Parikh, Christine Lee, Peronne Joseph, Sergio Marchini, Alessia Baccarini, Valentin Kolev, Chiara Romualdi, Robert Fruscio, Hardik Shah, Wang Feng, Gavriel Mullokandov, David Fishman, Maurizio D9Incalci, Jamal Rahaman, Tamara Kalir, Raymond Redline, Brian D. Brown, Goutham Narla, Analisa DiFeo. microRNA-181a plays a critical role in ovarian cancer progression through the regulation of epithelial-mesenchymal transition. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B47.