Vijayaraghavan J, Maggi EC, Crabtree JS. miR-24 regulates menin in the endocrine pancreas. Am J Physiol Endocrinol Metab 307: E84 -E92, 2014. First published May 13, 2014; doi:10.1152/ajpendo.00542.2013.-Menin, the product of the MEN1 gene, functions as a tumor suppressor and was first identified in 1997 due to its causative role in the endocrine tumor disorder multiple endocrine neoplasia, type 1 (MEN1). More recently, menin has been identified as a key player in pancreatic islet biology with the observation of an inverse relationship between menin levels and pancreatic islet proliferation. However, the factors regulating menin and the MEN1 gene in the pancreas are poorly understood. Here, we describe the regulation of menin by miR-24 and demonstrate that miR-24 directly decreases menin levels and impacts downstream cell cycle inhibitors in MIN6 insulinoma cells and in ␤lox5 immortalized ␤-cells. This regulation of menin impacts cell viability and proliferation in ␤lox5 cells. Furthermore, our data show a feedback regulation between miR-24 and menin that is present in the pancreas, suggesting that miR-24 regulates menin levels in the pancreatic islet. menin; MEN1; pancreatic islet; miR-24; miRNA THE ENDOCRINE PANCREAS is responsible for maintaining normal glucose homeostasis through the tightly controlled, regulated release of insulin from the pancreatic islet. Pancreatic islets have the unique ability to dynamically and reversibly expand to adapt to changes in insulin demand through careful balance of cell growth and renewal (including ␤-cell replication, neogenesis, transdifferentiation, and hypertrophy), and cell death (apoptosis, atrophy, and autophagy). When this process becomes compromised, it leads to the clinical manifestation of gestational diabetes or type 2 diabetes (T2D). However, detailed knowledge of the mechanisms underlying this process is lacking.MicroRNAs (miRNAs), an evolutionarily conserved class of posttranscriptional regulators, are short ϳ22-nucleotide noncoding RNA sequences that regulate the expression of mRNA targets by binding to the 3=-UTR and inhibiting translation and/or targeting the mRNA for degradation. There is growing evidence that miRNAs regulate key biological processes in the pancreatic islet (13,14,17,27,33,49). For example, miR-375, the first miRNA identified in ␤-cell function, regulates insulin secretion and is essential for normal glucose homeostasis and turnover of ␣-and ␤-cells (40, 41). Other recent studies indicate a role for miR-338-3p and miR-181a in ␤-cell differentiation and insulin sensitivity, respectively (7, 23). p38 (MAPK) is downregulated by miR-24 through the insulinresponsive glucose transporter 4 (GLUT4) to affect peripheral insulin resistance (21). miR-24 also regulates insulin production by targeting the insulin transcriptional repressors Sox6 and Bhlhe22 (35).

Neuroendocrine tumors (NETs), which can have survival rates as low as 4%, currently have limited therapeutic interventions available highlighting the dire need for the identification of novel biological targets for use as new potential drug targets. One such potential target is retinoblastoma-binding protein 2 (RBP2), an H3K4 demethylase whose overexpression has been linked to cancer formation and metastasis in non-endocrine tumor types. We measured RBP2 mRNA and protein levels in enteropancreatic NETs by measuring RBP2 in matched human normal and NET tissue samples. Further, proliferation, migration, invasion and colony formation assays were performed in the physiologically relevant NET cell lines βlox5, H727 and QGP-1 to understand the role of RBP2 and its demethylase activity on end points of tumorigenesis. Our data indicate a strong correlation between RBP2 mRNA and protein expression in NET specimens. RBP2 was overexpressed relative to tissue-matched normal controls in 80% of the human tumors measured. In vitro studies showed RBP2 overexpression significantly increased proliferation, migration, invasion and colony formation, whereas knockdown significantly decreases the same parameters in a demethylase-independent manner. The cell cycle inhibitors p21 and p57 decreased with RBP2 overexpression and increased upon its depletion, suggesting a regulatory role for RBP2 in cellular proliferation. Taken together, our results support the hypothesis that the aberrant overexpression of RBP2 is a frequent contributing factor to tumor formation and metastasis in enteropancreatic NETs.

Multiple endocrine neoplasia type 1 (MEN1) syndrome results from mutations in the MEN1 gene and causes tumor formation via largely unknown mechanisms. Using a novel genome-wide methylation analysis, we studied tissues from MEN1-parathyroid tumors, Men1 knockout (KO) mice, and Men1 null mouse embryonic fibroblast (MEF) cell lines. We demonstrated that inactivation of menin (the protein product of MEN1) increases activity of DNA (cytosine-5)-methyltransferase 1 (DNMT1) by activating retinoblastoma-binding protein 5 (Rbbp5). The increased activity of DNMT1 mediates global DNA hypermethylation, which results in aberrant activation of the Wnt/β-catenin signaling pathway through inactivation of Sox regulatory genes. Our study provides important insights into the role of menin in DNA methylation and its impact on the pathogenesis of MEN1 tumor development.

Rhabdomyosarcoma, one of the most common childhood sarcomas, is comprised of two main subtypes, embryonal and alveolar (ARMS). ARMS, the more aggressive subtype, is primarily characterized by the t(2;13)(p35;p14) chromosomal translocation, which fuses two transcription factors, PAX3 and FOXO1 to generate the oncogenic fusion protein PAX3-FOXO1. Patients with PAX3-FOXO1-postitive tumors have a poor prognosis, in part due to the enhanced local invasive capacity of these cells, which leads to the increased metastatic potential for this tumor. Despite this knowledge, little is known about the role that the oncogenic fusion protein has in this increased invasive potential. In this report we use large-scale comparative transcriptomic analyses in physiologically relevant primary myoblasts to demonstrate that the presence of PAX3-FOXO1 is sufficient to alter the expression of 70 mRNA and 27 miRNA in a manner predicted to promote cellular invasion. In contrast the expression of PAX3 alters 60 mRNA and 23 miRNA in a manner predicted to inhibit invasion. We demonstrate that these alterations in mRNA and miRNA translate into changes in the invasive potential of primary myoblasts with PAX3-FOXO1 increasing invasion nearly 2-fold while PAX3 decreases invasion nearly 4-fold. Taken together, these results allow us to build off of previous reports and develop a more expansive molecular model by which the presence of PAX3-FOXO1 alters global gene regulatory networks to enhance the local invasiveness of cells. Further, the global nature of our observed changes highlights the fact that instead of focusing on a single-gene target, we must develop multi-faceted treatment regimens targeting multiple genes of a single oncogenic phenotype or multiple genes that target different oncogenic phenotypes for tumor progression.

The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

The B7 family, and their receptors, the CD28 family, are major immune checkpoints that regulate T-cell activation and function. In the present study, we explore the role of two B7 immune-checkpoints: HERV-H LTR-Associating Protein 2 (HHLA2) and B7 Family Member, H4 (B7x), in the progression of gastrointestinal and pancreatic neuroendocrine tumors (GINETs and PNETs). We demonstrated that both HHLA2 and B7x were expressed to a high degree in human GINETs and PNETs. We determined that the expression of B7x and HHLA2 correlates with higher grade and higher incidence of nodal and distant spread. Furthermore, we confirmed that HIF-1 overexpression is associated with the upregulation of B7x both in our in vivo (animal model) and in vitro (cell culture) models. When grown in vitro, islet tumor β-cells lack B7x expression, unless cultured under hypoxic conditions, which results in both hypoxia inducible factor 1 subunit alpha (HIF-1α) and B7x upregulation. In vivo, we demonstrated that Men1/B7x double knockout (KO) mice (with loss of B7x expression) exhibited decreased islet β-cell proliferation and tumor transformation accompanied by increased T-cell infiltration compared with Men1 single knockout mice. We have also shown that systemic administration of a B7x mAb to our Men1 KO mice with PNETs promotes an antitumor response mediated by increased T-cell infiltration. These findings suggest that B7x may be a critical mediator of tumor immunity in the tumor microenvironment of NETs. Therefore, targeting B7x offers an attractive strategy for the immunotherapy of patients suffering from NETs.

Aneuploidy has been reported to occur at remarkably high levels in normal somatic tissues using Fluorescence In Situ Hybridization (FISH). Recently, these reports were contradicted by single-cell low-coverage whole genome sequencing (scL-WGS) analyses, which showed aneuploidy frequencies at least an order of magnitude lower. To explain these seemingly contradictory findings, we used both techniques to analyze artificially generated mock aneuploid cells and cells with natural random aneuploidy. Our data indicate that while FISH tended to over-report aneuploidies, a modified 2-probe approach can accurately detect low levels of aneuploidy. Further, scL-WGS tends to underestimate aneuploidy levels, especially in a polyploid background.

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