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PURPOSE Clinical outcomes of patients with neuroblastoma range from spontaneous tumor regression to fatality. Hence, understanding the mechanisms that cause tumor progression is crucial for the treatment of patients. In this study, we show that FOXR2 activation identifies a subset of neuroblastoma tumors with unfavorable outcome and we investigate the mechanism how FOXR2 relates to poor outcome in patients. MATERIALS AND METHODS We analyzed three independent transcriptional data sets of in total 1030 primary neuroblastomas with full clinical annotation. We performed immunoprecipitation for FOXR2 and MYCN and silenced FOXR2 expression in two neuroblastoma cell lines to examine the effect on cellular processes, transcriptome, and MYCN protein levels. Tumor samples were analyzed for protein levels of FOXR2 and MYCN. RESULTS In three combined neuroblastoma data sets, 9% of tumors show expression of FOXR2 but have low levels of MYCN mRNA. FOXR2 expression identifies a group of patients with unfavorable outcome, showing 10-year overall survival rates of 53%-59%, and proves to be an independent prognostic factor compared with established risk factors. Transcriptionally, FOXR2-expressing tumors are very similar to MYCN-amplified tumors, suggesting that they might share a common mechanism of tumor initiation. FOXR2 knockdown in FOXR2-expressing neuroblastoma cell lines resulted in cell cycle arrest, reduced cell growth, cell death, and reduced MYCN protein levels, all indicating that FOXR2 is essential for these tumors. Finally, we show that FOXR2 binds and stabilizes MYCN protein and MYCN protein levels are highly increased in FOXR2-expressing tumors, in several cases comparable with MYCN-amplified samples. CONCLUSION The stabilization of MYCN by FOXR2 represents an alternative mechanism to MYCN amplification to increase MYCN protein levels. As such, FOXR2 expression identifies another subset of neuroblastoma patients with unfavorable clinical outcome.

In addition to regulatory or accessory proteins, some complex retroviruses gain a repertoire of micro-RNAs (miRNAs) to regulate and control virus&ndash, host interactions for efficient replication and spread. In particular, bovine and simian foamy viruses (BFV and SFV) have recently been shown to express a diverse set of RNA polymerase III-directed miRNAs, some with a unique primary miRNA double-hairpin, dumbbell-shaped structure not known in other viruses or organisms. While the mechanisms of expression and structural requirements have been studied, the functional importance of these miRNAs is still far from understood. Here, we describe the in silico identification of BFV miRNA targets and the subsequent experimental validation of bovine Ankyrin Repeat Domain 17 (ANKRD17) and Bax-interacting factor 1 (Bif1) target genes in vitro and, finally, the suppression of ANKRD17 downstream genes in the affected pathway. Deletion of the entire miRNA cassette in the non-coding part of the U3 region of the long terminal repeats attenuated replication of corresponding BFV mutants in bovine cells. This repression can be almost completely trans-complemented by the most abundant miRNA BF2-5p having the best scores for predicted and validated BFV miRNA target genes. Deletion of the miRNA cassette does not grossly affect particle release and overall particle composition.

The retroviral subfamily of Spumaretrovirinae consists of five genera of foamy (spuma) viruses (FVs) that are endemic in some mammalian hosts. Closely related species may be susceptible to the same or highly related FVs. FVs are not known to induce overt disease and thus do not pose medical problems to humans and livestock or companion animals. A robust lab animal model is not available or is a lab animal a natural host of a FV. Due to this, research is limited and often focused on the simian FVs with their well-established zoonotic potential. The authors of this review and their groups have conducted several studies on bovine FV (BFV) in the past with the intention of (i) exploring the risk of zoonotic infection via beef and raw cattle products, (ii) studying a co-factorial role of BFV in different cattle diseases with unclear etiology, (iii) exploring unique features of FV molecular biology and replication strategies in non-simian FVs, and (iv) conducting animal studies and functional virology in BFV-infected calves as a model for corresponding studies in primates or small lab animals. These studies gained new insights into FV-host interactions, mechanisms of gene expression, and transcriptional regulation, including miRNA biology, host-directed restriction of FV replication, spread and distribution in the infected animal, and at the population level. The current review attempts to summarize these findings in BFV and tries to connect them to findings from other FVs.