Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.Electronic supplementary materialThe online version of this article (10.1007/s00401-019-02062-4) contains supplementary material, which is available to authorized users.

Human tumors show altered patterns of protein isoforms that can be related to the dysregulation of messenger RNA alternative splicing also observed in transformed cells. Although somatic mutations in core spliceosome components and their associated factors have been described in some cases, almost nothing is known about the contribution of distorted epigenetic patterns to aberrant splicing. Herein, we show that the splicing RNA-binding protein CELF2 is targeted by promoter hypermethylation-associated transcriptional silencing in human cancer. Focusing on the context of breast cancer, we also demonstrate that CELF2 restoration has growth-inhibitory effects and that its epigenetic loss induces an aberrant downstream pattern of alternative splicing, affecting key genes in breast cancer biology such as the autophagy factor ULK1 and the apoptotic protein CARD10. Furthermore, the presence of CELF2 hypermethylation in the clinical setting is associated with shorter overall survival of the breast cancer patients carrying this epigenetic lesion.

Time-lapse monitoring of somatic cell nuclear transfer (SCNT) embryos may help to predict developmental success and increase birth and embryonic stem cells (ESC) derivation rates. Here, the development of ICSI fertilized embryos and of SCNT embryos, non-treated or treated with either psammaplin A (PsA) or vitamin C (VitC), was monitored, and the ESC derivation rates from the resulting blastocysts were determined. Blastocyst rates were similar among PsA-treated and VitC-treated SCNT embryos and ICSI embryos, but lower for nontreated SCNT embryos. ESC derivation rates were higher in treated SCNT embryos than in non-treated or ICSI embryos. Time-lapse microscopy analysis showed that non-treated SCNT embryos had a delayed development from the second division until compaction, lower number of blastomeres at compaction and longer compaction and cavitation durations compared with ICSI ones. Treatment of SCNT embryos with PsA further increased this delay whereas treatment with VitC slightly reduced it, suggesting that both treatments act through different mechanisms, not necessarily related to their epigenetic effects. Despite these differences, the time of completion of the third division, alone or combined with the duration of compaction and/or the presence of fragmentation, had a strong predictive value for blastocyst formation in all groups. In contrast, we failed to predict ESC derivation success from embryo morphokinetics. Time-lapse technology allows the selection of SCNT embryos with higher developmental potential and could help to increase cloning outcomes. Nonetheless, further studies are needed to find reliable markers for full-term development and ESC derivation success.

Colon Cancer is the third most common cancer in the world and a major cause of morbidity and mortality. Molecular mechanisms of colon carcinoma have been deeply studied and current evidences indicate that microRNAs play a pivotal role in its tumorigenesis and progression. MicroRNAs (miRNAs) are small 19 to 22 nucleotides of RNA classified as non-coding RNAs that negatively regulate gene expression at the post-translational level controlling numerous biological processes including development, cell proliferation, apoptosis, differentiation and cell migration. Aberrant expression of miRNAs due to epigenetic alterations has been associated with carcinogenesis. Aimed to identify epigenetically regulated miRNAs involved in colon cancer, we analyzed DNA methylation profiles available in The Cancer Genome Atlas. One of the main hits identified was hsa-miR-3663, hypermethylated in 26% of colon cancer patients (n=286, p< 0.001) in comparison with complete lack of methylation in normal tissues (n=38), suggesting its potential function as a putative tumor suppressor. In order to study the role of DNA methylation controlling hsa-miR-3663, HumanMethylation450K methylation profiles were generated for a panel of colon cancer cell lines (n=10), including the HCT116-DNA methyltransferase knock-out model (DKO, double knockout of DNMT1 and DNMT3B). We found a significant correlation between methylation status of hsa-miR-3663 and expression in colon cancer cell lines. Furthermore, epigenetic regulation of hsa-miR-3663 was confirmed in HCT116 methylated cell line by restored expression upon treatment with DNA demethylating agent 5-aza-2′-deoxycytidine and in the HCT116-DKO model. In order to elucidate the function of hsa-miR-3663, we stably expressed hsa-miR-3663 in a panel of five methylated colorectal cancer cell lines (HCT116, RKO, DLD1, SW48 and SW480). Our initial functional assays revealed that hsa-miR-3663 is not involved in cell proliferation or migration. We are now performing additional assays to further study hsa-miR-3663 and its potential role in colon cancer. Note: This abstract was not presented at the meeting. Citation Format: Laia Pique, Humberto Jorge Ferrerira, Manel Esteller. Epigenetic regulation of hsa-miR-3663 in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 484. doi:10.1158/1538-7445.AM2017-484

Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like and epithelial-to-mesenchymal transition (EMT)-like features. However, it is still unclear how transformed melanocytes assume a metastatic melanoma cell state. Here, we define DNA methylation changes that accompany metastatic progression in melanoma patients and discover Nuclear Receptor Subfamily 2 Group F, Member 2 – isoform 2 (NR2F2-Iso2) as an epigenetically regulated metastasis driver. NR2F2-Iso2 is transcribed from an alternative transcriptional start site (TSS) and it is truncated at the N-terminal end which encodes the NR2F2 DNA-binding domain. We find that NR2F2-Iso2 expression is turned off by DNA methylation when NCCs differentiate into melanocytes. Conversely, this process is reversed during metastatic melanoma progression, when NR2F2-Iso2 becomes increasingly hypomethylated and re-expressed. Our functional and molecular studies suggest that NR2F2-Iso2 drives metastatic melanoma progression by modulating the activity of full-length NR2F2 (Isoform 1) over EMT- and NCC-associated target genes. Our findings indicate that DNA methylation changes play a crucial role during metastatic melanoma progression, and their control of NR2F2 activity allows transformed melanocytes to acquire NCC-like and EMT-like features. This epigenetically regulated transcriptional plasticity facilitates cell state transitions and metastatic spread.