RNA molecules undergo a vast array of chemical post-transcriptional modifications (PTMs) that can affect their structure and interaction properties. In recent years, a growing number of PTMs have been successfully mapped to the transcriptome using experimental approaches relying on high-throughput sequencing. Oxford Nanopore direct-RNA sequencing has been shown to be sensitive to RNA modifications. We developed and validated Nanocompore, a robust analytical framework that identifies modifications from these data. Our strategy compares an RNA sample of interest against a non-modified control sample, not requiring a training set and allowing the use of replicates. We show that Nanocompore can detect different RNA modifications with position accuracy in vitro, and we apply it to profile m6A in vivo in yeast and human RNAs, as well as in targeted non-coding RNAs. We confirm our results with orthogonal methods and provide novel insights on the co-occurrence of multiple modified residues on individual RNA molecules.

Estrogen receptor-α (ERα) has central role in hormone-dependent breast cancer and its ligand-induced functions have been extensively characterized. However, evidence exists that ERα has functions that are independent of ligands. In the present work, we investigated the binding of ERα to chromatin in the absence of ligands and its functions on gene regulation. We demonstrated that in MCF7 breast cancer cells unliganded ERα binds to more than 4,000 chromatin sites. Unexpectedly, although almost entirely comprised in the larger group of estrogen-induced binding sites, we found that unliganded-ERα binding is specifically linked to genes with developmental functions, compared with estrogen-induced binding. Moreover, we found that siRNA-mediated down-regulation of ERα in absence of estrogen is accompanied by changes in the expression levels of hundreds of coding and noncoding RNAs. Down-regulated mRNAs showed enrichment in genes related to epithelial cell growth and development. Stable ERα down-regulation using shRNA, which caused cell growth arrest, was accompanied by increased H3K27me3 at ERα binding sites. Finally, we found that FOXA1 and AP2γ binding to several sites is decreased upon ERα silencing, suggesting that unliganded ERα participates, together with other factors, in the maintenance of the luminal-specific cistrome in breast cancer cells.

Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ERα plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ERα down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ERα-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ERα+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ERα, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ERα-dependent lncRNA set representing a novel luminal signature in breast cancer cells.

The transcription factor signal transducer and activator of transcription 3 (STAT3) acts downstream of many pro-oncogenic signals, including cytokines, growth factors and oncogenes, and is accordingly constitutively active in a wide variety of tumors that often become addicted to it. Moreover, STAT3 is a key player in mediating inflammation-driven tumorigenesis, where its aberrant continuous activation is typically triggered by local or systemic production of the pro-inflammatory cytokine IL-6. We recently showed that mouse embryonic fibroblasts (MEFs) derived from STAT3C k/in mice, which express physiological levels of the constitutively active mutant STAT3C, display features of transformed cells such as increased proliferation, resistance to apoptosis and senescence, and aerobic glycolysis. Here, we show that pre-existing constitutively active STAT3 is sufficient to prime primary MEFs for malignant transformation upon spontaneous immortalization. Transformation is strictly STAT3-dependent and correlates with high resistance to apoptosis and enhanced expression of anti-apoptotic/pro-survival genes. Additionally, hypoxia inducible factor (HIF)-1a level is elevated by twofold and contributes to STAT3 oncogenic activity by supporting high rates of aerobic glycolysis. Thus, constitutively active STAT3, an accepted essential factor for tumor growth/ progression, can also act as a first hit in multistep carcinogenesis; this ability to predispose cells to malignant transformation may be particularly relevant in the pro-oncogenic niche represented by chronically inflamed tissues. Signal transducers and activators of transcription (STAT) factors mediate the signaling downstream of cytokine and growth factor receptors. 1,2 Once activated by tyrosinephosphorylation by receptor-associated JAK kinases, STATs concentrate into the nucleus and regulate the expression of target genes. 3 STAT3 can be activated by a wide variety of cytokines and growth factors and by a number of oncogenes, 1 and is accordingly constitutively phosphorylated on tyrosine in many tumors that often become addicted to its activity. 1,2,4 Indeed, STAT3 is required for cell transformation downstream of v-Src and many other oncogenes, 4-6 and overexpression of a constitutively active mutant form can transform immortalized fibroblasts and epithelial cells. 7,8 Even though STAT3 is often referred to as an oncogene, only recently a naturally activating mutation has been found in human hepatocellular adenomas, 9 and its aberrant activity in tumors mostly occurs downstream of multiple activated pro-oncogenic pathways. Importantly, STAT3 is a key player in mediating inflammationdriven tumorigenesis, being constitutively activated by chronically high levels of the pro-inflammatory cytokine In tumors, STAT3 is known to enhance cell survival and proliferation and to promote immune escape and angiogenesis, invasion and metastasis. 11,12 In an effort to characterize pro-oncogenic functions of continuous, weak STAT3 activation, we have recently generated knock-in mi...

Demethylation of the long interspersed nuclear element (LINE-1; L1) antisense promoter can result in transcription of neighboring sequences as for the L1-MET transcript produced by the L1 placed in the second intron of MET. To define the role of L1-MET, we investigated the sequence and the transcription of L1-MET in vitro models and heterogeneous breast cancers, previously reported to show other L1-derived transcripts. L1-MET expressing cell lines were initially identified in silico and investigated for L1-MET promoter methylation, cDNA sequence and cell fraction mRNA. The transcriptional level of L1-MET and MET were then evaluated in breast specimens, including 9 cancer cell lines, 41 carcinomas of different subtypes, and 11 normal tissues. In addition to a L1-MET transcript ending at MET exon 21, six novel L1-MET splice variants were identified. Normal breast tissues were negative for the L1-MET expression, whereas the triple-negative breast cancer (TNBC) and the high-grade carcinomas were enriched with the L1-MET mRNA (p = 0.005 and p = 0.018, respectively). In cancer cells and tissues the L1-MET expression was associated with its promoter hypomethylation (ρ = -0.8 and -0.9, respectively). No correlation was found between L1-MET and MET mRNA although L1-MET expressing tumors with higher L1-MET/MET ratio were negative for the MET protein expression (p = 0.006). Besides providing the first identification and detailed description of L1-MET in breast cancer, we clearly demonstrate that higher levels of this transcript specifically recognize a subset of more aggressive carcinomas, mainly TNBC. We suggest the possible evaluation of L1-MET in the challenging diagnosis of early TNBCs.