Michele De Bortoli - Academia.edu (original) (raw)

Papers by Michele De Bortoli

International Journal of Molecular Sciences, Jul 16, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

The activation of the LINE-1 sequence located within the second intron of MET leads to the onset ... more The activation of the LINE-1 sequence located within the second intron of MET leads to the onset of L1-MET transcript. We recently characterized the full L1-MET structure in breast cancer and showed that high levels of this transcript recognize a subset of more aggressive breast carcinomas, mainly of triple negative phenotype. However, at present, the relationship between L1-MET and MET is still poorly understood. In order to elucidate this function, we silenced L1-MET transcription using cells expressing different levels of L1-MET/MET, including lung cancer (A549 and EBC1), gastric cancer (GTL16), and breast cancer (MDA-MB231), that were transiently transfected with Gapmers-LNA (Exiqon), specifically targeting L1-MET sequence. Cell viability and apoptosis were evaluated after 24 h by cell count, Cell Titer Glow (Promega) and propidium iodide/annexin based-cytofluorimeter assays. RNA was purified from sample and control cells to assess the L1-METsilencing by qRT-PCR and to evaluate the gene-expression of a subset of cancer-related genes using Nanostring Technology, whereas western blot analyses were carried out to measure the protein expressions. A significant decrease of cell viability was detected in A549, EBC1 and GTL16, but not in MDA-MB231 cells, characterized by the lowest level of L1-MET overall. In parallel, the highly expressing L1-MET cells showed an increased rate of early and late apoptosis together with a strong reduction of MET gene and its protein expression. On the contrary, in MDA-MB231 cells L1-MET silencing induced only a slight MET gene and protein impairment. Overall, L1-MET knock-down caused a decrease expression of a conserved gene cluster, including a marked reduction of EGFR protein expression. Moreover, L1-MET silenced cells showed lower MET and EGFR phosphorylation, with a downstream silencing effect on pERK and pAKT. Results of cell treatment with the inhibitors of lysosome and proteasome activity bafilomycin and MG-132 ruled out the interaction of L1-MET silencing with protein degradation pathway. This is the first study investigating the function of the L1-MET transcript in cancer models. Our results show that although L1-MET is unable to encode for a protein, its silencing exerts a strong phenotypic effect on different tumor cell types, suggesting potential regulations at the transcriptional level. Note: This abstract was not presented at the meeting. Citation Format: Enrico Berrino, Umberto Miglio, Valentina Miano, Letizia Lanzetti, Silvia Benvenuti, Carla Debernardi, MIchele De Bortoli, Caterina Marchio, Tiziana Venesio, Anna Sapino. L1-MET transcription silencing modulates MET and EGFR gene and their protein expression and induces apoptosis and cell-death in different types of cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 261.

We previously reported that the long noncoding RNA (lncRNA) DSCAM-AS1 is one of the most interest... more We previously reported that the long noncoding RNA (lncRNA) DSCAM-AS1 is one of the most interesting functional molecules in the Estrogen Receptor alpha (ERα) pathway in breast cancer cells, among a set of lncRNAs showing dependency on ERα in MCF7 cells (Miano et al., Oncotarget Jan 19, 2016. DOI: 10.18632/oncotarget.6420). Importantly, these lncRNAs were identified as dependent on ERα expression in absence of hormones, and DSCAM-AS1 was the most representative of them, presenting a clear ERα ChIP-Seq signal on the DSCAM-AS1 promoter and responding sharply to ERα silencing, but not to estradiol treatment. This behavior was shared with some other, but not all, ERα-dependent lncRNAs. We showed also that DSCAM-AS1 expression was strongly related to the luminal B > A tumor subtype and strongly related to ER+, in all datasets examined. Together with other lncRNAs we identified, they constituted a sharp luminal-specific gene signature. All this was confirmed more recently by another group (Niknafs et al., Nat. Comm. Sept 26, 2016. DOI: 10.1038/ncomms12791), also showing that DSCAM-AS1 may be related to endocrine resistance in breast cancer. We report here that DSCAM-AS1 silencing in MCF7 cells evokes a response very similar to what we observed by knocking down ERα (Caizzi et al., PNAS 2009. DOI: 10.1073/pnas.1315445111), i.e. growth arrest, morphological changes and cell death. Noteworthy, ERα expression was not altered by DSCAM-AS1 silencing, thus indicating that DSCAM-AS1 is downstream ERα. Thus, we were interested in evaluating the overall transcriptional response to DSCAM-AS1 silencing. LNA-mediated DSCAM-AS1 down-regulation led to changes in the expression level of 436 protein-coding genes, as determined by RNA-seq and the following sample validation by qRT-PCR. Data analysis by means of IPA and EnrichR indicated that DSCAM-AS1 silencing regulated genes of cell growth and proliferation, cell signaling, cell death and survival and cellular movement. On the other side, there was also a clear stress response with involvement of the interferon signaling pathway. As in the case of ERα silencing, the overall picture is that DSCAM-AS1 may have a function in the maintenance of the luminal epithelial phenotype in breast cancer cells. Interestingly, genes related to cell movement were actually activated by DSCAM-AS1 knock-down and, in this respect, our result may be somehow contrasting with those shown by Niknafs et al. (above) who reported that stable DCAM-AS1 silencing by shRNA led to decreased migration and invasiveness. Differences in RNA-mediated long-term downregulation versus shorter term, LNA-mediated downregulation may account for discrepancies, but the matter clearly deserves more investigation. Finally, we present further data on the association of DSCAM-AS1 with ERα in breast tumors and clinical data. We suggest that its high level of expression, tissue-of-origin specificity and breast tumor phenotype specificity make DSCAM-AS1 an extremely interesting novel biomarker of luminal breast cancer.We previously reported that the long noncoding RNA (lncRNA) DSCAM-AS1 is one of the most interesting functional molecules in the Estrogen Receptor alpha (ERα) pathway in breast cancer cells, among a set of lncRNAs showing dependency on ERα in MCF7 cells (Miano et al., Oncotarget Jan 19, 2016. DOI: 10.18632/oncotarget.6420). Importantly, these lncRNAs were identified as dependent on ERα expression in absence of hormones, and DSCAM-AS1 was the most representative of them, presenting a clear ERα ChIP-Seq signal on the DSCAM-AS1 promoter and responding sharply to ERα silencing, but not to estradiol treatment. This behavior was shared with some other, but not all, ERα-dependent lncRNAs. We showed also that DSCAM-AS1 expression was strongly related to the luminal B > A tumor subtype and strongly related to ER+, in all datasets examined. Together with other lncRNAs we identified, they constituted a sharp luminal-specific gene signature. All this was confirmed more recently by another group (Niknafs et al., Nat. Comm. Sept 26, 2016. DOI: 10.1038/ncomms12791), also showing that DSCAM-AS1 may be related to endocrine resistance in breast cancer. We report here that DSCAM-AS1 silencing in MCF7 cells evokes a response very similar to what we observed by knocking down ERα (Caizzi et al., PNAS 2009. DOI: 10.1073/pnas.1315445111), i.e. growth arrest, morphological changes and cell death. Noteworthy, ERα expression was not altered by DSCAM-AS1 silencing, thus indicating that DSCAM-AS1 is downstream ERα. Thus, we were interested in evaluating the overall transcriptional response to DSCAM-AS1 silencing. LNA-mediated DSCAM-AS1 down-regulation led to changes in the expression level of 436 protein-coding genes, as determined by RNA-seq and the following sample validation by qRT-PCR. Data analysis by means of IPA and EnrichR indicated that DSCAM-AS1 silencing regulated genes of cell growth and proliferation, cell signaling, cell…

Cancers

Background: The transcriptional activity of estrogen receptor α (ERα) in breast cancer (BC) is ex... more Background: The transcriptional activity of estrogen receptor α (ERα) in breast cancer (BC) is extensively characterized. Our group has previously shown that ERα controls the expression of a number of genes in its unliganded form (apoERα), among which a large group of RNA-binding proteins (RBPs) encode genes, suggesting its role in the control of co- and post-transcriptional events. Methods: apoERα-mediated RNA processing events were characterized by the analysis of transcript usage and alternative splicing changes in an RNA-sequencing dataset from MCF-7 cells after siRNA-induced ERα downregulation. Results: ApoERα depletion induced an expression change of 681 RBPs, including 84 splicing factors involved in translation, ribonucleoprotein complex assembly, and 3′end processing. ApoERα depletion results in 758 isoform switching events with effects on 3′end length and the splicing of alternative cassette exons. The functional enrichment of these events shows that post-transcriptional r...

The International Journal of Biological Markers, 2018

Years of molecular research have described the qualitative and quantitative changes subverting ce... more Years of molecular research have described the qualitative and quantitative changes subverting cell physiology in pathological states. In cancer, accessibility to tumor biopsies has granted detailed analysis of the mechanisms leading to uncontrolled cell growth, spreading, and metastasis, allowing focused diagnosis and innovative treatments. For public health, a decisive step forward will be the availability of non-invasive tests to detect early signs of disease, establish the responsiveness to drugs, and anticipate relapses. This paradigm could be applied to any kind of human disease. A recently proposed blood test, which has wide press echoes, measures a "profile" of DNA mutations and proteins in blood and claimed a 55% success rate for early cancer detection. 1 Among novelties, the recent findings of galaxies of non-coding RNAs (ncRNAs) in human tissues and in body liquids has generated a flurry of reports proposing these molecules as biomarkers. Besides undoubted interest, we suggest that more stringent and adequate quality criteria should be established in order to consider and publish reports on ncRNAs as biomarkers.

Experimental and Molecular Therapeutics, 2019

Frontiers in Immunology, 2019

International Journal of Cancer, 2018

Demethylation of the long interspersed nuclear element (LINE‐1; L1) antisense promoter can result... more 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 carcin...

Oncotarget, Mar 6, 2018

Circular RNAs are highly stable molecules present in all eukaryotes generated by distinct transcr... more Circular RNAs are highly stable molecules present in all eukaryotes generated by distinct transcript processing. We have exploited poly(A-) RNA-Seq data generated in our lab in MCF-7 breast cancer cells to define a compilation of exonic circRNAs more comprehensive than previously existing lists. Development of a novel computational tool, named , allowed us to more accurately characterize circRNAs and to quantitatively evaluate their expression in publicly available RNA-Seq data from breast cancer cell lines and tumor tissues. We observed and confirmed, by ChIP analysis, that exons involved in circularization events display significantly higher levels of the histone post-transcriptional modification H3K36me3 than non-circularizing exons. This result has potential impact on circRNA biogenesis since H3K36me3 has been involved in alternative splicing mechanisms. By analyzing an Ago-HITS-CLIP dataset we also found that circularizing exons overlapped with an unexpectedly higher number of ...

International journal of molecular sciences, Jan 16, 2018

Estrogen receptor-α (ERα) is a ligand-inducible protein which mediates estrogenic hormones signal... more Estrogen receptor-α (ERα) is a ligand-inducible protein which mediates estrogenic hormones signaling and defines the luminal BC phenotype. Recently, we demonstrated that even in absence of ligands ERα (apoERα) binds chromatin sites where it regulates transcription of several protein-coding and lncRNA genes. Noteworthy, apoERα-regulated lncRNAs marginally overlap estrogen-induced transcripts, thus representing a new signature of luminal BC genes. By the analysis of H3K27ac enrichment in hormone-deprived MCF-7 cells, we defined a set of Super Enhancers (SEs) occupied by apoERα, including one mapped in proximity of the DSCAM-AS1 lncRNA gene. This represents a paradigm of apoERα activity since its expression is largely unaffected by estrogenic treatment, despite the fact that E2 increases ERα binding on DSCAM-AS1 promoter. We validated the enrichment of apoERα, p300, GATA3, FoxM1 and CTCF at both DSCAM-AS1 TSS and at its associated SE by ChIP-qPCR. Furthermore, by analyzing MCF-7 ChIA-P...

Scientific reports, Jan 17, 2017

In the study of genomic regulation, strategies to integrate the data produced by Next Generation ... more In the study of genomic regulation, strategies to integrate the data produced by Next Generation Sequencing (NGS)-based technologies in a meaningful ensemble are eagerly awaited and must continuously evolve. Here, we describe an integrative strategy for the analysis of data generated by chromatin immunoprecipitation followed by NGS which combines algorithms for data overlap, normalization and epigenetic state analysis. The performance of our strategy is illustrated by presenting the analysis of data relative to the transcriptional regulator Estrogen Receptor alpha (ERα) in MCF-7 breast cancer cells and of Glucocorticoid Receptor (GR) in A549 lung cancer cells. We went through the definition of reference cistromes for different experimental contexts, the integration of data relative to co-regulators and the overlay of chromatin states as defined by epigenetic marks in MCF-7 cells. With our strategy, we identified novel features of estrogen-independent ERα activity, including FoxM1 in...

The International Journal of Biological Markers, 2003

PLOS ONE, 2016

Tab2, originally described as a component of the inflammatory pathway, has been implicated in phe... more Tab2, originally described as a component of the inflammatory pathway, has been implicated in phenomena of gene de-repression in several contexts, due to its ability to interact with the NCoR corepressor. Tab2 interacts also with steroid receptors and dismisses NCoR from antagonist-bound Estrogen and Androgen Receptors on gene regulatory regions, thus modifying their transcriptional activity and leading to pharmacological resistance in breast and prostate cancer cells. We demonstrated previously that either Tab2 knock-down, or a peptide mimicking the Estrogen Receptor alpha domain interacting with Tab2, restore the antiproliferative response to Tamoxifen in Tamoxifen-resistant breast cancer cells. In this work, we map the domain of Tab2 responsible of Estrogen Receptor alpha interaction. First, using both co-immunoprecipitation and pull-down with recombinant proteins, we found that the central part of Tab2 is primarily responsible for this interaction, and that this region also interacts with Androgen Receptor. Then, we narrowed down the essential interaction region by means of competition assays using recombinant protein pull-down. The interaction motif was finally identified as a small region adjacent to, but not overlapping, the Tab2 MEKK1 phosphorylation sites. A synthetic peptide mimicking this motif efficiently displaced Tab2 from interacting with recombinant Estrogen Receptor alpha in vitro, prompting us to test its efficacy using derivatives of the MCF7 breast carcinoma cell lines that are spontaneously resistant to Tamoxifen. Indeed, we observed that this mimic peptide, made cell-permeable by addition of the TAT minimal carrier domain, reduced the growth of Tamoxifen-resistant MCF7 cells in the presence of Tamoxifen. These data indicate a novel functional domain of the Tab2 protein with potential application in drug design.

Frontiers in Cellular Neuroscience, 2016

Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB) is one ... more Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB) is one of the estrogen target genes involved in neuroprotection, but little is known about its transcriptional regulation. Estrogen genomic pathway in gene expression regulation is mediated by estrogen receptors (ERα and ERβ) that bind to specific regulatory genomic regions. We focused our attention on 17β-estradiol (E2)-induced NGB expression in human differentiated neuronal cell lines (SK-N-BE and NT-2). Previously, using bioinformatics analysis we identified a putative enhancer in the first intron of NGB locus. Therefore, we observed that E2 increased the enrichment of the H3K4me3 epigenetic marks at the promoter and of the H3K4me1 and H3K27Ac at the intron enhancer. In these NGB regulatory regions, we found estrogen receptor alpha (ERα) binding suggesting that ERα may mediate chromatin remodeling to induce NGB expression upon E2 treatment. Altogether our data show that NGB expression is regulated by ERα binding on genomic regulatory regions supporting hormone therapy applications for the neuroprotection against neurodegenerative diseases.

Oncotarget, Jan 28, 2015

Estrogen Receptor alpha (ERα) activation by estrogenic hormones induces luminal breast cancer cel... more 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 ...

Proceedings of the National Academy of Sciences, 2014

Significance Estrogen receptor-α (ERα) is a key protein in breast cancer and treatments targeting... more Significance Estrogen receptor-α (ERα) is a key protein in breast cancer and treatments targeting ERα are among the most widely used and effective in clinics. Although the role of estrogen-stimulated ERα in breast cancer has been exhaustively described, the functions of ERα in the absence of estrogen is hill-defined. In this work, we show that ERα binds extensively to the genome of breast cancer cells in the absence of estrogen, where it regulates the expression of hundreds of genes endowed with developmental functions. Our data suggest that ERα has a fundamental role in the homeostasis of luminal epithelial cells also when estrogen is ablated physiologically or pharmacologically.