Regulatory modules discovery and mesenchymal stem cells characterization from high-throughput cancer genomics data

2013/2014Il tumore è una malattia caratterizzata da un’estrema complessità molecolare. Gli approcci di tipo “omic”, collezionando dati sull’intero genoma, sui trascritti e proteine in dataset pubblici, permettono di superare questa complessità e di trovare moduli funzionali che eseguono le funzioni coinvolte nei processi tumorali. Ad esempio, i profili di espressione genica da tessuti vengono usati per definire firme di geni e testarne la rilevanza clinica. Ho usato questo tipo di informazione per caratterizzare specifici geni di interesse in modelli di tumore al seno. Uno dei più recenti progetti di tipo “omic” è il FANTOM5. Questo progetto ha generato una risorsa unica: il primo atlante di espressione in mammifero basato su sequenziamento a singola molecola. Il sistema CAGE (Cap Analysis of Gene Expression) è stato usato per misurare i siti di inizio trascrizione (TSS) e l’utilizzo dei promotori in una collezione di campioni umani: in questo modo sono stati misurati i livelli di espressione di gran parte dei trascritti codificanti e non-codificanti nel genoma umano. Ho usato questo tipo di informazione per caratterizzare una linea staminale mesenchimale/stromale (MSC) derivante da tumori sierosi ovarici di alto grado (HG-SOC-MSCs) o da tessuti normali (N-MSCs) inclusi nel dataset FANTOM5. Ho messo in luce programmi funzionali condivisi tra le due linee cellulari e osservato che le differenze principali tra le funzioni attivate nelle due linee sono di tipo quantitativo più che qualitativo. I risultati suggeriscono inoltre che le HG-SOC-MSCs sono simili alle cellule mesoteliali e alle cellule del tessuto muscolare liscio. Inoltre, ho analizzato l’intero dataset usando ScanAll, un nuovo software utile a predire ab initio la presenza di elementi arricchiti nelle regioni geniche che circondano i promotori trovati del progetto FANTOM5. Ho individuato moduli di regolazione, ossia gruppi di motif che si trovano a distanze predefinite sul genoma uno rispetto all’altro. Questi moduli sono arricchiti in regioni del genoma co-espresse rispetto a sequenze generate casualmente. Infine ho creato un compendio di fattori di trascrizione espressi e che partecipano ad interazione proteina-proteina.Cancer is a disease characterized by an extreme molecular complexity. Omics approaches, collecting data in public databases for all the genome, transcripts and proteins, attempt to overcome this complexity and find the functional modules that perform the functions involved in tumour related processes. For instance, cancer tissues gene expression profiles are widely used to define genes signatures and test their clinical relevance. I used this kind information in order to characterise interesting genes in breast cancer models. On the other hand, cellular models datasets could provide data that permits to focus on specific molecular mechanisms and probe the effects of molecules in a specific cancer model. One of the most recent omics project is the FANTOM5 project, that has generated a unique resource, the first single molecule sequencing-based expression atlas in mammalian systems. Cap analysis of gene expression (CAGE) was used to measure transcription start sites (TSS) and promoter usage across a wide collection of human samples thereby identifying and measuring levels of the majority of coding and non-coding transcripts in the human genome. I used this information to characterize a mesenchymal/stromal stem cell line (MSC) derived from high-grade serous ovarian cancer (HG-SOC-MSCs) or derived from normal tissue (N-MSCs) included in the entire FANTOM5 human dataset. I highlighted shared functional programs between HG-SOC-MSCs and N-MSCs suggesting that the global differences between the two cell lines are based on quantitative levels of transcriptional output rather than on qualitative differences. The results suggested that HG-SOC-MSCs are close relatives of mesothelial cells and smooth muscle cells. Furthermore, we analysed the entire dataset using ScanAll, a newly developed software, to ab initio predict the presence of enriched elements in the genomic regions surrounding FANTOM5 promoters. I pinpointed regulatory modules, i.e. groups of enriched motifs co-occurring in co-expressed regions within a fixed distance. These modules are enriched in the co-expressed sequences in each sample respect to random generated sequences. Finally, I created a Compendium of putative expressed and directly interacting transcription factors.XXVII Ciclo198

A novel HMGA1-CCNE2-YAP axis regulates breast cancer aggressiveness

High Mobility Group A1 (HMGA1) is an architectural chromatin factor that promotes neoplastic transformation and progression. However, the mechanism by which HMGA1 exerts its oncogenic function is not fully understood. Here, we show that cyclin E2 (CCNE2) acts downstream of HMGA1 to regulate the motility and invasiveness of basal-like breast cancer cells by promoting the nuclear localization and activity of YAP, the downstream mediator of the Hippo pathway. Mechanistically, the activity of MST1/2 and LATS1/2, the core kinases of the Hippo pathway, are required for the HMGA1- and CCNE2-mediated regulation of YAP localization. In breast cancer patients, high levels of HMGA1 and CCNE2 expression are associated with the YAP/TAZ signature, supporting this connection. Moreover, we provide evidence that CDK inhibitors induce the translocation of YAP from the nucleus to the cytoplasm, resulting in a decrease in its activity. These findings reveal an association between HMGA1 and the Hippo pathway that is relevant to stem cell biology, tissue homeostasis, and cancer

Translating proteomic into functional data: An high mobility group A1 (HMGA1) proteomic signature has prognostic value in breast cancer

Cancer is a very heterogeneous disease, and biological variability adds a further level of complexity, thus limiting the ability to identify new genes involved in cancer development. Oncogenes whose expression levels control cell aggressiveness are very useful for developing cellular models that permit differential expression screenings in isogenic contexts. HMGA1 protein has this unique property because it is a master regulator in breast cancer cells that control the transition from a nontumorigenic epithelial-like phenotype toward a highly aggressive mesenchymal-like one. The proteins extracted from HMGA1-silenced and control MDA-MB-231 cells were analyzed using label-free shotgun mass spectrometry. The differentially expressed proteins were cross-referenced with DNA microarray data obtained using the same cellular model and the overlapping genes were filtered for factors linked to poor prognosis in breast cancer gene expression meta-data sets, resulting in an HMGA1 protein signature composed of 21 members (HRS, HMGA1 reduced signature). This signature had a prognostic value (overall survival, relapse-free survival, and distant metastasis-free survival) in breast cancer. qRT-PCR, Western blot, and immunohistochemistry analyses validated the link of three members of this signature (KIFC1, LRRC59, and TRIP13) with HMGA1 expression levels both in vitro and in vivo and wound healing assays demonstrated that these three proteins are involved in modulating tumor cell motility. Combining proteomic and genomic data with the aid of bioinformatic tools, our results highlight the potential involvement in neoplastic transformation of a restricted list of factors with an as-yet-unexplored role in cancer. These factors are druggable targets that could be exploited for the development of new, targeted therapeutic approaches in triple-negative breast cancer

Effects of Pin1 Loss in Hdh(Q111) Knock-in Mice

Huntington's disease (HD) is a fatal, dominantly inherited, neurodegenerative disorder due to a pathological expansion of the CAG repeat in the coding region of the HTT gene. In the quest for understanding the molecular basis of neurodegeneration, we have previously demonstrated that the prolyl isomerase Pin1 plays a crucial role in mediating p53-dependent apoptosis triggered by mutant huntingtin (mHtt) in vitro. To assess the effects of the lack of Pin1 in vivo, we have bred Pin1 knock-out mice with Hdh(Q111) knock-in mice, a genetically precise model of HD. We show that Pin1 genetic ablation modifies a portion of Hdh(Q111) phenotypes in a time-dependent fashion. As an early event, Pin1 activity reduces the DNA damage response (DDR). In midlife mice, by taking advantage of next-generation sequencing technology, we show that Pin1 activity modulates a portion of the alterations triggered by mHtt, extending the role of Pin1 to two additional Hdh(Q111) phenotypes: the unbalance in the "synthesis/concentration of hormones", as well as the alteration of "Wnt/\u3b2-catenin signaling". In aging animals, Pin1 significantly increases the number of mHtt-positive nuclear inclusions while it reduces gliosis. In summary, this work provides further support for a role of Pin1 in HD pathogenesis. \ua9 2016 Agostoni, Michelazzi, Maurutto, Carnemolla, Ciani, Vatta, Roncaglia, Zucchelli, Leanza, Mantovani, Gustincich, Santoro, Piazza, Del Sal and Persichetti

Functional annotation of human long noncoding RNAs via molecular phenotyping

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-todate lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.Peer reviewe

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution

HMGA1 promotes metastatic processes in basal-like breast cancer regulating EMT and stemness

9In the present study, we show that the High Mobility Group A1 (HMGA1) protein plays a fundamental role in basal-like breast cancer subtype. HMGA1 knockdown induces the mesenchymal to epithelial transition and dramatically decreases stemness and self-renewal. Notably, HMGA1 depletion in basal-like breast cancer cell lines reduced migration and invasion in vitro and the formation of metastases in vivo.noneopenPegoraro S.; Ros G.; Piazza S.; Sommaggio R.; Ciani Y.; Rosato A.; Sgarra R.; Del Sal G.; Manfioletti G.Pegoraro, Silvia; Ros, Gloria; Piazza, S.; Sommaggio, R.; Ciani, Yari; Rosato, A.; Sgarra, Riccardo; DEL SAL, Giannino; Manfioletti, Guidalbert

HMGA1 regulates the Plasminogen activation system in the secretome of breast cancer cells

Abstract Cancer cells secrete proteins that modify the extracellular environment acting as autocrine and paracrine stimulatory factors and have a relevant role in cancer progression. The HMGA1 oncofetal protein has a prominent role in controlling the expression of an articulated set of genes involved in various aspect of cancer cell transformation. However, little is known about its role in influencing the secretome of cancer cells. Performing an iTRAQ LC–MS/MS screening for the identification of secreted proteins, in an inducible model of HMGA1 silencing in breast cancer cells, we found that HMGA1 has a profound impact on cancer cell secretome. We demonstrated that the pool of HMGA1–linked secreted proteins has pro–migratory and pro-invasive stimulatory roles. From an inspection of the HMGA1–dependent secreted factors it turned out that HMGA1 influences the presence in the extra cellular milieu of key components of the Plasminogen activation system (PLAU, SERPINE1, and PLAUR) that has a prominent role in promoting metastasis, and that HMGA1 has a direct role in regulating the transcription of two of them, i.e. PLAU and SERPINE1. The ability of HMGA1 to regulate the plasminogen activator system may constitute an important mechanism by which HMGA1 promotes cancer progression

Two distinct immunopathological profiles in autopsy lungs of COVID-19

Coronavirus Disease 19 (COVID-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has grown to a worldwide pandemic with substantial mortality. Immune mediated damage has been proposed as a pathogenic factor, but immune responses in lungs of COVID-19 patients remain poorly characterized. Here we show transcriptomic, histologic and cellular profiles of post mortem COVID-19 (n = 34 tissues from 16 patients) and normal lung tissues (n = 9 tissues from 6 patients). Two distinct immunopathological reaction patterns of lethal COVID-19 are identified. One pattern shows high local expression of interferon stimulated genes (ISGhigh) and cytokines, high viral loads and limited pulmonary damage, the other pattern shows severely damaged lungs, low ISGs (ISGlow), low viral loads and abundant infiltrating activated CD8+ T cells and macrophages. ISGhigh patients die significantly earlier after hospitalization than ISGlow patients. Our study may point to distinct stages of progression of COVID-19 lung disease and highlights the need for peripheral blood biomarkers that inform about patient lung status and guide treatment