Cancer evolution requires precise patterns of gene expression on which cancer cells rely for their growth and survival. These transcriptional programs are established through a complex regulatory landscape involving the hierarchical interaction of multiple regulatory elements. Transcription factors (TFs) have a pivotal role in orchestrating these regulatory circuitries, in a way that makes cancer cells dependent on their activities. The Runt-related Transcription Factor 2 (RUNX2) was found to be involved in the metastatic progression of many types of cancer, including thyroid cancer (TC). Studying the mechanism underlining RUNX2-upregulation in cancer we identified RAIN, a new chromatin-associated long non-coding RNA (lncRNA) that is expressed from RUNX2-active enhancers and is involved in RUNX2 regulation during TC progression. Preliminary data also suggest that RAIN may have additional RUNX2-independent transcriptional activities. Although the association of RUNX2 with cancer aggressiveness has been well demonstrated, the details of the architecture of the RUNX2-transcriptional landscape and the mechanism explaining how this TF and its associated lncRNA contribute to TC biology are still largely unexplored. In this work, we used a multidimensional approach to reconstruct the transcriptional landscape of RUNX2 in TC and to clarify its contribution to regulating biological processes that support cancer progression. We focused on both the upstream genomic activity of this TF and the downstream effect on biological processes that sustain cancer evolution. We also investigated how RAIN partakes in these activities and contributes to the overall transcriptional program underlining TC progression. By integrating ChIP-seq and RNA-seq data, we functionally characterized the RUNX2-associated genomic elements and got insights into its network of cooperators and its dependent gene program. All these pieces of -omics information were combined with clinical, mutational, and transcriptional data retrieved from the TCGA-THCA project in a computational hierarchical model that recapitulates the RUNX2-transcriptional network in TC. This analysis identified a series of RUNX2-dependent gene modules corresponding to specific biological functions that influence the clinical behavior of these neoplasms. Among these, the module of genes partaking in metabolism regulation resulted to be correlated with TC metastasis, showing for the first time a direct contribution of RUNX2 to cancer cell metabolic rewiring, by promoting lipid synthesis while inhibiting oxidative phosphorylation. A parallel multi-omics approach was applied to study the molecular functions of RAIN. We unveiled that, as for RUNX2, RAIN directly controls a core of other cancer-related TFs and cooperates with them in regulating the expression of downstream targets. We also showed that in cooperation with RUNX2, RAIN is required for the expression of a subset of genes that promote TC metastatization. Overall, these data define the RUNX2 transcriptional landscape in TC, providing a model in which RUNX2 affects the expression of specific categories of genes linked to specific biological functions by differentiating its activity and network of interactions. Besides some well-established biological processes that have been already associated with RUNX2 activity, we identified for the first time the direct role of RUNX2 in the complex metabolic rewiring of cancer cells. Furthermore, our data demonstrate that RAIN, besides its in-cis effect on the RUNX2 locus, influences TC progression through a widespread regulatory activity throughout the genome, underlining the importance of coding and non-coding functional interplay in cancer biology.

La progressione tumorale richiede programmi genici che garantiscono la crescita e la sopravvivenza delle cellule cancerose. Tali programmi dipendono da reti regolatorie instaurate dall’interazione gerarchica di molteplici elementi e governate dai fattori di trascrizione (TF). Il fattore di trascrizione RUNX2 promuove lo sviluppo metastatico di numerosi tumori, inclusi i carcinomi tiroidei. Il nostro gruppo di ricerca ha recentemente chiarito il meccanismo di riattivazione di RUNX2 nei tumori della tiroide. Questi studi hanno portato all’identificazione di RAIN, un long non-coding RNA (lncRNA) cromatinico trascritto a partire da due enhancer di RUNX2 e coinvolto nella regolazione dell’espressione del TF. In aggiunta a questo ruolo, i nostri dati preliminari suggeriscono delle funzioni di RAIN legate alla regolazione della trascrizione e svincolate dal suo effetto su RUNX2. Sebbene l’associazione tra RUNX2 e aggressività tumorale sia stata ben dimostrata, il meccanismo molecolare tramite cui questo TF e il lncRNA ad esso associato supportano lo sviluppo metastatico è ancora sconosciuto. In questo lavoro abbiamo utilizzato un approccio integrativo per ricostruire la complessa rete regolatoria governata da RUNX2 e per chiarire il suo contributo nello sviluppo metastatico dei tumori tiroidei, focalizzandoci sia sullo studio della sua attività genomica che sull’analisi dei processi biologici influenzati da tale attività. Abbiamo inoltre condotto indagini per chiarire il ruolo di RAIN nella definizione di un programma trascrizionale alla base dell’evoluzione dei tumori della tiroide. In particolare, tramite integrazione di dati di ChIP-seq e RNA-seq abbiamo identificato e caratterizzato le regioni genomiche occupate da RUNX2, la sua rete di cooperatori e il programma trascrizionale dipendente dalla sua attività. Combinando i nostri dati omici con i dati clinici, genetici e trascrittomici di pazienti, abbiamo costruito un modello computazionale dell’attività trascrizionale di RUNX2 nei tumori tiroidei, identificando moduli di geni target associati a specifici processi biologici e correlati a diversi gradi di aggressività delle lesioni. In particolare, dalle nostre analisi è emersa una correlazione tra il modulo di geni implicati nella regolazione del metabolismo cellulare e la presenza di metastasi a distanza, evidenziando per la prima volta la partecipazione di RUNX2 nella riprogrammazione metabolica delle cellule cancerose, contesto in cui il fattore stimola la sintesi lipidica e reprime la fosforilazione ossidativa. Tramite un approccio multi-omico parallelo a quello seguito per RUNX2, abbiamo dimostrato come RAIN regoli, oltre a RUNX2, un insieme di TF oncogenici con cui coopera nella regolazione dell’espressione di geni a valle. Abbiamo inoltre mostrato come RAIN in cooperazione con RUNX2 partecipi alla regolazione dell’espressione di geni che favoriscono lo sviluppo delle metastasi. Nel complesso, i dati presentati in questo lavoro forniscono informazioni sulla struttura della rete regolatoria di RUNX2 nei tumori tiroidei, supportando un modello in cui RUNX2 regola diversi processi specifici andando a differenziare la propria attività e diversificando i cooperatori con cui interagisce. Inoltre, i nostri dati evidenziano per la prima volta una connessione diretta tra l’attività di RUNX2 e il rimodellamento metabolico che caratterizza lo sviluppo dei tumori. In aggiunta a ciò, i nostri dati dimostrano come RAIN, aldilà del suo effetto in cis sul locus di RUNX2, influenzi l’evoluzione tumorale tramite un’azione regolatoria estesa all’intero genoma, sottolineando l’importanza dell’associazione funzionale tra elementi codificanti e non-codificanti per la biologia dei tumori.

Caratterizzazione approfondita della rete trascrizionale di RUNX2 nei tumori tiroidei: meccanismo d'azione e conseguenze biologiche / Emanuele Vitale , 2024 May 24. 36. ciclo, Anno Accademico 2022/2023.

Caratterizzazione approfondita della rete trascrizionale di RUNX2 nei tumori tiroidei: meccanismo d'azione e conseguenze biologiche

VITALE, EMANUELE
2024

Abstract

Cancer evolution requires precise patterns of gene expression on which cancer cells rely for their growth and survival. These transcriptional programs are established through a complex regulatory landscape involving the hierarchical interaction of multiple regulatory elements. Transcription factors (TFs) have a pivotal role in orchestrating these regulatory circuitries, in a way that makes cancer cells dependent on their activities. The Runt-related Transcription Factor 2 (RUNX2) was found to be involved in the metastatic progression of many types of cancer, including thyroid cancer (TC). Studying the mechanism underlining RUNX2-upregulation in cancer we identified RAIN, a new chromatin-associated long non-coding RNA (lncRNA) that is expressed from RUNX2-active enhancers and is involved in RUNX2 regulation during TC progression. Preliminary data also suggest that RAIN may have additional RUNX2-independent transcriptional activities. Although the association of RUNX2 with cancer aggressiveness has been well demonstrated, the details of the architecture of the RUNX2-transcriptional landscape and the mechanism explaining how this TF and its associated lncRNA contribute to TC biology are still largely unexplored. In this work, we used a multidimensional approach to reconstruct the transcriptional landscape of RUNX2 in TC and to clarify its contribution to regulating biological processes that support cancer progression. We focused on both the upstream genomic activity of this TF and the downstream effect on biological processes that sustain cancer evolution. We also investigated how RAIN partakes in these activities and contributes to the overall transcriptional program underlining TC progression. By integrating ChIP-seq and RNA-seq data, we functionally characterized the RUNX2-associated genomic elements and got insights into its network of cooperators and its dependent gene program. All these pieces of -omics information were combined with clinical, mutational, and transcriptional data retrieved from the TCGA-THCA project in a computational hierarchical model that recapitulates the RUNX2-transcriptional network in TC. This analysis identified a series of RUNX2-dependent gene modules corresponding to specific biological functions that influence the clinical behavior of these neoplasms. Among these, the module of genes partaking in metabolism regulation resulted to be correlated with TC metastasis, showing for the first time a direct contribution of RUNX2 to cancer cell metabolic rewiring, by promoting lipid synthesis while inhibiting oxidative phosphorylation. A parallel multi-omics approach was applied to study the molecular functions of RAIN. We unveiled that, as for RUNX2, RAIN directly controls a core of other cancer-related TFs and cooperates with them in regulating the expression of downstream targets. We also showed that in cooperation with RUNX2, RAIN is required for the expression of a subset of genes that promote TC metastatization. Overall, these data define the RUNX2 transcriptional landscape in TC, providing a model in which RUNX2 affects the expression of specific categories of genes linked to specific biological functions by differentiating its activity and network of interactions. Besides some well-established biological processes that have been already associated with RUNX2 activity, we identified for the first time the direct role of RUNX2 in the complex metabolic rewiring of cancer cells. Furthermore, our data demonstrate that RAIN, besides its in-cis effect on the RUNX2 locus, influences TC progression through a widespread regulatory activity throughout the genome, underlining the importance of coding and non-coding functional interplay in cancer biology.
Deep characterization of the RUNX2 transcriptional landscape in Thyroid Cancer: mechanism of action and biological consequences.
24-mag-2024
LUMINARI, Stefano
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/1342588
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