Tumors are complex ecosystems composed by heterogeneous populations of cancer cells embedded in a dynamic tumor microenvironment (TME). Communication of cancer cells with the TME displays both local and systemic tumor-promoting effects, including angiogenesis, ECM remodeling, and modulation of immune/inflammatory cells, to support tumor growth and progression, and escape from immune surveillance. Understanding immune evasion mechanisms that generate non-immunogenic “cold” tumors represents a key issue for improving the efficacy of anticancer immune therapies. Accumulating evidence has established that oncogenic drivers, such as mutant p53 and HIF1α, contribute to tumor progression and immune evasion by attenuating the cGAS/STING/IFN-I pathway in cancer cells. This cascade involves cGAS-dependent sensing of cell-intrinsic DNA damage with consequent induction of STING ER-Golgi trafficking, activation of the transcription factor IRF3 and expression of downstream type-I interferons (IFN) response target genes, thus engaging anti-tumor immune surveillance. Recently, our research group highlighted an oncogenic axis affecting tumor-stroma crosstalk. We discovered that miR-30d, a secreted onco-miRNA cooperatively induced by HIF1α and mutp53 oncoproteins, regulates targets involved in the secretory pathway, causing structural alterations of ER and Golgi compartments. This promotes the release of a pro-malignant secretome, which alters the TME and fosters tumor growth and metastatic colonization. Transcriptomic analysis in metastatic breast cancer (BC) cells upon downregulation of miR-30d highlighted a putative inhibitory effect of miR-30d on the categories of “cellular response to DNA damage”, “type-I interferon production” and “antiviral innate immune response”, leading to the hypothesis that high levels of miR-30d might inhibit the expression of type-I IFN target genes, a condition that could contribute to establish an immune “cold” microenvironment. In this work I have investigated the impact of miR-30d on the regulation of IFN response and dissected the mechanisms by which ablation of miR-30d leads to upregulation of IFN signaling in BC cells. By using an LNA inhibitor and a miR-30d Decoy construct, I have demonstrated that inhibition of miR-30d in BC cells led to activation of the main components of the cGAS/STING signaling machinery, in particular phosphorylation of TBK1 and STING, nuclear translocation of the IRF3 transcription factor, and consequent secretion of type-I IFNs. The effects of miR-30d inhibition included normalization of fragmented Golgi structure, and concomitant activation of STING at Golgi apparatus in BC cells, thus suggesting that miR-30d might attenuate the cGAS/STING pathway by inducing structural alterations of the secretory pathway, in particular of the Golgi. Furthermore, I found that miR-30d inhibition in BC cells promoted accumulation of nuclear DNA damage and of cytoplasmic dsDNA with activation of cGAS, which acts upstream of the STING DNA-sensing pathway. In sum, this evidence is consistent with a model in which inhibition of miR-30d may both trigger upstream induction of the cGAS/STING pathway in cancer cells, by causing release of dsDNA in the cytosol, and further sustain its activity by normalizing the structure of the secretory pathway. In addition, I observed that when miR-30d inhibition was combined with conventional chemotherapeutic agents in metastatic BC cells, the treatment led to a much stronger effect on the activation of the IFN signaling. Experiments with ex-vivo and in-vivo preclinical models are currently in progress to investigate whether inhibition of miR-30d could reactivate immune surveillance as well as to test synergies between miR-30d inhibition and cGAS/STING-inducing chemotherapeutic treatments.

I tumori sono ecosistemi complessi, nei quali popolazioni eterogenee di cellule tumorali sono immerse in un microambiente dinamico. La comunicazione delle cellule tumorali con il microambiente causa effetti protumorigenici sia locali che sistemici, tra cui l’angiogenesi, il rimodellamento della matrice extracellulare, e la modulazione della componente immunitaria/infiammatoria, supportando la progressione tumorale e l’evasione dalla sorveglianza immunitaria. La comprensione dei meccanismi di evasione dal sistema immunitario è di particolare importanza per migliorare l'efficacia delle immunoterapie antitumorali. È noto che oncogeni, quali le forme mutate di p53 (mut-p53) e HIF1α, contribuiscono all'evasione immunitaria attraverso l’inibizione della cascata di segnalazione di cGAS/STING/IFN-I nelle cellule tumorali. Questa via è iniziata dall’attivazione di cGAS da DNA citoplasmatico, con conseguente traslocazione di STING dal reticolo endoplasmatico al Golgi, attivazione del fattore di trascrizione IRF3 ed espressione dei geni della risposta all’interferone di tipo-I (IFN-I), i quali codificano per immunomodulatori che innescano la sorveglianza immunitaria antitumorale. Recentemente, il nostro gruppo di ricerca ha evidenziato un meccanismo oncogenico che influenza la comunicazione tra le cellule tumorali e quelle stromali. Abbiamo scoperto che il miR-30d, un miRNA oncogenico secreto indotto da HIF1α e mut-p53, causa alterazioni strutturali dell’apparato di Golgi. Ciò promuove la secrezione tumorale ed altera il microambiente favorendo la crescita tumorale e la colonizzazione metastatica. L'analisi trascrittomica di cellule di carcinoma mammario (CM) metastatico in seguito al silenziamento del miR-30d ha suggerito l'ipotesi che livelli elevati di miR-30d possano inibire l'espressione dei geni IFN-I, una condizione che potrebbe contribuire a inibire la risposta immune antitumorale. In questo lavoro di tesi ho studiato l'impatto del miR-30d nella regolazione della cascata di segnalazione di cGAS/STING/IFN-I e i meccanismi attraverso i quali il silenziamento del miR-30d porta all’induzione di tale segnalazione in cellule di CM. In particolare, ho dimostrato che l'inibizione del miR-30d porta all'attivazione dei principali componenti del macchinario di segnalazione cGAS/STING, in particolare induce la fosforilazione di TBK1 e STING, la traslocazione nucleare di IRF3 e la conseguente secrezione di interferoni di tipo¬-I. Gli effetti dell'inibizione del miR-30d includono la normalizzazione della struttura del Golgi e la concomitante attivazione di STING nel Golgi. Ciò suggerisce che il miR-30d potrebbe inibire la via cGAS/STING inducendo alterazioni strutturali dell’apparato del Golgi. Inoltre, abbiamo riscontrato che l'inibizione del miR-30d nelle cellule di CM promuove danno al DNA nucleare e accumulo di DNA nel citoplasma, con conseguente attivazione di cGAS. In sintesi, i nostri risultati sono coerenti con un modello in cui l'inibizione del miR-30d contribuisce ad innescare l'induzione della via cGAS/STING in cellule tumorali attraverso il rilascio di DNA nel citosol, ed inoltre a promuoverne l’attivazione attraverso la normalizzazione della struttura del Golgi. Ho inoltre osservato che la combinazione di inibitori del miR-30d con agenti chemioterapici in cellule di CM ha un effetto sinergico nell’attivazione di IFN-I. Sono attualmente in corso esperimenti mediante modelli preclinici ex-vivo e in-vivo per verificare se l'inibizione di miR-30d possa riattivare la sorveglianza immunitaria, e per valutare l’effetto della combinazione di inibitori di miR-30d con trattamenti chemioterapici che inducono cGAS/STING/IFN-I.

Analisi degli effetti del miR-30d sulla via di segnalazione immune cGAS/STING/IFN-I in cellule di carcinoma mammario

DI CAMILLO, FEDERICA
2023-03-24

Abstract

Tumors are complex ecosystems composed by heterogeneous populations of cancer cells embedded in a dynamic tumor microenvironment (TME). Communication of cancer cells with the TME displays both local and systemic tumor-promoting effects, including angiogenesis, ECM remodeling, and modulation of immune/inflammatory cells, to support tumor growth and progression, and escape from immune surveillance. Understanding immune evasion mechanisms that generate non-immunogenic “cold” tumors represents a key issue for improving the efficacy of anticancer immune therapies. Accumulating evidence has established that oncogenic drivers, such as mutant p53 and HIF1α, contribute to tumor progression and immune evasion by attenuating the cGAS/STING/IFN-I pathway in cancer cells. This cascade involves cGAS-dependent sensing of cell-intrinsic DNA damage with consequent induction of STING ER-Golgi trafficking, activation of the transcription factor IRF3 and expression of downstream type-I interferons (IFN) response target genes, thus engaging anti-tumor immune surveillance. Recently, our research group highlighted an oncogenic axis affecting tumor-stroma crosstalk. We discovered that miR-30d, a secreted onco-miRNA cooperatively induced by HIF1α and mutp53 oncoproteins, regulates targets involved in the secretory pathway, causing structural alterations of ER and Golgi compartments. This promotes the release of a pro-malignant secretome, which alters the TME and fosters tumor growth and metastatic colonization. Transcriptomic analysis in metastatic breast cancer (BC) cells upon downregulation of miR-30d highlighted a putative inhibitory effect of miR-30d on the categories of “cellular response to DNA damage”, “type-I interferon production” and “antiviral innate immune response”, leading to the hypothesis that high levels of miR-30d might inhibit the expression of type-I IFN target genes, a condition that could contribute to establish an immune “cold” microenvironment. In this work I have investigated the impact of miR-30d on the regulation of IFN response and dissected the mechanisms by which ablation of miR-30d leads to upregulation of IFN signaling in BC cells. By using an LNA inhibitor and a miR-30d Decoy construct, I have demonstrated that inhibition of miR-30d in BC cells led to activation of the main components of the cGAS/STING signaling machinery, in particular phosphorylation of TBK1 and STING, nuclear translocation of the IRF3 transcription factor, and consequent secretion of type-I IFNs. The effects of miR-30d inhibition included normalization of fragmented Golgi structure, and concomitant activation of STING at Golgi apparatus in BC cells, thus suggesting that miR-30d might attenuate the cGAS/STING pathway by inducing structural alterations of the secretory pathway, in particular of the Golgi. Furthermore, I found that miR-30d inhibition in BC cells promoted accumulation of nuclear DNA damage and of cytoplasmic dsDNA with activation of cGAS, which acts upstream of the STING DNA-sensing pathway. In sum, this evidence is consistent with a model in which inhibition of miR-30d may both trigger upstream induction of the cGAS/STING pathway in cancer cells, by causing release of dsDNA in the cytosol, and further sustain its activity by normalizing the structure of the secretory pathway. In addition, I observed that when miR-30d inhibition was combined with conventional chemotherapeutic agents in metastatic BC cells, the treatment led to a much stronger effect on the activation of the IFN signaling. Experiments with ex-vivo and in-vivo preclinical models are currently in progress to investigate whether inhibition of miR-30d could reactivate immune surveillance as well as to test synergies between miR-30d inhibition and cGAS/STING-inducing chemotherapeutic treatments.
24-mar-2023
DEL SAL, GIANNINO
35
2021/2022
Settore BIO/11 - Biologia Molecolare
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/3042422
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