Malignant pleural mesothelioma (MPM) is a highly aggressive and rapidly progressive tumor that affect the mesothelium componing the pleura; it can acquire different histological subtypes (mainly epithelioid, biphasic, and sarcomatoid MPM), which are of prognostic significance. Epigenetic modifications occurring during MPM initiation and progression may play a relevant role in negatively regulating the crosstalk between the tumor and the immune system, as well as contributing to the highly immunosuppressive microenvironment. A better understanding of MPM epigenetics will contribute to refine antitumor strategies, laying the ground for epigenetic-based immunotherapy. The present study evaluated, in the first instance, changes in the gene expression fingerprint of 10 MPM cell lines of different phenotype treated with the second-generation DNA hypomethylating agent (DHA) guadecitabine, through the Nanostring Oncology panel with nCounter readout. Ingenuity pathway analysis results revealed that guadecitabine induced the activation of natural killer and dendritic cells signaling pathways in 50% of MPM cell lines, followed by the activation of other components involved in the immune system response to infections and inflammation. Besides, the most frequently activated upstream regulators belonging to the interferon (IFN)-γ signaling pathway. Also, the up- regulation (mean fold change (mFC) ≥ 1.5) of key immune-related molecules, such as the NY-ESO-1 (mFC=13.16), MAGE-B2 (mFC=13.09), CD70 (mFC=5.27), and CTLA-4 (mFC=4.81) was reported. We also performed histological type-specific investigations to explore molecular changes induced by guadecitabine among the 3 histotypes. Guadecitabine induced the up-regulation of the expression of epithelial markers (e.g., CDH1, EPCAM, PECAM1), observed at higher levels in sarcomatoid cell lines; this was accompanied by the down-regulation of mesenchymal origin molecules (e.g., CDH2, NCAM), and inductor of metastatic signals (e.g., CDH11). Secondly, the immunomodulatory effects of guadecitabine were compared to those of different epigenetic drugs (the histone deacetylase (HDAC) inhibitors VPA and SAHA, or the EZH2 EPZ- 6438), alone or in combination with guadecitabine, in 5 MPM cell lines (two sarcomatoid, one biphasic, and two epithelioid). We performed cytofluorimetric and molecular qRT-PCR analyses and, in this regard, results showed that guadecitabine up-regulated the expression of immune-related molecules, such as HLA class I antigens (mFC=1.59), ICAM-1 (mFC=3.27), PD-L1 (mFC=2.13), and NKG2DLs (MIC-A mFC=1.88, MIC-B mFC=2.42, and ULBP2 mFC=3.16), and up-regulated/induced Cancer Testis Antigens (CTA: NY-ESO-1, MAGE-A1, and MAGE-A3) expression; VPA up-regulated the expression of HLA class I antigens (mFC=1.50), PD-L1 (mFC=2.76), NKG2DLs (MIC-A mFC=1.69, MIC-B mFC=2.67, and ULBP2 mFC=3.26), and the expression of CTA MAGE-A1 and MAGE-A3 in 2/5 and 3/5 MPM cell lines, respectively; SAHA up- regulated the expression of MICA (mFC=1.57), MICB (mFC=4.05), MAGE-A1 and MAGE-A3 in 2/5and 4/5 MPM cell lines, respectively; conversely, EPZ-6438 induced minimal immunomodulatory effects, inducing only NY-ESO-1 and up-regulating PD-L1, MIC-B, and ULBP2 expression in 1 MPM cell line each. Despite the heterogeneous activities of single epigenetic drugs, the addition of both VPA, SAHA, and EPZ-6438 to guadecitabine strengthened the immunomodulatory effects of the latter, by affecting the expression of all investigated molecules. Specifically, guadecitabine plus VPA, SAHA, or EPZ-6438 upregulated the expression of HLA class I antigens mFC=2.21, 2.03, or 2.29; ICAM-1 mFC=4.09, 4.63, or 5.33; PD-L1 mFC=6.95, 2.42, or 2.50; MIC-A mFC=3.48, 2.00, or 2.23; MIC-B mFC=6.80, 2.48, or 2.81; ULBP2 mFC=13.45, 3.40, or 4.11, respectively. Lastly, higher levels of upregulated/induced CTA expression were observed after all 3 combination treatments versus guadecitabine alone. Cadherins modulation was MPM histotype-related: CDH1 expression was induced in the 2 constitutive-negative sarcomatoid MPM cell lines by guadecitabine alone or combined with VPA, SAHA, or EPZ-6438; CDH2 expression was upregulated by VPA or SAHA in 1/5 cell lines, and by guadecitabine plus VPA or SAHA in 3/5 or in 1/5 MPM cell lines, respectively; however, no induction of CDH2 have been reported in the constitutive negative epithelioid cell lines. Overall, from comprehensive gene expression panel analyses, we confirmed that guadecitabine induced/up-regulated the expression of immune and immune-related molecules, pivotal in the tumor- immune system crosstalk; also, we highlighted that guadecitabine-induced activation of IFN-related genes, especially in the sarcomatoid phenotype, supporting the hypothesis that DHA could increase the immune response against MPM, potentially also with sarcomatoid features; moreover, the modulation of adhesion molecules towards the epithelial type suggests the possibility to revert the epithelial-to- mesenchymal transition (EMT) event, crucial in the invasion-metastasis cascade. Also, combining guadecitabine with HDACi/EZH2i strengthened its immunomodulatory capabilities, laying the rationale for epigenetic drugs-based immunotherapies, to enhance efficacy of these strategy in the MPM clinic.

Il mesotelioma pleurico maligno (MPM) è un tumore molto aggressivo e rapidamente progressivo che si sviluppa a livello del mesotelio che compone la pleura; questa neoplasia può assumere diversi sottotipi istologici (epitelioide, bifasico e sarcomatoide), i quali sono strettamente correlati alla prognosi. Le modificazioni epigenetiche che avvengono nelle fasi di iniziazione e progressione del MPM possono svolgere un ruolo fondamentale nel regolare negativamente il crosstalk tra tumore e sistema immunitario, contribuendo a mantenere un microambiente tumorale immunosoppressivo. Conoscere più dettagliatamente il panorama epigenetico del MPM può contribuire a definire il razionale per nuove terapie antitumorali e porre le basi per studi di combinazione che prevedano l’utilizzo di farmaci epigenetici con farmaci immunoterapeutici. Con il presente studio abbiamo voluto valutare, in un primo momento, le modificazioni nel profilo di espressione genica di 10 linee di MPM, di diverso istotipo, trattate con la guadecitabina, un agente demetilante il DNA di seconda generazione, tramite la piattaforma nCounter di Nanostring. I risultati ottenuti tramite Ingenuity Pathway Analysis (IPA) hanno mostrato che la guadecitabina era in grado di indurre l’attivazione dei geni coinvolti nel crosstalk tra cellule dendritiche e natural killer nel 50% delle linee cellulari di MPM indagate, accompagnata dall’attivazione di altre componenti coinvolte nella risposta immunitaria a infezioni e infiammazioni. I fattori trascrizionali “upstream” più frequentemente attivati appartenevano al pathway di segnalazione dell’interferon (IFN)-γ. Inoltre, è stata riscontrata l’up-regolazione (fold change medio (mFC) ≥ 1.5) di molecole immuno-relate, come NY-ESO-1 (mFC=13.16), MAGE-B2 (mFC=13.09), CD70 (mFC=5.27) e CTLA-4 (mFC=4.81). Abbiamo inoltre effettuato analisi istotipo-specifiche per esplorare le modificazioni molecolari indotte dalla guadecitabina nei 3 sottotipi di MPM. La guadecitabina ha indotto l’up-regolazione dell’espressione di marcatori del fenotipo epiteliale (es. CDH1, EPCAM e PECAM1), osservata ad alti livelli nelle linee cellulari sarcomatoidi; ciò è stato associato alla down-regolazione di molecole di origine mesenchimale (es. CDH2 e NCAM) e induttori della cascata metastatica (es. CDH11). Successivamente abbiamo comparato gli effetti immunomodulatori della guadecitabina con quelli di altri farmaci epigenetici (gli inibitori delle iston acetiltransferasi (HDAC) VPA e SAHA o l’inibitore di EZH2 EPZ-6438) da soli o in combinazione con la guadecitabina in 5 linee cellulari di MPM (2 sarcomatoidi, 1 bifasica e 2 epitelioidi). Analisi citofluorimetriche e molecolari hanno rivelato che la guadecitabina up-regolava l’espressione delle molecole immuno-relate, quali HLA di classe I (mFC=1.59), ICAM-1 (mFC=3.27), PD-L1 (mFC=2.13), e NKG2DL (MICA mFC=1.88, MICB mFC=2.42, ULBP2 mFC=3.16), inducendo/up-regolando l’espressione dei Cancer Testit Antigens (CTA) NY-ESO-1, MAGE-A1 e MAGE-A3; il VPA up-regolava l’espressione degli antigeni di HLA di classe I (mFC=1.50), PD-L1 (mFC=2.76), NKG2DL (MICA mFC=1.69, MICB mFC=2.67, ULBP2 mFC=3.26) e quella dei CTA MAGE-A1 e MAGE-A3, rispettivamente in 2/5 e 3/5 linee cellulari di MPM; il SAHA up-regolava l’espressione di MICA (mFC=1.57), MICB (mFC=4.05) e MAGE-A1 e MAGE-A3, rispettivamente in 2/5 e 4/5 linee cellulari; per contro, l’EPZ-6438 ha mostrato minime capacità immunomodulanti, inducendo solamente NY-ESO-1 e up-regolando l’espressione di PD-L1, MICB e ULBP2 in 1 linea cellulare ciascuno. Contrariamente ai risultati eterogenei ottenuti dai singoli farmaci, l’associazione di VPA, SAHA o EPZ-6438 alla guadecitabine ha rafforzato le capacità immunomodulanti di quest’ultima, influenzando l’espressione di tutte le molecole indagate. Specificatamente, le combinazioni di guadecitabine con VPA, SAHA o EPZ-6438 up-regolavano l’espressione degli antigeni HLA di classe I (mFC=2.21, 2.03, o 2.29 rispettivamente), di ICAM-1 (mFC=4.09, 4.63, o 5.33), di PD-L1 (mFC=6.95, 2.42, o 2.50), di MIC-A (mFC=3.48, 2.00, o 2.23), di MIC-B (mFC=6.80, 2.48, o 2.81) e di ULBP2 (mFC=13.45, 3.40, o 4.11). Infine, livelli di up- regolazione/induzione maggiori sono stati osservati per i CTA a seguito di tutti e 3 i trattamenti combinati rispetto alla guadecitabina in singolo. La modulazione delle caderine è stata influenzata dal sottotipo istologico di MPM: l’espressione di CDH1 è stata indotta dalla guadecitabina in singolo e dalla sua combinazione con VPA, SAHA e EPZ-6438 nelle 2 linee cellulari sarcomatoidi, costitutivamente negative per l’espressione del gene; l’espressione di CDH2 è stata up-regolata dal VPA e dal SAHA singoli in 1/5 linee cellulari e dalle combinazioni di guadecitabina con VPA o SAHA, rispettivamente in 3/5 o 1/5 linee cellulari di MPM; ciononostante, non è stata osservata alcuna up-regolazione del gene nelle 2 linee cellulari epiteliodi, costitutivamente negative per l’espressione di CDH2. In conclusione, dalle analisi approfondite del pannello di espressione genica abbiamo confermato che la guadecitabina è in grado di up-regolare/indurre l’espressione di molecole immunitarie e immuno- relate cruciali per il crosstalk tra il tumore e il sistema immunitario; inoltre, abbiamo dimostrato che essa induce l’attivazione di geni correlati all’IFN, soprattutto nel fenotipo sarcomatoide, supportando l’ipotesi che i demetilanti possano aumentare la risposta immunitaria contro il MPM, potenzialmente anche del tipo istologico più aggressivo; la modulazione delle molecole di adesione tendente verso il fenotipo epitelioide suggerisce la possibilità di revertire la transizione epitelio-mesenchima, cruciale nel processo di metastatizzazione. Infine, combinando la guadecitabina con farmaci inibitori delle HDAC/EZH2 ha rafforzato la sua attività immunomodulante, fornendo il razionale per studi di associazione di farmaci epigenetici e agenti immunoterapici in modo da aumentare l’efficacia di questi ultimi nel trattamento del mesotelioma.

Cannito, S. (2020). Modeling of cancer immune phenotype by new epigenetic drugs: a strategy to improve efficacy of immunotherapy [10.25434/cannito-sara_phd2020].

Modeling of cancer immune phenotype by new epigenetic drugs: a strategy to improve efficacy of immunotherapy

Cannito, Sara
2020-01-01

Abstract

Malignant pleural mesothelioma (MPM) is a highly aggressive and rapidly progressive tumor that affect the mesothelium componing the pleura; it can acquire different histological subtypes (mainly epithelioid, biphasic, and sarcomatoid MPM), which are of prognostic significance. Epigenetic modifications occurring during MPM initiation and progression may play a relevant role in negatively regulating the crosstalk between the tumor and the immune system, as well as contributing to the highly immunosuppressive microenvironment. A better understanding of MPM epigenetics will contribute to refine antitumor strategies, laying the ground for epigenetic-based immunotherapy. The present study evaluated, in the first instance, changes in the gene expression fingerprint of 10 MPM cell lines of different phenotype treated with the second-generation DNA hypomethylating agent (DHA) guadecitabine, through the Nanostring Oncology panel with nCounter readout. Ingenuity pathway analysis results revealed that guadecitabine induced the activation of natural killer and dendritic cells signaling pathways in 50% of MPM cell lines, followed by the activation of other components involved in the immune system response to infections and inflammation. Besides, the most frequently activated upstream regulators belonging to the interferon (IFN)-γ signaling pathway. Also, the up- regulation (mean fold change (mFC) ≥ 1.5) of key immune-related molecules, such as the NY-ESO-1 (mFC=13.16), MAGE-B2 (mFC=13.09), CD70 (mFC=5.27), and CTLA-4 (mFC=4.81) was reported. We also performed histological type-specific investigations to explore molecular changes induced by guadecitabine among the 3 histotypes. Guadecitabine induced the up-regulation of the expression of epithelial markers (e.g., CDH1, EPCAM, PECAM1), observed at higher levels in sarcomatoid cell lines; this was accompanied by the down-regulation of mesenchymal origin molecules (e.g., CDH2, NCAM), and inductor of metastatic signals (e.g., CDH11). Secondly, the immunomodulatory effects of guadecitabine were compared to those of different epigenetic drugs (the histone deacetylase (HDAC) inhibitors VPA and SAHA, or the EZH2 EPZ- 6438), alone or in combination with guadecitabine, in 5 MPM cell lines (two sarcomatoid, one biphasic, and two epithelioid). We performed cytofluorimetric and molecular qRT-PCR analyses and, in this regard, results showed that guadecitabine up-regulated the expression of immune-related molecules, such as HLA class I antigens (mFC=1.59), ICAM-1 (mFC=3.27), PD-L1 (mFC=2.13), and NKG2DLs (MIC-A mFC=1.88, MIC-B mFC=2.42, and ULBP2 mFC=3.16), and up-regulated/induced Cancer Testis Antigens (CTA: NY-ESO-1, MAGE-A1, and MAGE-A3) expression; VPA up-regulated the expression of HLA class I antigens (mFC=1.50), PD-L1 (mFC=2.76), NKG2DLs (MIC-A mFC=1.69, MIC-B mFC=2.67, and ULBP2 mFC=3.26), and the expression of CTA MAGE-A1 and MAGE-A3 in 2/5 and 3/5 MPM cell lines, respectively; SAHA up- regulated the expression of MICA (mFC=1.57), MICB (mFC=4.05), MAGE-A1 and MAGE-A3 in 2/5and 4/5 MPM cell lines, respectively; conversely, EPZ-6438 induced minimal immunomodulatory effects, inducing only NY-ESO-1 and up-regulating PD-L1, MIC-B, and ULBP2 expression in 1 MPM cell line each. Despite the heterogeneous activities of single epigenetic drugs, the addition of both VPA, SAHA, and EPZ-6438 to guadecitabine strengthened the immunomodulatory effects of the latter, by affecting the expression of all investigated molecules. Specifically, guadecitabine plus VPA, SAHA, or EPZ-6438 upregulated the expression of HLA class I antigens mFC=2.21, 2.03, or 2.29; ICAM-1 mFC=4.09, 4.63, or 5.33; PD-L1 mFC=6.95, 2.42, or 2.50; MIC-A mFC=3.48, 2.00, or 2.23; MIC-B mFC=6.80, 2.48, or 2.81; ULBP2 mFC=13.45, 3.40, or 4.11, respectively. Lastly, higher levels of upregulated/induced CTA expression were observed after all 3 combination treatments versus guadecitabine alone. Cadherins modulation was MPM histotype-related: CDH1 expression was induced in the 2 constitutive-negative sarcomatoid MPM cell lines by guadecitabine alone or combined with VPA, SAHA, or EPZ-6438; CDH2 expression was upregulated by VPA or SAHA in 1/5 cell lines, and by guadecitabine plus VPA or SAHA in 3/5 or in 1/5 MPM cell lines, respectively; however, no induction of CDH2 have been reported in the constitutive negative epithelioid cell lines. Overall, from comprehensive gene expression panel analyses, we confirmed that guadecitabine induced/up-regulated the expression of immune and immune-related molecules, pivotal in the tumor- immune system crosstalk; also, we highlighted that guadecitabine-induced activation of IFN-related genes, especially in the sarcomatoid phenotype, supporting the hypothesis that DHA could increase the immune response against MPM, potentially also with sarcomatoid features; moreover, the modulation of adhesion molecules towards the epithelial type suggests the possibility to revert the epithelial-to- mesenchymal transition (EMT) event, crucial in the invasion-metastasis cascade. Also, combining guadecitabine with HDACi/EZH2i strengthened its immunomodulatory capabilities, laying the rationale for epigenetic drugs-based immunotherapies, to enhance efficacy of these strategy in the MPM clinic.
2020
Cannito, S. (2020). Modeling of cancer immune phenotype by new epigenetic drugs: a strategy to improve efficacy of immunotherapy [10.25434/cannito-sara_phd2020].
Cannito, Sara
File in questo prodotto:
File Dimensione Formato  
phd_unisi_076556.pdf

accesso aperto

Descrizione: Tesi di dottorato di Sara Cannito
Tipologia: PDF editoriale
Licenza: PUBBLICO - Pubblico con Copyright
Dimensione 5.16 MB
Formato Adobe PDF
5.16 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11365/1120775