Il ruolo funzionale della via cGAS-STING/IFN-Beta nel controllo della sottopopolazione di cellule staminali tumorali del sarcoma dei tessuti molli

Nowadays, the solely treatment option for advanced and metastatic soft tissue sarcomas (STS) not amenable with surgery is chemotherapy. Our research group previously proposed a novel drug combination strategy based on a peculiar DNA damage induction by the chemotherapeutic agent trabectedin (TR) and the concomitant blockade of DNA repair by the PARP1 inhibitor olaparib (OL). However, in the phase Ib study, 50% of treated patients manifested progressive disease and relapsed. Drug resistance, treatment failures, and disease relapse are attributable to a peculiar cancer cell subpopulation, the so-called cancer stem cells (CSCs), endowed with stemness features and stem cell markers (e.g. OCT4, CD133, SOX2, ALDH1). Unraveling the molecular mechanism involved in the promotion of CSC is crucial to further developing curative treatment options. We preliminary showed that TR+OL increases the cytosolic DNA content triggering the cellular innate immunity through the cGAS/STING/IRF3/IFNβ signaling cascade. This thesis project aims at investigating the involvement of this pathway in the control of CSC feature in STS cells. The experimental plan was developed through two main steps: 1) evaluation of CSC markers after drug-induced DNA damage, and 2) functional validation of the role of cGAS/STING/IRF3/IFNβ pathway in the CSCs control. As a first step, the expression of the OCT4 marker was investigated in three STS cell lines (leiomyosarcoma DMR, fibrosarcoma HT1080, and undifferentiated pleomorphic sarcoma S018) both at mRNA and protein levels by qRT-PCR and flow cytometry after the treatment with TR and OL, as a single agent and in combination showing a statistically significant OCT4 upregulation. Moreover, a significant OCT4 downregulation was obtained after silencing the key transducer IRF3. In the next step, STS cells were treated directly with IFNβ1 revealing a significant increase of the OCT4-positive fraction. Moreover, IFNβ silencing significantly decreased the OCT4 marker. Furthermore, the expression of OCT4 was studied in STS cells treated with the STING agonist 2’3’-cGAMP, in presence or absence of the IFNβ downstream pathway inhibitor, itacitinib. We observed that itacitinib abolished the cGAMP-induced OCT4 upregulation. In conclusion, this study showed that the peculiar DNA damage inflicted by TR+OL treatment promotes the CSC sub-population in STS cells, by activating the cGAS/STING pathway and the IFNβ production, which in turn upregulates the CSC marker OCT4, through its downstream JAK/STAT signaling pathway. These insights pave the way for the future development of molecular targeting of the CSC compartment as a potentially curative treatment option for advanced STS.