Titolo della tesi: EXPLOITING THE THERAPEUTIC POTENTIAL OF ADAR1 SILENCING TO ENHANCE NK CELL-MEDIATED RESPONSES AGAINST CERVICAL CANCER
Cervical cancer is the fourth cause of cancer death among women worldwide, mainly resulting from human papillomavirus (HPV) infection. Disease progression from premalignant lesions to aggressive cancer can span up to twenty years after HPV infection, reflecting a prolonged inability of the immune system to control tumor development, yet providing a window of opportunity for therapeutic interventions. Notably, ADAR1 expression is upregulated in cervical cancer along with many other tumors, where its editing activity on double-stranded (ds) RNAs modulates the type I IFN pathway, preventing aberrant IFN activation and autoimmunity. Since type I IFN signaling is pivotal for anti-tumor immunity, strategies that restore or enhance its proper activity are crucial. Importantly, reactivation of the type I IFN response promotes the production of proinflammatory cytokines and chemokines, thereby enhancing NK cell recruitment and effector function. This project aims to shed light on the therapeutic potential of ADAR1 inhibition to restore type I IFN signaling and boost NK cell-mediated antitumor immunity in cervical cancer. Immunohistochemistry and real-time RT-PCR analysis performed on patient-derived cervical biopsies confirmed that ADAR1 expression level increases from normal mucosa to tumor, along with a gradual depletion of infiltrating innate lymphocytes as indicated by flow cytometry analysis. Of note, NK cells upregulated CD49a, CD94, NKp44, and TIGIT, while downregulating CD16 and Granzyme B, delineating an altered phenotypic profile. Transcriptomic analysis of SiHa and CaSki cervical cancer-derived cell lines revealed the production of type I IFNs, chemokines and cytokines upon ADAR1 silencing. Consistently, ADAR1 silencing in CC-derived cell lines resulted in reduced proliferation evaluated by Incucyte. On the other hand, their conditioned media increased both the degranulation and migration capacity of freshly purified NK cells from healthy donors. CC xenograft RAG2-/- C57BL/6 mice subjected to NK cell depletion showed bigger tumors with increased ADAR1 expression compared with wild-type controls. In addition, ADAR1 silencing by siRNA or inducible shRNA slowed tumor growth and reshaped NK-cell phenotype, as evidenced by increased CD11b and decreased Ki67 expression, along with an increasing trend of Granzyme A expression. Then, fluorescent microscopy showed the efficiency of the lipid nanoparticle-based delivery system that we explored, also leading to ADAR1 knockdown. Finally, we evaluated the replacement of siRNAs with aptamers to be generated via SELEX technique, which required the production of the ADAR1 recombinant protein using bacterial cultures. Our findings identify ADAR1 as a promising therapeutic target and pave the way for novel strategies in the treatment of cervical cancer and other malignancies, combining direct inhibition of tumor proliferation with the enhancement of antitumor immune responses.