ROLE OF CAF-1 PROTEIN IN THE PROGRESSION OF HEAD AND NECK SQUAMOUS CELL CARCINOMA

HEAD AND NECK SQUAMOUS CELL CARCINOMA (HNSCC) IS AN AGGRESSIVE CANCER WHOSE TREATMENTS SIGNIFICANTLY IMPACTS PATIENTS’ PROGNOSIS AND QUALITY OF LIFE. THUS, IDENTIFYING NEW BIOMARKERS AND THERAPEUTIC TARGETS, AS WELL AS ADJUVANT APPROACHES, REMAINS CRUCIAL TO IMPROVE EFFICACY OF TRADITIONAL APPROACHES, INCLUDING SURGERY, RADIOTHERAPY AND CHEMOTHERAPY, AND TO REDUCE RESISTANCE AND COMPLICATIONS RELATED THERETO. CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) RECENTLY EMERGED AS A NEW POTENTIAL TARGET IN ORAL SQUAMOUS CELL CARCINOMA (OSCC), THE MOST COMMON SUBTYPE OF HNSCC. IT IS A COMPLEX, ACTING AS HISTONE CHAPERONE INVOLVED IN CHROMATIN REMODELLING DURING DNA REPLICATION AND REPAIR, AND WHOSE OVEREXPRESSION IS ASSOCIATED WITH POOR PROGNOSIS AND ITS DOWNMODULATION INCREASED RESPONSE TO IONIZING RADIATION. IN THE CONTEXT OF ADJUVANT APPROACHES, METFORMIN, A FIRST-LINE ANTIDIABETIC DRUG, IS STANDING OUT AS A REPOSITIONED DRUG IN CANCER THERAPY, WITH A COMPLEX MECHANISM OF ACTION, WHICH ALSO INCLUDE CHROMATIN REMODELLING. THE STUDY AIMED TO INVESTIGATE CAF-1 AS A POTENTIAL BIOMARKER AND THERAPEUTIC TARGET, AND TO EXPLORE HOW ITS MODULATION - PHARMACOLOGICAL OR GENETIC - MAY INFLUENCE CANCER PROGRESSION AND RADIOSENSITIVITY. TO ADDRESS THESE ISSUES, IN VITRO EXPERIMENTS WERE PERFORMED IN HPV-NEGATIVE (CAL27) AND HPV-POSITIVE (SCC154) OSCC CELL LINES IN BOTH MONOLAYERS AND THREE-DIMENSIONAL SPHEROIDS. FOR THE FIRST TIME, METFORMIN WAS SHOWN TO STRONGLY DECREASE CAF-1 EXPRESSION IN A TIME-DEPENDENT MANNER THROUGH WESTERN BLOT. MOREOVER, THE ANTITUMORAL EFFECTS OF THIS DRUG WERE CONFIRMED BY THE REDUCTION OF COLONY FORMATION, CELL ABILITY TO MIGRATE AND INVADE, AND BY REVERTING THE EXPRESSION OF EPITHELIAL–MESENCHYMAL TRANSITION (EMT) MARKERS. TO CONFIRM THAT THESE EFFECTS COULD BE INDUCED ALSO BY CAF-1 DOWNMODULATION, THE DIRECT SILENCING OF ITS MAJOR SUBUNITS WAS REALIZED BY SIRNA. THIS LATTER PRODUCED METFORMIN-LIKE EFFECTS ON CELL AGGRESSIVENESS. ADDITIONALLY, IT TURNED OUT THAT METFORMIN SENSITIZED OSCC BI- AND THREE-DIMENSIONAL MODELS TO IONIZING RADIATION, LOWERING THE IC50 DOSE AND SUGGESTING A SYNERGISTIC POTENTIAL WITH RADIOTHERAPY. TO BETTER UNDERSTAND THE ROLE OF CAF-1 IN CANCER PROGRESSION, STABLE CELL LINES OVEREXPRESSING INDIVIDUAL CAF-1 SUBUNITS WERE GENERATED THROUGH LENTIVIRAL TRANSDUCTION AND USED TO TEST METFORMIN EFFICACY AND ANALYSE TRANSCRIPTOMIC ALTERATIONS. RESULTS DEMONSTRATED THAT OVEREXPRESSION OF CHAF1A/P150 AND RBBP4/P48 REDUCED METFORMIN’S ANTIPROLIFERATIVE EFFECT, CONFIRMING THEIR POTENTIAL CONTRIBUTION TO ITS MECHANISM OF ACTION. TRANSCRIPTOMIC PROFILE SUPPORTED THEIR PRO-ONCOGENIC ROLE, NOT ONLY RELATED TO AGGRESSIVE TRAITS OF THE TUMOUR, BUT ALSO TO IMMUNE ESCAPE. IN CONCLUSION, THE FINDINGS OF THIS RESEARCH DEMONSTRATED FOR THE FIRST TIME THE ABILITY OF METFORMIN TO DOWNMODULATE CAF-1 EXPRESSION, UPREGULATED IN OSCC AND ASSOCIATED TO POOR PROGNOSIS, AND SUGGEST THAT CAF-1 DEPLETION COULD CONTRIBUTE THE ANTITUMOUR ACTIVITY OF THIS DRUG. FROM A THERAPEUTIC PERSPECTIVE, THIS STUDY LAYS THE PRECLINICAL FOUNDATION FOR COMBINING METFORMIN AND RADIOTHERAPY IN THE TREATMENT OF OSCC, BASED ON THE DRUG’S PROPERTIES PROBABLY CAF-1-MEDIATED. MOREOVER, THE RESULTS DESCRIBED IN THIS THESIS ADDED IMPORTANT TILES IN THE CHARACTERIZATION OF THE ROLE PLAYED BY CAF-1 IN OSCC PROGRESSION, PAVING THE WAY TO FURTHER INVESTIGATIONS RELATING TO THE POSSIBLE USE OF SIRNA AGAINST CAF-1 AS THERAPEUTIC STRATEGY IN THIS CANCER.