Adaptation to Arginine Deprivation Leads to a More Aggressive, Therapy-Resistant Phenotype in HNSCC Cells

<i>Purpose:</i> The development of acquired resistance to arginine deprivation therapy (ADT) is a major barrier to its efficacy. This study aimed to elucidate the possible mechanisms underlying the resistance to ADT. <i>Methods:</i> We applied repeated ADT and established a subline SAS-R9 of the human head and neck squamous cell carcinoma (HNSCC) cells semi-resistant to arginine (Arg) deprivation <i>in vitro</i>. This subline was compared to the parental SAS cell lines for its relative clonogenic proliferation, aggregation, adhesion, and migration capacities. The transcriptomic changes were assessed by RNA sequencing. Signaling pathway alterations were confirmed by RT-PCR and Western blotting. Relative cell radioresistance was analyzed by radiobiological clonogenic survival assay. DNA double-strand break (DSB) repair was assessed by γH2A.X foci analysis. <i>Results:</i> SAS-R9 cells showed higher survival in response to ADT and radiotherapy, elevated clonogenic proliferation rate, cell-cell aggregation, and cell-matrix adhesion, along with increased epithelial-mesenchymal transition (EMT) markers and enhanced DNA DSB repair, potentially related to a more aggressive and therapy-resistant phenotype. <i>Conclusions:</i> While acute ADT has radiosensitizing potential, this new study suggests that long-term, repeated ADT is associated with cell selection and reprogramming, resulting in resistance to radiotherapy-induced DNA damage and higher tumor cell aggressiveness.