Rapalogs induce non‐apoptotic, autophagy‐dependent cell death in HPV‐negative TP53 mutant head and neck squamous cell carcinoma

• Published in: Molecular carcinogenesis 2022-01, Vol.61 (1), p.33-44
• Main Authors: Alam, Md Maksudul, Marin Fermin, Janmaris, Spiller, Patrick T., Burnett, Chaning, Rong, Xiaohua, Moore‐Medlin, Tara, Maxwell, Caden O., Khandelwal, Alok R., Nathan, Cherie‐Ann O.
• Format: Article
• Language: English
• Subjects: 1-Phosphatidylinositol 3-kinase; AKT protein; Animals; Apoptosis; Autophagic Cell Death - drug effects; Autophagy; Autophagy-Related Protein-1 Homolog - genetics; Autophagy-Related Protein-1 Homolog - metabolism; Cell death; Cell Line, Tumor; Cell Proliferation - drug effects; Cell Survival; Cell viability; Everolimus - administration & dosage; Everolimus - pharmacology; Gene Expression Regulation, Neoplastic - drug effects; Head & neck cancer; Head and neck carcinoma; Head and Neck Neoplasms - drug therapy; Head and Neck Neoplasms - genetics; Head and Neck Neoplasms - metabolism; HNSCC; Humans; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Kinases; Mice; mTOR; MTOR Inhibitors - administration & dosage; MTOR Inhibitors - pharmacology; Mutants; Mutation; p53 Protein; Phagocytosis; rapalog; Signal transduction; Sirolimus - administration & dosage; Sirolimus - analogs & derivatives; Sirolimus - pharmacology; Squamous cell carcinoma; Squamous Cell Carcinoma of Head and Neck - drug therapy; Squamous Cell Carcinoma of Head and Neck - genetics; Squamous Cell Carcinoma of Head and Neck - metabolism; TOR protein; TP53; Tumor Suppressor Protein p53 - genetics; Tumors; Xenograft Model Antitumor Assays; Xenografts
• ISSN: 0899-1987; 1098-2744
• DOI: 10.1002/mc.23357

TP53 is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). Patients with HPV‐negative TP53 mutant HNSCC have the worst prognosis, necessitating additional agents for treatment. Since mutant p53 causes sustained activation of the PI3K/AKT/mTOR signaling pathway, we investigated the effect of rapalogs RAD001 and CCI‐779 on HPV‐negative mutTP53 HNSCC cell lines and xenografts. Rapalogs significantly reduced cell viability and colony formation. Interestingly, rapalogs‐induced autophagy with no effect on apoptosis. Pretreatment with autophagy inhibitors, 3‐methyladenine (3‐MA) and ULK‐101 rescued the cell viability by inhibiting rapalog‐induced autophagy, suggesting that both RAD001 and CCI‐779 induce non‐apoptotic autophagy‐dependent cell death (ADCD). Moreover, rapalogs upregulated the levels of ULK1 and pULK1 S555 with concomitant downregulation of the mTORC1 pathway. However, pretreatment of cells with rapalogs prevented the ULK‐101‐mediated inhibition of ULK1 to sustained autophagy, suggesting that rapalogs induce ADCD through the activation of ULK1. To further translate our in vitro studies, we investigated the effect of RAD001 in HPV‐negative mutTP53 (HN31 and FaDu) tumor cell xenograft model in nude mice. Mice treated with RAD001 exhibited a significant tumor volume reduction without induction of apoptosis, and with a concomitant increase in autophagy. Further, treatment with RAD001 was associated with a considerable increase in pULK1 S555 and ULK1 levels through the inhibition of mTORC1. 3‐MA reversed the effect of RAD001 on FaDu tumor growth suggesting that RAD001 promotes ACDC in HPV‐negative mutTP53 xenograft. This is the first report demonstrating that rapalogs promote non‐apoptotic ADCD in HPV‐negative mutTP53 HNSCC via the ULK1 pathway. Further studies are required to establish the promising role of rapalogs in preventing the regrowth of HPV‐negative mutTP53 HNSCC.