Secretome proteomics-based targeting of inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2) and clusterin (Clu) mitigates p53 amyloid–mediated tumorigenesis in oral carcinoma

Oral cancer, a major form of head and neck cancer (HNC), remains clinically challenging due to its aggressiveness, and therapeutic resistance. Tumor microenvironment (TME) is one of the key factors of cancer progression which strongly influences how cancer cells survive, spread, and respond to treatment. However, the molecular factors underlying its dysregulation are not fully understood. Recent data suggest that oncogenic forms of p53, particularly the mutant R273H and its amyloidogenic conformer, display gain-of-functon(GOF) phenotype. In this study, we investigated how oncogenic p53 remodel the secretome to affect the extracellular matrix (ECM) organization. Proteomic analysis of conditioned media from AW13516 (tongue carcinoma) cells show distinct ECM-related secreted proteins. Among the differentially enriched proteins, Inter-Alpha-Trypsin Inhibitor Heavy Chain H2 (ITIH2) and Clusterin (CLU) were secreted in amyloid p53 and mutant p53 expressing cells. Super-resolution microscopy revealed spatial co-localization of both ITIH2 and CLU with intracellular mutant p53 aggregates. Further, exosome isolation and analysis confirmed that both proteins are actively packaged and released through exosomes. Functional studies showed that ITIH2 silencing induced apoptosis through inhibition of the AKT/NF-κB pathway. Similarly, the CLU silencing reduced Vimentin and mTOR levels, while increasing BAX and E-cadherin levels, indicating loss of stemness, induction of apoptosis and partial restoration of epithelial features. To identify potential therapeutic vulnerabilities, molecular docking analysis revealed that methotrexate (MTX) binds strongly and specifically to both ITIH2 and CLU. Consistent with these predictions, MTX treatment combined with ITIH2 or CLU silencing produced a synergistic effect on the suppression of proliferation and migration. Overall, our findings identify ITIH2 and CLU as exosome-associated downstream effectors of oncogenic p53 that contribute to tumor microenvironment dysregulation. Thus, targeting this secretome axis, particularly, in combination with MTX may offer a promising strategy to limit oral cancer progression.