The expression pattern, prognostic significance and pharmacologic targeting of the eukaryotic translation initiation factor 2alpha in head and neck squamous cell carcinoma

Abstract

Study background: Drug resistance is a common cause of therapy failure in head and neck squamous cell carcinoma (HNSCC). One approach to tackle it is by targeting fundamental cellular processes, such as translation. The eukaryotic translation initiation factor 2 (EIF2) integrates diverse stress signals with key cellular processes. It consists of 3 subunits: alpha, beta and gamma. Phosphorylation of the alpha subunit is primarily a cytoprotective mechanism, curbing global protein synthesis in response to stress, however, prolonged phosphorylation is deleterious to cells. Methods: Publicly available datasets were analyzed to determine the RNA and protein expression patterns of eIF2 complex factors in HNSCC, with focus on the regulatory subunit EIF2alpha, encoded by EIF2S1. Immunohistochemical staining (IHC) was used to determine EIF2alpha abundance in metastasis, carcinoma, premalignant lesions and adjacent non-neoplastic tissue. Intraoperative samples were used to evaluate steady-state phosphorylation levels in tumor and adjacent normal tissue. A small-molecule inhibitor of EIF2alpha dephosphorylation, salubrinal, was tested in vitro, followed by viability assays, flow cytometry, and immunoblot analyses. Patient-derived 3D tumor spheroids (PD3DS) were cultured with salubrinal, and their viability assessed. Lastly, salubrinal was evaluated in combination with standard-of-care chemotherapeutics. Results: The analysis of RNA and proteomics data shows that many EIF2 complex factors are deregulated in HNSCC. EIF2S1 overexpression negatively impacts overall and disease-free survival and is associated with clinicopathological parameters indicating disease aggressiveness and severity. The elevation of EIF2S1 transcript and protein expression, though present irrespective of etiology, is significantly more pronounced in human papillomavirus (HPV) negative HNSCC. Mutations in the gene are infrequent, but its promoter methylation is decreased. Further, IHC staining reveals increasing EIF2alpha abundance from premalignant lesions to invasive and metastatic carcinoma. In immunoblots from intraoperative samples, EIF2alpha expression and steady-state phosphorylation are higher in HNSCC than in neighboring normal tissue. Inhibition of EIF2alpha dephosphorylation decreases HNSCC cell viability, clonogenic survival, and impairs G1/S transition. Salubrinal also decreases the viability of PD3DS and acts synergistically with cisplatin, 5-fluorouracil, and proteasome inhibitors. Conclusion: The results indicate that pharmacological inhibition of EIF2alpha dephosphorylation is a potential therapeutic and preventive strategy in HNSCC.