Establishment and characterization of a new immortalized human oral keratinocyte (HOK) cell line harboring various...

Head and neck squamous cell carcinoma (HNSCC) is characterized by frequentTP53mutations, which include gain-of-function (GOF) variants that drive tumor progression and chemoresistance. While genomic evidence across multiple specimens ranging from oral pre-malignant lesions (OPL) through invasive and metastatic HNSCC indicates that TP53 loss or mutation occurs relatively early in HNSCC carcinogenesis, it has been difficult to discern how mutation of this tumor suppressor drives further tumor cell evolution and influences the tumor microenvironment to drive tumor progression. To address this question, we generated an immortalized human oral keratinocyte (iHOK) cell line expressing wt TP53 as well as TP53 mutant forms to determine how mutations impact functional and genomic characteristics of HOKs that can in turn drive their evolution to neoplastic cells. We established a novel panel of iHOK cell lines harboring distinctTP53mutations, including the high-risk C238F variant. Key results revealed significant phenotypic and molecular divergence: high-risk lines exhibited enhanced invasiveness and chemoresistance compared to low-risk counterparts. Weighted gene co-expression network analysis (WGCNA) linked high-risk mutations to pro-metastatic pathways and stress-response signatures, with the C238F line uniquely enriched for p53-related GOF mechanisms. Conversely, low-risk lines remained chemosensitive. These findings underscore the clinical relevance of mutation-specific iHOK models in determining the transcriptomic events that drive invasiveness and drug resistance and identify ways to intercept these phenotypes. By mirroring theTP53diversity observed in patient tumors, this cell line panel bridges a critical gap in head and neck carcinogenesis research, enabling mechanistic dissection of GOF phenotypes and preclinical evaluation of therapies in an array of TP53mutant cell models. Its significance lies in providing a validated, annotated resource that accelerates drug discovery and clarifies the role ofTP53mutational subtypes in HNSCC development and progression, offering a framework for future translational studies in genetically complex malignancies.