Abstract:

Pathogenic germline variants in TP53 constitute the central etiological driver of hereditary tumor predisposition disorders syndromes. The oncogenic mechanisms of hotspot mutations in the DNA-binding domain of p53 are well-established. However, the functional consequences of non-hotspot missense mutations remain incompletely understood. In this study, we characterized the molecular pathogenesis and clinical significance of the p53 non-hotspot mutation p.Arg267Trp (p.R267W). In addition, evolutionary conservation analysis, structural prediction, and functional assays including CCK-8 cell proliferation, clonogenic assay, Transwell migration, wound healing assay, qPCR, Western blot, single luciferase reporter assay, and flow cytometry techniques were performed to assess the impact of R267W on TP53 target gene (CDKN1A) regulation and tumor-suppressive phenotypes (proliferation, colony formation, migration) in non-small cell lung cancer models (A549/NCI-H1299). Experiments confirmed that the mutant does not affect the p53 protein stability. The impairment of protein function is hypothesized to result from the disruption of the DNA-binding domain conformation. Experimental evidence suggested that the mutant TP53 exhibited significantly reduced transcriptional activity (P<0.001), resulting in a concomitant reduction in CDKN1A mRNA expression and diminished cell cycle arrest capability when compared to wild type. At the tumor-suppressive functional level, the R267W mutant significantly reduced inhibition rates of non-small cell lung cancer cell proliferation, colony formation, and migration relative to wild type (P<0.05). This study reveals that the R267W variant drives cell cycle dysregulation and malignant phenotypes in lung cancer by disrupting TP53’s transcriptional functions. These findings establish a molecular basis for the pathogenic classification of TP53 variants of uncertain clinical significance.

Key words: p53 (p.R267W), DNA-binding domain, missense mutation, transcriptional repression, lung cancer