N-(1,3-dimethylbutyl)-N’-phenyl-p-phenylenediamine (6PPD) and its transformation product 6PPD-quinone (6PPDQ) are emerging tire-derived environmental contaminants, showing a potential tumor-promoting effect. However, their relevance to cutaneous squamous cell carcinoma (cSCC) remains unclear. Here, we used network toxicology and experimental validation to investigate their potential involvement in cSCC progression. A total of 91 candidate genes were identified, and enrichment analysis revealed their participation in oxidative stress response, epithelial proliferation, extracellular matrix degradation, and oncogenic pathways, including PI3K-Akt, MAPK, and IL-17 pathways. Machine learning identified six hub genes: MMP1, MMP9, RECK, LGALS3, GSTP1, and FOXM1. Immune profiling and single-cell RNA-sequencing analysis linked hub-gene expression patterns and tumor microenvironment features, with an emphasis on macrophages, T cells, and keratinocytes. Molecular docking prioritized MMP9 among the six candidate proteins, and molecular dynamics simulations characterized predicted MMP9-ligand complexes. Furthermore, 6PPDQ was found to promote cSCC cell proliferation and tumor growth in both in vitro and in vivo experiments. At the molecular level, 6PPDQ exposure was accompanied by increased PI3K p110α expression, elevated p-AKT/AKT ratio, and MMP9 upregulation, consistent with the computational predictions. Pharmacological AKT inhibition attenuated MMP9 upregulation and the 6PPDQ-associated growth response, while MMP9 silencing reduced 6PPDQ-enhanced invasion and wound closure. Overall, this bioinformatics-led exploratory study suggests that 6PPDQ has tumor-promoting potential in experimental cSCC models and proposes PI3K-Akt/MMP9 activation as a preliminary mechanistic hypothesis. These findings provide candidate targets for future mechanistic and environmental risk studies.

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