Engineered SERPINE1 Overexpression Activates the pp53/p21 Pathway and Promotes a Growth-Restricted, Migratory Phenotype in KRAS-Transformed Renal Proximal Tubular Cells

Oncogenic Ras signaling drives cell growth and tumor progression through acquired changes in gene expression that promote the malignant phenotype. One such gene, SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), is a poor prognostic biomarker in human cancers and a p53 target gene. This study utilizes v-KRAS-transformed renal epithelial (KNRK) cells to define how PAI-1 overexpression influences cell morphology, growth dynamics, and migratory behavior. KNRK cells were engineered to express PAI-1 using a CMV-driven lentiviral expression vector, with a CMV drug resistance only vector as a control, followed by stable selection with puromycin. Cellular morphology and growth characteristics were assessed by microscopic imaging and cell density analysis; cell migration was evaluated using a scratch-wound assay. Western blotting and immunofluorescence served to assess expression and subcellular localization of PAI-1, p21, and p53 in CMV-control and PAI-1 cells. CMV-PAI-1 cells exhibited a shift to a mesenchymal, more pleomorphic and motile phenotype, with increased spreading, irregular borders and decreased cell–cell adhesion. Wound closure in CMV-PAI-1 cells was significantly increased compared to CMV-control cells (p < 0.001), indicating enhanced migratory capacity, with prominent deposition of PAI-1 into cellular migration trails. CMV-PAI-1 cells had a reduced growth rate, an approximately 4-fold increase in p-p53 and a shift in p21 from a perinuclear localization in controls to predominantly cytoplasmic in CMV-PAI-1 cells. These findings highlight PAI-1 as a potential driver of mesenchymal cell traits in KRAS-transformed renal epithelial cells.