Panoply™ Human PAK1 Over-expressing Stable Cell Line

Non-small cell lung cancer (NSCLC) is a leading cause of cancer death, highlighting the urgent need to identify new therapeutic targets. P21-activated kinase-1 (PAK1) plays a crucial role in the development and progression of various cancers, including NSCLC. T-cell factor 1 (TCF1) is an anti-tumor factor that influences T-cell biological function. However, the specific mechanisms by which PAK1 promotes NSCLC progression by regulating TCF1 remain unclear. Here, researchers found that PAK1 expression was elevated in NSCLC tissues and cells, while TCF1 expression was significantly reduced. PAK1 expression was significantly negatively correlated with TCF1 mRNA expression in NSCLC. Silencing PAK1 using shRNA or inhibiting PAK1 with the small molecule inhibitor IPA-3 dose-dependently suppressed the malignant proliferation of NSCLC cells and upregulated TCF1 expression, and vice versa. Enhancing TCF1 expression using the small molecule TWS119 dose-dependently inhibited the proliferation, migration, and invasion of NSCLC cells without affecting PAK1 expression. Immunoprecipitation analysis confirmed an interaction between PAK1 and TCF1 in NSCLC. Combined survival analysis indicated that high PAK1 expression and low TCF1 expression were associated with poor prognosis in NSCLC patients. Finally, TCF1 expression was significantly correlated with immune cell infiltration (CD8+ T cells and tumor-infiltrating lymphocytes (TILs)) and immune checkpoint inhibitors (PD1, PDL1), and could accurately predict the efficacy of immunotherapy.

MTT assay results showed that the proliferation of PAK1-overexpressing A549 cells was significantly increased, with significantly higher absorbance at 490 nm at 24, 48, 72, and 96 hours. Conversely, the proliferation of PAK1-knockdown A549 cells was significantly inhibited at all time points (Figure 1c). Wound healing assays showed that PAK1-overexpressing A549 cells were almost completely healed at 18 hours (72.62%) and completely healed at 30 hours (100%), while PAK1-knockdown A549 cells showed slower healing, reaching only 43.94% healing rate at 18 hours and 76.72% at 40 hours, while the control group was completely healed at 40 hours (Figure 1d). Subsequently, Transwell migration assays showed that the number of migrating PAK1-overexpressing A549 cells increased by 2.68 times, while the number of migrating PAK1-knockdown cells was significantly reduced (54.17%) (Figure 1e). Furthermore, to assess the effect of PAK1 on anchorage-independent growth, soft agar colony formation assays were performed. The results showed that shRNA-mediated knockdown of PAK1 significantly reduced the colony-forming ability of A549 cells by 68.63% (Figure 1f). In addition, when studying the effect of PAK1 on A549 cell apoptosis, flow cytometry analysis showed a higher proportion of Annexin V-positive cells in PAK1-knockdown cells compared to control cells (Figure 1g). In summary, these results indicate that PAK1 promotes cell proliferation, induces cell migration, and inhibits cell apoptosis.

Figure 1. PAK1 plays a critical role in tumorigenesis of A549 lung cancer cell line. (Lu C, et al., 2025)