Figure 5.

p53^R248W mutant selectively regulates STAT3 phosphorylation and activity in PDAC cells. (A, B) STAT3 knockdown phenocopies mutp53 knockdown in migration assays. MIA-PACA-2 (A) and PANC-1 (B) cells were transfected with two different siRNAs against STAT3 mRNA (siSTAT3-1, -2) or scrambled control (scr). Seventy-two-hour post-transfection cells were seeded into transwell inserts to assess their migration. After 24 h, cells were fixed, stained, and counted at the membrane underside. Scale bars, 200 µm. MIA-PACA-2 cells: 4 biological replicates (n = 4), PANC-1 cells: 3 biological replicates, 2 out of 3 with 2 technical replicates (n = 5). Cells were calculated relative to scrambled control. Immunoblot analysis to confirm knockdown of STAT3. HSC70, loading control. (C) Cell viability assays of the indicated PDAC cell lines. Dose response curve after treatment with increasing concentrations of the STAT3 inhibitor Stattic or solvent control for 24 h. For each cell line, three to four biological replicates were measured. Diagram represents means ± SEM. From these curves, IC₅₀ values were determined, indicated in the table. Of note, MIA-PACA-2 cells are the most sensitive to Stattic treatment, indicated by the dashed line. (D) STAT3 inhibition phenocopies mutp53 knockdown in migration assays. Transwell migration assays of MIA-PACA-2, PANC-1, and PA-TU-8988T cells treated with the indicated concentrations of Stattic for 24 h. Scale bars, 200 µm. For all cell lines, quantification of two biological replicates, one of them with two technical replicates (n = 3 total), calculated relative to 0 µM control treatment. (E) Survival curve of PDAC patients harboring TP53 R248 mutations versus patients harboring TP53 nonsense or frameshift (NS/FS) mutations. Number of patients and mean overall survival in months as indicated. TCGA data. Kaplan–Meier statistic, log-rank test. (A, B, D) Diagrams represent the means ± SEM. Student’s t test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns, not significant.