Fig. 3: Invasive phenotype of PTEN edge mutants.

U87MG cells and PTEN-null PDCs (P089 and P090) were used to overexpress PTEN edge mutants. PDCs with endogenous PTEN edge mutation were also used for validation (P045 with R130Q mutation; P087 with H93Y mutation). A CLUMP analysis of PTEN mutations identified in SMC and TCGA cohorts. A total of 68 residues were finally selected for CLUMP analysis and CLUMP analysis revealed four distinct subgroups of PTEN mutations. According to CLUMP analysis, PTEN mutations within the phosphatase domain were further divided into distinct subgroups, which correlated with the 3D structure of PTEN protein. B 3D crystal structure of the PTEN protein: all residues changed by PTEN edge mutations (H93, C124, and R130) are located within the same pocket of the phosphatase domain. C Comparison of survival outcomes of orthotopic xenografts established by U87MG cells with distinct PTEN mutants. The mice with edge mutations showed poor survival outcome compared to PTEN-null mice (P-value <0.001, Log-rank test). Three PTEN-null xenografts were used as control group, while six PTEN-mutant xenografts were made per each mutation of interest. D–F In vitro invasion assay to assess the enhanced invasion capacity of PTEN edge mutation compared to nuclear mutation. Data are shown as the means of triplicates of experiments ± s.d. The P-value was calculated by two-sided t test. D In vitro trans-well invasion assay using U87MG cells with exogenous mutants. All tumor cells with edge mutants exhibited significantly increased invasion capacity compared to tumors with nuclear mutants, except H93Y (p-value=0.076, 0.005, and 0.008 for H93Y, C124S, and R130Q, respectively, two-sided t test). Their enhanced invasion capacity was also significantly superior to PTEN-null U87MG cells (p-value=0.203, 0.001, and 0.015 for H93Y, C124S, and R130Q, respectively, two-sided t test). E in vitro 3D sphenoid invasion assay using PTEN-null PDCs (P090) with exogenous mutants. All PDCs (P090) with edge mutants exhibited significantly enhanced invasion capacity compared to PDCs with nuclear mutation (p-value=0.028, <0.001, and <0.001 for H93Y, C124S, and R130Q, respectively, two-sided t test). F In vitro microfluid assay to measure the development of invasion in response to chemotactic stimuli in PTEN-null PDCs (P089) with exogenous mutants. PDCs expressing edge mutants showed a more invasive phenotype than PTEN-null PDCs (p-value=0.135, <0.001, and 0.023 for H93Y, C124S, and R130Q, respectively, two-sided t test). G, H Co-localization of F-actin and PTEN edge mutants at the cell periphery of motile PDCs upon chemotactic stimuli. I Development of cellular projections (black arrow) of PDCs (P089) with exogenous PTEN edge mutations. NT, null-type; Del, deletion.