FIG 6.

Plk1 phosphorylation of axin2 mediates degradation of β-catenin in human prostate cancer cells. (A to C) Introduction of the S311A mutation antagonizes axin2 expression-induced degradation of β-catenin. PC3 (A) and LNCaP (B and C) cells were transfected with Flag-axin2 (WT or S311A) constructs for 48 h in the absence (A and B) or presence (C) of R1881. (D and E) Inhibition of Plk1 activity by BI2536 inhibits axin2 expression-induced degradation of β-catenin. PC3 (D) and LNCaP (E) cells were transfected with axin2 (WT or S311A) constructs for 48 h and treated with or without BI2536. (F) Axin2-S311A-expresssing cells have a greater colony formation ability than do axin2-expressing cells. PC3 or LNCaP cells (2 × 10³) expressing axin2 constructs were seeded into plates for 7 days, followed by colony formation assays. (G) Quantification of the colonies in panel F. The bar graph was obtained by calculating the percentages of colonies relative to those of WT-axin2, defined as 100% (means ± standard deviations; n = 3 independent experiments). **, P < 0.01. (H to K) Axin2-expressing PC3 cells show reduced tumor formation ability compared to cells stably expressing axin2-S311A. PC3 cells (1 × 10⁶) stably expressing axin2 (WT or S311A) were inoculated into nude mice and grown for 42 days. (H) Tumor sizes were measured every 3 days (means ± standard errors of the means; n = 4 mice from each experiment group). *, P < 0.05. (I) Images of tumors at the end of the study. (J) Representative images of IHC staining for Ki67 and β-catenin in harvested tumors. (K) Average percentages of Ki67-positive cells from multiple tumor sections. Data represent means ± standard errors of the means (n = 4). **, P < 0.01. (L) Expression of Plk1, axin2, and β-catenin in human normal and malignant prostate tissues. (M) Statistical analysis of IHC staining for panel L. “+++, ++” represents overexpression or high relative expression levels; “+/−” represents negative expression or low relative expression levels. P values were determined by a χ² test. Western blots in panels A to E are representative of data from three or more experiments.