Table 1.
Immunohistochemcal scoring system.
Figure 1.
Immunohistochemically stained tissues from CRC patients and adjacent normal control tissues.
(A) YAP-positive tumor; (B) YAP-negative tumor; (C) YAP-positive normal tissue; (D) YAP-negative normal tissue; (E) TAZ-positive tumor; (F) TAZ-negative tumor; (G) TAZ-positive normal tissue; (H) TAZ-negative normal tissue. Representative images were taken under a microscope (x10). These results indicate the clinical significance of YAP and TAZ overexpression in CRC tissue.
Table 2.
Clinical correlation of YAP and/or TAZ expression in colorectal cancer.
Table 3.
Cox regression analysis of overall survival (n = 168).
Figure 2.
Kaplan-Meier analysis of overall survival (cumulative survival) of CRC patients relative to YAP and TAZ expression.
(A) Correlation of YAP expression with overall survival; (B) correlation of TAZ expression with overall survival; (C) correlation of combined YAP and TAZ expression with overall survival. A statistically significant difference is shown in overall survival outcome between the different groups of patients, with those having positive co-overexpression of YAP and TAZ having the worst overall survival.
Figure 3.
Assessment of YAP and TAZ expression in six human colon adenocarcinoma cell lines.
(A) QRT-PCR analysis of YAP and TAZ expressions; (B) Western blot analysis of YAP and TAZ expressions. β-actin is used as an internal loading control. HCT116 cells show the highest expression levels of both YAP and TAZ.
Figure 4.
Effect of YAP and TAZ expression on proliferation and apoptosis of HCT116 cells.
(A) Western blot analysis of cell lines with reduced YAP and TAZ levels after transfection with siRNAs: parental HCT116 cells carrying no siRNA (parental); control siRNA (si-Con); siRNA to YAP (si-YAP); siRNA to TAZ (si-TAZ); and co-transfected with siRNA to YAP and siRNA to TAZ (si-YAP-TAZ). (B) MTT cell growth assays of control cells (parental, si-Con); cells with reduced YAP or TAZ expression (si-YAP, si-TAZ); or cells with reduced YAP and TAZ expression (si-YAP-TAZ). Data are presented as mean ± SD, N = 3, *P<0.05. (C) Flow cytometric analyses of cells stained with Annexin-V-FITC and PI: cells carrying no siRNA (parental); control siRNA (si-Con); siRNA to YAP (si-YAP); siRNA to TAZ (si-TAZ); and cells with reduced YAP and TAZ expression (si-YAP-TAZ). These results indicate that YAP expression has a greater effect on cell proliferation and suppression of apoptosis compared to TAZ expression; however, a synergistic role between YAP and TAZ increases their combined individual effects.
Figure 5.
Effect of YAP and TAZ expression on the migration and invasion of HCT116 colon cancer cells.
(A) Representative images (×10) of migration assays of HCT116 cells with normal levels of YAP and TAZ expression (parental and si-Con); cells with suppressed levels of YAP or TAZ expression (si-YAP or si-TAZ); and cells with suppressed YAP and TAZ expression (si-YAP-TAZ). (B) Mean number of cells from the five independent migration assays described in previously. (C) Invasion assay of parental HCT116 cells; si-Con cells; si-YAP; si-TAZ; and si-YAP-TAZ cells in modified Boyden chambers with Matrigel-coated membranes. After 24 h, invasive cells that had moved through the Matrigel membrane were stained and counted under a microscope at ×10 magnification. (D) Graphical representation of invasive cells calculated as mean value ± SD from five fields. These show statistically significantly reduced migration in both YAP and TAZ siRNA transfected cells compared to control cells; the effect is greatest in the group transfected with both YAP and TAZ. [Asterisks indicate statistically significant differences in YAP or TAZ siRNA transfected cells vs. si-Con or parental cells, (*, P<0.05; **, P<0.01).].