Figure 6.

Decreased DUSP6 expression is associated with human colorectal cancer progression. HT29 and HCT116 colorectal carcinoma cells were stably infected with lentiviruses encoding for a control shRNA or a shRNA against DUSP6. (a) ERK phosphorylation, MEK phosphorylation, and DUSP6 expression were analyzed by western blot. ERK2 and MEK2 expression were used as loading control. Representative immunoblots are shown. (b) HT29 and HCT116 cells were cultured in soft agarose for 14 days before MTT staining. Graphs are representative of three independent experiments. Data are expressed as mean ± SEM. Student’s t test; *p ≤ 0.05. (c) Invasion of shControl‐ and shDUSP6‐expressing HT29 and HCT116 cells was evaluated in Matrigel‐coated transwells. After 72 hr for HT29 cells and 48 hr for HCT116 cells, invading cells were fixed, stained with 1% crystal violet and counted (n = 3 independent experiments for HT29 cells and n = 5 for HCT116 cells). Data are expressed as mean ± SEM. Student’s t test; *p ≤ 0.05. (d) Relative DUSP6 mRNA expression was analyzed by qPCR with RNAs isolated from human colorectal tumor specimens compared to paired adjacent healthy tissue. Relative expression was normalized to housekeeping genes MRPL19, SDHA, and YWHAZ expression (n = 66). Data are expressed as a median ± interquartile range. The Wilcoxon signed‐rank test; **p ≤ 0.01 (e) Relative DUSP6 mRNA expression was assessed by qPCR with RNAs isolated from colorectal tumors and paired healthy adjacent tissue separated by stage (n = 7 for adenomas and Stage 1, n = 22 for Stage 2, n = 27 for Stage 3 and n = 10 for Stage 4). Relative expression was normalized to housekeeping genes MRPL19, SDHA, and YWHAZ expression. Data are expressed as a median ± interquartile range. The Wilcoxon signed‐rank test; *p ≤ 0.05. (f) Ratio of DUSP6 mRNA expression (tumor/paired adjacent healthy tissue) was compared between MSS (n = 50) and MSI (n = 16) patients. Relative expression was normalized to housekeeping genes MRPL19, SDHA, and YWHAZ expression. Data are expressed as a median ± interquartile range. Mann–Whitney U test; *p ≤ 0.05. (g) Ratio of DUSP6 mRNA expression (colorectal tumor/paired adjacent healthy tissue) was compared between wild‐type (n = 38), KRAS‐mutated (n = 17), and BRAF‐mutated (n = 11) patients. Relative expression was normalized to housekeeping genes MRPL19, SDHA, and YWHAZ expression. Data are expressed as a median ± interquartile range. Mann–Whitney U test. DUSP6: dual‐specificity phosphatase 6; ERK: extracellular signal‐regulated kinase; MEK: mitogen‐activated protein kinase/Erk kinase; mRNA: messenger RNA; qPCR: quantitaive polymerase chain reaction; shRNA: short hairpin RNA