Slug promotes p53 and p21 protein degradation by inducing Mdm2 expression in HCT116 colon cancer cells

Abstract

Our previous study revealed that the tumor suppressor/transcription factor p53 directly binds to its transcriptional target, p21, and that the p53/p21 complex binds to zinc finger protein SNAI2 (Slug), a tumor promoter/transcription factor; thereby promoting the degradation of Slug by Mdm2, an E3 ligase. The present study demonstrated that Slug reduced the cellular expression levels of p53 and p21 in HCT116 colon cancer by decreasing their protein stability. In parallel, Slug increased the mRNA and protein expression levels of Mdm2 in these cells. Moreover, knockdown of Mdm2 using specific small interfering RNAs abolished the ability of Slug to induce the degradation of p53 and p21. Considering the well-known function of Mdm2 in facilitating p53 and p21 degradation, these data suggested that Slug promoted p53 and p21 degradation by inducing Mdm2 expression. Moreover, Slug increased ubiquitination levels of p53 in HCT116 cells. This is consistent with the fact that Mdm2 induces p53 degradation by ubiquitinating p53, and further confirmed that Mdm2 acted downstream of Slug. Comparative studies using HCT116 cells and their p53- or p21-knockout variants have revealed that Slug requires p21 to induce p53 degradation. This result is consistent with our previous study, which revealed that Mdm2 acts more efficiently on p53 in the p53/p21 complex compared with on p53 alone. By contrast, Slug did not require p53 to induce p21 degradation, suggesting that p53 was dispensable in Mdm2-mediated p21 degradation. Notably, the ability of Slug to increase/decrease Mdm2/p53 and p21 levels, respectively, was not confined to HCT116 cells alone, but was also confirmed in A549 and H460 lung cancer cells. Collectively, the results of the present study suggested that Slug could counter p53 and p21. The balance between these two opposing groups (Slug vs. p53/p21) may depend on environmental stresses and the internal physiology of cells.

Keywords: Mdm2; oncogene; p21; p53; protein stability; tumor suppressor; zinc finger protein SNAI2.

Figure 1.

Slug decreases cellular levels of p53 and p21. HCT116 cells were treated either with a control or two sets of Slug-targeting siRNAs. After 24 h of incubation, cellular expression levels of p53, p21 and Slug were compared using western blotting. β-actin was used as the loading control. siRNA, small interfering RNA; Slug, zinc finger protein SNAI2.

Figure 2.

Slug promotes p53 protein degradation in a p21-dependent manner. HCT116 cells and their p21-knockout variants were treated with control or Slug siRNA. After 24 h of recovery, the cells were additionally treated with CHX (10 µM) and further incubated for the indicated periods. p53 levels were analyzed by western blotting and normalized to those of β-actin. Western blotting results are representative of both short and long exposures for more precise comparison. Their mean values and standard deviations are shown in the plots. *P<0.005, **P<0.05 si-control vs. si-Slug in HCT116 cells. ns, non-significant; siRNA, small interfering RNA; Slug, zinc finger protein SNAI2; CHX, cycloheximide.

Figure 3.

Slug promotes p21 protein degradation in a p53-independent manner. HCT116 cells and their p53-knockout variants were treated with control or Slug siRNA for 24 h, and then incubated in the presence of CHX (10 µM) for the indicated periods. p21 and Slug levels were analyzed. *P<0.005, **P<0.05 si-control vs. si-Slug in HCT116 cells. #P<0.005 si-control vs. si-Slug in p53-knockout variants. siRNA, small interfering RNA; Slug, zinc finger protein SNAI2; CHX, cycloheximide.

Figure 4.

Slug increases Mdm2 expression. (A) HCT116 cells were treated with siRNAs targeting Slug or Mdm2 in the combination indicated. After 24 h of incubation, expression levels of the specified proteins were compared using western blotting. (B) Mdm2 and Slug mRNA levels in HCT1116 cells, treated with control or Slug siRNA, were compared by reverse transcription-quantitative PCR. (C) HCT116 cells were transfected with the expression vector for HA-tagged ubiquitin along with siRNAs targeting Slug or Mdm2 in the indicated combinations. The transfectants were treated with MG132 (10 µM) for 2 h. The precipitated proteins were analyzed by western blotting using anti-HA and anti-p53 antibodies. Effects of siRNAs were confirmed by analyzing cell lysates using anti-Mdm2 and anti-Slug antibodies (input). (D) A549 and H460 cells were treated with control or Slug siRNA for 24 h. Levels of the indicated proteins were compared. *P<0.005, si-control vs. si-Slug for Mdm2 mRNA. **P<0.05, si-control vs. si-Slug for Slug mRNA. siRNA, small interfering RNA; Slug, zinc finger protein SNAI2; HA-Ub, HA-ubiquitin; IP, immunoprecipitation; IB, immuonoblotting.

Figure 5.

Schematic model representing the functional relationship between Slug, Mdm2, p53 and p21. Slug increased cellular levels of Mdm2 mRNA and protein. As Mdm2 could stabilize Slug mRNA by binding to it, an amplification loop between Slug and Mdm2 expression appeared to exist (indicated by two-headed arrow). Mdm2 could act on p53 and p21, inducing their degradation. While Mdm2 was not required p53 for p21 degradation, its action on p53 required p21 since the p53/p21 interaction promoted the p53/Mdm2 interaction. This oncogenic situation is indicated by red arrows. Mdm2 could also induce proteolytic cleavage of Slug, which was facilitated by the p53/p21 complex. This tumor-suppressing pathway is indicated by black arrows. This model suggested that Mdm2 could exert dual functions by mediating either the tumor-promoting function of Slug or tumor-suppressing function of the p53/p21 complex; the balance between these two opposing pathways may change depending on the external environment and internal physiology of cells. Slug, zinc finger protein SNAI2.