The mTOR inhibitor rapamycin down-regulates the expression of the ubiquitin ligase subunit Skp2 in breast cancer cells

Abstract

Introduction: Loss of the cyclin-dependent kinase inhibitor p27 is associated with poor prognosis in breast cancer. The decrease in p27 levels is mainly the result of enhanced proteasome-dependent degradation mediated by its specific ubiquitin ligase subunit S phase kinase protein 2 (Skp2). The mammalian target of rapamycin (mTOR) is a downstream mediator in the phosphoinositol 3' kinase (PI3K)/Akt pathway that down-regulates p27 levels in breast cancer. Rapamycin was found to stabilize p27 levels in breast cancer, but whether this effect is mediated through changes in Skp2 expression is unknown.

Methods: The expression of Skp2 mRNA and protein levels were examined in rapamycin-treated breast cancer cell lines. The effect of rapamycin on the degradation rate of Skp2 expression was examined in cycloheximide-treated cells and in relationship to the anaphase promoting complex/Cdh1 (APC\C) inhibitor Emi1.

Results: Rapamycin significantly decreased Skp2 mRNA and protein levels in a dose and time-dependent fashion, depending on the sensitivity of the cell line to rapamycin. The decrease in Skp2 levels in the different cell lines was followed by cell growth arrest at G1. In addition, rapamycin enhanced the degradation rate of Skp2 and down-regulated the expression of the APC\C inhibitor Emi1.

Conclusion: These results suggest that Skp2, an important oncogene in the development and progression of breast cancer, may be a novel target for rapamycin treatment.

Figure 1

Effect of rapamycin on cell growth. (a) The dose-effect curve of rapamycin on cell growth. The breast cancer cell lines T47D and MDA-MB-231 were treated with different concentrations of rapamycin for 72 h and the decrease in growth rate compared to control cells was assessed. (b) Time dependence of the effect of rapamycin on cell growth. The breast cancer cell lines T47D (left) and MDA-MB-231 (right) were treated with rapamycin (20 nM) for different time periods and the decrease in growth rate compared to control cells was assessed. Results are means ± standard error of the mean, with n = 3 for each treatment. *p < 0.05 between the two conditions.

Figure 2

The effect of rapamycin on cell cycle profile and mTOR activation. (a) The effect of rapamycin on cell cycle distribution. T47D cells were treated with rapamycin (20 nM) or DMSO (0.02%) for 24 h and subjected to FACS analysis to determine the distribution of the cell cycle. (b) The effect of rapamycin on the phosphorylation of the mTOR effectors p-S6K1 and p-4E-BP1. The breast cancer cell lines T47D and MDA-MB-231 were treated with rapamycin (20 nM) for different time periods and the phosphorylated forms of the proteins was determined by western blot analysis. Skp1 was used as a loading control. Arrows indicate the specific bands.

Figure 3

The effect of rapamycin on Skp2 protein levels and cell cycle distribution. (a) The effect of rapamycin on Skp2 protein levels. The breast cancer cell lines T47D and MDA-MB-231 were treated with rapamycin (20 nM) for different time periods and the expression of the protein was determined by western blot analysis. Skp1 was used as a loading control. (b) The time-dependent effect of rapamycin on G1 distribution in T47D and MDA-MB-231 cells. Cells were treated with rapamycin (20 nM) for different time periods and the percentage of cells in G1 was determined by FACS analysis using Modfit LT 2.0 software.

Figure 4

The effect of rapamycin on Skp2 mRNA levels. T47D cells were treated with rapamycin (20 nM) for 8 h and the change in Skp2 mRNA levels was determined by real-time RT-PCR. PGK, phosphoglycerate kinase.

Figure 5

The effect of rapamycin on Skp2 degradation. (a) The effect of rapamycin on Skp2 degradation rate. T47D cells were treated with rapamycin (20 nM) or DMSO (0.02%) for 24 h followed by treatment with cycloheximide (CHX; 100 μg/ml) for the indicated time periods. Levels of Skp2 protein levels were determined by western blot analysis. (b) The effect of rapamycin on Emi1 protein levels. T47D cells were treated with rapamycin (20 nM) and levels of Emi1 were determined by western blot analysis. Skp1 was used as a loading control.

Figure 6

T47D cells were transiently transfected with a Skp2 insert or an empty plasmid (vehicle) and treated with rapamycin (20 nM) for 48 h. Levels of Skp2 protein levels were determined by western blot analysis. Optic Density (od) of the Skp2 band as measured by chemiluminesence – blot background (Bkg).