Androgens upregulate Cdc25C protein by inhibiting its proteasomal and lysosomal degradation pathways
Cdc25C is a crucial cell cycle regulator belonging to the dual specificity phosphatase family, essential for activating the cdk1/Cyclin B1 complex as cells enter mitosis. Given that alterations in the cell cycle are a hallmark of many cancers, we aimed to explore the regulation of Cdc25C by androgens in human prostate cancer (PCa) cells. Our study focused on androgen-sensitive (AS) LNCaP C-33 cells, androgen-independent (AI) LNCaP C-81 cells, and PC-3 cells, which vary in their response to androgen signaling.
Under normal culture conditions with fetal bovine serum (FBS), we observed that Cdc25C protein levels were comparable across the different PCa cell lines. However, when cultured in a steroid-depleted medium, the Cdc25C protein was significantly reduced in AS C-33 cells, but remained unchanged in AI C-81 and PC-3 cells. Upon androgen treatment of C-33 cells, Cdc25C protein levels were markedly increased in a dose- and time-dependent manner, which correlated with enhanced cell proliferation. This androgen-induced upregulation of Cdc25C was effectively blocked by Casodex, an androgen receptor (AR) antagonist. In contrast, epidermal growth factor (EGF), a known stimulant of PCa cell growth, resulted in only a modest increase (about 1.5-fold) in Cdc25C protein levels.
Further investigations into the altered expression of Cdc25C in C-33 and PC-3 cells via cDNA and/or shRNA transfection revealed that changes in Cdc25C levels were closely associated with corresponding alterations in cell growth and Cyclin B1 protein levels. Interestingly, treatments with actinomycin D and cycloheximide, inhibitors of transcription and translation respectively, only partially blocked the androgen-induced increase in Cdc25C protein. However, both proteasomal and lysosomal inhibitors significantly elevated Cdc25C protein levels, suggesting that Cdc25C degradation is tightly regulated through these pathways.
Immunoprecipitation assays revealed that androgens decrease the ubiquitination of Cdc25C, suggesting that the reduced degradation of Cdc25C under androgen treatment contributes to the increased protein levels. These findings represent the first evidence that Cdc25C plays a critical role in regulating PCa cell growth and that androgen treatments, unlike EGF, substantially increase Cdc25C protein levels in AS PCa cells. This increase is, in part, due to the suppression of its degradation.
These results provide valuable insights into the mechanisms by which androgens regulate Cdc25C and open potential therapeutic avenues for advanced prostate cancer by targeting the degradation pathways of Cdc25C. Upregulating the degradation of Cdc25C could potentially offer a new strategy for controlling PCa cell proliferation and progression. Aloxistatin