Vitamin D Inhibits G₁ to S Progression in LNCaP Prostate Cancer Cells through p27^Kip1 Stabilization and Cdk2 Mislocalization to the Cytoplasm

1,25-(OH)₂ vitamin D₃ (1,25-(OH)₂D ₃) exerts antiproliferative effects via cell cycle regulation in a variety of tumor cells, including prostate. We have previously shown that in the human prostate cancer cell line LNCaP, 1,25-(OH)₂D₃ mediates an increase in cyclin-dependent kinase inhibitor p27^Kip1 levels, inhibition of cyclin-dependent kinase 2 (Cdk2) activity, hypophosphorylation of retinoblastoma protein, and accumulation of cells in G₁. In this study, we investigated the mechanism whereby 1,25-(OH)₂D₃ increases p27 levels. 1,25-(OH) ₂D₃ had no effect on p27 mRNA levels or on the regulation of a 3.5-kb fragment of the p27 promoter. The rate of p27 protein synthesis was not affected by 1,25-(OH)₂D₃ as measured by luciferase activity driven by the 5′- and 3′-untranslated regions of p27 that regulate p27 protein synthesis. Pulse-chase analysis of ³⁵S-labeled p27 revealed an increased p27 protein half-life with 1,25-(OH)₂D ₃ treatment. Because Cdk2-mediated phosphorylation of p27 at Thr ¹⁸⁷ targets p27 for Skp2-mediated degradation, we examined the phosphorylation status of p27 in 1,25-(OH)₂D₃-treated cells. 1,25-(OH)₂D₃ decreased levels of Thr¹⁸⁷ phosphorylated p27, consistent with inhibition of Thr¹⁸⁷ phosphorylation-dependent p27 degradation. In addition, 1,25-(OH) ₂D₃ reduced Skp2 protein levels in LNCaP cells. Cdk2 is activated in the nucleus by Cdk-activating kinase through Thr¹⁶⁰ phosphorylation and by cdc25A phosphatase via Thr¹⁴ and Tyr ¹⁵ dephosphorylation. Interestingly, 1,25-(OH)₂D ₃ decreased nuclear Cdk2 levels as assessed by subcellular fractionation and confocal microscopy. Inhibition of Cdk2 by 1,25-(OH) ₂D₃ may thus involve two mechanisms: 1) reduced nuclear Cdk2 available for cyclin binding and activation and 2) impairment of cyclin E-Cdk2-dependent p27 degradation through cytoplasmic mislocalization of Cdk2. These data suggest that Cdk2 mislocalization is central to the antiproliferative effects of 1,25-(OH)₂D₃.