Inhibition of F-Box Protein p45SKP2 Expression and Stabilization of Cyclin-Dependent Kinase Inhibitor p27KIP1 in Vitamin D Analog-Treated Cancer Cells

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Abstract
Treatment of cancer cells with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or its analogs induces growth arrest and expression of the cyclin-dependent kinase inhibitor p27KIP1. Although 1,25(OH)2D3 transiently enhances p27kip1 gene transcription in some cells, its effects on p27KIP1 protein levels are generally more gradual and sustained. This suggests that 1,25(OH)2D3 treatment may be stabilizing p27KIP1 protein, which is sensitive to modification by the SCFSKP2 protein ubiquitin ligase and proteosomal degradation. Here, we show that treatment of AT-84 head and neck squamous carcinoma cells with the 1,25(OH)2D3 analog EB1089 increases p27KIP1 protein levels without significantly affecting expression of its mRNA. EB1089 treatment repressed expression of mRNAs encoding the F-box protein p45SKP2, a marker of poor head and neck cancer prognosis, and the cyclin kinase subunit CKS1, which is essential for targeting p45SKP2 to p27KIP1. This coincided with a reduction of total p45SKP2 protein, and p45SKP2 associated with p27KIP1. Consistent with these findings, turnover of p27KIP1 protein was strongly inhibited in the presence of EB1089. A similar reduction in p45SKP2 expression and stabilization of p27KIP1 protein was observed in 1,25(OH)2D3-sensitive UF-1 promyelocytic leukemia cells, which also respond by transiently increasing p27kip1 gene transcription. Our results reveal that 1,25(OH)2D3 analogs increase levels of p27KIP1 in different cell types by inhibiting expression of SCFSKP2 subunits and reducing turnover of p27KIP1 protein.