Nuclear Targeting of Cyclin-Dependent Kinase 2 Reveals Essential Roles of Cyclin-Dependent Kinase 2 Localization and Cyclin E in Vitamin D-Mediated Growth Inhibition

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Abstract
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), inhibits proliferation of a variety of cell types including adenocarcinoma of the prostate. We have previously shown that 1,25-(OH)2D3 increases the stability of the cyclin-dependent kinase inhibitor p27KIP1, decreases cyclin-dependent kinase 2 (CDK2) activity, and promotes G1 phase accumulation in human prostate cancer cells. These effects correlate with cytoplasmic relocalization of CDK2. In this study, we investigated the role of CDK2 cytoplasmic relocalization in the antiproliferative effects of 1,25-(OH)2D3. CDK2 was found to be necessary for prostate cancer cell proliferation. Although induced by 1,25-(OH)2D3, the cyclin-dependent kinase inhibitor p27KIP1 was dispensable for 1,25-(OH)2D3-mediated growth inhibition. Reduction in CDK2 activity by 1,25-(OH)2D3 was associated with decreased T160 phosphorylation, a residue whose phosphorylation in the nucleus is essential for CDK2 activity. Ectopic expression of cyclin E was sufficient to overcome 1,25-(OH)2D3-mediated cytoplasmic mislocalization of CDK2 and all antiproliferative effects of 1,25-(OH)2D3, yet endogenous levels of cyclin E or binding to CDK2 were not affected by 1,25-(OH)2D3. Similarly, knockdown of the CDK2 substrate retinoblastoma, which causes cyclin E up-regulation, resulted in resistance to 1,25-(OH)2D3-mediated growth inhibition. Human prostate cancer cells resistant to growth inhibition by 1,25-(OH)2D3 but retaining fully functional vitamin D receptors were developed. These cells did not exhibit 1,25-(OH)2D3-mediated cytoplasmic relocalization of CDK2. Targeting CDK2 to the nucleus of 1,25-(OH)2D3-sensitive cancer cells blocked G1 accumulation and growth inhibition by 1,25-(OH)2D3. These data establish central roles for CDK2 nuclear-cytoplasmic trafficking and cyclin E in the mechanism of 1,25-(OH)2D3-mediated growth inhibition in prostate cancer cells.