Histone deacetylase inhibitors increase p27Kip1 by affecting its ubiquitin-dependent degradation through Skp2 down-regulation

Histone deacetylase inhibitors (HDACIs) represent a promising class of pharmacologically active compounds. Currently, some HDACIs are FDA approved for cancer therapy and many others are in clinical trials, showing important activities against hematologic and solid tumors at doses generally well tolerated by the patients. HDACIs are also able to interfere with the aging process and have been involved in the control of inflammation and oxidative stress. In vitro, HDACIs induce different cellular responses including growth arrest, differentiation and apoptosis. Although the molecules frequently prevent G1->S transition, the mechanisms underlying G1 arrest remain still undefined. Here, we evaluated the effects of HDACIs on p27Kip1, a key cyclin-dependent kinase inhibitor (CKI). We observed that HDACI-dependent antiproliferative activity is associated with p27Kip1 accumulation. The CKI increase occurs rapidly and is not due to enhanced gene expression or to p27Kip1 cellular relocalization. Conversely, p27Kip1 up-regulation is due to the elongation of its half-life, suggesting that HDACIs interfere with the CKI degradation process. p27Kip1 removal requires a preliminary ubiquitination step due to the Skp2-SCF E3 ligase complex. We demonstrated that HDACIs increase p27Kip1 stability through the down-regulation of Skp2 protein levels. Accordingly, we also observed that HDACIs up-regulate p27Kip1 but not its ubiquitinated bands. HDACI-associated Skp2 decline is only partially due to a negative effect on Skp2 gene expression. Indeed, the decrease of the protein is most profound and enduring than the changes of Skp2 transcript. This argues for HDACI-dependent activities on Skp2 protein post-translational modifications and/or on its removal. In summary, we demonstrate in human cells of different origin that HDACIs increase p27Kip1 by hampering its degradation. We also present evidence demonstrating that HDACIs affect the activity of specific ubiquitination complexes. The findings might be of relevance in the phenotypic effects of these compounds, including their anticancer and aging-modulating activities.