Skp2 is an F-box protein involved in the ubiquitination and subsequent degradation of the cyclin-dependent kinase (Cdk) inhibitor p27. Skp2 has been reported to be overexpressed in a variety of cancer types and to correlate with poor prognosis. We have identified a novel isoform of Skp2 we named Skp2B, which differs from Skp2 only in the C-terminal domain and unlike Skp2 localizes to the cytoplasm. Here, we describe the relative expression of both Skp2 and Skp2B in breast cancer cell lines and in primary breast cancers using quantitative real time RT-PCR. We show that Skp2B mRNA is expressed 10-fold less than Skp2 mRNA in the immortalized but nontransformed breast cell line, 184B5. However, Skp2B is overexpressed as frequently as Skp2, and to higher levels than Skp2 in breast cancer cell lines and primary cancers. Further, we show that cytoplasmic staining is frequent in primary breast cancers. In addition, we found that xenografts expressing Skp2B grow faster than xenografts expressing low levels of Skp2B, and that this effect is independent of p27 degradation. These findings therefore suggest that Skp2B overexpression is also observed in breast cancers and identify Skp2B as a putative oncogene.

The mechanism by which all-trans retinoic acid (ATRA) leads to a G 1 arrest of the cell cycle remains unclear. We show here that the decrease in D-type cyclin levels observed following ATRA treatment correlates with an increase in the rate of cyclin D1 ubiquitylation in both T-47D and MCF-7 breast cancer cell lines. However, MCF-7 cells are more resistant to ATRA than T-47D cells indicating that cyclin D1 degradation is not sufficient for ATRA-mediated arrest. We found a striking difference between these cells in that while ATRA induces an elevation in the cdk inhibitor p27 in T-47D cells, this is not observed in the ATRA-resistant MCF-7 cells. Furthermore, we demonstrate that ATRA promotes the ubiquitylation of Skp2, an F-box protein that targets p27 for degradation. Moreover, overexpression of Skp2 in T-47D cells prevents accumulation of p27 and promotes resistance to ATRA. In addition, overexpression of cyclin D1 in T-47D cells also promotes ATRA resistance. We found that the mechanism of ATRA-induced ubiquitylation of cyclin D1 and Skp2 is independent of CUL-1 expression and that ATRA can rescue cyclin D1 degradation in the uterine cell line SK-UT-1, where D-type cyclins are stabilized due to a specific defect in proteolysis. These data suggest that ATRA induces a novel pathway of ubiquitylation and that the degradation of the F-box protein Skp2 is the mechanism underlying p27 accumulation and cyclin E-cdk2 inactivation following ATRA treatment.It has been known for more than 50 years that retinoids are potent agents for the control of cellular differentiation and proliferation. Several studies have shown that retinoids can suppress the process of tumorigenesis both in vitro and in vivo (1-3) and that cells exposed to all-trans retinoic acid (ATRA) 1 arrest in the G 1 phase of the cell cycle. However, the molecular mechanism underlying this arrest remains unclear.Cyclin-dependent kinases (cdk) are key cell cycle regulators. Their activities are regulated at several levels including phosphorylation, binding to their regulatory subunits, the cyclins, and binding to small inhibitory proteins called the cyclin-dependent kinase inhibitors (CKI). Among the cyclin subunits, D-type cyclins associate with CDK 4 and 6 to phosphorylate the retinoblastoma (Rb) protein. Hyperphosphorylation of Rb promotes the release of the E2F family of transcription factors that then promote entry into S phase through activation of key target genes. Cyclin E-CDK2 complexes act downstream of cyclin D-CDK4/6 to maintain Rb phosphorylation and events leading to the premature activation of cyclin E-CDK2 complex trigger inappropriate entry into S phase. However, the activity of cyclin E-CDK2 complexes are restricted by binding to the CDK inhibitors p27 and p21. The importance of these proteins is best illustrated by the fact that mutations leading to either accumulation of cyclin D1, or elevated cyclin E-associated kinase activity due to loss of p27 expression, are both frequent events contributing to tumorigenesis (4 -7). Therefore, agents...

Cyclin D1 repression of STAT3 expression may explain the association between cyclin D1 overexpression and improved outcome in breast cancer. In addition, bortezomib can amplify the proapoptotic function of cyclin D1, raising the possibility that cyclin D1 levels may be a marker for predicting the response to this novel drug.