Differential expression of the F-box proteins Skp2 and Skp2B in breast cancer

Abstract

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.

TGFβ prevents proteasomal degradation of the cyclin-dependent kinase inhibitor p27kip1 for cell cycle arrest

“…Primers for the Skp2 variant (p45-Skp2), the specific E3 ligase for p27 were used. 106 The PCR reactions consisted of the following: 11 μl H 2 O, 12.5 μl iQ SYBR Green Supermix, 0.25 μl of each primer (10 μM) and 1 μl cDNA. The PCR conditions for Skp2 was: 95°C for 3 min, 95°C for 15 s/66°C for 30 s/72°C for 30 s (40 cycles).…”

Nuclear translocation of Skp2 facilitates its destruction in response to TGFβ signaling

“…As shown in Figure 2A, the majority of Skp2 in the absence of TGFβ is localized in the cytosol, which is consistent with known previous Skp2 localization in prostate, melanoma, colon, lymphoma and breast cancer tissues. [23][24][25][26][27] To examine the potential effect of TGFβ stimulation on cellular translocation of Cdh1 and Skp2, a similar study was performed as described in the above. As shown in Figure 2A, TGFβ stimulation results in significant translocation of Skp2 from the cytosol to the nucleus, while no obvious redistribution of Cdh1 was observed.…”

“…21,22 Localization of Skp2 has been variable depending on cell type. [23][24][25][26][27] To assess the localization of Cdh1 and Skp2 in Mv1Lu cell, we performed immunocytochemistry in the presence and absence of TGFβ. Localization of Cdh1 was measured by using monoclonal antibody against Cdh1 and FITC conjugated anti-mouse secondary antibody.…”

Downregulation of the KIP family members p27KIP1 and p57KIP2 by SKP2 and the role of methylation in p57KIP2 inactivation in nonsmall cell lung cancer

“…For SKP2 and JAB1 we used previously described positive controls. 21,37,38 Evaluation. For each antigen, the nuclear IHC signal was scored, respectively.…”