The F-Box Protein Skp2 Regulates Expression of p27 in BCR-ABL Transformed Cell Lines and Is Required for Induction of Myeloproliferative Disease in a Murine Model.

Background:The cyclin-dependent kinase inhibitor p27 is a central regulator of cell cycle progression, whose function is perturbed in many human cancers, either due to decreased expression or abnormal localization. p27 levels are negatively correlated with Skp2, the F-box protein of SCF^SKP2, a E3 ubiquitin ligase targeting nuclear p27 for degradation. Skp2 has been shown to cooperate with mutant Ras in in vitro transformation assays, implicating Skp2 as a bona fide oncogene. In chronic myeloid leukemia cell lines, p27 is down-regulated in a Bcr-Abl dependent fashion, while cytoplasmic accumulation has been described in primary CML cells. We herefore hypothesized that Bcr-Abl may regulate p27 via Skp2.

Experimental approach and results:Mo7e-p210^BCR-ABL treated with 2.5 μM imatinib arrested in G0/G1 in a time-dependent manner (53.6±2, 58.3±2, 71.9±1% at 4, 8 and 16h), correlated with reduced in vitro kinase activity of Cdk2 (32% of controls at 16h). Western blot analysis showed a marginal increase of cytoplasmic p27 and 2.5-fold accumulation of nuclear p27 that preceded G_0/1 arrest. Despite the reduced Cdk2 activity, most p27 was phosphorylated on T187, a target of cdk2/cyclinE, suggesting reduced degradation of phospo-p27 (T187). Degradation of nuclear p27 is mediated by SCF^SKP2 and degradation of cytoplasmic p27 by the recently discovered KPC complex. We therefore assayed expression SCF^SKP2 components and KPC1/2 by immunoblot analysis of imatinib-treated cells. Skp2 expression was greatly reduced compared to controls while expression of other SCF^SKP2 components and KPC1/2 was unchanged, consistent with up-regulation of nuclear but not cytoplasmic p27 and suggesting a central role of Skp2 in mediating p27 degradation in Bcr-Abl positive cells. To test whether Skp2 is crucial for Bcr-Abl-driven leukemogenesis, we infected bone marrow of Skp2+/+ and Skp2−/− mice with BCR-ABL retrovirus. No consistent difference was observed in B-cell transformation assay (Whitlock-Witte cultures). However, formation of myeloid colonies in semisolid media was reduced in Skp2−/− compared to Skp2+/+ marrow [46.4±10% of controls (p=0.002) without and 76.6±9% of controls (p=0.008) with cytokines, n=6]. Skp2+/+ mice transplanted with BCR-ABL infected Skp2−/− marrow had significantly longer median survival (19days, range 12–60days, n=8) compared to recipients of Skp2+/+ marrow (13days, range 12–22days, n=10) (p=0.0034) with significant reduction of spleen weight (0.42±0.07g vs. 0.28±0.09g, p=0.004) and white blood cell counts (median 59x10³/μl, range 9.6–142x10³/μl, vs. 7.9x10³/μl, range 0.8–87x10³/μl, p=0.02). Histology and immunophenotyping of tissues (blood, marrow, spleen) revealed no signinificant differences between Skp2+/+ and Skp2−/− mice.

Conclusions:

1. Our data suggest that the primary cell cycle effect of Bcr-Abl kinase is up-regulation of Skp2. This leads to increased activity of SCF^SKP2, inducing degradation of T187 phosphorylated p27 which in turn promotes cell cycle progression by relieving suppression of Cdk2.

2. Skp2 is required for Bcr-Abl to fully realize its potential to induce myeloproliferative disease, providing the first in vivo evidence that SKP2 is an oncogene.

3. Targeting Skp2-p27 interactions to prevent p27 degradation may be therapeutically useful in malignancies with a high Skp2/p27 ratio.