BCR-ABL Induces the Expression of Skp2 through the PI3K Pathway to Promote p27Kip1 Degradation and Proliferation of Chronic Myelogenous Leukemia Cells

Abstract

Chronic myelogenous leukemia (CML) is characterized by the expression of the BCR-ABL tyrosine kinase, which results in increased cell proliferation and inhibition of apoptosis. In this study, we show in both BCR-ABL cells (Mo7e-p210 and BaF/3-p210) and primary CML CD34+ cells that STI571 inhibition of BCR-ABL tyrosine kinase activity results in a G 1 cell cycle arrest mediated by the PI3K pathway. This arrest is associated with a nuclear accumulation of p27Kip1 and down-regulation of cyclins D and E. As a result, there is a reduction of the cyclin E/Cdk2 kinase activity and of the retinoblastoma protein phosphorylation. By quantitative reverse transcription-PCR we show that BCR-ABL/PI3K regulates the expression of p27Kip1 at the level of transcription. We further show that BCR-ABL also regulates p27Kip1 protein levels by increasing its degradation by the proteasome. This degradation depends on the ubiquitinylation of p27Kip1 by Skp2-containing SFC complexes: silencing the expression of Skp2 with a small interfering RNA results in the accumulation of p27Kip1 . We also demonstrate that BCR-ABL cells show transcriptional up-regulation of Skp2. Finally, expression of a p27Kip1 mutant unable of being recognized by Skp2 results in inhibition of proliferation of BCR-ABL cells, indicating that the degradation of p27Kip1 contributes to the pathogenesis of CML. In conclusion, these results suggest that BCR-ABL regulates cell cycle in CML cells at least in part by inducing proteasome-mediated degradation of the cell cycle inhibitor p27Kip1 and provide a rationale for the use of inhibitors of the proteasome in patients with BCR-ABL leukemias. (Cancer Res 2005; 65(8): 3264-72)

Bortezomib decreases Rb phosphorylation and induces caspase-dependent apoptosis in Imatinib-sensitive and -resistant Bcr-Abl1-expressing cells

“…We compared this effect with that of the Bcr-Abl1-kinase inhibitor IM. As earlier reported (Andreu et al, 2005), IM also increases the level of hypo-phosphorylated Rb, finally inducing the disappearance of the hyper-phosphorylated forms of the protein (Figure 4a). …”

“…It is known that these two proteins are among the many factors that regulate the levels and activity of the cell-cycle inhibitors p21Cip1 and p27Kip1 (Perez-Roger et al, 1999;Kossatz et al, 2004). On the other hand, Bcr-Abl1-kinase activity inhibition with IM results in the downregulation of both Skp2 and c-Myc, indicating that these two proteins are molecular targets of Bcr-Abl1 (Sawyers, 1993;Andreu et al, 2005). Therefore, we analyzed the effect of Btz on Skp2 and c-Myc and found that both show lower levels in Btztreated cells expressing Bcr-Abl1, with no significant change in BaF/3 control cells (Figure 5b).…”

“…In the case of the BaF/3 cells, 10% WEHIconditioned medium was added to the culture medium as a source of murine IL-3, whereas growth of Bcr-Abl1-expressing BaF/3 cells was independent of growth factors. Primary CML CD34 þ cells and control CD34 þ cells from umbilical cord blood, obtained after informed consent, were selected and cultured as described (Andreu et al, 2005). All media, serum, and supplements were from Invitrogen Life Technologies (Paisley, UK).…”

“…We have earlier reported that Bcr-Abl1 induces the degradation of the cell-cycle inhibitor p27Kip1 by the proteasome, through the induction of the F-box protein Skp2 (Andreu et al, 2005). These results prompted us to explore the possibility of using the proteasome as a potential therapeutic target in the treatment of CML.…”

Exposure of neuroblastoma cell lines to imatinib results in the upregulation of the CDK inhibitor p27KIP1 as a consequence of c-Abl inhibition

“…This effect was dependent on the upregulation of the CDK inhibitor p27 KIP1 in the nuclear compartment as demonstrated by siRNA-mediated depletion of p27 KIP1 , which prevented the effects of imatinib on BrdU incorporation. Regulation of p27 KIP1 protein levels is mainly achieved by ubiquitin dependent proteolysis [27,33]; however, increased p27 KIP1 mRNA levels in imatinib-treated cells have been described [34]. Real-time PCR assays ruled-out increased p27 KIP1 mRNA levels in neuroblastoma cells exposed to imatinib.…”