Bcr-Abl Promotes CML Cell Proliferation through the Suppression of PHLPP Expression.

[Background] The proto-oncogene Akt/PKB is activated in many human cancers including leukemias, and regulates the cell survival and apoptosis. Akt/PKB is activated by growth factors or the phosphatidylinositol-3,4,5-triphosphate via PI3K, phosphorylates many substrates for cell survival or proliferation. The tumor suppressor PTEN prevents Akt/ PKB phosphorylation and activation. On the other hand, the PH domain leucine-rich repeat protein phosphatase (PHLPP) dephosphorylates Akt/PKB, induces apoptosis, and suppresses tumor growth. It has been reported that the expression levels of PHLPP are significantly reduced in several colon cancer and glioblastoma cell lines, and Akt/PKB phosphorylation is elevated. However, its function is little known in CML. We found that the expression of PHLPP gene and protein was suppressed in CML cells. In this study, we have investigated the expression and the function of the PHLPP in CML cells.

[Methods] The cells used in this study were human CML cell lines, K562 and Meg01 cells. Primary CML (CP; n=10) cells were obtained from the peripheral blood or bone marrow. Human normal mononuclear cells (MNCs) were isolated from peripheral blood or bone marrow of healthy volunteers after obtaining informed consents. For analysis of PHLPP mRNA expression, quantitative RTPCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the levels of Akt phosphorylation in CML cells, MTT assays and western blot were performed in all cell lines transfected with Bcr-Abl or PHLPP siRNA, respectively. For colony analysis, the colonies of CFU-GEMM, CFUGM, and BFU-E were counted in CML stem/progenitor cells transfected with PHLPP siRNA or treated with Abl kinase inhibitors.

[Results] In CML cell lines, the expressions of PHLPP mRNA and protein were significantly increased more than normal MNCs by treatment with Abl kinase inhibitors or transfection with Bcr-Abl siRNA. On the other hands, in CML cells transfected with the PHLPP siRNA, it is shown that the phosphorylation levels of Akt were markedly increased compared to the untransfected cells. In CML stem/progenitor cells obtained from patients with CML, the expression of PHLPP gene was suppressed in 10/10 (100 %) of CML. This higher expression was not associated to the counts of blasts of PB (P < 0.001). The transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of PHLPP mRNA and protein, and decreased the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusions] Our results suggest that the Bcr- Abl suppressed the expression of PHLPP mRNA and protein, and the phosphorylation of Akt in CML cells to induce the proliferation of CML cells. Moreover, induction of PHLPP expression might regulate the proliferation of CML stem/progenitor cells.