Analysis of the Expression of an Endogenous Tumor Suppressor Gene, PHLPP, and Elevation of PHLPP as an Ideal Target for MRD Monitoring in Leukemia.

[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. Therefore, in these cell lines, the reduction of PHLPP causes to promote tumor growth. In this study, we have investigated the expression and function of PHLPP in leukemia cells.

[Methods] The cells used in this study were human leukemia cell lines, K562, HL60, U937, CEM, and MOLT4 cells. Primary acute lymphoblastic leukemia (6ALL (6 L2)), myelodysplastic syndromes (8 MDS (8 RAEB)), and acute myeloblastic (12 AML (3 M1, 4 M2, 2 M3, 2 M4, 1 M5)) cells were obtained from the peripheral blood. Human normal mononuclear cells (MNCs) were isolated from peripheral blood (PB) of healthy volunteers after obtaining informed consents. For analysis of PHLPP mRNA, RQ-PCR was performed in all cell lines and clinical samples before and after chemotherapy. For analysis of proliferation in all cell lines, MTT assays and western blot were performed in all cell lines untransfected or transfected with siRNA pHLPP.

[Results] In all cell lines and clinical samples obtained from patients with leukemia, the expression of PHLPP mRNA were significantly higher than normal MNCs. In K562, HL60, U937, CEM and MOLT4 cells transfected with the siRNA PHLPP, it is shown that the proliferation of these cells was stimulated and the phosphorylation state of Akt was markedly increased compared to the untransfected cells. The mean PHLPP gene expression was for AML: 3.57, MDS: 2.18, and ALL 3.14 relative to normal MNCs. Mean PHLPP expression was significantly higher in AML, MDS and ALL compared to normal MNCs (P < 0.01). Moreover, when CR status after chemotherapy, mean PHLPP expression levels were significantly reduced compared to unCR status after chemotherapy in MNCs from PB of patients with leukemia.

[Conclusions] In this study, we showed in the first time that the PHLPP expression levels were high compared to normal MNCs in leukemia cells. Quantitive analysis of PHLPP gene expression in PB appeared to be a useful tool in discriminating abnormal levels in AML, MDS and ALL patients. Moreover, it is serves as an ideal target for MRD monitoring in PB. However, it is unclear why PHLPP, which is reported that promotes apoptosis and suppresses tumor growth, is at high expression level in leukemia cells.