Analysis of FoxM1 Expression and Regulation of Cell Cycle, and Anti-Leukemic Effectss by FoxM1 Knockdown on Leukemia Cells Transfected with siRNA FoxM1.

[Background] FoxM1, a member of the Fox transcription factor family, plays an important cell cycle regulator of both the transition from G1 to S phase and progression to mitosis. FoxM1 expression was also found to be up-regulated in some solid tumors (basal cell carcinomas, hepatocellular carcinoma, and primary breast cancer). These results suggested that FoxM1 plays a role in the oncogenesis of malignancies. However, it is unknown whether FoxM1 expression contributes to the development or progression of leukemia cells. Therefore, we investigated whether and how FoxM1 regulated the cell cycle of leukemia cells.

[Methods] The cells used in this study were human leukemia cell lines, K562, HL60, U937 cells. For analysis of FoxM1 mRNA, RT-PCR was performed in all cell lines. For analysis of proliferation and mitotic regulatory proteins (p27, p21, Skp2, Cdc25B, Cyclin D1, Survivin, and Aurora kinase B) in leukemia cells, MTT assays and western blot were performed in all cell lines untransfected or transfected with siRNA FoxM1, respectively. For cell cycle analysis, flow cytometory analysis was performed in leukemia cells untransfected or transfected with siRNAFoxM1 by PI staining.

[Results] In all leukemia cell lines, the expression of FoxM1B mRNA were significantly higher than normal MNCs. In K562, HL60, and U937 cells transfected with the siRNA FoxM1, suppression of FoxM1 caused a mean 71% (range 62 to 80%) reduction in S phase cells and a mean 4.4-fold (range 3.2 to 5.6-fold) increase in G2/M phase cells compared to untransfected cells. MTT assay demonstrated that the proliferation of the siRNA FoxM1 transfected cells was inhibited compared to the untransfected cells at 2, 3, 4, or 5 days after siRNA FoxM1 transfection. FoxM1 has been reported to regulate transcription of essential mitotic regulatory genes. We showed that FoxM1 knockdown by siRNA in leukemia cells reduced protein and mRNA expression of Aurora kinase B, Survivin, Cyclin D1, Skp2 and Cdc25B, while increased protein expression of p21and p27 in RT-PCR and western blot analysis.

[Conclusions] In this study, we report in the first time that FoxM1 is overexpressed in myeloid leukemia cells. These results demonstrated that expression of FoxM1 is an essential transcription factor for growth of leukemia cells, and regulate expression of the mitotic regulators, Cdc25B, Cyclin D1, Survivin, Aurora kinase B, and p21. Moreover, we showed that FoxM1 regulated the expression of Skp2 protein, which is known to promote degradation of the cell cycle regulator p27. Our study found that inhibition of FoxM1 expression in leukemia cells suppressed their growth in vitro. Therefore, FoxM1 might be a new potential target of therapy for leukemias. We will have further study whether the level of FoxM1 expression in leukemia cells is correlated with patient survival or sensitivity for chemotherapy.