The Expression And Clinical Significance Of FoxM1in Acute Myeloid Leukemia

Background:Acute myeloid leukemia (AML) is a clonal malignant disease characterized by maturational arrest of hematopoietic precursors in an early stage of development and proliferation, which accounts for about70%of all acute leukemia cases. With the development of chemotherapy regimens, the strengthening of supporting treatment and the widespread usage of hematopoietic stem cell transplantation, the effect of treatment has been improved. However, the five-year survival rate for patients below60years is only around30-40%, which could be even lower than10%for the patients aged over60years. Hence further study on underlying mechanism for new molecular target is urgently required.FoxMl is a member of the forkhead family of transcription factors, which earlier publications have found to be able to regulate the transcriptional program of the G2phase of the cell cycle and function in maintaining genomic stability. It has recently been found that FoxMl is overexpressed in many solid human tumors and involved in different aspects of carcinogenesis and tumor progression by regulating tumor angiogenesis, invasion and metastasis. Therefore, FoxMl is considered as an important molecular marker and potentially therapeutic target in many solid tumors. However, the function of FoxM1and the signaling involved in leukemia are poorly reported. Objectives:1. To examine the expression of FoxM1in AML patient with different types and to analyze its role in AML development and clinical significance.2. To get more understanding of significance of FoxM1overexpression in AML, through examining the expression of FoxM1downstream targets and activity of caspase-3when FoxM1is inhibited by siRNA transfection in AML cell lines.Methods and Results:1. qRT-PCR was used to detect the expression of FoxMl in bone marrow samples from30cases of newly diagnosed AML patients,21cases of patients with complete remission and13cases of normal donators. In group of newly diagnosed patients, the expression of FoxM1was much higher compared with control group, the difference was significant (P<0.01). Meanwhile, the level of FoxM1expression in CR group, in which the patients reached complete remission, was much lower than the one in group of newly diagnosed patients, the difference was significant as well (P<0.01), but the FoxM1expression has no significant difference between CR group and control group (P>0.05).2. qRT-PCR was used to detect the expression of FoxM1in bone marrow samples from patients with different types of AML, including M3, M4and M5. For each type, the expression of FoxM1was higher in the newly diagnosed patients’group than that of control group (P<0.01); while in patients with complete remission, the FoxM1expression level was significantly lower compared with the newly diagnosed patients (P<0.01). No significant difference was detected between different types of AML.3. By using siRNA transfection technique, FoxM1was knocked down in K562and HL60cells, and then Caspase-3Activity Assay Kit was used to detect the activity of caspase-3(an apoptosis related enzyme) by spectrophotometry. Our results showed, after FoxM1was knocked down, both in K562and HL60cells, the activity of caspase-3was elevated (P<0.05). 4. By using siRNA technique to knock down FoxMl in K562, HL60and THP-1cells, qRT-PCR was employed to detect the expression level of FoxMl and its downstream targets, including c-myc, hTERT, skp2and p27. The result showed that FoxM1, c-myc, hTERT, skp2expression level were downregulated in all three cell lines significantly (P<0.05); and p27expression had a significant increase in both K562and HL60cell lines (P<0.05), while in THP-1cells, p27was unregulated but without significant difference (P>0.05).5. With the treatment of four kinds of clinical drugs, including Bortezomib, Arsenious acid, Homoharringtonine (HTT) and all trans retinoic acid (ATRA) on HL60cells. qRT-PCR and Western Blot were used to detect the FoxMl expression in both gene and protein level. Results showed that FoxM1gene expression level were all decreased in a significant degree compared with control sample (treated by PBS)(P<0.05); and the protein level of FoxMl decreased sharply after drug treatment.Conclusions:1. FoxM1was expressed higher in newly diagnosed AML patients’group than control group; In patients who has reached complete remission after standard treatment, FoxM1expression level was much lower than in patients newly diagnosed, while there was no significant difference detected between CR group and control group. This could suggest FoxMl might promote the development of AML.2. After FoxMl was knocked down in K562and HL60cells, the activity of caspase-3, an apoptosis related enzyme, was elevated, which could be a sign that FoxM1promotes AML development by inhibiting apoptosis.3. When FoxM1was knocked down in K562, HL60cells and THP-1cells, FoxM1downstream targets, such as c-myc, hTERT and skp2expression level were all decreased; While the expression level of p27(the tumor suppresser gene) went up, which hints that FoxMl is involved in leukemogenesis by regulating apoptosis and cell cycle through these genes. It provides us initial information for further study upon the complicated mechanism of AML.4. By using four different clinical drugs on HL60cells, we could see the obvious effect on suppressing FoxMl expression in both gene and protein level. Our results suggest that these drugs might exert the therapeutic function through inhibiting FoxM1, which could provide us initial experimental evidence that FoxMl could be the potential molecular target for AML treatment.