Abstract
The FoxO family of transcription factors is regulated by PI3K/Akt induced phosphorylation resulting in nuclear exclusion and degradation. Nuclear FoxO transcribes proapoptotic molecules and cell cycle inhibitors. Although multiple mechanisms regulate FoxO activity, Akt seems to be crucial to its regulation and function. PI3K/Akt pathway has been reported to be abnormally activated in AML blast cells. The aim of this study was to investigate the function of FoxO in AML blast cells and the presence of alternative pathways responsible for FoxO3 inactivation other than PI3K-Akt. BM cells were collected from 35 AML patients at diagnosis and after chemotherapy and from 20 healthy donors. The expression levels of FoxO1, FoxO3, FoxO4 were tested by RQ-PCR, FoxO3 protein amount and localization by Western blot and immunofluorescence and the DNA binding activity by EMSA. Furthermore, downstream target genes transcribed by FoxO3 were quantified. Among these, Spred1 which codes for a negative regulator of RTK signal, including Ras mediated pathway triggered by FLT3. We have previously described the absence of Spred1 is AML patients and we have demonstrated that it promotes growth arrest and apoptosis in haematopoietic cells. Finally, BM cells were incubated with 5 mM of the PI3K inhibitor LY294002 and 20 mM PS1145, the inhibitor of IKK kinase also responsible for FoxO phosphorylation and with the combination LY294002 plus PS1145. We found that the amount of FoxO1, FoxO3 and FoxO4 mRNA are similar in AML patients and controls. Interestingly, while FoxO3 in control cells is localized in both, nucleous and cytoplasm, is completely cytoplasmatic in AML cells and it enters the nucleous after chemotherapy. The quantification of FoxO fluorescent signal in controls shows a mean value of intensity of 21.4±2 in the nucleous and 14,6±1.7 in the cytoplasm. By contrast, in AML cells is 8,2±4 in the nucleous and 18.1±4,6 in the cytoplasm. Additionally, FoxO3 DNA binding activity in AML patients is completely absent at diagnosis and reappears after therapy. Also the mRNA of the target gene Spred1 is rather undetectable at diagnosis (mean value 2−ΔΔCt= 0,009±0,3) and is upregulated during remission (mean value 2−ΔΔ= 2±1,5) or after LY29400 incubation (mean value =0,8±0,3). LY294002 and PS1145 results in FoxO partial nuclear relocalization with a nuclear signal of 15±3 and 12±3 respectively. Interestingly, the association of PS1145 and LY294002 induces a complete nuclear shuttle with a nuclear signal of 25±4, suggesting that both pathways are implicated in FoxO inactivation. Taken together these observations suggest that FoxO inactivation may be crucial for the apoptosis arrest observed in AML. These data demonstrate that also IKK pathway contributes to this effect, providing the rationale for a therapeutic strategy based on the combination of selective inhibitors such as FLT3 or Akt inhibitors or standard chemotherapy and the IKK inhibitor.
Direct activation of Sex-lethal transcription by the Drosophila runt protein