FoxO transcription factor is delocalized in CML patients by Bcr-Abl induced PI3K/AKT activation and IKK pathway and can be reactivated by Imatinib treatment
Authors: Francesca Arruga, Cristina Panuzzo, Francesca Messa, Paolo Nicoli, Emanuela Messa, Simona Soverini, Ilaria Iacobucci, Giovanni Martinelli, Michele Baccarani, Antonia Rotolo, Enrico Bracco, Thea Kalebic, Giuseppe Saglio, Daniela Cilloni
Journal: Cancer Research (2008)

4397  Background: The FoxO transcription factor promotes apoptosis and triggers cell cycle inhibition. Although multiple mechanisms regulate FoxO activity, the PI3K/Akt induced phosphorylation seems to be crucial, resulting in nuclear exclusion and degradation. In Chronic Myeloid Leukemia (CML) the TK activity of Bcr-Abl leads to the abnormal activation of downstream effectors including PI3K/Akt. Aim: we aimed to clarify the role of FoxO in Bcr-Abl induced apoptotic arrest and cell growth, the effect of imatinib (IM) treatment on FoxO activity and to investigate alternative pathways eventually responsible for FoxO3 inactivation. Methods: FoxO3 protein amount and localization were analysed by Western blot and immunofluorescence and the DNA binding activity by EMSA in BM samples from 20 CML patients at diagnosis and during IM therapy and in 20 healthy donors. Furthermore, Spred1 a downstream target gene transcribed by FoxO3, which codes for a negative regulator of RTK signal including Ras mediated pathway triggered by FLT3, was quantified. We previously described the absence of Spred1 in CML patients thus promoting growth arrest and apoptosis in haematopoietic cells. BM cells and BV173 Ph+ cell line were incubated with 1 M IM, 5 M of the PI3K inhibitor LY294002 and 20 M PS1145, the inhibitor of IKK kinase also responsible for FoxO phosphorylation, and with the combination of IM + PS1145 and LY294002 + PS1145. Results: we found that, while FoxO3 in control cells is localized in both nucleous and cytoplasm is completely cytoplasmatic in Ph+ CML cells and it enters the nucleous during IM treatment. 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 CML cells is 6.6 0.8 in the nucleous and 16.7 1.1 in the cytoplasm. Additionally, FoxO3 DNA binding activity in CML patients is completely absent at diagnosis and reappears during therapy or after IM incubation. Also the Spred1 mRNA is rather undetectable at diagnosis (mean value 2- Ct= 0,001 0,09) and is restored during remission (mean value 2- Ct= 1,2 1,8) or after IM incubation (mean value = 0,7 0,9) or LY294002(1 0,7). Exposure to IM, LY294002 and PS1145 results in FoxO partial nuclear relocalization with a nuclear signal of 15 5, 17 3 and 12 2 respectively. The association of PS1145 and IM or PS1145 and LY294002 induces a complete nuclear shuttle with a nuclear signal of 23 4 and 24 4 respectively, suggesting that both pathways are implicated in FoxO inactivation. Conclusions: the data suggest that FoxO inactivation may be crucial for Bcr-Abl induced proliferation and apoptosis arrest. The antiproliferative activity of IM may be mediated by FoxO3 re-localization. Moreover, we demonstrated the involvement of IKK pathway, providing the rationale for a therapeutic strategy based on the combination of IM and IKK inhibitor.