NOTCH Signaling Pathway Regulates Glioma Stem-like Cell Proliferation and Self-renewal Abilities via PI3K/AKT Activity

Objective To investigate the underlying mechanism of NOTCH signaling regulating glioblastoma (GBM) cell proliferation and self-renewal abilities.

Methods We cultured LN-229, U118-MG, and A172 GBM neurospheres in serum-free medium supplemented with growth factors. MTT assay and single cell sphere formation assay were performed to test glioma stem-like cell (GSC) proliferation and self-renewal abilities. To investigate the NOTCH dependency upon PI3K/AKT pathway, both lentiviral shRNA targeting AKT1 and PI3K inhibitor LY294002 were introduced to inhibit PI3K/AKT activity. We detected NOTCH and PI3K/AKT signaling activity through Western blot. BLISS independence model was used to evaluate the interaction between LY294002 and NOTCH inhibitor DAPT.

Results We obtained LN-229, U118-MG, and A172 GBM neurospheres in vitro. DAPT reduced NOTCH and PI3K/AKT signaling activity. The downregulation of PI3K/AKT activity by NOTCH was independent of PTEN. DAPT inhibited GSC proliferation and self-renewal abilities, which could be partially rescued by shAKT1 or LY294002-induced PI3K/AKT inhibition (P < 0.05). DAPT and LY294002 showed the antagonistic effect upon GSC proliferation.

Conclusion NOTCH signaling requires PI3K/AKT pathway to stimulate GSC proliferation and maintain self-renewal ability.