Levels of
vascular endothelial growth factor (
VEGF) are regulated, in part, through activation of the
phosphatidylinositol 3'-kinase/Akt pathway. Using pharmacologic inhibitors, we have examined the relative contributions of Akt and
mammalian target of rapamycin (mTOR) signaling to
VEGF production in
neuroblastoma and
rhabdomyosarcoma cells growing under normoxic (21% O(2)) or hypoxic (1% O(2)) conditions. Exogenous
VEGF stimulated both Akt and
extracellular signal-regulated kinase 1/2 phosphorylation in six of seven
rhabdomyosarcoma cell lines but in only one of seven
neuroblastoma cells, suggesting autocrine stimulation predominantly in
rhabdomyosarcoma cell lines. In general, under normoxic conditions,
neuroblastoma cells produced more
VEGF (120-1,180 pg/10(6) cells/24 h) compared with
rhabdomyosarcoma lines (0-200 pg/10(6) cells/24 h).
Rapamycin, a selective inhibitor of mTOR, reduced
VEGF production in
rhabdomyosarcoma cells under normoxic conditions and partially suppressed
hypoxia-driven increases in
VEGF. However, it poorly inhibited
VEGF production under either condition in the majority of
neuroblastoma cell lines despite inhibition of mTOR signaling.
Rapamycin failed to modulate levels of
hypoxia-inducible factor 1alpha (HIF-1alpha) under normoxic conditions and modestly reduced
hypoxia-driven increases in HIF-1alpha only in
rhabdomyosarcoma cells. In contrast to
rapamycin, inhibition of Akt by
A-443654 completely blocked signaling to
glycogen synthase kinase 3beta and had more dramatic effects on
VEGF production. Notably,
A-443654 significantly inhibited
VEGF production in
rapamycin-refractory
neuroblastoma cell lines. Importantly, whereas combining
A-443654 with
rapamycin had variable effect on cell proliferation, the combination essentially blocked
hypoxia-driven increases in
VEGF in all cell lines examined, suggesting that dual blockade at different levels in the
phosphatidylinositol 3'-kinase-initiated signaling pathway may be a reasonable strategy for preventing
VEGF production in
cancer cells derived from pediatric solid
tumors. However, this will require formal testing in vivo using animal models of childhood
cancer.