Based on prior work showing that human
pituitary tumors overexpress epidermal and
fibroblast growth factor receptors, we hypothesized that downstream components of
growth factor signaling pathways may also be dysregulated.
Epidermal growth factor pathway substrate number 8 (Eps8) was identified as a transcript overexpressed (5.9-fold) in human
pituitary tumors compared with normal pituitary by
DNA microarrays. Eps8
mRNA up-regulation was confirmed by semiquantitative RT-PCR. Immunoblot analysis showed that Eps8
protein levels and its downstream target phosphorylated ERK were also up-regulated in human
pituitary tumors. Stable overexpression of Eps8 in LbetaT2 gonadotrope pituitary cells augmented colony formation in soft
agar at d 21. Eps8 cells proliferated more robustly compared with controls in
growth factor replete as well as growth-restricted conditions. In addition, the Eps8 overexpressing cells were protected from serum withdrawal-induced apoptosis compared with controls as assessed by
caspase-3 cleavage.
Epidermal growth factor activated a robust amplification of ERK and modest up-regulation of Akt in Eps8-overexpressing pituitary cells compared with vector controls.
MAPK kinase inhibition or silencing of Eps8 blunted the proliferation of the cells in response to
growth factor stimulation. Blockade of the
phosphatidylinositol 3-kinase pathway or silencing of Eps8 resulted in a loss of the Eps8 protection from
growth factor withdrawal-induced apoptosis. Together these data support a role of Eps8 in amplifying
growth factor receptor signaling in human
pituitary tumors to promote proliferation and cell survival.