Activation of the PI3K/AKT signal pathway is a known driving force for the progression to
castration-recurrent
prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic
shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP
metastasis suppressor. FOXO4
protein levels inversely correlate with the invasive potential of a panel of
human CaP cell lines, with decreased
mRNA levels correlating with increased incidence of clinical
metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN)
metastasis in vivo without affecting indices of proliferation or apoptosis. Increased
Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary
tumors and in LN
metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by
siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter
RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased
metastasis-free survival in
human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness.