FOXO1 reduces tumorsphere formation capacity via reciprocal down-regulation of LGR5 in gastric cancer cells

Abstract

Background: FOXO1 inactivation is detected in a variety of human cancers, including gastric cancer (GC). Although the role of FOXO1 in cancer stem cells has been reported in pancreatic cancer and glioblastoma, the implication of FOXO1 in GC cell stemness has been elusive. The present study investigated the implication of FOXO1 in GC cell stemness and its association with LGR5.

Methods: The expressions of FOXO1 and LGR5 were modulated in GC cell lines (SNU-638, MKN45, AGS and MKN28) by stable transfections of their shRNA or gene construct. The effects of transfection on GC stemness were evaluated in vitro and in animal models. In addition, the relationship between FOXO1 and LGR5 was analyzed using GC clinical specimens, cell lines and xenografts.

Results: Tumorspheres obtained from SNU-638 and MKN45 GC cells were cultured under serum-free culture conditions. Western blot showed that the expressions of GC stem cell markers, LGR5, CD44 and CD133, were higher in tertiary tumorsphere cells than those in adherent cells, whereas FOXO1 expression was significantly lower. More importantly, FOXO1 silencing in SUN-638 and MKN45 cells increased tumorsphere formation, whereas FOXO1 overexpression in AGS and MKN28 GC cells produced the opposite results. Additionally, immunohistochemical tissue array analysis showed an inverse relationship between FOXO1 and LGR5 in GC specimens. Further analyses using in vitro and in vivo experiments showed that FOXO1 activity in GC cells negatively controlled and was controlled by LGR5. Also, double knockdown of both FOXO1 and LGR5 in GC cells revealed that LGR5 silencing reversed the FOXO1 shRNA-induced tumorsphere formation capacity even without the FOXO1 restoration. Consistently, FOXO1 silencing did not change cyclin D1 expression in the absence of change in LGR5 expression.

Conclusions: My results suggest that FOXO1 inhibits tumorsphere formation capacity of GC cells through interaction with LGR5. Thus, FOXO1/LGR5 signaling pathway may provide a novel targeted therapy for GC.