The transcription factor FOXO4 is down-regulated and inhibits tumor proliferation and metastasis in gastric cancer

Abstract

Background: FOXO4, a member of the FOXO family of transcription factors, is currently the focus of intense study. Its role and function in gastric cancer have not been fully elucidated. The present study was aimed to investigate the expression profile of FOXO4 in gastric cancer and the effect of FOXO4 on cancer cell growth and metastasis.

Methods: Immunohistochemistry, Western blotting and qRT-PCR were performed to detect the FOXO4 expression in gastric cancer cells and tissues. Cell biological assays, subcutaneous tumorigenicity and tail vein metastatic assay in combination with lentivirus construction were performed to detect the impact of FOXO4 to gastric cancer in proliferation and metastasis in vitro and in vivo. Confocal and qRT-PCR were performed to explore the mechanisms.

Results: We found that the expression of FOXO4 was decreased significantly in most gastric cancer tissues and in various human gastric cancer cell lines. Up-regulating FOXO4 inhibited the growth and metastasis of gastric cancer cell lines in vitro and led to dramatic attenuation of tumor growth, and liver and lung metastasis in vivo, whereas down-regulating FOXO4 with specific siRNAs promoted the growth and metastasis of gastric cancer cell lines. Furthermore, we found that up-regulating FOXO4 could induce significant G1 arrest and S phase reduction and down-regulation of the expression of vimentin.

Conclusion: Our data suggest that loss of FOXO4 expression contributes to gastric cancer growth and metastasis, and it may serve as a potential therapeutic target for gastric cancer.

Figure 1

FoxO4 is significantly down-regulated in GC tissues and cell lines. (A1) IHC analysis of FOXO4 expression in 75 paired GC and adjacent non-tumorous tissues. (A2) Statistical analysis of FOXO4 expression in GC tissues and adjacent non-tumorous stomach tissues. (A3) Representative FOXO4expression in primary and metastatic GC tissues detected by IHC methods. (A4) Statistical analysis of FOXO4 expression between GC tissues with and without node metastasis. (B1-B2) Real-time PCR and western blot analysis of FOXO4 expression in 8 pairs of GC and adjacent non-tumorous tissues. (C1-C2) Real-time PCR and western blotting analysis of FOXO4 expression in different GC cell lines.

Figure 2

Effect of FOXO4 on regulating GC cell proliferation. (A1-A2) Relative expression of FOXO4 in SGC-7901 cells transfected with LV-FOXO4 or LV-control, which was confirmed by real-time PCR and western blot analysis. The values represent the means from three separate experiments, and the error bars represent the SEM (**P < 0.01). (A3) The proliferation rates of cells were measured using the MTT assay. The values represent the means from three separate experiments, and the error bars represent the SEM (*P < 0.05). (B1-B2) Relative expression of FOXO4 in BGC-823 cells transfected with FOXO4 oligo nucleotide inhibitor or oligo nucleotide control, which was confirmed by real-time PCR and western blot analysis. The values represent the means from three separate experiments, and the error bars represent the SEM (**P < 0.01). (B3) The proliferation rates of cells were measured using the MTT assay. The values represent the means from three separate experiments and the error bars represent the SEM (*P < 0.05). (C1-C2) Colony formation of SGC07901 cells transfected with LV-FOXO4 and LV-control was carried out by seeding cells onto plates for 2 weeks, and the number of colonies was then counted. The values represent the means from three separate experiments, and the error bars represent the SEM (*P < 0.05). (D1-D2) Cell cycle distribution of SGC-7901 cells transfected with LV-FOXO4 or LV-control. Cell cycle analysis was performed 24 h after transfection. The cell cycle distribution was calculated and expressed as the mean ± SD of three separate experiments. *P < 0.05. (E1-E2) Relative expression of CyclinD1 in SGC-7901 cells transfected with LV-FOXO4 or LV-control, which was confirmed western blot analysis. The values represent the means from three separate experiments, and the error bars represent the SEM(*P < 0.05).

Figure 3

Effect of FOXO4 in regulating GC cell metastasis. (A1-A2) Up-regulation of FOXO4 expression in LV-FOXO4 cells decreased SGC-7901 cell migration and invasion in vitro, whereas the inhibition of FOXO4 expression using the oligo nucleotide inhibitor of FOXO4 enhanced BGC-823 cell migration and invasion. (B) Cell migration capacity was evaluated by performing a high-content assay in SGC-7901 cells transfected with LV-FOXO4 or LV-control. *P < 0.05 (C) Cell migration capacity was also tested by performing a wound-healing assay in SGC-7901 cells transfected with LV-FOXO4 or LV-control. *P < 0.05.

Figure 4

In vivo proliferation and metastasis assay. (A1-A3) SGC-7901 cells transfected with LV-FOXO4 or LV-control were transplanted under the skin. Six weeks later, tumors were more clearly seen in mice implanted with 7901-NC cells as compared to the 7901-FOXO4 groups: (6/6 in the 7901-NC groups and 3/6 in 7901-FOXO4 groups). The tumors were dissected and measured. (B1-B3) SGC-7901 cells transfected with LV-FOXO4 or LV-control were injected into the tail veins of nude mice. Ten weeks later, mice implanted with 7901-NC cells showed lung and liver metastases, whereas few metastases were detected in mice implanted with 7901-FOXO4 cells: (for lung metastasis, 6/10 in the 7901-NC groups and 1/10 in the 7901-FoxO4 groups, For liver metastasis, 3/10 in 7901-NC groups and 0/10 in 7901-FoxO4 groups. (C) Images showing representative hematoxylin and eosin staining of lung and liver tissue samples from the different experimental groups *P < 0.05. (D) Overall survival of the nude mice in each group.

Figure 5

FOXO4 inhibits EMT in GC cells. (A1-A2) Real-time PCR showed up-regulated expression of epithelial markers (E-cadherin) and down-regulated expression of mesenchymal markers (vimentin) in 7901-FOXO4 cells. (B1-B2) Immunofluorescence staining showed up-regulated expression of epithelial markers (E-cadherin) and down-regulated expression of mesenchymal markers (vimentin) in 7901-FOXO4 cells.