Background: Metastasis is the major cause of cancer-related death. Forkhead Box M1 (FoxM1) is a master regulator of tumor metastasis. This study aims to identify new FoxM1 targets in regulating tumor metastasis using bioinformatics tools as well as biological experiments.
Methods: Illumina microarray was used to profile WT and PTTG1 knockout HCT116 cells. R2 Genomics Analysis was used to identify PTTG1 as a potential FoxM1 targeted gene. Luciferase reporter array, EMSA and Chromatin Immunoprecipitation (ChIP) were used to determine the binding of FoxM1 to PTTG1 promoter. Boyden chamber assay was used to evaluate the effects of FoxM1-PTTG1 on cell migration and invasion. Splenic-injection induced liver metastasis model was used to evaluate the effects of FoxM1-PTTG1 on liver metastasis of colorectal cancer.
Results: Analyses of multiple microarray datasets derived from human colorectal cancer indicated that correlation levels of FoxM1 and pituitary tumor transforming gene (PTTG1) are highly concordant (R = 0.68 ~ 0.89, p = 2.1E-226 ~ 9.6E-86). FoxM1 over-expression increased and knock-down decreased PTTG1 expression. Luciferase reporter assay identified that the -600 to -300 bp region of PTTG1 promoter is important for FoxM1 to enhance PTTG1 promoter activity. EMSA and ChIP assays confirmed that FoxM1 directly binds to PTTG1 promoter at the -391 to -385 bp region in colorectal cancer cells. Boyden chamber assay indicated that both FoxM1 and PTTG1 regulate migration and invasion of HCT116 and SW620 colorectal cancer cells. Further in vivo assays indicated that PTTG1 knock out decreased the liver metastasis of FoxM1 over-expressing HCT116 cells. Microarray analyses identified 662 genes (FDR < 0.05) differentially expressed between WT and PTTG1(-/-) HCT116 cells. Among them, dickkopf homolog 1 (DKK1), a known WNT pathway inhibitor, was suppressed by PTTG1 and FoxM1.
Conclusions: PTTG1 is a FoxM1 targeted gene. FoxM1 binds to PTTG1 promoter to enhance PTTG1 transcription, and FoxM1-PTTG1 pathway promotes colorectal cancer migration and invasion.