Investigation of G-quadruplex formation in the FGFR2 promoter region and its transcriptional regulation by liensinine.

BACKGROUND: Fibroblast growth factor receptor 2 (FGFR2) is overexpressed in breast cancer tissues and cells, and has been shown to be a susceptibility factor for breast cancer. In this study, we found that the G-rich sequences in the FGFR2 promoter region can form G-quadruplexes, which could be the target and inhibitor of the FGFR2 gene.

METHODS: Initially, the formation of G-quadruplexes was confirmed by ESI-MS and CD, and DMS footprinting experiments gave the folding pattern of the G-quadruplexes. After luciferase reporter assays revealed that the G-quadruplex could inhibit the activity of the FGFR2 promoter, MS and SPR showed binding affinity and selectivity of the ligand. Then cell culture experiments and ChIP assay showed the bioactivity of the ligand.

RESULTS: We found that three G-rich sequences (S1-S3) in the FGFR2 promoter region can form G-quadruplex structures. And a natural alkaloid, liensinine, was found to bind to the S1 G-quadruplex with relative high affinity and selectivity. Cell culture experiments showed that liensinine inhibits FGFR2 activity at both the transcriptional and translational levels. Moreover, chromatin immunoprecipitation assay (ChIP) results showed that liensinine blocks the binding of E2F1 at the transcription factor binding site (TFBS) in the S1 sequence, which is the mechanism through which liensinine inhibits the FGFR2 gene.

CONCLUSIONS: A natural alkaloid was discovered to selectively bind to the S1 G-quadruplex with relative high affinity, and therefore inhibited FGFR2 transcription and translation.

GENERAL SIGNIFICANCE: Our discovery offers a useful strategy to inhibit FGFR2 transcription, i.e., targeting the G-quadruplex with a natural alkaloid.