Figure 6. SLPI regulates binding of FoxM1 to its target MMP2 gene and increases cancer cell transmigration through an endothelial cell layer.

A. SLPI regulated FoxM1 binding to the MMP2 promoter sequence in gel shift assay (EMSA). All lanes contained biotin end-labeled MMP2 duplex DNA. NP (lane 1) is a control without protein. Lanes 2 and 3 contained protein extract without and with addition of SLPI protein. Lane 4: non stimulated with SLPI protein extract. Lane 5: supeshift with cells stimulated by SLPI and protein extract was incubated with FoxM1 antibodies. Arrows indicate bands shifted and supershifted by antibodies against FoxM1. Lane 6: the loss of FoxM1-specific DNA-binding in the presence of FoxM1 oligo competitor. B. SLPI increases transmigration of breast cancer cells through an endothelial layer in a transwell assay. 4T1 cells with down-regulated SLPI (SLPI shRNA) had decreased transmigration across an endothelial monolayer compared to control 4T1 cells. Error bars represent SEM; n = 10, * P < 0.001. C. Schematic representation of SLPI’s effects on the regulation of the Rb/FoxM1 complex. In the presence of low levels of SLPI, Rb protein binds to FoxM1 and represses the activity of this transcriptional factor. Inhibition of FoxM1 leads to repression of FoxM1 target genes that are associated with tumor growth and metastasis. In cancer cells that express high levels of SLPI, it physically interacts with Rb to facilitate Rb release from FoxM1. This makes FoxM1 available to initiate transcription of target genes involved in tumor growth and metastasis.