The Transcription Factor Sp3 Regulates the Expression of a Metastasis-Related Marker of Sarcoma, Actin Filament-Associated Protein 1-Like 1 (AFAP1L1)

We previously identified actin filament-associated protein 1-like 1 (AFAP1L1) as a metastasis-predicting marker from the gene-expression profiles of 65 spindle cell sarcomas, and demonstrated the up-regulation of AFAP1L1 expression to be an independent risk factor for distant metastasis in multivariate analyses. Little is known, however, about how the expression of AFAP1L1 is regulated. Luciferase reporter assays showed tandem binding motives of a specificity protein (Sp) located at -85 to -75 relative to the transcriptional start site to be essential to the promoter activity. Overexpression of Sp1 and Sp3 proteins transactivated the proximal AFAP1L1 promoter construct, and electrophoretic mobility shift assays showed that both Sp1 and Sp3 were able to bind to this region in vitro. Chromatin immunoprecipitation experiments, however, revealed that Sp3 is the major factor binding to the proximal promoter region of the AFAP1L1 gene in AFAP1L1- positive cells. Treatment with mithramycin A, an inhibitor of proteins binding to GC-rich regions, prevented Sp3 from binding to the proximal promoter region of AFAP1L1 and decreased its expression in a dose-dependent manner. Finally, knocking down Sp3 using small inhibitory RNA duplex (siRNA) reduced AFAP1L1 expression significantly, which was partially restored by expressing siRNA-resistant Sp3. These findings indicate a novel role for Sp3 in sarcomas as a driver for expression of the metastasis-related gene AFAP1L1.