Pokemon inhibits transforming growth factor β-smad4-related cell proliferation arrest in breast cancer through specificity protein 1

Abstract

Purpose: Pokemon, also known as ZBTB7A, belongs to the POZ and Krüppel (POK) family of transcription repressors and is implicated in tumor progression as a key proto-oncogene. This present study aimed at determining the mechanism by which Pokemon inhibits transforming growth factor β (TGFβ)-Smad4 pathway-dependent proliferation arrest of breast cancer cells via specificity protein 1 (SP1). Methods: Over-expressing plasmid or small interfering RNA (siRNA) transfection was used to regulate Pokemon levels. The EdU incorporation assay, MTS assay, and clone formation were used to identify the inhibitory effect of Pokemon siRNA on cell proliferation. Quantitative real-time polymerase chain reaction assay confirmed that Pokemon deletion inhibited the expression of proliferation-associated genes. The dual-luciferase reporter assay, electrophoretic mobility shift assay, and co-immunoprecipitation assay were used to analyze binding between Pokemon, Smad4, and SP1. Results: Pokemon deletion induced proliferation arrest of breast cancer cells and inhibited the expression of proliferation-associated genes, especially Smad4. Pokemon bound with SP1 to interdict Smad4 promoter activity. Information on clinical samples was obtained from The Cancer Genome Atlas data, in which the Pokemon mRNA levels showed a negative correlation with Smad4 levels in different subtypes of breast cancer in two independent datasets. Conclusion: We demonstrated that Pokemon binds to SP1 to down-regulate Smad4 expression, thereby promoting proliferation of breast cancer cells. This suggests that Pokemon is a potential TGFβ-signaling participant in breast cancer progression.