Evening Primrose Extract Modulates TYMS Expression via SP1 Transcription Factor in Malignant Pleural Mesothelioma

SIMPLE SUMMARY: Malignant pleural mesothelioma belongs to the aggressive tumor of pleura commonly recognized at the advanced, chemoresistant stage. Thymidylate synthase (TYMS) is a critical therapeutic target because it determines the susceptibility to folate-related drug treatment. We have previously reported that a natural extract isolated from defatted evening primrose seeds (EPE) reduces cell proliferation and the invasiveness of MPM cells. Therefore, we aimed to expound the mechanism of EPE in downregulating TYMS expression. Our bioinformatic analysis of an MPM patient genes data set confirmed the correlation of overexpressed TYMS with epithelial-to-mesenchymal transition biomarkers. Mechanistic studies of the culture cell have provided evidence for the direct effect of EPE compounds on the transcriptional activity of tyms promoter to its transcription factor, specificity protein 1. EPE mixture is a complex intervention related to the occupancy of SP1 motifs, blocking TYMS expression and triggering invasiveness of MPM cells. ABSTRACT: Purpose: To determine the mechanism of EPE in downregulating TYMS in MPM cancer. Methods: The TYMS mRNA expression with epithelial-to-mesenchymal transition biomarkers and nuclear factor SP1 was assessed using the GEO database in a data set of MPM patients (GSE51024). Invasive MPM cell lines were in vitro models for the investigation of TYMS expression after EPE treatment. The tyms promoter SP1 binding sequences were determined using Genomatix v 3.4 software Electrophoretic mobility shift and dual-luciferase reporter assays revealed specific SP1 motifs in the interaction of EPE and reference compounds. Chromatin immunoprecipitation and Re-ChIP were used for the co-occupancy study. Results: In MPM patients, a positive correlation of overexpressed TYMS with mesenchymal TWIST1, FN1 and N-cadherin was observed. EPE and its major components, gallic and ellagic acid (GA and EA, respectively), downregulated TYMS in invasive MPM cells by interacting with particular SP1 motifs on the tyms promoter. The luciferase constructs confirmed the occupation of two SP1 regulatory regions critical for the promotion of TYMS expression. Both EPE and reference standards influenced SP1 translocation into the nucleus. Conclusion: EPE components reduced TYMS expression by occupation of SP1 motifs on the tyms promoter and reversed the EMT phenotype of invasive MPM cells. Further in-depth analysis of the molecular docking of polyphenol compounds with SP1 regulatory motifs is required.