Repression of WT1-Mediated LEF1 Transcription by Mangiferin Governs β-Catenin-Independent Wnt Signalling Inactivation in Hepatocellular Carcinoma

Background/Aims: The development of hepatocellular carcinoma (HCC) is a complex process which involves deregulation of multiple signalling pathways. The hyper-activation of Wnt signalling promotes sustained expansion, invasion, and neovascularization of HCC. Mangiferin, a natural small molecule present in Mangifera indica L. has been shown to inactivate β-catenin, which is an indispensable regulator in Wnt pathway. Our study aimed to determine whether mangiferin has any inhibitory effect on HCC and examine how it modulates Wnt signalling. Methods: The tumour inhibitory effect of mangiferin was examined by in vitro cellular models and an in vivo orthotopic HCC implantation model. The genes responsible for mangiferin-mediated anti-HCC were delineated by polymerase chain reaction (PCR) microarray. The expression of target genes was further determined by quantitative PCR and immuno-blotting assays. The binding capacity of Wilms’ tumour 1 (WT1) to the lymphoid enhancer-binding factor 1 (LEF1) promoter was confirmed by chromatin immunoprecipitation-qPCR. Results: Oral administration of mangiferin inhibited orthotopic tumour growth. Cellular investigations confirmed the dose-dependent inhibition of mangiferin on HCC expansion and invasion. PCR array combined with Gene Ontology analysis revealed that the Wnt pathway was the predominant target of mangiferin and LEF1 was the most reduced gene in the Wnt pathway. Overexpression of LEF1 diminished repression of Wnt signalling and reduced proliferation activity in mangiferin-treated HCC cells. The mangiferin-mediated down-regulation of LEF1 was independent of β-catenin but associated with WT1 protein. WT1 knock-in in HCC cells further enhanced LEF1 expression. Chromatin immunoprecipitation assays revealed that the mangiferin induced repression of LEF1 was associated with decreased occupancy of WT1 on the LEF1 promoter. Conclusion: Our study identifies a novel mechanism of hepatocellular carcinoma inhibition through β-catenin-independent Wnt signalling, which is regulated by WT1-associated LEF1 repression. The study also highlights mangiferin as a promising Wnt inhibitor for HCC treatment