MRP4 sustains Wnt/β-catenin signaling for pregnancy, endometriosis and endometrial cancer

Rationale: Abnormal Wnt/β-catenin signaling in the endometrium can lead to both embryo implantation failure and severe pathogenic changes of the endometrium such as endometrial cancer and endometriosis. However, how Wnt/β-catenin signaling is regulated in the endometrium remains elusive. We explored possible regulation of Wnt/β-catenin signaling by multi-drug resistance protein 4 (MRP4), a potential target in cancer chemotherapy, and investigated the mechanism. Methods: Knockdown of MRP4 was performed in human endometrial cells in vitro or in a mouse embryo-implantation model in vivo. Immunoprecipitation, immunoblotting and immunofluorescence were used to assess protein interaction and stability. Wnt/β-catenin signaling was assessed by TOPflash reporter assay and quantitative PCR array. Normal and endometriotic human endometrial tissues were examined. Data from human microarray or RNAseq databases of more than 100 participants with endometriosis, endometrial cancer or IVF were analyzed. In vitro and in vivo tumorigenesis was performed. Results: MRP4-knockdown, but not its transporter-function-inhibition, accelerates β-catenin degradation in human endometrial cells. MRP4 and β-catenin are co-localized and co-immunoprecipitated in mouse and human endometrium. MRP4-knockdown in mouse uterus reduces β-catenin levels, downregulates a series of Wnt/β-catenin target genes and impairs embryo implantation, which are all reversed by blocking β-catenin degradation. Analysis of human endometrial biopsy samples and available databases reveals significant and positive correlations of MRP4 with β-catenin and Wnt/β-catenin target genes in the receptive endometrium in IVF, ectopic endometriotic lesions and endometrial cancers. Knockdown of MRP4 also inhibits in vitro and in vivo endometrial tumorigenesis. Conclusion: A previously undefined role of MRP4 in stabilizing β-catenin to sustain Wnt/β-catenin signaling in endometrial cells is revealed for both embryo implantation and endometrial disorders, suggesting MRP4 as a theranostic target for endometrial diseases associated with Wnt/β-catenin signaling abnormality.