RSPO4 exerts tumor suppression through antagonizing canonical and non-canonical Wnt signaling

R-spondins are a family of four secretory proteins reported to be Wnt agonists. Among them, R-spondin 4 (RSPO4) is unique, with the lowest binding affinity towards ZNRF3/RNF43 and the lowest efficacy in regulating Wnt/β-catenin signaling. RSPO4 has been shown to play important roles in nail development, liver fibrogenesis and periodontitis, while its role in cancerous context remains largely unknown. In this study, we performed multi-omic analysis on transcriptional expression and methylation pattern of <i>RSPO4</i>. <i>In vitro</i> cell-based assays were performed to evaluate the functionality of RSPO4. Through cancer epigenomics, we identified RSPO4 as a candidate tumor suppressor with tumor-specific epigenetic inactivation. We further found that <i>RSPO4</i> is readily expressed in human normal tissues, but frequently downregulated or silenced in multiple cancer types due to its promoter CpG methylation. Functional studies showed that RSPO4 inhibited tumor cell proliferation, migration, invasion and stemness, through antagonizing canonical and non-canonical Wnt signaling. Mechanistically, RSPO4 exerted suppressive effects on Wnt signaling in an LGR4/5- and ZNRF3- dependent manner, through promoting LRP6 degradation and ZNRF3 stabilization. Our study revealed a novel role of RSPO4 as a tumor suppressor through antagonizing Wnt signaling, which provides important implications for development of diagnostic biomarkers and targeted therapy.