WNT3A Upregulates the RIP1/HIF-1α/GDF-15 Pathway to Induce EMT and Thereby Increase Colorectal Cancer Cell Migration and Invasion.

Abstract

Purpose: We previously demonstrated that WNT signaling, a key regulator of epithelial-mesenchymal transition (EMT), promotes malignancy via RIP1 in colorectal cancer (CRC). In this study, we aimed to reveal the novel signaling pathway through which WNT3A induces EMT in CRC.

Materials and methods: CRC cells (DLD-1, HCT116) and mouse embryonic fibroblasts (MEFs; wtMEF and RIP1-/- MEF) were used in this study. Expression levels of GDF-15 and GFRAL were assessed by RT-qPCR, immunoblotting, and ELISA. RIP1 and HIF-1α were silenced using siRNA or shRNA. Cytokine profiling and ELISA were performed to quantify GDF-15 secretion. Migration and invasion assays were conducted in GDF-15-treated cells. Expression of HIF-1α within the pathway was analyzed with RT-qPCR and immunoblotting. In vivo expressions of GDF-15 and GFRAL were detected in human blood and CRC tissue specimens.

Results: WNT3A treatment significantly upregulated both mRNA and protein levels of GDF-15 and GFRAL in CRC cells. Silencing of RIP1 with siRIP1 or shRIP1 decreased the expression and secretion of GDF-15 and GFRAL. Cytokine profiling and ELISA confirmed that RIP1 facilitates GDF-15 secretion. Treatment with GDF-15 led to enhanced cell migration, invasion, and increased EMT marker expression. We also detected increases of GDF-15 in blood specimens and metastatic tissues of CRC patients. Furthermore, HIF-1α was identified as a key transcription factor downstream of RIP1 that regulates GDF-15 expression.

Conclusion: Our findings demonstrate that the novel WNT3A/RIP1/HIF-1α/GDF-15 signaling axis induces EMT, migration, and invasion in CRC. This pathway might represent a potential therapeutic target for limiting metastatic progression in CRC.