Characterization of RNF43 frameshift mutations that drive Wnt ligand‐ and R‐spondin‐dependent colon cancer

Abstract Loss‐of‐function mutations in RNF43 induce activation of Wnt ligand‐dependent Wnt/β‐catenin signaling through stabilization of the Frizzled receptor, which is often found in microsatellite instability (MSI)‐type colorectal cancer (CRC) that develops from sessile serrated adenomas. However, the mechanism underlying how RNF43 mutations promote tumorigenesis remains poorly understood. In this study, we established nine human CRC‐derived organoids and found that three organoid lines carried RNF43 frameshift mutations associated with MSI‐high and BRAF V600E mutations, suggesting that these CRCs developed through the serrated pathway. RNF43 frameshift mutant organoids required both Wnt ligands and R‐spondin for proliferation, indicating that suppression of ZNRF3 and retained RNF43 function by R‐spondin are required to achieve an indispensable level of Wnt activation for tumorigenesis. However, active β‐catenin levels in RNF43 ‐mutant organoids were lower than those in APC two‐hit mutant CRC, suggesting a lower threshold for Wnt activation in CRC that developed through the serrated pathway. Interestingly, transplantation of RNF43 ‐mutant organoids with intestinal myofibroblasts accelerated the β‐catenin nuclear accumulation and proliferation of xenograft tumors, indicating a key role of stromal cells in the promotion of the malignant phenotype of RNF43 ‐mutant CRC cells. Sequencing of subcloned organoid cell‐expressed transcripts revealed that two organoid lines carried monoallelic RNF43 cis ‐mutations, with two RNF43 frameshift mutations introduced in the same allele and the wild‐type RNF43 allele remaining, while the other organoid line carried two‐hit biallelic RNF43 trans ‐mutations. These results suggest that heterozygous RNF43 frameshift mutations contribute to CRC development via the serrated pathway; however, a second‐hit RNF43 mutation may be advantageous in tumorigenesis compared with a single‐hit mutation through further activation of Wnt signaling. Finally, treatment with the PORCN inhibitor significantly suppressed RNF43 ‐mutant cell‐derived PDX tumor development. These results suggest a novel mechanism underlying RNF43 mutation‐associated CRC development and the therapeutic potential of Wnt ligand inhibition against RNF43 ‐mutant CRC. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.