Lack of dominant-negative activity for tumor-associated ZNRF3 missense mutations at endogenous expression levels

ZNRF3, a negative regulator of β-catenin signaling, removes Wnt receptors from the membrane. Currently, it is unknown which tumor-associated variants can be considered driver mutations and through which mechanisms they contribute to cancer. Here we show that all truncating mutations analyzed at endogenous levels exhibit loss-of-function, with longer variants retaining partial activity. Regarding missense mutations, we show that 27/82 ZNRF3 variants in the RING and R-Spondin domain structures, lead to (partial) loss-of-function/hyperactivation. Mechanistically, defective R-spondin domain variants appear to undergo endoplasmic-reticulum-associated degradation due to protein misfolding. They show reduced stability and fail to reach the membrane correctly, which can be partially restored for several variants by culturing cells at 27°C. Although RING and R-spondin domain mutations in RNF43/ZNRF3 are often considered to possess dominant-negative oncogene-like activity in cancers, our findings challenge this notion. When representative variants are heterozygously introduced into endogenous ZNRF3, their impact on β-catenin signaling mirrors that of heterozygous knockout, suggesting that the supposed dominant-negative effect is non-existent. In other words, so-called “hyperactivating” ZNRF3/RNF43 mutations behave as classical loss-of-function mutations at endogenous levels. Taken together, our findings provide valuable information on ZNRF3 mutation impact in tumorigenesis and clarify their mechanism of action.