Lack of dominant-negative activity for tumor-related ZNRF3 missense mutations at endogenous 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, leading to reduced protein levels. They 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.