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Updated: Jun 5, 2025
In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila
Published on: August 20, 2019
Lack of dominant-negative activity for tumor-related ZNRF3 missense mutations at endogenous levels
Shanshan Li1, Jiahui Niu1, Ruyi Zhang1
1Department of Gastroenterology and Hepatology, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, The Netherlands.
Abstract:
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.
Insights
Tumor-associated ZNRF3 variants, including truncating and missense mutations, primarily cause loss-of-function. These mutations impact beta-catenin signaling, challenging the notion of dominant-negative activity in cancer.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- ZNRF3 negatively regulates beta-catenin signaling by internalizing Wnt receptors.
- The role of tumor-associated ZNRF3 variants in cancer development remains unclear.
- Understanding ZNRF3 variant mechanisms is crucial for cancer research.
Purpose of the Study:
- To investigate the functional impact of tumor-associated ZNRF3 variants.
- To elucidate the mechanisms by which ZNRF3 variants contribute to cancer.
- To re-evaluate the oncogenic potential of ZNRF3 mutations.
Main Methods:
- Analysis of endogenous truncating and missense ZNRF3 mutations.
- Functional assessment of ZNRF3 variants in beta-catenin signaling.
- Investigation of protein degradation pathways and cellular localization.
- Introduction of variants into endogenous ZNRF3 in cell models.
Main Results:
- All analyzed truncating ZNRF3 mutations exhibit loss-of-function.
- 27/82 missense variants in RING and R-Spondin domains cause partial or complete loss-of-function.
- Defective R-Spondin variants undergo ER-associated degradation due to misfolding.
- Supposed dominant-negative effects of ZNRF3 variants are not observed at endogenous levels.
Conclusions:
- Tumor-associated ZNRF3 variants predominantly act through loss-of-function mechanisms.
- The proposed dominant-negative oncogenic activity of ZNRF3 mutations is likely non-existent.
- ZNRF3 variants contribute to cancer by impairing Wnt pathway regulation.
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