Academic Background and Paper Introduction

Background
The Wnt/β-catenin signaling pathway is dysregulated in various cancers, particularly in colorectal and endometrial cancers. Abnormal activation of this pathway is often associated with mutations in tumor suppressor genes such as APC, AXIN1/2, and β-catenin itself. Additionally, RNF43 and ZNRF3, as negative regulators of Wnt receptors, limit Wnt-induced β-catenin signaling by removing Wnt receptors from the cell membrane. However, it remains unclear which tumor-associated ZNRF3 mutations are driver mutations and how they contribute to cancer development. Therefore, this study aims to systematically analyze tumor-associated ZNRF3 mutations, particularly missense and truncating mutations, to reveal their functional significance in cancer.

Paper Source
The paper was co-authored by Shanshan Li, Jiahui Niu, Ruyi Zhang, and others from the Department of Gastroenterology and Hepatology at the Erasmus MC Cancer Institute in Rotterdam, Netherlands. It was published in 2024 in the journal Oncogene, with the DOI 10.1038/s41388-024-03253-4.

Research Process and Results

Research Process

1. Analysis of ZNRF3 Mutation Distribution
   Using the cBioPortal database, the authors analyzed the distribution of ZNRF3 mutations across various cancer types. The results showed that truncating and missense mutations were evenly distributed across the ZNRF3 coding region, except for the first exon (encoding the first 100 amino acids), which had fewer mutations. Additionally, ZNRF3 mutations were more common in endometrial and bowel cancers, with missense mutations being predominant.

2. Functional Validation of ZNRF3 Long Isoform
   The authors identified two ZNRF3 isoforms: a long isoform (936aa) and a short isoform (836aa). Through β-catenin reporter assays, they demonstrated that the long isoform significantly inhibited Wnt-induced β-catenin signaling, while the short isoform did not. Further qPCR experiments showed that the long isoform was highly expressed in various cancer cell lines and contained a signal peptide, enabling it to effectively localize to the cell membrane.

3. Functional Analysis of ZNRF3 Truncating Mutations
   The authors constructed various ZNRF3 truncating mutation expression vectors and validated their function in HEK293T cells. The results showed that all truncating mutations partially inhibited β-catenin signaling when overexpressed, but at endogenous expression levels, all truncating mutations exhibited partial loss of function, with longer truncations retaining more functionality.

4. Functional Analysis of ZNRF3 Missense Mutations
   The authors systematically analyzed 82 tumor-associated ZNRF3 missense mutations and found that 27 mutations in the RING and R-spondin domains led to loss of function or partial loss of function. Further experiments revealed that missense mutations in the R-spondin domain resulted in reduced protein stability and impaired membrane localization. Interestingly, lowering the cell culture temperature to 27°C partially restored the function of some mutations.

5. Validation of “Dominant-Negative” Effects
   Using CRISPR-Cas9 gene editing, the authors validated the “dominant-negative” effects of ZNRF3 missense mutations at endogenous expression levels. The results showed that these mutations did not exhibit significant dominant-negative effects, and their loss of function was similar to that of heterozygous ZNRF3 knockout.

Key Results

1. Loss of Function in ZNRF3 Truncating Mutations
   All tested ZNRF3 truncating mutations exhibited partial loss of function at endogenous expression levels, with longer truncations retaining more functionality.

2. Functional Impact of ZNRF3 Missense Mutations
   In the RING and R-spondin domains, ²⁷⁄₈₂ missense mutations led to loss of function or partial loss of function. Missense mutations in the R-spondin domain resulted in reduced protein stability and impaired membrane localization.

3. Temperature-Dependent Functional Rescue
   Lowering the cell culture temperature to 27°C partially restored the function of some R-spondin domain missense mutations, suggesting that these mutations may involve protein misfolding defects.

4. Negation of “Dominant-Negative” Effects
   At endogenous expression levels, ZNRF3 missense mutations did not exhibit significant dominant-negative effects, and their loss of function was similar to that of heterozygous ZNRF3 knockout.

Conclusions and Significance

This study systematically analyzed tumor-associated ZNRF3 mutations, revealing their functional significance in cancer. The main conclusions include:

1. Loss of Function in ZNRF3 Truncating Mutations
   All tested truncating mutations exhibited partial loss of function at endogenous expression levels, with longer truncations retaining more functionality.

2. Functional Impact of ZNRF3 Missense Mutations
   In the RING and R-spondin domains, some missense mutations led to loss of function or partial loss of function, and these mutations may involve protein misfolding defects.

3. Negation of “Dominant-Negative” Effects
   At endogenous expression levels, ZNRF3 missense mutations did not exhibit significant dominant-negative effects, and their loss of function was similar to that of heterozygous ZNRF3 knockout.

Research Highlights

1. Systematic Analysis of ZNRF3 Mutations
   This study is the first to systematically analyze tumor-associated ZNRF3 mutations, revealing their functional significance in cancer.

2. Temperature-Dependent Functional Rescue
   Lowering the cell culture temperature partially restored the function of some R-spondin domain missense mutations, suggesting that these mutations may involve protein misfolding defects.

3. Negation of “Dominant-Negative” Effects
   This study negates the dominant-negative effects of ZNRF3 missense mutations at endogenous expression levels, providing new insights for cancer treatment strategies.

Research Value

This study provides important clues for understanding the role of ZNRF3 mutations in cancer, particularly their regulatory role in the Wnt/β-catenin signaling pathway. Additionally, the findings offer a theoretical basis for developing cancer treatment strategies targeting ZNRF3 mutations.