Distinct Mutations in the Same Gene Drive Cancer Differently

By employing a CRISPR-based approach, scientists introduced hundreds of variants in a gene to study how they fuel tumor growth.

Cells have a carefully orchestrated mechanism consisting of several proteins to ensure healthy replication and division. One of the key players that participates in regulating tissue growth and repair is β-catenin.

However, mutations in CTNNB1, the gene encoding β-catenin, increase its signaling and result in a cascade of events driving tumor growth. Missense mutations—which cause a different amino acid to be encoded at a particular position—within a specific region of CTNNB1 are among the most common mutations in several cancers.¹ While scientists previously found that different mutations within this region of the gene result in distinct tumor progression, the functional implications of these variants remained unknown.²

Now, using a high-throughput CRISPR-based approach in mouse stem cells, scientists individually introduced each of the 342 possible missense mutations in a CTNNB1 region to establish the extent to which each mutation drove uncontrolled cell growth.³ Their work, published in Nature Genetics, provides a map to understand how mutational diversity within a hotspot impacts cancer progression, with implications for targeted anticancer therapies.

“The new map provides a powerful tool for predicting how specific CTNNB1 mutations affect cancer behavior,” said study coauthor Andrew Wood, a geneticist at the University of Edinburgh, in a statement. “As the first study to experimentally test every possible mutation in this critical hotspot, it gives scientists a clearer picture of how β-catenin drives tumor growth across different cancer types.”

For their study, Wood and his colleagues generated transgenic mice containing a fluorescent reporter of β-catenin signaling activity. They then isolated embryonic stem cells from the animals and introduced each of the 342 possible missense mutations in the CTNNB1 region in different cultures. Using fluorescence-activated cell sorting, the scientists then quantified the output of each edited allele.

Using this data, the researchers assigned mutational effect scores to distinct variants, which helped them predict the extent to which each of the mutations activated β-catenin and its target genes. While some mutations increased β-catenin activity only slightly, others had a stronger effect: Substitution of serine at the 45^th position activated β-catenin weakly, while mutations between positions 32-37 led to its very strong activation.

Moreover, different substitutions at the same position had different effects on β-catenin activity: Threonine at the 41^st position converted to asparagine or proline activated β-catenin signaling weakly, but its conversion to alanine or isoleucine had a stronger effect.

Up to 30 percent of people with liver cancer carry mutations in the CTNNB1 hotspot.⁴ So, Wood and his colleagues examined data from hepatocellular carcinoma patients to investigate how different variants influenced signaling and immune landscapes. They observed that those with weaker mutation effect scores on β-catenin activity had upregulation of more genes linked with immune cell infiltration and vice versa, suggesting that mutation strengths may influence anticancer therapy outcomes.

Despite this, the authors noted in the paper that further work is required to establish whether the mutation effect scores can accurately predict β-catenin signaling outputs across all different cell types other than mouse embryonic stem cells.