Oncogenic β-catenin-driven liver cancer is susceptible to methotrexate-mediated disruption of nucleotide synthesis.

Background: Liver cancer is largely resistant to chemotherapy. This study aimed to identify the effective chemotherapeutics for β-catenin-activated liver cancer which is caused by gain-of-function mutation of catenin beta 1 ( CTNNB1 ), the most frequently altered proto-oncogene in hepatic neoplasms.

Methods: Constitutive β-catenin-activated mouse embryonic fibroblasts (MEFs) were established by deleting exon 3 ( β-catenin Δ(ex3)/+ ), the most common mutation site in CTNNB1 gene. A screening of 12 widely used chemotherapy drugs was conducted for the ones that selectively inhibited β-catenin Δ(ex3)/+ but not for wild-type MEFs. Untargeted metabolomics was carried out to examine the alterations of metabolites in nucleotide synthesis. The efficacy and selectivity of methotrexate (MTX) on β-catenin-activated human liver cancer cells were determined in vitro . Immuno-deficient nude mice subcutaneously inoculated with β-catenin wild-type or mutant liver cancer cells and hepatitis B virus ( HBV ); β-catenin lox(ex3)/+ mice were used, respectively, to evaluate the efficacy of MTX in the treatment of β-catenin mutant liver cancer.

Results: MTX was identified and validated as a preferential agent against the proliferation and tumor formation of β-catenin-activated cells. Boosted nucleotide synthesis was the major metabolic aberration in β-catenin-active cells, and this alteration was also the target of MTX. Moreover, MTX abrogated hepatocarcinogenesis of HBV ; β-catenin lox(ex3)/+ mice, which stimulated concurrent Ctnnb1- activated mutation and HBV infection in liver cancer.

Conclusion: MTX is a promising chemotherapeutic agent for β-catenin hyperactive liver cancer. Since repurposing MTX has the advantages of lower risk, shorter timelines, and less investment in drug discovery and development, a clinical trial is warranted to test its efficacy in the treatment of β-catenin mutant liver cancer.