肝癌在化疗中往往有很高的耐药性。本研究旨在找到有效的化疗药物应用于β-连接蛋白激活的肝癌，该肝癌由β-连接蛋白1基因（CTNNB1）突变引起的功能增强所致，而CTNNB1是肝脏肿瘤中最常见的原癌基因。通过删除外显子3（β-连接蛋白Δ（ex3）／+），在CTNNB1基因中最常见的突变位点，建立了β-连接蛋白激活的胚胎纤维母细胞（MEFs）。对12种广泛使用的化疗药物进行筛查，以选择能选择性抑制β-连接蛋白Δ（ex3）／+的化疗药物，但不对野生型MEFs起作用。进行非靶向代谢组学分析，检测核苷酸合成方面的代谢物变化。通过体外实验方法检测了甲氨蝶呤（MTX）对β-连接蛋白激活的人体肝癌细胞的疗效和选择性。分别以β-连接蛋白野生型或突变型肝癌细胞和乙型肝炎病毒（HBV）植入体内的裸鼠和β-连接蛋白lox（ex3）／+小鼠作为模型，评估MTX在治疗β-连接蛋白突变型肝癌方面的疗效。MTX被确定并验证为抑制β-连接蛋白激活细胞增殖和肿瘤形成的优选药物。核苷酸合成的增强是β-连接蛋白激活细胞主要的代谢异常，而这种变化也是MTX的靶点。此外，MTX还能抑制HBV鼠和β-连接蛋白lox（ex3）／+小鼠的肝癌发生，这两种模型分别模拟了肝癌中β-连接蛋白活化突变和HBV感染。MTX是治疗β-连接蛋白高活化肝癌的有希望的化疗药物。由于重新应用MTX具有风险较低、时间较短以及在药物发现和开发方面的投资较少等优势，因此应进行临床试验以测试其在治疗β-连接蛋白突变型肝癌方面的疗效。 版权所有 © 2023 中国医学会，由Wolters Kluwer公司在CC-BY-NC-ND许可下制作。

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.Constitutive β-catenin-activated mouse embryonic fibroblasts (MEFs) were established by deleting exon 3 (β-cateninΔ(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-cateninΔ(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-cateninlox(ex3)/+ mice were used, respectively, to evaluate the efficacy of MTX in the treatment of β-catenin mutant liver cancer.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-cateninlox(ex3)/+ mice, which stimulated concurrent Ctnnb1-activated mutation and HBV infection in liver cancer.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.Copyright © 2023 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.