by Ruyi Zhang, Shanshan Li, Kelly Schippers, Boaz Eimers, Jiahui Niu, Bastian V. H. Hornung, Mirjam C. G. N. van den Hout, Wilfred F. J. van Ijcken, Maikel P. Peppelenbosch, Ron Smits
BackgroundHepatocellular carcinoma (HCC) is a highly aggressive liver cancer with significant morbidity and mortality rates. AXIN1 is one of the top-mutated genes in HCC, but the mechanism by which AXIN1 mutations contribute to HCC development remains unclear.
MethodsIn this study, we utilized CRISPR/Cas9 genome editing to repair AXIN1-truncated mutations in five HCC cell lines.
ResultsFor each cell line we successfully obtained 2–4 correctly repaired clones, which all show reduced β-catenin signaling accompanied with reduced cell viability and colony formation. Although exposure of repaired clones to Wnt3A-conditioned medium restored β-catenin signaling, it did not or only partially recover their growth characteristics, indicating the involvement of additional mechanisms. Through RNA-sequencing analysis, we explored the gene expression patterns associated with repaired AXIN1 clones. Except for some highly-responsive β-catenin target genes, no consistent alteration in gene/pathway expression was observed. This observation also applies to the Notch and YAP/TAZ-Hippo signaling pathways, which have been associated with AXIN1-mutant HCCs previously. The AXIN1-repaired clones also cannot confirm a recent observation that AXIN1 is directly linked to YAP/TAZ protein stability and signaling.
ConclusionsOur study provides insights into the effects of repairing AXIN1 mutations on β-catenin signaling, cell viability, and colony formation in HCC cell lines. However, further investigations are necessary to understand the complex mechanisms underlying HCC development associated with AXIN1 mutations.
