Abstract
Comprehensive analysis of clinical samples has recently identified molecular and immunological classification of hepatocellular carcinoma (HCC), and the CTNNB1 (β-catenin)-mutated subtype exhibits distinctive characteristics of immunosuppressive tumor microenvironment. For clarifying the molecular mechanisms, we first established human and mouse HCC cells with exon 3 skipping of β-catenin, which promoted nuclear translocation and activated the Wnt/β-catenin signaling pathway, by using newly developed multiplex CRISPR/Cas9-based genome engineering system. Gene set enrichment analysis indicated downregulation of immune-associated gene sets in the HCC cells with activated β-catenin signaling. Comparative analysis of gene expression profiles between HCC cells harboring wild-type and exon 3 skipping β-catenin elucidated that the expression levels of four cytokines were commonly decreased in human and mouse β-catenin-mutated HCC cells. Public exome and transcriptome data of 373 human HCC samples showed significant downregulation of two candidate cytokine genes, CCL20 and CXCL2, in HCC tumors with β-catenin hotspot mutations. T cell killing assays and immunohistochemical analysis of grafted tumor tissues demonstrated that the mouse Ctnnb1Δex3 HCC cells evaded immunosurveillance. Taken together, this study discovered that cytokine controlled by β-catenin signaling activation could contribute to immune evasion, and provided novel insights into cancer immunotherapy for the β-catenin-mutated HCC subtype.
Highlights
Hepatocellular carcinoma (HCC) is a complex disease with various risk factors, that is, chronic infection with hepatitis B virus and hepatitis C virus, alcohol abuse, metabolic disease including obesity and diabetes, and dietary toxins such as aflatoxins and aristolochic a cid[1]
We newly developed a highly efficient multiplex CRISPR/Cas9-based genome engineering system for exon skipping by modifying the lentiGuide-Puro plasmid (Nat Methods), originally provided from Feng Zhang’s laboratory[18]
To the human hepatocellular carcinoma (HCC) cells, mouse HCC cells expressing active form β-catenin were derived from the 3H3 cell line, which was a HrasQ61L-mutated mouse HCC cell line isolated from the C57BL6/J MC4R-KO mouse m odel[19], and termed as 3H3-Ctnnb1Δex[3]
Summary
Hepatocellular carcinoma (HCC) is a complex disease with various risk factors, that is, chronic infection with hepatitis B virus and hepatitis C virus, alcohol abuse, metabolic disease including obesity and diabetes, and dietary toxins such as aflatoxins and aristolochic a cid[1]. Activation of the Wnt/β-catenin signaling pathway[8,9] For examining this biological process, two genetically engineered mouse models have been developed; one is a transgenic mouse model with ectopic expression of mutated and stabilized β-catenin in which Ser[33], Ser[37], Thr[41] and Ser[45] are substituted by alanine r esidues[10] or N-terminal d eletion[11]; the other is a Cre/loxP-based mouse model harboring a mutant Ctnnb[1] allele with loxP sequences in intron 2 and intron 3 for intrinsically skipping of exon 3 12. Luke et al have recently addressed that the Wnt/β-catenin signaling pathway is activated, by CTNNB1 mutation, in non-T cell-inflamed tumors across cancer types including HCC13. We established a novel model of intrinsically active β-catenin signaling by CRISPR/Cas9-mediated exon skipping in human and mouse HCC cells, and investigated how tumor β-catenin signaling evades the immune system in HCC
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