ß-Catenin mutations as determinants of hepatoblastoma phenotypes in mice

Hepatoblastoma (HB) is the most common pediatric liver cancer. While long-term survival is generally favorable, it is dependent upon clinical stage, tumor histology and a variety of biochemical and molecular features. HB appears almost exclusively before the age of three years, is represented by seven histologic subtypes and is usually associated with highly heterogeneous somatic mutations in the CTNNB1 gene, which encodes b-catenin, a Wnt ligand-responsive transcriptional co-factor. Numerous recurrent b-catenin mutations, not previously documented in HB, have also been identified in various other pediatric and adult cancer types. Little is known regarding the underlying factors that determine the above HB features and behaviors or whether non-HB-associated b-catenin mutations are tumorigenic when expressed in hepatocytes. Here, we investigated the oncogenic properties of 14 different HB- and non-HB-associated b-catenin mutants encoded by Sleeping Beauty vectors following their delivery into the liver by hydrodynamic tail vein injection. We show that all b-catenin mutations, as well as wild-type b-catenin are tumorigenic when co-expressed with a mutant form of yes-associated protein. However, tumor growth rates, histologies, nuclear:cytoplasmic partitioning and metabolic and transcriptional landscapes were strongly influenced by the identities of the b-catenin mutations. These findings provide a context for understanding at the molecular level the notable biological diversity of this important pediatric cancer. Overall design: RNA purification from five representative tumors of each group was performed using Qiagen RNAeasy columns (Qiagen, Inc., Valencia, CA) followed by DNase digestion. Sample integrity was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Foster City, CA). All samples had RIN values of 8.5-10 prior to any further processing. Samples for sequencing were prepared using an NEB NEBNext Ultra Directional RNA Library Prep kit according to the directions provided by the vendor (New England Biolab, Beverly, MA) and sequencing was performed on a NovaSeq 6000 Instrument (Illumina, Inc., San Diego, CA) by Novagene, Inc. (Sacramento, CA).

肝母细胞瘤（Hepatoblastoma, HB）是最常见的儿童肝脏恶性肿瘤。尽管其长期生存预后总体良好，但仍取决于临床分期、肿瘤组织学类型以及多种生化与分子特征。HB几乎仅在3岁前发病，存在7种组织学亚型，且通常伴随CTNNB1基因高度异质性的体细胞突变——该基因编码β-连环蛋白（β-catenin），一种响应Wnt配体的转录辅因子。此前未在HB中报道的多种复发性β-连环蛋白突变，也已在其他多种儿童及成人癌症类型中被发现。目前，学界对于决定上述HB特征与行为的潜在因素，以及非HB相关的β-连环蛋白突变在肝细胞中表达时是否具有致瘤性，所知甚少。本研究通过水动力尾静脉注射法，将睡美人（Sleeping Beauty）载体编码的14种不同HB相关及非HB相关β-连环蛋白突变体递送至肝脏，探究其致瘤特性。结果显示，所有β-连环蛋白突变体与野生型β-连环蛋白，在与yes相关蛋白（yes-associated protein, YAP）突变体共表达时均具有致瘤性。然而，肿瘤生长速率、组织学表型、核质分布比例以及代谢与转录谱特征，均受β-连环蛋白突变体类型的显著影响。这些发现为在分子水平理解这一重要儿童癌症的显著生物学多样性提供了研究框架。整体实验设计：从每组的5个代表性肿瘤中提取总RNA，使用凯杰（Qiagen, Inc.）RNAeasy柱式试剂盒（美国加利福尼亚州瓦伦西亚）完成纯化，随后进行DNase消化处理。使用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer，美国加利福尼亚州福斯特城）检测样本完整性，所有样本在进一步处理前的RNA完整性数值（RIN）均介于8.5至10之间。测序文库使用新英格兰生物实验室（New England Biolabs, NEB）的NEBNext Ultra Directional RNA文库制备试剂盒，严格遵循供应商提供的操作指南构建；随后由诺禾致源（Novagene, Inc.，美国加利福尼亚州萨克拉门托）在Illumina NovaSeq 6000测序仪（美国加利福尼亚州圣地亚哥）上完成测序。