β-Catenin mutations as determinants of hepatoblastoma phenotypes in mice. β-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）是儿童最常见的原发性肝脏恶性肿瘤。尽管其长期生存率整体尚可，但仍取决于临床分期、肿瘤组织学亚型及多种生化与分子特征。肝母细胞瘤几乎仅见于3岁以下儿童，共包含7种组织学亚型，且通常伴随编码β-连环蛋白（β-catenin，一种响应Wnt配体的转录辅因子）的CTNNB1基因发生高度异质性体细胞突变。此前未在肝母细胞瘤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肿瘤类型中被检出。目前学界对决定肝母细胞瘤上述特征与生物学行为的潜在因素，以及非肝母细胞瘤相关的β-连环蛋白突变在肝细胞中表达时是否具有致瘤性，仍所知有限。
本研究针对睡美人（Sleeping Beauty）载体编码的14种不同肝母细胞瘤相关及非肝母细胞瘤相关β-连环蛋白突变体，通过水动力尾静脉注射将其递送至小鼠肝脏，探究其致瘤特性。研究结果显示，所有β-连环蛋白突变体与野生型β-连环蛋白，在与突变型yes相关蛋白（yes-associated protein, YAP）共表达时均具有致瘤性。但肿瘤生长速率、组织学表型、核质分布比例以及代谢与转录谱特征，均显著受β-连环蛋白突变类型的影响。本研究为从分子层面解析这一重要儿童肿瘤的显著生物学多样性提供了研究基础。
实验设计：采用Qiagen RNAeasy柱式提取试剂盒（Qiagen公司，美国加利福尼亚州巴伦西亚），从每组5个代表性肿瘤样本中提取总RNA，随后进行DNase消化处理。使用Agilent 2100生物分析仪（安捷伦科技公司，美国加利福尼亚州福斯特城）检测样本RNA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。测序文库构建采用NEB NEBNext Ultra Directional RNA文库制备试剂盒，严格遵循供应商（新英格兰生物实验室，美国马萨诸塞州贝弗利）提供的操作指南；测序工作由诺禾致源公司（Novagene, Inc.，美国加利福尼亚州萨克拉门托）在NovaSeq 6000测序仪（Illumina公司，美国加利福尼亚州圣地亚哥）上完成。