LIM homeobox-2 suppresses hallmarks of adult and pediatric liver cancers by controlling MAPK/ERK and Wnt/beta-catenin pathways. LIM homeobox-2 suppresses hallmarks of adult and pediatric liver cancers by controlling MAPK/ERK and Wnt/beta-catenin pathways

Hepatocellular carcinoma (HCC) and Hepatoblastoma (HB) are two liver cancers characterized by high gene deregulation, chromosomal rearrangements and typical mutations in genes related to the Wnt/β-catenin (Wnt) pathway. LHX2, a transcriptional factor member of the LIM homeobox gene family, has important functions in embryogenesis, liver development but also tumorigenesis. LHX2 is an oncogene in may solid tumors and leukemia but its role in liver cancer is unknown. To address this question we analyzed the expression of LHX2 in HCC and HB using various transcriptomic datasets and found a strong connection between LHX2 down-regulation and Wnt activation in these two liver cancers. In HB LHX2 downregulation correlated with multiple poor outcome parameters including higher patient age, intermediate- and high-risk tumors and patients’ survival. A forced expression of LHX2 reduced the proliferation, the migration and the survival of hepatoma cells in vitro through the inactivation of MAPK/ERK and Wnt signalings. In vivo, LHX2 impeded the development of tumors in the chick embryo and repressed the Wnt pathway in Xenopus embryo. RNA-sequencing data and bioinformatic analyses confirmed the deregulation of many biological functions and molecular processes associated with cell migration, cell survival and liver carcinogenesis in LHX2-expressing hepatoma cells. At a mechanistic level, LHX2 induced the disassembling of beta-catenin and T-cell factor 4 and the expression of multiple inhibitors of Wnt (e.g. TLE/Groucho) and MAPK/ERK (e.g. DUSPs) pathways. Collectively, our findings demonstrate the tumor suppressive function of LHX2 in adult and pediatric liver cancers. Overall design: Transcriptome profile in Huh7 cell overexpressing LHX2 vs CTRL cells

肝细胞癌（Hepatocellular carcinoma, HCC）与肝母细胞瘤（Hepatoblastoma, HB）是两类以基因广泛失调、染色体重排，以及Wnt/β-连环蛋白（Wnt/β-catenin, Wnt）通路相关基因出现典型突变为特征的肝脏恶性肿瘤。LIM同源框基因家族（LIM homeobox gene family）的转录因子（transcriptional factor）成员LHX2在胚胎发生、肝脏发育及肿瘤发生过程中均发挥关键作用。LHX2在多种实体瘤与白血病中被证实为癌基因（oncogene），但其在肝癌中的功能仍未明确。

为阐明LHX2在肝脏肿瘤中的作用，本研究利用多套转录组数据集（transcriptomic datasets）分析了其在HCC与HB中的表达模式，发现LHX2下调（down-regulation）与这两类肝脏肿瘤的Wnt通路激活存在显著相关性。在肝母细胞瘤中，LHX2下调与多项不良预后指标相关，包括患者更高的发病年龄、中高危肿瘤分型以及更差的总体生存率。

体外实验显示，过表达LHX2可通过抑制MAPK/ERK与Wnt信号通路，降低肝癌细胞的增殖、迁移与存活能力。体内实验中，LHX2可阻碍鸡胚内的肿瘤生成，并抑制非洲爪蟾胚胎中的Wnt通路活性。RNA测序（RNA-sequencing）与生物信息学分析（bioinformatic analyses）证实，在过表达LHX2的肝癌细胞中，诸多与细胞迁移、细胞存活及肝脏癌变相关的生物学功能与分子过程均出现失调。

从机制层面来看，LHX2可诱导β-连环蛋白与T细胞因子4（T-cell factor 4）的解离，并上调Wnt通路（如TLE/Groucho家族）与MAPK/ERK通路（如双特异性磷酸酶DUSPs家族）的多种抑制剂的表达。综上，本研究结果证实LHX2在成人与儿童肝脏恶性肿瘤中发挥肿瘤抑制功能。

本研究的整体实验设计为：对比过表达LHX2的Huh7细胞（Huh7 cell）与对照（CTRL）细胞的转录组特征。