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

Introduction: Hepatocellular carcinoma and hepatoblastoma are two liver cancers characterized by gene deregulations, chromosomal rearrangements, and mutations in Wnt/beta-catenin (Wnt) pathway-related genes. LHX2, a transcriptional factor member of the LIM homeobox gene family, has important functions in embryogenesis and liver development. LHX2 is oncogenic in many solid tumors and leukemia but its role in liver cancer is unknown. Methods: We analyzed the expression of LHX2 in hepatocellular carcinoma and hepatoblastoma samples using various transcriptomic datasets and biological samples. The role of LHX2 was studied using lentiviral transduction, in vitro cell-based assays (growth, migration, senescence, apoptosis), molecular approaches (phospho-kinase arrays, RNA-seq), bioinformatics and two in vivo models in chicken and Xenopus embryos. Results: We found a strong connection between LHX2 down-regulation and Wnt activation in these two liver cancers. In hepatoblastoma, LHX2 downregulation correlated with multiple poor outcome parameters including higher patient age, intermediate- and high-risk tumors and low patients’ survival. Forced expression of LHX2 reduced the proliferation, migration and survival of hepatoma cells in vitro through the inactivation of MAPK/ERK and Wnt signals. In vivo, LHX2 impeded the development of tumors in chick embryos and repressed the Wnt pathway in Xenopus embryos. 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 mediated the disassembling of beta-catenin/T-cell factor 4 complex and induced expression of multiple inhibitors of Wnt (e.g. TLE/Groucho) and MAPK/ERK (e.g. DUSPs) pathways. Conclusion: Collectively, our findings demonstrate a tumor suppressive function of LHX2 in adult and pediatric liver cancers.

Kindlin2 regulates neural crest specification via integrin-independent regulation of the FGF signaling pathway

The focal adhesion protein Kindlin2 is essential for integrin activation, a process that is fundamental to cell-extracellular matrix adhesion. Kindlin2 is widely expressed in mouse embryos, and its absence causes lethality at the peri-implantation stage due to the failure to trigger integrin activation. The function of kindlin2 during embryogenesis has not yet been fully elucidated as a result of this early embryonic lethality. Here, we showed that kindlin2 is essential for neural crest (NC) formation in Xenopus embryos. Loss-of-function assays performed with kindlin2-specific morpholino antisense oligos (MOs) or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 techniques in Xenopus embryos severely inhibit the specification of NC. Moreover, integrin-binding deficient mutants of Kindlin2 rescued the phenotype caused by loss of kindlin2, suggesting that the function of kindlin2 during NC specification is independent of integrins. Mechanistically, we found that Kindlin2 regulates the fibroblast growth factor (FGF) pathway, and promotes the stability of FGF receptor 1. Our study reveals a novel function of Kindlin2 in regulating FGF signaling pathway and provides mechanistic insights into the function of Kindlin2 during NC specification.

Whole-Mount RNA In Situ Hybridization and Immunofluorescence of Xenopus Embryos and Tadpoles

A major advantage of experimentation in Xenopus is the ability to query the localization of endogenous proteins and RNAs in situ in the entire animal during all of development. Here I describe three variations of staining to visualize mRNAs and proteins in developing Xenopus embryos and tadpoles. The first section outlines a traditional colorimetric staining for mRNAs that is suitable for all stages of development, and the second extends this protocol for fluorescence-based detection for higher spatial and quantitative resolution. The final section details detection of proteins by immunofluorescence, optimized for tadpole stages but widely applicable to others. Finally, optimization strategies are provided.