FAT4 loss initiates hepatocarcinogenesis through the switching of canonical to noncanonical WNT signaling pathways

Abstract

<h3>Background: </h3><p>Mutation and downregulation of FAT atypical cadherin 4 (FAT4) are frequently detected in HCC, suggesting a tumor suppressor role of FAT4. However, the underlying molecular mechanism remains elusive.</p><h3>Methods: </h3><p>CRISPR-Cas9 system was used to knockout FAT4 (<em>FAT4</em>-KO) in a normal human hepatic cell line L02 to investigate the impact of FAT4 loss on the development of HCC. RNA-sequencing and xenograft mouse model were used to study gene expression and tumorigenesis, respectively. The mechanistic basis of FAT4 loss on hepatocarcinogenesis was elucidated using <em>in vitro</em> experiments.</p><h3>Results: </h3><p>We found that <em>FAT4</em>-KO disrupted cell-cell adhesion, induced epithelial-mesenchymal transition, and increased expression of extracellular matrix components. <em>FAT4</em>-KO is sufficient for tumor initiation in a xenograft mouse model. RNA-sequencing of <em>FAT4</em>-KO cells identified PAK6-mediated WNT/β-catenin signaling to promote tumor growth. Suppression of PAK6 led to β-catenin shuttling out of the nucleus for ubiquitin-dependent degradation and constrained tumor growth. Further, RNA-sequencing of amassed <em>FAT4</em>-KO cells identified activation of WNT5A and ROR2. The noncanonical WNT5A/ROR2 signaling has no effect on β-catenin and its target genes (CCND1 and c-Myc) expression. Instead, we observed downregulation of receptors for WNT/β-catenin signaling, suggesting the shifting of β-catenin-dependent to β-catenin-independent pathways as tumor progression depends on its receptor expression. Both PAK6 and WNT5A could induce the expression of extracellular matrix glycoprotein, laminin subunit alpha 4. Laminin subunit alpha 4 upregulation in HCC correlated with poor patient survival.</p><h3>Conclusions: </h3><p>Our data show that FAT4 loss is sufficient to drive HCC development through the switching of canonical to noncanonical Wingless-type signaling pathways. The findings may provide a mechanistic basis for an in-depth study of the two pathways in the early and late stages of HCC for precise treatment.</p>