SCYL3, as a novel binding partner and regulator of ROCK2, promotes hepatocellular carcinoma progression.

Background & Aims: SCY1-like pseudokinase 3 (SCYL3) was identified as a binding partner of ezrin, implicating it in metastasis. However, the clinical relevance and functional role of SCYL3 in cancer remain uncharacterized. In this study, we aimed to elucidate the role of SCYL3 in the progression of hepatocellular carcinoma (HCC). Methods: The clinical significance of SCYL3 in HCC was evaluated in publicly available datasets and by qPCR analysis of an in-house HCC cohort. The functional significance and mechanistic consequences of SCYL3 were examined in SCYL3-knockdown/overexpressing HCC cells. In vivo tumor progression was evaluated in Tp53 KO/c-Myc OE mice using the sleeping beauty transposon system. Potential downstream pathways were investigated by co-immunoprecipitation, western blotting analysis and immunofluorescence staining. Results: SCYL3 is often overexpressed in HCC; it is preferentially expressed in metastatic human HCC tumors and is associated with worse patient survival. Suppression of SCYL3 in HCC cells attenuated cell proliferation and migration as well as in vivo metastasis. Intriguingly, endogenous SCYL3 overexpression increased tumor development and metastasis in Tp53 KO/c-Myc OE mice. Mechanistic investigations revealed that SCYL3 physically binds and regulates the stability and transactivating activity of ROCK2 (Rho kinase 2) via its C-terminal domain, leading to the increased formation of actin stress fibers and focal adhesions. Conclusions: These findings reveal that SCYL3 plays a critical role in promoting the progression of HCC and have implications for developing new therapeutic strategies to tackle metastatic HCC. Impact and implications: SCYL3 was first reported to be a binding partner of a metastasis-related gene, ezrin. To date, the clinical relevance and functional role of SCYL3 in cancer remain uncharacterized. Herein, we uncover its crucial role in liver cancer progression. We show that it physically binds and regulates the stability and transactivating activity of ROCK2 leading to HCC tumor progression. Our data provide mechanistic insight that SCYL3-mediated ROCK2 protein stability plays a pivotal role in growth and metastasis of HCC cells. Targeting SCYL3/ROCK2 signaling cascade may be a novel therapeutic strategy for treatment of HCC patients.

ZBTB20 regulates WNT/CTNNB1 signalling pathway by suppressing PPARG during hepatocellular carcinoma tumourigenesis.

BACKGROUND & AIMS: Zinc finger and BTB domain containing 20 (ZBTB20) has been implicated as a potential oncogene in liver cancer. However, knockout studies have shown it to be a transcriptional repressor of the alpha-foetoprotein (Afp) gene in adult liver, and reduced levels of ZBTB20 allow for upregulation of AFP with increased tumour severity in certain cases of hepatocellular carcinoma (HCC). As there are many discrepancies in the literature regarding its role in liver tumourigenesis, the aim of this study was to elucidate the role of ZBTB20 in HCC tumourigenesis. METHODS: A reverse genetic study using the Sleeping Beauty (SB) transposon system in mice was performed to elucidate the role of ZBTB20 in HCC tumourigenesis. In vitro ZBTB20 gain- and loss-of-function experiments were used to assess the relationship amongst ZBTB20, peroxisome proliferator activated receptor gamma (PPARG) and catenin beta 1 (CTNNB1). RESULTS: Transgenic overexpression of ZBTB20 in hepatocytes and in the context of transformation related protein (T r p53) inactivation induced hepatic hypertrophy, activation of WNT/CTNNB1 signalling, and development of liver tumours. In vitro overexpression and knockout experiments using CRISPR/Cas9 demonstrated the important role for ZBTB20 in downregulating PPARG, resulting in activation of the WNT/CTNNB1 signalling pathway and its downstream effectors in HCC tumourigenesis. CONCLUSIONS: These findings demonstrate a novel interaction between ZBTB20 and PPARG, which leads to activation of the WNT/CTNNB1 signalling pathway in HCC tumourigenesis. LAY SUMMARY: ZBTB20 has been implicated as a potential oncogene in liver cancer. Herein, we uncover its important role in liver cancer development. We show that it interacts with PPARG to upregulate the WNT/CTNNB1 signalling pathway, leading to tumourigenesis.

Novel vectors and approaches for gene therapy in liver diseases.

Gene therapy is becoming an increasingly valuable tool to treat many genetic diseases with no or limited treatment options. This is the case for hundreds of monogenic metabolic disorders of hepatic origin, for which liver transplantation remains the only cure. Furthermore, the liver contains 10-15% of the body's total blood volume, making it ideal for use as a factory to secrete proteins into the circulation. In recent decades, an expanding toolbox has become available for liver-directed gene delivery. Although viral vectors have long been the preferred approach to target hepatocytes, an increasing number of non-viral vectors are emerging as highly efficient vehicles for the delivery of genetic material. Herein, we review advances in gene delivery vectors targeting the liver and more specifically hepatocytes, covering strategies based on gene addition and gene editing, as well as the exciting results obtained with the use of RNA as a therapeutic molecule. Moreover, we will briefly summarise some of the limitations of current liver-directed gene therapy approaches and potential ways of overcoming them.

Sleeping Beauty transposon system for genetic etiological research and gene therapy of cancers.

Carcinogenesis is etiologically associated with somatic mutations of critical genes. Recently, a number of somatic mutations and key molecules have been found to be involved in functional networks affecting cancer progression. Suitable animal models are required to validate cancer-promoting or -inhibiting capacities of these mutants and molecules. Sleeping Beauty transposon system consists of a transposon that carries gene(s) of interest and a transposase that recognizes, excises, and reinserts genes in given location of the genome. It can create both gain-of-function and loss-of-function mutations, thus being frequently chosen to investigate the etiological mechanisms and gene therapy for cancers in animal models. In this review, we summarized current advances of Sleeping Beauty transposon system in revealing molecular mechanism of cancers and improving gene therapy. Understanding molecular mechanisms by which driver mutations contribute to carcinogenesis and metastasis may pave the way for the development of innovative prophylactic and therapeutic strategies against malignant diseases.