A transposon-based insertional mutagenesis screen for identifying cancer genes and sorafenib resistance mechanisms in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most common primary liver cancer with high incidence and mortality rates. The development of HCC is a multistep process involving activation of oncogenic signaling pathways and inactivation of tumor suppressor genes. Importantly, HCC exhibits complicated genetic heterogeneity resulting in various alterations of signaling pathways. The genetic mechanism(s) of HCC is still unclear, raising the difficulties for the improvement of diagnosis and prognosis for HCC patients. Moreover, majority of HCC patients are diagnosed at late-stage leading to the limitation for the optimal outcome for therapy. Sorafenib is one of the first-line drugs used in treatment for late-stage HCC. Unfortunately, drug resistance is usually observed in HCC patients which limits the therapeutic efficacy. Therefore, this research study aims to elucidate tumorigenic and drug resistant mechanisms in HCC. Zine finger and BTB domain containing 20 (Zbtb20) was previously identified in forward genetic screens as a candidate driver gene for liver cancer using the Sleeping Beauty (SB) transposon insertional mutagenesis system. Transgenic overexpression of ZBTB20 in hepatocytes and in the context of transformation related protein (Trp53) inactivation induced hepatic hypertrophy, activation of WNT/CTNNB1 signaling, and development of liver tumors. In vitro overexpression and knockout experiments using CRISPR/Cas9 demonstrated the important role for ZBTB20 in downregulating peroxisome proliferator activated receptor gamma (PPARG), resulting in activation of the WNT/CTNNB1 signaling pathway in HCC tumorigenesis. Interestingly, hepatic hypertrophy was observed in liver tumors of our transgenic mouse model with ZBTB20 overexpression and the context of Trp53 inactivation. WNT/CTNNB1 signaling and Hippo-YAP/TAZ signaling play an important role in hepatic hypertrophy and exhibit crosstalk by positively regulation. Ubiquitin domain containing 1 (UBTD1) and MOB kinase activator 2 (MOB2) were identified from our ChIP-seq data as potential targets of ZBTB20 in the regulation of Hippo-YAP/TAZ signaling. Our preliminary results showed that yes-associated protein 1 (YAP1) protein and downstream genes of Hippo-YAP/TAZ signaling were upregulated in ZBTB20-overexpressing cells and conversely downregulated in ZBTB20-knoutout cells. Increased HOP/HIP luciferase activity also confirmed the activation of YAP/TAZ/TEAD activity by ZBTB20. Moreover, larger sphere size and anti-apoptotic effect were observed in ZBTB20-overexpressing cells. Another focus of this study was to understand the molecular mechanisms of Sorafenib resistance. Sorafenib resistant PLC/PRF/5 cells (PLC/PRF/5Sor-R) was established using SB transposon insertional mutagenesis, which resulted in higher clonogenic survival ability and reduced drug sensitivity to Sorafenib. Interleukin 7 (IL7) and T cell differentiation protein 2 (MAL2) were revealed as candidate genes of Sorafenib-associated resistance by regulating JAK/signal transducer and activator of transcription-3 (STAT3) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways. Our preliminary results showed that overexpressing IL7 and/or MAL2 in PLC/PRF/5 cells exhibited higher clonogenic survival ability, decreased anti-apoptotic effect to Sorafenib and activated levels of PI3K/AKT/STAT3 by Western blot analyses.