AXL Knock-Out in SNU475 Hepatocellular Carcinoma Cells Provides Evidence for Lethal Effect Associated with G2 Arrest
Tugce Batur1,2, Ayse Argundogan1,3, Umur Keles1
1Izmir Biomedicine and Genome Center, Dokuz Eylul University Health Campus, Izmir 35330, Turkey.
Abstract:
AXL, a member of the TAM family, is a promising therapeutic target due to its elevated expression in advanced hepatocellular carcinoma (HCC), particularly in association with acquired drug resistance. Previously, RNA interference was used to study its role in cancer, and several phenotypic changes, including attenuated cell proliferation and decreased migration and invasion, have been reported. The mechanism of action of AXL in HCC is elusive. We first studied the AXL expression in HCC cell lines by real-time PCR and western blot and showed its stringent association with a mesenchymal phenotype. We then explored the role of AXL in mesenchymal SNU475 cells by CRISPR-Cas9 mediated gene knock-out. AXL-depleted HCC cells displayed drastic phenotypic changes, including increased DNA damage response, prolongation of doubling time, G2 arrest, and polyploidization in vitro and loss of tumorigenicity in vivo. Pharmacological inhibition of AXL by R428 recapitulated G2 arrest and polyploidy phenotype. These observations strongly suggest that acute loss of AXL in some mesenchymal HCC cells is lethal and points out that its inhibition may represent a druggable vulnerability in AXL-high HCC patients.
Insights
AXL protein is crucial for hepatocellular carcinoma (HCC) cell survival. Inhibiting AXL in AXL-high HCC may offer a new therapeutic strategy by causing cell death and reducing tumor growth.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- AXL receptor tyrosine kinase is overexpressed in advanced hepatocellular carcinoma (HCC).
- AXL expression is linked to drug resistance and a mesenchymal phenotype in HCC.
- The precise role and mechanism of AXL in HCC remain unclear.
Purpose of the Study:
- To investigate the role and mechanism of AXL in HCC.
- To determine if AXL is a druggable target in HCC.
Main Methods:
- Real-time PCR and Western blot to assess AXL expression in HCC cell lines.
- CRISPR-Cas9 gene editing to knock out AXL in SNU475 HCC cells.
- Pharmacological inhibition of AXL using R428.
Main Results:
- AXL expression correlated with a mesenchymal phenotype in HCC cells.
- AXL knockout led to increased DNA damage, prolonged doubling time, G2 arrest, and polyploidization in vitro.
- AXL depletion resulted in loss of tumorigenicity in vivo.
- R428 treatment mimicked the G2 arrest and polyploidy phenotype.
Conclusions:
- Acute loss of AXL is lethal in some mesenchymal HCC cells.
- AXL inhibition represents a potential therapeutic vulnerability in AXL-high HCC patients.
