Abstract
BackgroundAberrant activation of Axl is implicated in the progression of hepatocellular carcinoma (HCC). We explored the biologic significance and preclinical efficacy of Axl inhibition as a therapeutic strategy in sorafenib-naive and resistant HCC.MethodsWe evaluated Axl expression in sorafenib-naive and resistant (SR) clones of epithelial (HuH7) and mesenchymal origin (SKHep-1) using antibody arrays and confirmed tissue expression. We tested the effect of Axl inhibition with RNA-interference and pharmacologically with R428 on a number of phenotypic assays.ResultsAxl mRNA overexpression in cell lines (n = 28) and RNA-seq tissue datasets (n = 373) correlated with epithelial-to-mesenchymal transition (EMT). Axl was overexpressed in HCC compared to cirrhosis and normal liver. We confirmed sorafenib resistance to be associated with EMT and enhanced motility in both HuH7-SR and SKHep-1-SR cells documenting a 4-fold increase in Axl phosphorylation as an adaptive feature of chronic sorafenib treatment in SKHep-1-SR cells. Axl inhibition reduced motility and enhanced sensitivity to sorafenib in SKHep-1SR cells. In patients treated with sorafenib (n = 40), circulating Axl levels correlated with shorter survival.ConclusionsSuppression of Axl-dependent signalling influences the transformed phenotype in HCC cells and contributes to adaptive resistance to sorafenib, providing a pre-clinical rationale for the development of Axl inhibitors as a measure to overcome sorafenib resistance.
Highlights
The majority of patients with a new diagnosis of hepatocellular carcinoma (HCC), the third cause of cancer-related death, present with incurable disease.[1]
Axl is overexpressed in HCC and is associated with epithelial-tomesenchymal transition (EMT) We used the Cell Line Encyclopedia (CCLE) database to investigate the expression of Axl and its ligand growth arrest signal 6 (Gas-6) in a panel of 28 immortalised HCC cell lines
Gene set enrichment analysis (GSEA) analysis on the CCLE RNA-seq dataset inclusive of all the 28 HCC cell lines confirmed enrichment of transcripts pertaining to EMT in Axloverexpressing cell lines (FDR q = 0.20, Fig. 1d), which was validated in the The Cancer Genome Atlas (TCGA) dataset (FDR q < 0.001, Fig. 1e)
Summary
The majority of patients with a new diagnosis of hepatocellular carcinoma (HCC), the third cause of cancer-related death, present with incurable disease.[1]. We explored the biologic significance and preclinical efficacy of Axl inhibition as a therapeutic strategy in sorafenib-naive and resistant HCC. METHODS: We evaluated Axl expression in sorafenib-naive and resistant (SR) clones of epithelial (HuH7) and mesenchymal origin (SKHep-1) using antibody arrays and confirmed tissue expression. RESULTS: Axl mRNA overexpression in cell lines (n = 28) and RNA-seq tissue datasets (n = 373) correlated with epithelial-tomesenchymal transition (EMT). We confirmed sorafenib resistance to be associated with EMT and enhanced motility in both HuH7-SR and SKHep-1-SR cells documenting a 4-fold increase in Axl phosphorylation as an adaptive feature of chronic sorafenib treatment in SKHep-1-SR cells. Axl inhibition reduced motility and enhanced sensitivity to sorafenib in SKHep-1SR cells. CONCLUSIONS: Suppression of Axl-dependent signalling influences the transformed phenotype in HCC cells and contributes to adaptive resistance to sorafenib, providing a pre-clinical rationale for the development of Axl inhibitors as a measure to overcome sorafenib resistance
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