Background Aberrant 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. 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. We tested the effect of Axl inhibition with RNA-interference and pharmacologically with R428 on a number of phenotypic assays. Results Axl 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. 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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HCC is the third most frequent cancer-related death worldwide and incidence continues to rise. Most patients presenting with advanced stage disease where their treatment goal is palliation, emphasizing the requirement for new treatment options. HCC is characterized by arterialization of its blood supply deemed pathognomonic for its development. We investigated the neoangiogenic peptide apelin as a driver of carcinogenesis in HCC followed by the efficacy of novel apelin antagonists as a therapeutic strategy in-vitro. Firstly, meta-analysis conducted through the TCGA database showed that apelin is significantly upregulated in liver cancer compared to healthy tissue and this correlated with a worse mean overall survival. Additionally, enzyme-linked immunosorbent assay (ELISA) of patient serum samples corroborated that apelin concentration significantly increases from healthy liver to cirrhosis and then further through tumorigenesis. In-vitro studies across multiple hepatocyte cell lines in media conditioned with elevated serum apelin levels were then performed to investigate the effects of increased apelin. All cell-lines demonstrated an increase in migration and proliferative properties with naturally higher basal apelin expressing cell lines showing the most significant changes. Using knockdown models, we elucidated hypoxia a well-known driver of angiogenesis, up-regulates apelin expression through a HIF-1α dependent mechanism. Furthermore, increased apelin expression resulted in upregulation of its G-protein coupled receptor, APJ which initiates its downstream signaling pathways, together referred to as apelinergic signaling, demonstrating an autocrine loop. Co-culture of hepatocytes with hepatic stellate cells (HSCs), activated in cirrhosis, further elucidated a paracrine signaling loop whereby secreted chemokines from HSCs, which including apelin, enhanced hepatocyte proliferation and migration. Assessing apelin as a therapeutic target, several novel antagonists were designed in-silico and investigated across multiple hepatocyte cell lines. MM315 showed the most promise with significant cytostatic properties at nanomolar levels coupled with a high binding affinity to APJ at picomolar levels demonstrated through blocking studies. As HCC is a highly vascularized cancer, we explored the likely effects of these antagonists on endothelial cells, cultured in the presence of IC50 concentrations of each antagonist. MM315 treatment resulted in a significant reduction of abnormal blood vessel formation. Overall, our results suggest inhibiting apelinergic signaling is a promising therapeutic treatment for HCC, with MM315 being a lead compound. In-vivo work which will be presented. Citation Format: Jamie Beaumont, Eric Aboagye, Beata Wojciak-Stothard, Gilberto Serrano-De-Almeida, Robert Glen, Rohini Sharma. Apelinergic signalling in hepatocellular carcinoma (HCC): A new therapeutic treatment option [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5398.