The Role of Axl in Hepatocellular Carcinoma Progression

Metastasis is the leading cause of cancer mortality and represents a multi-step process including local tumor cell invasion, entry into the vasculature followed by the exit of carcinoma cells from the circulation and colonization at the distal sites. In hepatocellular carcinoma (HCC), intrahepatic metastasis frequently correlates with a preceding epithelial to mesenchymal transition (EMT) of malignant hepatocytes. EMT is therefore considered as a pivotal event in HCC progression towards late stages of hepatocarcinogenesis. Several mechanisms have been elaborated to be essentially involved in hepatocellular EMT such as the activation of the Ras signalling collaborating with tumor-promoting transforming growth factor-beta functions. Yet, the diversity and integration of signalling cascades that govern EMT in liver cancer progression is still poorly understood. Our recent analysis showed that the receptor tyrosine kinase Axl is upregulated as well as activated in human hepatocellular EMT which is reflected in a multitude of HCC patient samples. So far, very little is known about the implications of Axl in HCC development. In this project we aim to assess the functional impact of Axl in human EMT and HCC progression. Advanced cellular human tumor models such as 3-dimensional hepatosphere cultures are available to analyze the role of Axl in the migration and invasion of HCC cells. Loss- and gain-of-function studies will allow us to accurately accomplish these tasks and to investigate the relevance of Axl in cancer cell dissemination and metastatic colonization by xenografting in vivo. Moreover, we will tackle the issue on Axl signalling at the level of a putative receptor interaction with integrin components and the subsequent downstream signalling in EMT and HCC progression. In this experimental setting, we will make efforts to identify target genes downstream of Axl signalling in HCC that are crucially involved in the malignant progression of hepatocytes. A particular focus will further examine the importance of Axl in the chemosensitivity against novel and approved anticancer drugs. These studies will facilitate the determination of Axl function in EMT and HCC progression, which might be of particular relevance for the design of novel effective protocols in HCC therapy.

In hepatocellular carcinoma (HCC) progression, epithelial to mesenchymal (EMT) plays a crucial role in early steps of metastasis when cells lose cell-cell contacts and acquire increased motility to spread into surrounding or distant tissues. Transcriptome profiling and phospho-proteomics revealed upregulation and activation of the receptor tyrosine kinase Axl in EMT. The major goal of this project was to address the functional impact of Axl in hepatocellular EMT and HCC progression. Knockdown of Axl expression showed abrogation of invasive and transendothelial migratory abilities of EMT-transformed HCC cells in vitro and reduced metastatic colonization in vivo. Importantly, Axl knockdown severely impaired resistance to transforming growth factor (TGF)-?-mediated growth inhibition, which is a crucial regulatory event of EMT. Analysis of the Axl interactome revealed binding of Axl to 14-3-3zeta, which is essentially required for Axl-mediated cell invasion, transendothelial migration and resistance against TGF-?. Notably, Axl/14-3-3zeta signaling activated by Gas6 caused phosphorylation of Smad3 linker region (Smad3L) at Ser213, resulting in the upregulation of tumor-progressive TGF-? target genes such as PAI1, MMP9 and Snail as well as augmented TGF-?1 secretion in mesenchymal HCC cells. In HCC patients, high Axl expression or upregulation of both Axl and 14-3-3zeta showed strong phosphorylation of Smad3L, elevated vessel invasion and reduced overall survival, suggesting that Axl/14-3-3zeta signaling is central for HCC progression. Furthermore, analysis of blood samples from a large multicentric cohort of HCC patients and patients with chronic liver disease revealed that soluble Axl (sAxl), a cleavage product of the extracellular domain of Axl, represents an accurate biomarker of very early HCC as well as cirrhosis. Most notably, sAxl outperforms the diagnostic accuracy of the commonly used biomarker ?-fetoprotein. High sAxl levels correlated with decreased overall survival HCC patients and were not detected in patients with liver adenoma, cholangiocarcinoma, secondary hepatic malignancies and other types of carcinomas, suggesting that sAxl is a specific biomarker for routine clinical diagnosis of cirrhosis and HCC.