Role of Axl Receptor Shedding in Liver Cancer

Hepatocellular carcinoma (HCC) is the most common subtype of liver cancer, which shows the second highest cancer-related mortality worldwide. Chronic liver diseases (CLDs) such as virally-induced hepatitis or non-alcoholic fatty liver disease/steatohepatitis are the major risk factors for HCC. Activation of the receptor tyrosine kinase Axl by binding of Gas6 to the extracellular domain is essentially involved in the progression of liver fibrosis and the development of HCC. Cleavage of the Axl ectodomain, resulting in soluble Axl (sAxl), provides a highly accurate biomarker for the diagnosis of advanced liver fibrosis/cirrhosis, and early to late HCC in the absence and presence of cirrhosis. These observations raise the question whether the shedding of sAxl either (i) leads to Gas6-independing Axl signalling, or (ii) does not affect Axl signalling by abundant expression of wild type Axl together with available free Gas6, or (iii) dampens a tumor-suppressive Axl signal. In this project, we will address the role of Axl receptor shedding as a driver of liver fibrosis and HCC development by using genetically engineered mouse and human liver models as well as CLD/HCC patient samples. Comprehensive gene expression analysis will be performed to identify Gas6- dependent and Gas6-independent Axl targets which will be further analyzed in gain- and loss-of-functions studies to determine their mechanistic impact on liver fibrosis and HCC. Furthermore, chemical compound screening will be employed to identify novel Axl treatment targets that can be exploited for the reversion of liver fibrosis and ablation of HCC. Altogether, this project will yield novel insights into Axl receptor shedding that could be used for innovative strategies to efficiently combat liver fibrosis and HCC .

Tumor-intrinsic and extrinsic Gas6/Axl functions are poorly understood in hepatocellular carcinoma (HCC). We addressed the role of Axl in transplantation-based mouse models and in diethylnitrosamine (DEN)-induced liver tumors of systemic and hepatocyte-specific Axl-deficient mice. Axl expression enhanced invasion of HCC cells by epithelial to mesenchymal transition (EMT), increased metastasis and reduced survival. The tumor burden was elevated in DEN-treated Axl-deficient mice with CCl4-induced inflammation, showing higher proliferation of neoplastic hepatocytes. Tumors were less infiltrated with CD8+ T cells and showed increased abundance of FoxP3+ T cells and M2-macrophages suggesting that Axl-deficient tumors exhibit an immunosuppressive environment. These data suggest that tumor-intrinsic Gas6/Axl signaling triggers the metastatic phenotype of HCC cells while Axl deficiency in the microenvironment escapes immune surveillance and promotes liver tumorigenesis. We further identified and functionally analyzed targets of Gas6/Axl signaling in HCC. Exploitation of well-characterized HCC models expressing Axl or devoid of Axl allowed the identification of target genes including PRAME (PReferentially expressed Antigen in MElanoma). Intervention with Gas6/Axl or MAPK/ERK1/2 signaling resulted in reduced PRAME expression. PRAME levels associated with EMT and augmented cell motility. Interactions with pro-oncogenic proteins such as CCAR1 suggested further tumor-promoting functions of PRAME in HCC. Moreover, PRAME showed elevated expression in Axl-stratified HCC patients correlating with vascular invasion and lowered patient survival. These data demonstrate that PRAME is a bona fide target of Gas6/Axl/ERK signaling linked to EMT and cancer cell invasion in HCC. A major hindrance of HCC therapy at advanced stages is the development of resistance to tyrosine kinase inhibitors (TKIs). We examined Gas6/Axl in acquiring refractoriness to TKIs in HCC which was highest in HCC cells exhibiting an EMT phenotype. Gas6/Axl expression was particularly upregulated in Regorafenib-resistant HCC cells together with the induction of ErbB receptors, whereas HCC cells lacking Axl failed to stimulate ErbBs. In line, treatment of Regorafenib-insensitive HCC cells with the ErbB inhibitor Afatinib strongly reduced cell viability. Together, these data suggest that the inhibition of ErbBs is synthetic lethal with Regorafenib in Axl-expressing HCC cells, revealing a novel vulnerability of HCC. Additionally, we evaluated the diagnostic value of soluble Axl (sAxl) and Gas6 and their albumin (alb) ratios in chronic liver disease including 1111 patients. Gas6/alb showed high diagnostic accuracy for the detection of significant (F2: AUC 0.805) to advanced fibrosis (F3: AUC 0.818), and was superior to the Fib-4 score for the detection of cirrhosis. Notably, Gas6 exceeded the levels of sAxl. These data emphasize the availability of high levels of free Gas6 in fibrosis and HCC which bind to uncleaved Axl receptors and activate Axl signaling.