Tumor-Extrinsic Axl Expression Shapes an Inflammatory Microenvironment Independent of Tumor Cell Promoting Axl Signaling in Hepatocellular Carcinoma.

The activation of the receptor tyrosine kinase Axl by Gas6 is a major driver of tumorigenesis. Despite recent insights, tumor cell-intrinsic and -extrinsic Axl functions are poorly understood in hepatocellular carcinoma (HCC). Thus, we analyzed the cell-specific aspects of Axl in liver cancer cells and in the tumor microenvironment. We show that tumor-intrinsic Axl expression decreased the survival of mice and elevated the number of pulmonary metastases in a model of resection-based tumor recurrence. Axl expression increased the invasion of hepatospheres by the activation of Akt signaling and a partial epithelial-to-mesenchymal transition (EMT). However, the liver tumor burden of Axl+/+ mice induced by diethylnitrosamine plus carbon tetrachloride was reduced compared to systemic Axl-/- mice. Tumors of Axl+/+ mice were highly infiltrated with cytotoxic cells, suggesting a key immune-modulatory role of Axl. Interestingly, hepatocyte-specific Axl deficiency did not alter T cell infiltration, indicating that these changes are independent of tumor cell-intrinsic Axl. In this context, we observed an upregulation of multiple chemokines in Axl+/+ compared to Axl-/- tumors, correlating with HCC patient data. In line with this, Axl is associated with a cytotoxic immune signature in HCC patients. Together these data show that tumor-intrinsic Axl expression fosters progression, while tumor-extrinsic Axl expression shapes an inflammatory microenvironment.

Synergism of the receptor tyrosine kinase Axl with ErbB receptors mediates resistance to regorafenib in hepatocellular carcinoma.

Introduction: Hepatocellular carcinoma (HCC) patients at advanced stages receive immunotherapy or treatment with tyrosine kinase inhibitors (TKIs) such as Sorafenib (Sora) or Lenvatinib in frontline as well as Regorafenib (Rego) or Cabozantinib in second-line. A major hindrance of TKI therapies is the development of resistance, which renders drug treatment futile and results in HCC progression. Methods: In this study, we addressed the impact of the receptor tyrosine kinase Axl binding to its ligand Gas6 in acquiring refractoriness to TKIs. The initial responses of Axl-positive and Axl-negative cell lines to different TKIs were assessed. Upon inducing resistance, RNA-Seq, gain- and loss-of-function studies were applied to understand and intervene with the molecular basis of refractoriness. Secretome analysis was performed to identify potential biomarkers of resistance. Results: We show that HCC cells exhibiting a mesenchymal-like phenotype were less sensitive to drug treatment, linking TKI resistance to changes in epithelial plasticity. Gas6/Axl expression and activation were upregulated in Rego-resistant HCC cells together with the induction of ErbB receptors, whereas HCC cells lacking Axl failed to stimulate ErbBs. Treatment of Rego-insensitive HCC cells with the pan-ErbB family inhibitor Afatinib rather than with Erlotinib blocking ErbB1 reduced cell viability and clonogenicity. Genetic intervention with ErbB2-4 but not ErbB1 confirmed their crucial involvement in refractoriness to Rego. Furthermore, Rego-resistant HCC cells secreted basic fibroblast growth factor (bFGF) depending on Axl expression. HCC patients treated with Sora in first-line and with Rego in second-line displayed elevated serum levels of bFGF, emphasizing bFGF as a predictive biomarker of TKI treatment. Discussion: Together, these data suggest that the inhibition of ErbBs is synthetic lethal with Rego in Axl-expressing HCC cells, showing a novel vulnerability of HCC.

PRAME Is a Novel Target of Tumor-Intrinsic Gas6/Axl Activation and Promotes Cancer Cell Invasion in Hepatocellular Carcinoma.

(1) Background: Activation of the receptor tyrosine kinase Axl by Gas6 fosters oncogenic effects in hepatocellular carcinoma (HCC), associating with increased mortality of patients. The impact of Gas6/Axl signaling on the induction of individual target genes in HCC and its consequences is an open issue. (2) Methods: RNA-seq analysis of Gas6-stimulated Axl-proficient or Axl-deficient HCC cells was used to identify Gas6/Axl targets. Gain- and loss-of-function studies as well as proteomics were employed to characterize the role of PRAME (preferentially expressed antigen in melanoma). Expression of Axl/PRAME was assessed in publicly available HCC patient datasets and in 133 HCC cases. (3) Results: Exploitation of well-characterized HCC models expressing Axl or devoid of Axl allowed the identification of target genes including PRAME. Intervention with Axl signaling or MAPK/ERK1/2 resulted in reduced PRAME expression. PRAME levels were associated with a mesenchymal-like phenotype augmenting 2D cell migration and 3D cell invasion. 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, which correlates with vascular invasion and lowered patient survival. (4) Conclusions: PRAME is a bona fide target of Gas6/Axl/ERK signaling linked to EMT and cancer cell invasion in HCC.

The glucocorticoid receptor associates with RAS complexes to inhibit cell proliferation and tumor growth.

Mutations that activate members of the RAS family of GTPases are associated with various cancers and drive tumor growth. The glucocorticoid receptor (GR), a member of the nuclear receptor family, has been proposed to interact with and inhibit the activation of components of the PI3K-AKT and MAPK pathways downstream of RAS. In the absence of activating ligands, we found that GR was present in cytoplasmic KRAS-containing complexes and inhibited the activation of wild-type and oncogenic KRAS in mouse embryonic fibroblasts and human lung cancer A549 cells. The DNA binding domain of GR was involved in the interaction with KRAS, but GR-dependent inhibition of RAS activation did not depend on the nuclear translocation of GR. The addition of ligand released GR-dependent inhibition of RAS, AKT, the MAPK p38, and the MAPKK MEK. CRISPR-Cas9-mediated deletion of GR in A549 cells enhanced tumor growth in xenografts in mice. Patient samples of non-small cell lung carcinomas showed lower expression of NR3C1, the gene encoding GR, compared to adjacent normal tissues and lower NR3C1 expression correlated with a worse disease outcome. These results suggest that glucocorticoids prevent the ability of GR to limit tumor growth by inhibiting RAS activation, which has potential implications for the use of glucocorticoids in patients with cancer.

Intrinsic and Extrinsic Control of Hepatocellular Carcinoma by TAM Receptors.

Hepatocellular carcinoma (HCC) is the major subtype of liver cancer, showing high mortality of patients due to limited therapeutic options at advanced stages of disease. The receptor tyrosine kinases Tyro3, Axl and MerTK-belonging to the TAM family-exert a large impact on various aspects of cancer biology. Binding of the ligands Gas6 or Protein S activates TAM receptors causing homophilic dimerization and heterophilic interactions with other receptors to modulate effector functions. In this context, TAM receptors are major regulators of anti-inflammatory responses and vessel integrity, including platelet aggregation as well as resistance to chemotherapy. In this review, we discuss the relevance of TAM receptors in the intrinsic control of HCC progression by modulating epithelial cell plasticity and by promoting metastatic traits of neoplastic hepatocytes. Depending on different etiologies of HCC, we further describe the overt role of TAM receptors in the extrinsic control of HCC progression by focusing on immune cell infiltration and fibrogenesis. Additionally, we assess TAM receptor functions in the chemoresistance against clinically used tyrosine kinase inhibitors and immune checkpoint blockade in HCC progression. We finally address the question of whether inhibition of TAM receptors can be envisaged for novel therapeutic strategies in HCC.

Down-regulation of A20 promotes immune escape of lung adenocarcinomas.

Inflammation is a well-known driver of lung tumorigenesis. One strategy by which tumor cells escape tight homeostatic control is by decreasing the expression of the potent anti-inflammatory protein tumor necrosis factor alpha-induced protein 3 (TNFAIP3), also known as A20. We observed that tumor cell intrinsic loss of A20 markedly enhanced lung tumorigenesis and was associated with reduced CD8+ T cell-mediated immune surveillance in patients with lung cancer and in mouse models. In mice, we observed that this effect was completely dependent on increased cellular sensitivity to interferon-γ (IFN-γ) signaling by aberrant activation of TANK-binding kinase 1 (TBK1) and increased downstream expression and activation of signal transducer and activator of transcription 1 (STAT1). Interrupting this autocrine feed forward loop by knocking out IFN-α/ß receptor completely restored infiltration of cytotoxic T cells and rescued loss of A20 depending tumorigenesis. Downstream of STAT1, programmed death ligand 1 (PD-L1) was highly expressed in A20 knockout lung tumors. Accordingly, immune checkpoint blockade (ICB) treatment was highly efficient in mice harboring A20-deficient lung tumors. Furthermore, an A20 loss-of-function gene expression signature positively correlated with survival of melanoma patients treated with anti-programmed cell death protein 1. Together, we have identified A20 as a master immune checkpoint regulating the TBK1-STAT1-PD-L1 axis that may be exploited to improve ICB therapy in patients with lung adenocarcinoma.

JAK-STAT inhibition impairs K-RAS-driven lung adenocarcinoma progression.

Oncogenic K-RAS has been difficult to target and currently there is no K-RAS-based targeted therapy available for patients suffering from K-RAS-driven lung adenocarcinoma (AC). Alternatively, targeting K-RAS-downstream effectors, K-RAS-cooperating signaling pathways or cancer hallmarks, such as tumor-promoting inflammation, has been shown to be a promising therapeutic strategy. Since the JAK-STAT pathway is considered to be a central player in inflammation-mediated tumorigenesis, we investigated here the implication of JAK-STAT signaling and the therapeutic potential of JAK1/2 inhibition in K-RAS-driven lung AC. Our data showed that JAK1 and JAK2 are activated in human lung AC and that increased activation of JAK-STAT signaling correlated with disease progression and K-RAS activity in human lung AC. Accordingly, administration of the JAK1/2 selective tyrosine kinase inhibitor ruxolitinib reduced proliferation of tumor cells and effectively reduced tumor progression in immunodeficient and immunocompetent mouse models of K-RAS-driven lung AC. Notably, JAK1/2 inhibition led to the establishment of an antitumorigenic tumor microenvironment, characterized by decreased levels of tumor-promoting chemokines and cytokines and reduced numbers of infiltrating myeloid derived suppressor cells, thereby impairing tumor growth. Taken together, we identified JAK1/2 inhibition as promising therapy for K-RAS-driven lung AC.

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.