Milica Stefanovic

Growth arrest–specific protein 6 is hepatoprotective against murine ischemia/reperfusion injury†

Growth arrest–specific gene 6 (GAS6) promotes growth and cell survival during tissue repair and development in different organs, including the liver. However, the specific role of GAS6 in liver ischemia/reperfusion (I/R) injury has not been previously addressed. Here we report an early increase in serum GAS6 levels after I/R exposure. Moreover, unlike wild‐type (WT) mice, Gas6−/− mice were highly sensitive to partial hepatic I/R, with 90% of the mice dying within 12 hours of reperfusion because of massive hepatocellular injury. I/R induced early hepatic protein kinase B (AKT) phosphorylation in WT mice but not in Gas6−/− mice without significant changes in c‐Jun N‐terminal kinase phosphorylation or nuclear factor kappa B translocation, whereas hepatic interleukin‐1β (IL‐1β) and tumor necrosis factor (TNF) messenger RNA levels were higher in Gas6−/− mice versus WT mice. In line with the in vivo data, in vitro studies indicated that GAS6 induced AKT phosphorylation in primary mouse hepatocytes and thus protected them from hypoxia‐induced cell death, whereas GAS6 diminished lipopolysaccharide‐induced cytokine expression (IL‐1β and TNF) in murine macrophages. Finally, recombinant GAS6 treatment in vivo not only rescued GAS6 knockout mice from severe I/R‐induced liver damage but also attenuated hepatic damage in WT mice after I/R. Conclusion: Our data have revealed GAS6 to be a new player in liver I/R injury that is emerging as a potential therapeutic target for reducing postischemic hepatic damage. (HEPATOLOGY 2010;)

Gas6/Axl pathway is activated in chronic liver disease and its targeting reduces fibrosis via hepatic stellate cell inactivation

BACKGROUND & AIMS Liver fibrosis, an important health concern associated to chronic liver injury that provides a permissive environment for cancer development, is characterized by accumulation of extracellular matrix components mainly derived from activated hepatic stellate cells (HSCs). Axl, a receptor tyrosine kinase and its ligand Gas6, are involved in cell differentiation, immune response and carcinogenesis. METHODS HSCs were obtained from WT and Axl(-/-) mice, treated with recombinant Gas6 protein (rGas6), Axl siRNAs or the Axl inhibitor BGB324, and analyzed by western blot and real-time PCR. Experimental fibrosis was studied in CCl4-treated WT and Axl(-/-) mice, and in combination with Axl inhibitor. Gas6 and Axl serum levels were measured in alcoholic liver disease (ALD) and hepatitis C virus (HCV) patients. RESULTS In primary mouse HSCs, Gas6 and Axl levels paralleled HSC activation. rGas6 phosphorylated Axl and AKT prior to HSC phenotypic changes, while Axl siRNA silencing reduced HSC activation. Moreover, BGB324 blocked Axl/AKT phosphorylation and diminished HSC activation. In addition, Axl(-/-) mice displayed decreased HSC activation in vitro and liver fibrogenesis after chronic damage by CCl4 administration. Similarly, BGB324 reduced collagen deposition and CCl4-induced liver fibrosis in mice. Importantly, Gas6 and Axl serum levels increased in ALD and HCV patients, inversely correlating with liver functionality. CONCLUSIONS The Gas6/Axl axis is required for full HSC activation. Gas6 and Axl serum levels increase in parallel to chronic liver disease progression. Axl targeting may be a therapeutic strategy for liver fibrosis management.

Targeting glucosylceramide synthase upregulation reverts sorafenib resistance in experimental hepatocellular carcinoma

Evasive mechanisms triggered by the tyrosine kinase inhibitor sorafenib reduce its efficacy in hepatocellular carcinoma (HCC) treatment. Drug-resistant cancer cells frequently exhibit sphingolipid dysregulation, reducing chemotherapeutic cytotoxicity via the induction of ceramide-degrading enzymes. However, the role of ceramide in sorafenib therapy and resistance in HCC has not been clearly established. Our data reveals that ceramide-modifying enzymes, particularly glucosylceramide synthase (GCS), are upregulated during sorafenib treatment in hepatoma cells (HepG2 and Hep3B), and more importantly, in sorafenib-resistant cell lines. GCS silencing or pharmacological GCS inhibition sensitized hepatoma cells to sorafenib exposure. GCS inhibition, combined with sorafenib, triggered cytochrome c release and ATP depletion in sorafenib-treated hepatoma cells, leading to mitochondrial cell death after energetic collapse. Conversely, genetic GCS overexpression increased sorafenib resistance. Of interest, GCS inhibition improved sorafenib effectiveness in a xenograft mouse model, recovering drug sensitivity of sorafenib-resistant tumors in mice. In conclusion, our results reveal GCS induction as a mechanism of sorafenib resistance, suggesting that GCS targeting may be a novel strategy to increase sorafenib efficacy in HCC management, and point to target the mitochondria as the subcellular location where sorafenib therapy could be potentiated.

Angiogenin Secretion From Hepatoma Cells Activates Hepatic Stellate Cells To Amplify A Self-Sustained Cycle Promoting Liver Cancer

Hepatocellular carcinoma (HCC) frequently develops in a pro-inflammatory and pro-fibrogenic environment with hepatic stellate cells (HSCs) remodeling the extracellular matrix composition. Molecules secreted by liver tumors contributing to HSC activation and peritumoral stromal transformation remain to be fully identified. Here we show that conditioned medium from HCC cell lines, Hep3B and HepG2, induced primary mouse HSCs transdifferentiation, characterized by profibrotic properties and collagen modification, with similar results seen in the human HSC cell line LX2. Moreover, tumor growth was enhanced by coinjection of HepG2/LX2 cells in a xenograft murine model, supporting a HCC-HSC crosstalk in liver tumor progression. Protein microarray secretome analyses revealed angiogenin as the most robust and selective protein released by HCC compared to LX2 secreted molecules. In fact, recombinant angiogenin induced in vitro HSC activation requiring its nuclear translocation and rRNA transcriptional stimulation. Moreover, angiogenin antagonism by blocking antibodies or angiogenin inhibitor neomycin decreased in vitro HSC activation by conditioned media or recombinant angiogenin. Finally, neomycin administration reduced tumor growth of HepG2-LX2 cells coinjected in mice. In conclusion, angiogenin secretion by HCCs favors tumor development by inducing HSC activation and ECM remodeling. These findings indicate that targeting angiogenin signaling may be of potential relevance in HCC management.

Orthoxenografts of Testicular Germ Cell Tumors Demonstrate Genomic Changes Associated with Cisplatin Resistance and Identify PDMP as a Resensitizing Agent

Purpose: To investigate the genetic basis of cisplatin resistance as efficacy of cisplatin-based chemotherapy in the treatment of distinct malignancies is often hampered by intrinsic or acquired drug resistance of tumor cells. Experimental Design: We produced 14 orthoxenograft transplanting human nonseminomatous testicular germ cell tumors (TGCT) in mice, keeping the primary tumor features in terms of genotype, phenotype, and sensitivity to cisplatin. Chromosomal and genetic alterations were evaluated in matched cisplatin-sensitive and their counterpart orthoxenografts that developed resistance to cisplatin in nude mice. Results: Comparative genomic hybridization analyses of four matched orthoxenografts identified recurrent chromosomal rearrangements across cisplatin-resistant tumors in three of them, showing gains at 9q32-q33.1 region. We found a clinical correlation between the presence of 9q32-q33.1 gains in cisplatin-refractory patients and poorer overall survival (OS) in metastatic germ cell tumors. We studied the expression profile of the 60 genes located at that genomic region. POLE3 and AKNA were the only two genes deregulated in resistant tumors harboring the 9q32-q33.1 gain. Moreover, other four genes (GCS, ZNF883, CTR1, and FLJ31713) were deregulated in all five resistant tumors independently of the 9q32-q33.1 amplification. RT-PCRs in tumors and functional analyses in Caenorhabditis elegans (C. elegans) indicate that the influence of 9q32-q33.1 genes in cisplatin resistance can be driven by either up- or downregulation. We focused on glucosylceramide synthase (GCS) to demonstrate that the GCS inhibitor DL-threo-PDMP resensitizes cisplatin-resistant germline-derived orthoxenografts to cisplatin. Conclusions: Orthoxenografts can be used preclinically not only to test the efficiency of drugs but also to identify prognosis markers and gene alterations acting as drivers of the acquired cisplatin resistance. Clin Cancer Res; 24(15); 3755–66. ©2018 AACR.

Antiapoptotic BCL-2 proteins determine sorafenib/regorafenib resistance and BH3-mimetic efficacy in hepatocellular carcinoma

Sorafenib, systemic treatment for advanced hepatocellular carcinoma (HCC), and regorafenib, novel second line treatment after sorafenib failure, have efficacy limited by evasive mechanisms of acquired-drug resistance. BCL-2 proteins participate in the response to tyrosine kinase inhibitors; however, their role in HCC therapy with sorafenib/regorafenib remains uncertain. BH3-mimetic ABT-263 (navitoclax) enhanced sorafenib activity, inducing cell death via a mitochondrial caspase-dependent mechanism, after BCL-xL/BCL-2 inhibition. Sorafenib-resistant hepatoma cells (HepG2R and Hep3BR) exhibited altered mRNA expression of BCL-2 and other anti-apoptotic family members, such as MCL-1, priming drug-resistant cancer cells to death by BH3-mimetics. ABT-263 restored sorafenib efficacy in sorafenib-resistant cell lines and HCC mouse models. Moreover, in mice xenografts from patient-derived BCLC9 cells, better tumor response to sorafenib was associated to higher changes in the BCL-2 mRNA pattern. HCC non-treated patients displayed altered BCL-2, MCL-1 and BCL-xL mRNA levels respect to adjacent non-tumoral biopsies and an increased BCL-2/MCL-1 ratio, predictive of navitoclax efficacy. Moreover, regorafenib administration also modified the BCL-2/MCL-1 ratio and navitoclax sensitized hepatoma cells to regorafenib by a mitochondrial caspase-dependent mechanism. In conclusion, sorafenib/regorafenib response is determined by BCL-2 proteins, while increased BCL-2/MCL-1 ratio in HCC sensitizes drug resistant-tumors against ABT-263 co-administration. Thus, changes in the BCL-2 profile, altered in HCC patients, could help to follow-up sorafenib efficacy, allowing patient selection for combined therapy with BH3-mimetics or early switch them to second line therapy.

Targeting Mitochondrial Function with the BCL-2 Inhibitor ABT-263 Increases Therapy Efficacy and Evades Sorafenib Resistance

Trabajo presentado en el International Liver Congress, celebrado en Barcelona, Espana, del 13 al 17 de abril de 2016

P0299 : Sorafenib effect on mitochondrial function provides a target for increasing HCC therapy efficacy

Trabajo presentado en el 50th International Liver Congress, celebrado en Viena, Austria, del 22 al 26 de abril de 2015

P0293 : Targeting glucosylceramide synthase increases sorafenib efficacy and evades sorafenib resistance in HCC models

Trabajo presentado en el 50th International Liver Congress, celebrado en Viena, Austria, del 22 al 26 de abril de 2015

P611 GAS6/AXL AXIS AS PROGNOSTIC AND THERAPEUTIC TARGET IN LIVER DISEASE

Background and Aims: AXL, a receptor tyrosin kinase, and its ligand GAS6 are related to processes leading to cell differentiation and carcinogenesis. The role of GAS6/AXL signaling in hepatic stellate cell (HSC) transformation during liver disease has been insufficiently addressed. Methods: HSCs were isolated from wild-type and AXL knockout (KO) mice. HSCs and LX2 human activated HSCs were exposed to recombinant GAS6, AXL inhibitor (BGB324, BerGenBio) or siRNA silencing. p-AXL, p-AKT and HSC transformation were evaluated by western blotting and qPCR. CCl4-induced fibrosis was analyzed in wild-type and AXL KO mice. Gas6 and soluble AXL (sAXL) levels were measured by ELISA in serum from alcoholic patients with normal liver, compensated cirrhosis and decompensated cirrhosis, and in control individuals (n = 10, each group). Results: Recombinant GAS6 stimulated AXL and AKT activation, inducing phenotypic changes in primary HSCs. HSCs from AXL KO mice displayed reduced capacity to be activated in vitro, and AXL inhibition (BGB324, Bergenbio) reduced HSC activation and proliferation. Similar results were obtained in LX2 cells and after RNA silencing of AXL. Chronic CCl4 administration increased serum levels of sAXL and GAS6, while AXL deficient mice exhibited reduced liver fibrosis. More importantly, AXL inhibition (BGB324) efficiently diminished CCl4-induced liver fibrosis in wild-type mice. In parallel, GAS6 and sAXL levels in serum were increased in alcoholic liver disease (ALD) patients, inversely correlating with liver functionality. Conclusions: AXL inhibitors have clinical interest since AXL targeting is effective to treat experimental liver fibrosis. In addition, GAS6 and sAXL levels could be proposed as early prognostic markers in liver disease.