BI-3231, an enzymatic inhibitor of HSD17B13, reduces lipotoxic effects induced by palmitic acid in murine and human hepatocytes.

17-ß-hydroxysteroid dehydrogenase 13 (HSD17B13), a lipid droplet-associated enzyme, is primarily expressed in the liver and plays an important role in lipid metabolism. Targeted inhibition of enzymatic function is a potential therapeutic strategy for treating steatotic liver disease (SLD). The present study is aimed at investigating the effects of the first selective HSD17B13 inhibitor, BI-3231, in a model of hepatocellular lipotoxicity using human cell lines and primary mouse hepatocytes in vitro. Lipotoxicity was induced with palmitic acid in HepG2 cells and freshly isolated mouse hepatocytes and the cells were coincubated with BI-3231 to assess the protective effects. Under lipotoxic stress, triglyceride (TG) accumulation was significantly decreased in the BI-3231-treated cells compared with that of the control untreated human and mouse hepatocytes. In addition, treatment with BI-3231 led to considerable improvement in hepatocyte proliferation, cell differentiation, and lipid homeostasis. Mechanistically, BI-3231 increased the mitochondrial respiratory function without affecting ß-oxidation. BI-3231 inhibited the lipotoxic effects of palmitic acid in hepatocytes, highlighting the potential of targeting HSD17B13 as a specific therapeutic approach in steatotic liver disease.NEW & NOTEWORTHY 17-ß-Hydroxysteroid dehydrogenase 13 (HSD17B13) is a lipid droplet protein primarily expressed in the liver hepatocytes. HSD17B13 is associated with the clinical outcome of chronic liver diseases and is therefore a target for the development of drugs. Here, we demonstrate the promising therapeutic effect of BI-3231 as a potent inhibitor of HSD17B13 based on its ability to inhibit triglyceride accumulation in lipid droplets (LDs), restore lipid metabolism and homeostasis, and increase mitochondrial activity in vitro.

The PB1 and the ZZ domain of the autophagy receptor p62/SQSTM1 regulate the interaction of p62/SQSTM1 with the autophagosome protein LC3B.

Autophagy is a highly conserved cellular process that allows degradation of large macromolecules. p62/SQSTM1 is a key adaptor protein that interacts both with material to be degraded and with LC3 at the autophagosome, enabling degradation of cargos such as protein aggregates, lipid droplets and damaged organelles by selective autophagy. Dysregulation of autophagy contributes to the pathogenesis of many diseases. In this study, we investigated if the interaction of p62/SQSTM1 with LC3B could be regulated. We purified full-length p62/SQSTM1 and established an in vitro assay that measures the interaction with LC3B. We used the assay to determine the role of the different domains of p62/SQSTM1 in the interaction with LC3B. We identified a mechanism of regulation of p62/SQSTM1 where the ZZ and the PB1 domains regulate the exposure of the LIR-sequence to enable or inhibit the interaction with LC3B. A mutation to mimic the phosphorylation of a site on the ZZ domain leads to increased interaction with LC3B. Also, a small compound that binds to the ZZ domain enhances interaction with LC3B. Dysregulation of these mechanisms in p62/SQSTM1 could have implications for diseases where autophagy is affected. In conclusion, our study highlights the regulated nature of p62/SQSTM1 and its ability to modulate the interaction with LC3B through a LIR-sequence Accessibility Mechanism (LAM). Furthermore, our findings suggest the potential for pharmacological modulation of the exposure of LIR, paving the way for future therapeutic strategies.

Pathogenetic mechanisms and therapeutic approaches of acute-to-chronic liver failure.

Liver cirrhosis is the end stage of all chronic liver diseases and contributes significantly to overall mortality of 2% globally. The age-standardized mortality from liver cirrhosis in Europe is between 10 and 20% and can be explained by not only the development of liver cancer but also the acute deterioration in the patient's overall condition. The development of complications including accumulation of fluid in the abdomen (ascites), bleeding in the gastrointestinal tract (variceal bleeding), bacterial infections, or a decrease in brain function (hepatic encephalopathy) define an acute decompensation that requires therapy and often leads to acute-on-chronic liver failure (ACLF) by different precipitating events. However, due to its complexity and organ-spanning nature, the pathogenesis of ACLF is poorly understood, and the common underlying mechanisms leading to the development of organ dysfunction or failure in ACLF are still elusive. Apart from general intensive care interventions, there are no specific therapy options for ACLF. Liver transplantation is often not possible in these patients due to contraindications and a lack of prioritization. In this review, we describe the framework of the ACLF-I project consortium funded by the Hessian Ministry of Higher Education, Research and the Arts (HMWK) based on existing findings and will provide answers to these open questions.

Assessment of the Pharmacokinetics, Disposition, and Duration of Action of the Tumour-Targeting Peptide CEND-1.

CEND-1 (iRGD) is a bifunctional cyclic peptide that can modulate the solid tumour microenvironment, enhancing the delivery and therapeutic index of co-administered anti-cancer agents. This study explored CEND-1's pharmacokinetic (PK) properties pre-clinically and clinically, and assessed CEND-1 distribution, tumour selectivity and duration of action in pre-clinical tumour models. Its PK properties were assessed after intravenous infusion of CEND-1 at various doses in animals (mice, rats, dogs and monkeys) and patients with metastatic pancreatic cancer. To assess tissue disposition, [3H]-CEND-1 radioligand was administered intravenously to mice bearing orthotopic 4T1 mammary carcinoma, followed by tissue measurement using quantitative whole-body autoradiography or quantitative radioactivity analysis. The duration of the tumour-penetrating effect of CEND-1 was evaluated by assessing tumour accumulation of Evans blue and gadolinium-based contrast agents in hepatocellular carcinoma (HCC) mouse models. The plasma half-life was approximately 25 min in mice and 2 h in patients following intravenous administration of CEND-1. [3H]-CEND-1 localised to the tumour and several healthy tissues shortly after administration but was cleared from most healthy tissues by 3 h. Despite the rapid systemic clearance, tumours retained significant [3H]-CEND-1 several hours post-administration. In mice with HCC, the tumour penetration activity remained elevated for at least 24 h after the injection of a single dose of CEND-1. These results indicate a favourable in vivo PK profile of CEND-1 and a specific and sustained tumour homing and tumour penetrability. Taken together, these data suggest that even single injections of CEND-1 may elicit long-lasting tumour PK improvements for co-administered anti-cancer agents.

Adeno-associated virus serotype 2 capsid variants for improved liver-directed gene therapy.

BACKGROUND AND AIMS: Current liver-directed gene therapies look for adeno-associated virus (AAV) vectors with improved efficacy. With this background, capsid engineering is explored. Whereas shuffled capsid library screenings have resulted in potent liver targeting variants with one first vector in human clinical trials, modifying natural serotypes by peptide insertion has so far been less successful. Here, we now report on two capsid variants, MLIV.K and MLIV.A, both derived from a high-throughput in vivo AAV peptide display selection screen in mice. APPROACH AND RESULTS: The variants transduce primary murine and human hepatocytes at comparable efficiencies, a valuable feature in clinical development, and show significantly improved liver transduction efficacy, thereby allowing a dose reduction, and outperform parental AAV2 and AAV8 in targeting human hepatocytes in humanized mice. The natural heparan sulfate proteoglycan binding ability is markedly reduced, a feature that correlates with improved hepatocyte transduction. A further property that might contribute to the improved transduction efficacy is the lower capsid melting temperature. Peptide insertion also caused a moderate change in sensitivity to human sera containing anti-AAV2 neutralizing antibodies, revealing the impact of epitopes located at the basis of the AAV capsid protrusions. CONCLUSIONS: In conclusion, MLIV.K and MLIV.A are AAV peptide display variants selected in immunocompetent mice with improved hepatocyte tropism and transduction efficiency. Because these features are maintained across species, MLIV variants provide remarkable potential for translation of therapeutic approaches from mice to men.

Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors.

Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current-or develop novel-strategies for treating HCC.

Time-dependent changes in proliferation, DNA damage and clock gene expression in hepatocellular carcinoma and healthy liver of a transgenic mouse model.

Hepatocellular carcinoma (HCC) is highly resistant to anticancer therapy and novel therapeutic strategies are needed. Chronotherapy may become a promising approach because it may improve the efficacy of antimitotic radiation and chemotherapy by considering timing of treatment. To date little is known about time-of-day dependent changes of proliferation and DNA damage in HCC. Using transgenic c-myc/transforming growth factor (TGFα) mice as HCC animal model, we immunohistochemically demonstrated Ki67 as marker for proliferation and γ-H2AX as marker for DNA damage in HCC and surrounding healthy liver (HL). Core clock genes (Per1, Per2, Cry1, Cry2, Bmal 1, Rev-erbα and Clock) were examined by qPCR. Data were obtained from samples collected ex vivo at four different time points and from organotypic slice cultures (OSC). Significant differences were found between HCC and HL. In HCC, the number of Ki67 immunoreactive cells showed two peaks (ex vivo: ZT06 middle of day and ZT18 middle of night; OSC: CT04 and CT16). In ex vivo samples, the number of γ-H2AX positive cells in HCC peaked at ZT18 (middle of the night), while in OSC their number remained high during subjective day and night. In both HCC and HL, clock gene expression showed a time-of-day dependent expression ex vivo but no changes in OSC. The expression of Per2 and Cry1 was significantly lower in HCC than in HL. Our data support the concept of chronotherapy of HCC. OSC may become useful to test novel cancer therapies.

Tax1BP1 limits hepatic inflammation and reduces experimental hepatocarcinogenesis.

The nuclear factor kappa beta (NFκB) signaling pathway plays an important role in liver homeostasis and cancer development. Tax1-binding protein 1 (Tax1BP1) is a regulator of the NFκB signaling pathway, but its role in the liver and hepatocellular carcinoma (HCC) is presently unknown. Here we investigated the role of Tax1BP1 in liver cells and murine models of HCC and liver fibrosis. We applied the diethylnitrosamine (DEN) model of experimental hepatocarcinogenesis in Tax1BP1+/+ and Tax1BP1-/- mice. The amount and subsets of non-parenchymal liver cells in in Tax1BP1+/+ and Tax1BP1-/- mice were determined and activation of NFκB and stress induced signaling pathways were assessed. Differential expression of mRNA and miRNA was determined. Tax1BP1-/- mice showed increased numbers of inflammatory cells in the liver. Furthermore, a sustained activation of the NFκB signaling pathway was found in hepatocytes as well as increased transcription of proinflammatory cytokines in isolated Kupffer cells from Tax1BP1-/- mice. Several differentially expressed mRNAs and miRNAs in livers of Tax1BP1-/- mice were found, which are regulators of inflammation or are involved in cancer development or progression. Furthermore, Tax1BP1-/- mice developed more HCCs than their Tax1BP1+/+ littermates. We conclude that Tax1BP1 protects from liver cancer development by limiting proinflammatory signaling.

ACE2, the Receptor that Enables Infection by SARS-CoV-2: Biochemistry, Structure, Allostery and Evaluation of the Potential Development of ACE2 Modulators.

Angiotensin converting enzyme 2 (ACE2) is the human receptor that interacts with the spike protein of coronaviruses, including the one that produced the 2020 coronavirus pandemic (COVID-19). Thus, ACE2 is a potential target for drugs that disrupt the interaction of human cells with SARS-CoV-2 to abolish infection. There is also interest in drugs that inhibit or activate ACE2, that is, for cardiovascular disorders or colitis. Compounds binding at alternative sites could allosterically affect the interaction with the spike protein. Herein, we review biochemical, chemical biology, and structural information on ACE2, including the recent cryoEM structures of full-length ACE2. We conclude that ACE2 is very dynamic and that allosteric drugs could be developed to target ACE2. At the time of the 2020 pandemic, we suggest that available ACE2 inhibitors or activators in advanced development should be tested for their ability to allosterically displace the interaction between ACE2 and the spike protein.

TGF-ß2 silencing to target biliary-derived liver diseases.

OBJECTIVE: TGF-ß2 (TGF-ß, transforming growth factor beta), the less-investigated sibling of TGF-ß1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-ß2 in biliary-derived liver diseases. DESIGN: As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-ß2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels. RESULTS: TgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue. CONCLUSIONS: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-ß2 silencing and provide a direct rationale for TGF-ß2-directed drug development.

Theranostic Sorafenib-Loaded Polymeric Nanocarriers Manufactured by Enhanced Gadolinium Conjugation Techniques.

: Today, efficient delivery of sorafenib to hepatocellular carcinoma remains a challenge for current drug formulation strategies. Incorporating the lipophilic molecule into biocompatible and biodegradable theranostic nanocarriers has great potential for improving the efficacy and safety of cancer therapy. In the present study, three different technologies for the encapsulation of sorafenib into poly(d,l-lactide-co-glycolide) and polyethylene glycol-poly(d,l-lactide-co-glycolide) copolymers were compared. The particles ranged in size between 220 and 240 nm, with encapsulation efficiencies from 76.1 ± 1.7% to 69.1 ± 10.1%. A remarkable maximum drug load of approximately 9.0% was achieved. Finally, a gadolinium complex was covalently attached to the nanoparticle surface, transforming the nanospheres into theranostic devices, allowing their localization using magnetic resonance imaging. The manufacture of sorafenib-loaded nanoparticles alongside the functionalization of the particle surface with gadolinium complexes resulted in a highly efficacious nanodelivery system which exhibited a strong magnetic resonance imaging signal, optimal stability features, and a sustained release profile.

Stimulating brain recovery after stroke using theranostic albumin nanocarriers loaded with nerve growth factor in combination therapy.

For many years, delivering drug molecules across the blood brain barrier has been a major challenge. The neuropeptide nerve growth factor is involved in the regulation of growth and differentiation of cholinergic neurons and holds great potential in the treatment of stroke. However, as with many other compounds, the biomolecule is not able to enter the central nervous system. In the present study, nerve growth factor and ultra-small particles of iron oxide were co-encapsulated into a chemically crosslinked albumin nanocarrier matrix which was modified on the surface with apolipoprotein E. These biodegradable nanoparticles with a size of 212 ±â€¯1 nm exhibited monodisperse size distribution and low toxicity. They delivered NGF through an artificial blood brain barrier and were able to induce neurite outgrowth in PC12 cells in vitro. In an animal model of stroke, the infarct size was significantly reduced compared to the vehicle control. The combination therapy of NGF and the small-molecular MEK inhibitor U0126 showed a slight but not significant difference compared to U0126 alone. However, further in vivo evidence suggests that successful delivery of the neuropeptide is possible as well as the synergism between those two treatments.

Circulating hypoxia marker carbonic anhydrase IX (CA9) in patients with hepatocellular carcinoma and patients with cirrhosis.

BACKGROUND AND AIMS: Expression of carbonic anhydrase IX (CA9), an enzyme expressed in response to hypoxia, acidosis and oncogenic alterations, is reported to be a prognostic factor in HCC patients. Here we evaluated serum CA9 levels in HCC and cirrhosis patients. METHODS: HCC and cirrhosis patients were prospectively recruited and CA9 levels were determined. CA9 levels were compared to stages of cirrhosis and HCC stages. The association of the CA9 levels and overall survival (OS) was assessed. Furthermore, immunohistochemical CA9 expression in HCC and cirrhosis was evaluated. RESULTS: 215 patients with HCC were included. The median serum CA9 concentration in patients with HCC was 370 pg/ml and significantly higher than in a healthy cohort. Patients with advanced cancer stages (BCLC and ALBI score) had hid significant higher levels of CA9 in the serum. HCC patients with high serum CA9 concentrations (>400 pg/ml) had an increased mortality risk (hazard ratio (HR) 1.690, 95% confidence interval (CI) 1.017-2.809, P = 0.043). Serum CA9 concentration in cirrhotic patients did not differ significantly from HCC patients. Higher CA9 levels in cirrhotic patients correlated with portal hypertension and esophageal varices. Patients with ethanol induced cirrhosis had the highest CA9 levels in both cohorts. Levels of CA9 did not correlate with immunohistochemical expression. CONCLUSIONS: We conclude that a high CA9 level is a possible prognostic indicator for a poor outcome in HCC patients. The high CA9 levels are probably mainly associated with portal hypertension. Ductular reactions might be a possible source of serum CA9.

Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors.

BACKGROUND/AIMS: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. METHODS: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. RESULTS: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. CONCLUSION: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

Selective targeting of tumor associated macrophages in different tumor models.

Tumor progression largely depends on the presence of alternatively polarized (M2) tumor-associated macrophages (TAMs), whereas the classical M1-polarized macrophages can promote anti-tumorigenic immune responses. Thus, selective inhibition of M2-TAMs is a desirable anti-cancer approach in highly resistant tumor entities such as hepatocellular carcinoma (HCC) or breast cancer. We here examined whether a peptide that selectively binds to and is internalized by in vitro-differentiated murine M2 macrophages as compared to M1 macrophages, termed M2pep, could be used to selectively target TAMs in HCC and breast carcinoma. We confirmed selectivity of M2pep for in vitro M2 polarized macrophages. Upon incubation of suspended mixed 4T1 tumor cells with M2pep, high amounts of the TAMs were found to be associated with M2pep, whereas in mixed tumor cell suspensions from two HCC mouse models, M2pep showed only low-degree binding to TAMs. M2pep also showed low-degree targeting of liver macrophages. This indicates that the TAMs in different tumor entities show different targeting of M2pep and that M2pep is a very promising approach to develop selective M2-TAM-targeting in tumor entities containing M2-TAMs with significant amounts of the so far elusive M2pep receptor(s).

Hypoxia Causes Downregulation of Dicer in Hepatocellular Carcinoma, Which Is Required for Upregulation of Hypoxia-Inducible Factor 1α and Epithelial-Mesenchymal Transition.

Purpose: A role of Dicer, which converts precursor miRNAs to mature miRNAs, in the tumor-promoting effect of hypoxia is currently emerging in some tumor entities. Its role in hepatocellular carcinoma (HCC) is unknown.Experimental Design: HepG2 and Huh-7 cells were stably transfected with an inducible Dicer expression vector and were exposed to hypoxia/normoxia. HepG2-Dicer xenografts were established in nude mice; hypoxic areas and Dicer were detected in HCC xenografts and HCCs from mice with endogenous hepatocarcinogenesis; and epithelial-mesenchymal transition (EMT) markers were analyzed by immunohistochemistry or by immunoblotting. The correlation between Dicer and carbonic anhydrase 9 (CA9), a marker of hypoxia, was investigated in resected human HCCs.Results: Hypoxia increased EMT markers in vitro and in vivo and led to a downregulation of Dicer in HCC cells. The levels of Dicer were downregulated in hypoxic tumor regions in mice with endogenous hepatocarcinogenesis and in HepG2 xenografts. In human HCCs, the levels of Dicer correlated inversely with those of CA9, indicating that the negative regulation of Dicer by hypoxia also applies to HCC patients. Forced expression of Dicer prevented the hypoxia-induced increase in hypoxia-inducible factor 1α (HIF1α), HIF2α, hypoxia-inducible genes (CA9, glucose transporter 1), EMT markers, and cell migration.Conclusions: We here identify downmodulation of Dicer as novel essential process in hypoxia-induced EMT in HCC and demonstrate that induced expression of Dicer counteracted hypoxia-induced EMT. Thus, targeting hypoxia-induced downmodulation of Dicer is a promising novel strategy to reduce HCC progression. Clin Cancer Res; 23(14); 3896-905. ©2017 AACR.

An Allosteric Inhibitor Scaffold Targeting the PIF-Pocket of Atypical Protein Kinase C Isoforms.

There is a current and pressing need for improved cancer therapies. The use of small molecule kinase inhibitors and their application in combinatorial regimens represent an approach to personalized targeted cancer therapy. A number of AGC kinases, including atypical Protein Kinase C enzymes (PKCs), are validated drug targets for cancer treatment. Most drug development programs for protein kinases focus on the development of drugs that bind at the ATP-binding site. Alternatively, allosteric drugs have great potential for the development of future innovative drugs. However, the rational development of allosteric drugs poses important challenges because the compounds not only must bind to a given site but also must stabilize forms of the protein with a desired effect at a distant site. Here we describe the development of a new class of compounds targeting a regulatory site (PIF-pocket) present in the kinase domain and provide biochemical and crystallographic data showing that these compounds allosterically inhibit the activity of atypical PKCs. PS432, a representative compound, decreased the rate of proliferation of non-small cell lung cancer cells more potently than aurothiomalate, an atypical PKCι inhibitor currently under evaluation in clinical trials, and significantly reduced tumor growth without side effects in a mouse xenograft model. The druglike chemical class provides ample possibilities for the synthesis of derivative compounds, with the potential to allosterically modulate the activity of atypical PKCs and other kinases.

High levels of the soluble programmed death-ligand (sPD-L1) identify hepatocellular carcinoma patients with a poor prognosis.

AIM: Immunotherapy in cancer is a recent and very promising approach, namely the inhibition of the PD/programmed death-ligand 1 (PD-L1) axis. Here we aimed to investigate the prognostic value of a soluble form of PD-L1 in hepatocellular carcinoma (HCC) patients. METHODS: HCC patients were prospectively recruited and soluble programmed death-ligand 1 (sPD-L1) levels were determined. sPD-L1 levels were compared to stages of cirrhosis and HCC. The association of the sPD-L1 levels and overall survival (OS) was assessed. RESULTS: Two hundred fifteen patients with HCC were prospectively included. The median serum sPD-L1 concentration in patients with HCC was 0.5 ng/ml (range 0.03-6.04). Soluble PD-L1 levels positively correlated with the stage of cirrhosis and with stages of HCC. Furthermore, sPD-L1 correlated positively with a marker of macrophage activation (sCD163) and inflammation (C-reactive protein). The cut-off for high-level sPD-L1 (>0.8 ng/ml) was defined by sPD-L1 levels determined in a healthy control cohort. Patients with high serum sPD-L1 concentrations had an increased mortality risk (hazard ratio 3.340, 95 % confidence interval 1.609-6.934, P<0.001), while very low PD-L1 levels seem to come along with better prognosis. High sPD-L1 levels were associated with mortality independently from cirrhosis stage, alpha-fetoprotein and sCD163 levels in a multivariate Cox regression model. CONCLUSIONS: We conclude that a high sPD-L1 level is a possible prognostic indicator for a poor outcome in HCC patients. The predictive value of sPD-L1 levels for a successful anti-PD1/PD-L1 therapy should be investigated in the future.

Serum sphingolipidomic analyses reveal an upregulation of C16-ceramide and sphingosine-1-phosphate in hepatocellular carcinoma.

We have recently shown that major alterations of serum sphingolipid metabolites in chronic liver disease associate significantly with the stage of liver fibrosis in corresponding patients. In the current study we assessed via mass spectrometry serum concentrations of sphingolipid metabolites in a series of 122 patients with hepatocellular carcinoma (HCC) compared to an age- and sex-matched series of 127 patients with cirrhosis. We observed a highly significant upregulation of long and very long chain ceramides (C16-C24) in the serum of patients with HCC as compared to patients with cirrhosis (P < 0.001). Accordingly, dihydro-ceramides, synthetic precursors of ceramides and notably sphingosine, sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (SA1P) were upregulated in patients with HCC (P < 0.001). Especially the diagnostic accuracy of C16-ceramide and S1P, assessed by receiver operating curve (ROC) analysis, showed a higher area under the curve (AUC) value as compared to alpha fetoprotein (AFP) (0.999 and 0.985 versus 0.823, P < 0.001 respectively). In conclusion, serum levels of sphingolipid metabolites show a significant upregulation in patients with HCC as compared to patients with cirrhosis. Particularly C16-ceramide and S1P may serve as novel diagnostic markers for the identification of HCC in patients with liver diseases. Our data justify further investigations on the role of sphingolipids in HCC.