TGF-beta signaling in onset and progression of hepatocellular carcinoma

Transforming growth factor (TGF)-beta is a central regulator in chronic liver disease, contributing to all stages of disease progression from initial liver injury through inflammation and fibrosis to cirrhosis and hepatocellular carcinoma. Liver damage-induced levels of active TGF-beta enhance hepatocyte destruction and mediate hepatic stellate cell and fibroblast activation resulting in a wound-healing response, including myofibroblast generation and extracellular matrix deposition. Further evidence points to a decisive role of cytostatic and apoptotic functions mediated on hepatocytes, which is critical for the control of liver mass, with loss of TGF-beta activities resulting in hyperproliferative disorders and cancer. This concept is based on studies that describe a bipartite role of TGF-beta with tumor suppressor functions at early stages of liver damage and regeneration, whereas during cancer progression TGF-beta may turn from a tumor suppressor into a tumor promoter that exacerbates invasive and metastatic behavior. We have delineated this molecular switch of the pathway from cytostatic to tumor promoting in further detail and identify activation of survival signaling pathways in hepatocytes as a most critical requirement. Targeting the TGF-beta signaling pathway has been explored to inhibit liver disease progression. While interfering with TGF-beta signaling in various short-term animal models has demonstrated promising results, liver disease progression in humans is a process of decades with different phases in which TGF-beta or its targeting may have both beneficial and adverse outcomes. We emphasize that, in order to achieve therapeutic effects, targeting TGF-beta signaling in the right cell type at the right time is required. Copyright © 2012 S. Karger AG, Base

A multicenter open-label phase II trial to evaluate nivolumab and ipilimumab for 2nd line therapy in elderly patients with advanced esophageal squamous cell cancer (RAMONA)

Background: Advanced esophageal squamous cell cancer (ESCC) is frequently diagnosed in elderly patients. The impact of 2nd line chemotherapy is poorly defined. Recent data demonstrated effectiveness of checkpoint inhibitors in different squamous cell carcinomas. Therefore, we assess combined nivolumab/ipilimumab as 2nd line therapy in elderly ESCC patients. Methods: RAMONA is a multicenter open-label phase II trial. The primary objective is to demonstrate a significant survival benefit of nivolumab/ipilimumab in advanced ESCC compared to historical data of standard chemotherapy. Primary endpoint is therefore overall survival (OS). Major secondary objective is the evaluation of tolerability. Time to QoL deterioration will thus be determined as key secondary endpoint. Further secondary endpoints are tumor response, PFS and safety. We aim to recruit a total of n = 75 subjects that have to be > 65 years old. Eligibility is determined by the geriatric status (G8 screening and Deficit Accumulation Frailty Index (DAFI)). A safety assessment will be performed after a 3 cycle run-in phase of nivolumab (240 mg Q2W) to justify escalation for eligible patients to combined nivolumab (240 mg Q2W) and ipilimumab (1 mg/kg Q6W), while the other patients will remain on nivolumab only. RAMONA also includes translational research sub-studies to identify predictive biomarkers, including PD-1 and PD-L1 evaluation at different time points, establishment of organoid cultures and microbiome analyses for response prediction. Discussion: The RAMONA trial aims to implement checkpoint inhibitors for elderly patients with advanced ESCC as second line therapy. Novel biomarkers for checkpoint-inhibitor response are analyzed in extensive translational sub-studies. Trial registration: EudraCT Number 2017–002056-86; NCT03416244, registered: 31.1.2018

TGF-β1 and TGF-β2 abundance in liver diseases of mice and men

TGF-β1 is a major player in chronic liver diseases promoting fibrogenesis and tumorigenesis through various mechanisms. The expression and function of TGF-β2 have not been investigated thoroughly in liver disease to date. In this paper, we provide evidence that TGF-β2 expression correlates with fibrogenesis and liver cancer development. Using quantitative realtime PCR and ELISA, we show that TGF-β2 mRNA expression and secretion increased in murine HSCs and hepatocytes over time in culture and were found in the human-derived HSC cell line LX-2. TGF-β2 stimulation of the LX-2 cells led to upregulation of the TGF-β receptors 1, 2, and 3, whereas TGF-β1 treatment did not alter or decrease their expression. In liver regeneration and fibrosis upon CCl4 challenge, the transient increase of TGF-β2 expression was accompanied by TGF-β1 and collagen expression. In bile duct ligation-induced fibrosis, TGF-β2 upregulation correlated with fibrotic markers and was more prominent than TGF-β1 expression. Accordingly, MDR2-KO mice showed significant TGF-β2 upregulation within 3 to 15 months but minor TGF-β1 expression changes. In 5 of 8 hepatocellular carcinoma (HCC)/hepatoblastoma cell lines, relatively high TGF-β2 expression and secretion were observed, with some cell lines even secreting more TGF-β2 than TGF-β1. TGF-β2 was also upregulated in tumors of TGFα/cMyc and DEN-treated mice. The analysis of publically available microarray data of 13 human HCC collectives revealed considerable upregulation of TGF-β2 as compared to normal liver. Our study demonstrates upregulation of TGF-β2 in liver disease and suggests TGF-β2 as a promising therapeutic target for tackling fibrosis and HCC

Charge Pair Interactions in Transmembrane Helices and Turn Propensity of the Connecting Sequence Promote Helical Hairpin Insertion

α-Helical hairpins, consisting of a pair of closely spaced transmembrane (TM) helices that are connected by a short interfacial turn, are the simplest structural motifs found in multi-spanning membrane proteins. In naturally occurring hairpins, the presence of polar residues is common and predicted to complicate membrane insertion. We postulate that the pre-packing process offsets any energetic cost of allocating polar and charged residues within the hydrophobic environment of biological membranes. Consistent with this idea, we provide here experimental evidence demonstrating that helical hairpin insertion into biological membranes can be driven by electrostatic interactions between closely separated, poorly hydrophobic sequences. Additionally, we observe that the integral hairpin can be stabilized by a short loop heavily populated by turn-promoting residues. We conclude that the combined effect of TM¿TM electrostatic interactions and tight turns plays an important role in generating the functional architecture of membrane proteins and propose that helical hairpin motifs can be acquired within the context of the Sec61 translocon at the early stages of membrane protein biosynthesis. Taken together, these data further underline the potential complexities involved in accurately predicting TM domains from primary structures

MicroRNA-224 Targets SMAD Family Member 4 to Promote Cell Proliferation and Negatively Influence Patient Survival

10.1371/journal.pone.0068744PLoS ONE87-POLN

Asiatic Acid Inhibits Liver Fibrosis by Blocking TGF-beta/Smad Signaling In Vivo and In Vitro

Liver fibrosis is a major cause of liver failure, but treatment remains ineffective. In the present study, we investigated the mechanisms and anti-hepatofibrotic activities of asiatic acid (AA) in a rat model of liver fibrosis induced by carbon tetrachloride (CCl4) and in vitro in TGF-beta1-stimulated rat hepatic stellate cell line (HSC-T6). Treatment with AA significantly attenuated CCl4-induced liver fibrosis and functional impairment in a dosage-dependent manner, including blockade of the activation of HSC as determined by inhibiting de novo alpha smooth muscle actin (a-SMA) and collagen matrix expression, and an increase in ALT and AST (all p<0.01). The hepatoprotective effects of AA on fibrosis were associated with upregulation of hepatic Smad7, an inhibitor of TGF-beta signaling, thereby blocking upregulation of TGF-beta1 and CTGF and the activation of TGF-beta/Smad signaling. The anti-fibrosis activity and mechanisms of AA were further detected in vitro in HSC-T6. Addition of AA significantly induced Smad7 expression by HSC-T6 cells, thereby inhibiting TGF-beta1-induced Smad2/3 activation, myofibroblast transformation, and collagen matrix expression in a dosage-dependent manner. In contrast, knockdown of Smad7 in HSC-T6 cells prevented AA-induced inhibition of HSC-T6 cell activation and fibrosis in response to TGF-beta1, revealing an essential role for Smad7 in AA-induced anti-fibrotic activities during liver fibrosis in vivo and in vitro. In conclusion, AA may be a novel therapeutic agent for liver fibrosis. Induction of Smad7-dependent inhibition of TGF-beta/Smad-mediated fibrogenesis may be a central mechanism by which AA protects liver from injury

Liver Cancer-Derived Hepatitis C Virus Core Proteins Shift TGF-Beta Responses from Tumor Suppression to Epithelial-Mesenchymal Transition

International audienceBACKGROUND: Chronic hepatitis C virus (HCV) infection and associated liver cirrhosis represent a major risk factor for hepatocellular carcinoma (HCC) development. TGF-beta is an important driver of liver fibrogenesis and cancer; however, its actual impact in human cancer progression is still poorly known. The aim of this study was to investigate the role of HCC-derived HCV core natural variants on cancer progression through their impact on TGF-beta signaling. PRINCIPAL FINDINGS: We provide evidence that HCC-derived core protein expression in primary human or mouse hepatocyte alleviates TGF-beta responses in terms or growth inhibition or apoptosis. Instead, in these hepatocytes TGF-beta was still able to induce an epithelial to mesenchymal transition (EMT), a process that contributes to the promotion of cell invasion and metastasis. Moreover, we demonstrate that different thresholds of Smad3 activation dictate the TGF-beta responses in hepatic cells and that HCV core protein, by decreasing Smad3 activation, may switch TGF-beta growth inhibitory effects to tumor promoting responses. CONCLUSION/SIGNIFICANCE: Our data illustrate the capacity of hepatocytes to develop EMT and plasticity under TGF-beta, emphasize the role of HCV core protein in the dynamic of these effects and provide evidence for a paradigm whereby a viral protein implicated in oncogenesis is capable to shift TGF-beta responses from cytostatic effects to EMT development

Arginine in Membranes: The Connection Between Molecular Dynamics Simulations and Translocon-Mediated Insertion Experiments

Several laboratories have carried out molecular dynamics (MD) simulations of arginine interactions with lipid bilayers and found that the energetic cost of placing arginine in lipid bilayers is an order of magnitude greater than observed in molecular biology experiments in which Arg-containing transmembrane helices are inserted across the endoplasmic reticulum membrane by the Sec61 translocon. We attempt here to reconcile the results of the two approaches. We first present MD simulations of guanidinium groups alone in lipid bilayers, and then, to mimic the molecular biology experiments, we present simulations of hydrophobic helices containing single Arg residues at different positions along the helix. We discuss the simulation results in the context of molecular biology results and show that the energetic discrepancy is reduced, but not eliminated, by considering free energy differences between Arg at the interface and at the center of the model helices. The reduction occurs because Arg snorkeling to the interface prevents Arg from residing in the bilayer center where the energetic cost of desolvation is highest. We then show that the problem with MD simulations is that they measure water-to-bilayer free energies, whereas the molecular biology experiments measure the energetics of partitioning from translocon to bilayer, which raises the fundamental question of the relationship between water-to-bilayer and water-to-translocon partitioning. We present two thermodynamic scenarios as a foundation for reconciliation of the simulation and molecular biology results. The simplest scenario is that translocon-to-bilayer partitioning is independent of water-to-bilayer partitioning; there is no thermodynamic cycle connecting the two paths

Hepatic stellate cells:central modulators of hepatic carcinogenesis

Hepatocellular carcinoma (HCC) represents the second most common cause of cancer-related death worldwide, and is increasing in incidence. Currently, our therapeutic repertoire for the treatment of HCC is severely limited, and therefore effective new therapies are urgently required. Recently, there has been increasing interest focusing on the cellular and molecular interactions between cancer cells and their microenvironment. HCC represents a unique opportunity to study the relationship between a diseased stroma and promotion of carcinogenesis, as 90 % of HCCs arise in a cirrhotic liver. Hepatic stellate cells (HSC) are the major source of extracellular proteins during fibrogenesis, and may directly, or via secreted products, contribute to tumour initiation and progression. In this review we explore the complex cellular and molecular interplay between HSC biology and hepatocarcinogenesis. We focus on the molecular mechanisms by which HSC modulate HCC growth, immune cell evasion and angiogenesis. This is followed by a discussion of recent progress in the field in understanding the mechanistic crosstalk between HSC and HCC, and the pathways that are potentially amenable to therapeutic intervention. Furthermore, we summarise the exciting recent developments in strategies to target HSC specifically, and novel techniques to deliver pharmaceutical agents directly to HSC, potentially allowing tailored, cell-specific therapy for HCC