Primary and Secondary micro-RNA Modulation the Extrinsic Pathway of Apoptosis in Hepatocellular Carcinoma.

Abstract-One of the most common malignant liver diseases is hepatocellular carcinoma, which has a high recurrence rate and a low five-year survival rate. It is very heterogeneous both in structure and between patients, which complicates the diagnosis, prognosis and response to treatment. In this regard, an individualized, patient-centered approach becomes important, in which the use of mimetics and hsa-miRNA inhibitors involved in the pathogenesis of the disease may be determinative. From this point of view hsa-miRNAs are of interest, their aberrant expression is associated with poor prognosis for patients and is associated with tumor progression due to dysregulation of programmed cell death (apoptosis). However, the effect of hsa-miRNA on tumor development depends not only on its direct effect on expression of genes, the primary targets, but also on secondary targets mediated by regulatory pathways. While the former are actively studied, the role of secondary targets of these hsa-miRNAs in modulating apoptosis is still unclear. The present work summarizes data on hsa-miRNAs whose primary targets are key genes of the extrinsic pathway of apoptosis. Their aberrant expression is associated with early disease relapse and poor patient outcome. For these hsa-miRNAs, using the software package ANDSystem, we reconstructed the regulation of the expression of secondary targets and analyzed their impact on the activity of the extrinsic pathway of apoptosis. The potential effect of hsa-miRNAs mediated by action on secondary targets is shown to negatively correlate with the number of primary targets. It is also shown that hsa-miR-373, hsa-miR-106b and hsa-miR-96 have the highest priority as markers of hepatocellular carcinoma, whose action on secondary targets enhances their anti-apoptotic effect.

[Primary and Secondary micro-RNA Modulation the Extrinsic Pathway of Apoptosis in Hepatocellular Carcinoma].

One of the most common malignant liver diseases is hepatocellular carcinoma, which has a high recurrence rate and a low five-year survival rate. It is very heterogeneous both in structure and between patients, which complicates the diagnosis, prognosis and response to treatment. In this regard, an individualized, patient-centered approach becomes important, in which the use of mimetics and hsa-miRNA inhibitors involved in the pathogenesis of the disease may be determinative. From this point of view hsa-miRNAs are of interest, their aberrant expression is associated with poor prognosis for patients and is associated with tumor progression due to dysregulation of programmed cell death (apoptosis). However, the effect of hsa-miRNA on tumor development depends not only on its direct effect on expression of genes, the primary targets, but also on secondary targets mediated by regulatory pathways. While the former are actively studied, the role of secondary targets of these hsa-miRNAs in modulating apoptosis is still unclear. The present work summarizes data on hsa-miRNAs whose primary targets are key genes of the extrinsic pathway of apoptosis. Their aberrant expression is associated with early disease relapse and poor patient outcome. For these hsa-miRNAs, using the software package ANDSystem, we reconstructed the regulation of the expression of secondary targets and analyzed their impact on the activity of the extrinsic pathway of apoptosis. The potential effect of hsa-miRNAs mediated by action on secondary targets is shown to negatively correlate with the number of primary targets. It is also shown that hsa-miR-373, hsa-miR-106b and hsa-miR-96 have the highest priority as markers of hepatocellular carcinoma, whose action on secondary targets enhances their anti-apoptotic effect.

Prioritization of potential pharmacological targets for the development of anti-hepatocarcinoma drugs modulating the extrinsic apoptosis pathway: the reconstruction and analysis of associative gene networks help.

Hepatocellular carcinoma (HCC) is a common severe type of liver cancer characterized by an extremely aggressive course and low survival rates. It is known that disruptions in the regulation of apoptosis activation are some of the key features inherent in most cancer cells, which determines the pharmacological induction of apoptosis as an important strategy for cancer therapy. The computer design of chemical compounds capable of specifically regulating the external signaling pathway of apoptosis induction represents a promising approach for creating new effective ways of therapy for liver cancer and other oncological diseases. However, at present, most of the studies are devoted to pharmacological effects on the internal (mitochondrial) apoptosis pathway. In contrast, the external pathway induced via cell death receptors remains out of focus. Aberrant gene methylation, along with hepatitis C virus (HCV) infection, are important risk factors for the development of hepatocellular carcinoma. The reconstruction of gene networks describing the molecular mechanisms of interaction of aberrantly methylated genes with key participants of the extrinsic apoptosis pathway and their regulation by HCV proteins can provide important information when searching for pharmacological targets. In the present study, 13 criteria were proposed for prioritizing potential pharmacological targets for developing anti-hepatocarcinoma drugs modulating the extrinsic apoptosis pathway. The criteria are based on indicators of the structural and functional organization of reconstructed gene networks of hepatocarcinoma, the extrinsic apoptosis pathway, and regulatory pathways of virus-extrinsic apoptosis pathway interaction and aberrant gene methylation-extrinsic apoptosis pathway interaction using ANDSystem. The list of the top 100 gene targets ranked according to the prioritization rating was statistically significantly (p-value = 0.0002) enriched for known pharmacological targets approved by the FDA, indicating the correctness of the prioritization method. Among the promising potential pharmacological targets, six highly ranked genes (JUN, IL10, STAT3, MYC, TLR4, and KHDRBS1) are likely to deserve close attention.

Computer analysis of regulation of hepatocarcinoma marker genes hypermethylated by HCV proteins.

Hepatitis C virus (HCV) is a risk factor that leads to hepatocellular carcinoma (HCC) development. Epigenetic changes are known to play an important role in the molecular genetic mechanisms of virus-induced oncogenesis. Aberrant DNA methylation is a mediator of epigenetic changes that are closely associated with the HCC pathogenesis and considered a biomarker for its early diagnosis. The ANDSystem software package was used to reconstruct and evaluate the statistical significance of the pathways HCV could potentially use to regulate 32 hypermethylated genes in HCC, including both oncosuppressor and protumorigenic ones identified by genome-wide analysis of DNA methylation. The reconstructed pathways included those affecting protein-protein interactions (PPI), gene expression, protein activity, stability, and transport regulations, the expression regulation pathways being statistically significant. It has been shown that 8 out of 10 HCV proteins were involved in these pathways, the HCV NS3 protein being implicated in the largest number of regulatory pathways. NS3 was associated with the regulation of 5 tumor-suppressor genes, which may be the evidence of its central role in HCC pathogenesis. Analysis of the reconstructed pathways has demonstrated that following the transcription factor inhibition caused by binding to viral proteins, the expression of a number of oncosuppressors (WT1, MGMT, SOCS1, P53) was suppressed, while the expression of others (RASF1, RUNX3, WIF1, DAPK1) was activated. Thus, the performed gene-network reconstruction has shown that HCV proteins can influence not only the methylation status of oncosuppressor genes, but also their transcriptional regulation. The results obtained can be used in the search for pharmacological targets to develop new drugs against HCV-induced HCC.

Mathematical Modeling Reveals the Importance of the DED Filament Composition in the Effects of Small Molecules Targeting Caspase-8/c-FLIPL Heterodimer.

Procaspase-8 activation at the death-inducing signaling complex (DISC) triggers extrinsic apoptotic pathway. Procaspase-8 activation takes place in the death effector domain (DED) filaments and is regulated by c-FLIP proteins, in particular, by the long isoform c-FLIPL. Recently, the first-in-class chemical probe targeting the caspase-8/c-FLIPL heterodimer was reported. This rationally designed small molecule, FLIPin, enhances caspase-8 activity after initial heterodimer processing. Here, we used a kinetic mathematical model to gain an insight into the mechanisms of FLIPin action in a complex with DISC, in particular, to unravel the effects of FLIPin at different stoichiometry and composition of the DED filament. Analysis of this model has identified the optimal c-FLIPL to procaspase-8 ratios in different cellular landscapes favoring the activity of FLIPin. We predicted that the activity FLIPin is regulated via different mechanisms upon c-FLIPL downregulation or upregulation. Our study demonstrates that a combination of mathematical modeling with system pharmacology allows development of more efficient therapeutic approaches and prediction of optimal treatment strategies.

Isolation of High-Molecular-Weight Activation Complexes of Initiator Caspases in DNA Damage.

Initiation of apoptosis by chemotherapeutic drugs is one of the most effective approaches to the treatment of cancers. Caspases, the main enzymes of apoptosis, undergo activation to initiate cell death. Activation of initiator caspases requires their binding to special protein complexes. For elucidation of the mechanisms of apoptosis, these complexes should be isolated. However, their purification is challenging because they are formed in the cell in negligible amounts and rapidly degrade. We have developed an effective way to isolate caspase activation complexes formed in tumor cells in response to DNA damage. The method is based on combination of gel filtration with immunoprecipitation. The first stage is aimed at the separation of the high-molecular-weight caspase activation complexes and their monomeric forms, which allows increasing the efficiency of isolation of complexes at the second stage.

[Mechanisms of Procaspase-8 Activation in the Extrinsic Programmed Cell Death Pathway].

Caspase-8 performs initiatory functions during the induction of apoptosis through the extrinsic pathway. Apoptosis is a type of programmed cell death that plays an important role in regulating embryogenesis and maintaining homeostasis in the tissue of an adult organism, as well as differentiating and removing damaged cells. Dysregulation of the apoptosis mechanisms is associated with the pathogenesis and progression of a number of oncological and neurodegenerative diseases. Caspase-8 (also called СAP4, FLICE, MACH, MCH5) is one of two members of the death effector domain (DED)-containing caspases. Despite the fact that the role of caspase-8 in apoptosis has been well known since the mid 1990s, we are only now beginning to understand the subtle mechanisms of its activation and regulation in response to the activation of death receptors (DRs). In particular, it was demonstrated that the activation of caspase-8 requires the formation of specific oligomeric structures, which are named DED filaments. In this review, the recent data on the mechanisms of activating initiator caspase-8 in DED filaments are considered that allow us to better understand the subtle mechanisms of the initiation of the programmed cell death.

[Caspase-2 as an Oncosupressor and Metabolism Regulator: What Life Will Bring over the Long Run?]

Programmed cell death is governed by a set of gene networks, which define a variety of distinct molecular mechanisms essential for the maintenance of multicellular organisms. The most studied modality of programmed cell death is known as apoptosis. Caspase-2, as a member of the family of the cysteine-dependent protease, demonstrates both proapoptotic and tumor suppressive functions. This protease plays an essential role in the maintenance of genomic stability and induces apoptotic cell death in response to geno-toxic stress. Here we discuss the molecular mechanisms of caspase-2 regulation and its physiological role as a tumor suppressor and metabolic regulator.

[Nutrient restriction in combinatory therapy of tumors].

The main objective of anticancer treatment is the elimination of degenerated cells by the induction of programmed cell death. Various chemotherapy drugs and radiation are able to activate cell death mechanisms in tumors. However, unfortunately, monotherapy will always be insufficiently effective because of the variety and virulence of tumors, as well as their ability to develop resistance to drugs. Moreover, monotherapy might constrain many negative side effects. Therefore, the combination of different approaches and/or drugs will increase the efficiency of treatment. One such promising approach is the combination of nutrient restriction (NR) and various chemotherapeutic drugs. This approach may not only affect the autophagy but also influence apoptotic cell death. This review is focused on the potential of NR use in anticancer therapy, as well as the molecular mechanisms underlying this approach.

Mechanism of caspase-2 activation upon DNA damage.

The mechanism of caspase-2 activation in response to DNA damage was studied using human ovarian cancer cells Caov-4 treated with chemotherapeutic agent cisplatin. It was shown that mutations of the three cleavage sites of caspase-2 do not affect the assembly of the macromolecular complex of caspase-2 and its activation, but, conversely, stabilize this complex, most likely, via the inhibition of the dissociation of the active caspase-2.

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain.

The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

Systems biology of death receptor networks: live and let die.

The extrinsic apoptotic pathway is initiated by death receptor activation. Death receptor activation leads to the formation of death receptor signaling platforms, resulting in the demolition of the cell. Despite the fact that death receptor-mediated apoptosis has been studied to a high level of detail, its quantitative regulation until recently has been poorly understood. This situation has dramatically changed in the last years. Creation of mathematical models of death receptor signaling led to an enormous progress in the quantitative understanding of the network regulation and provided fascinating insights into the mechanisms of apoptosis control. In the following sections, the models of the death receptor signaling and their biological implications will be addressed. Central attention will be given to the models of CD95/Fas/APO-1, an exemplified member of the death receptor signaling pathways. The CD95 death-inducing signaling complex (DISC) and regulation of CD95 DISC activity by its key inhibitor c-FLIP, have been vigorously investigated by modeling approaches, and therefore will be the major topic here. Furthermore, the non-linear dynamics of the DISC, positive feedback loops and bistability as well as stoichiometric switches in extrinsic apoptosis will be discussed. Collectively, this review gives a comprehensive view how the mathematical modeling supported by quantitative experimental approaches has provided a new understanding of the death receptor signaling network.

[Caspase-2: what do we know today?].

Apoptosis (programmed cell death) is essential machinery for multicellular organisms. Apoptosis plays an important role in cell differentiation, damaged cell elimination and immune system homeostasis. This review is focused on various mechanisms of signal transduction through caspase-2 which believed to be one of the most enigmatical protease involved in apoptosis. Caspase-2 is activated upon stimulation by such agents as genotoxic stress, death receptors ligation, ER stress, metabolic changes, etc. In addition, caspase-2 may act as a tumor suppressor and has been implicated in cell response to oxidative stress and neurodegenerative progression during ischemic brain damage. Thus, variety of signal pathways triggered by caspase-2 place this protease apart from other members of the family and suggests a prominent role in apoptosis. Here, we analyse different functions of this unique caspase and discuss possible applications of accumulated knowledge in advanced oncology and medicine.

Regulation of CD95/Fas signaling at the DISC.

CD95 (APO-1/Fas) is a member of the death receptor (DR) family. Stimulation of CD95 leads to induction of apoptotic and non-apoptotic signaling pathways. The formation of the CD95 death-inducing signaling complex (DISC) is the initial step of CD95 signaling. Activation of procaspase-8 at the DISC leads to the induction of DR-mediated apoptosis. The activation of procaspase-8 is blocked by cellular FLICE-inhibitory proteins (c-FLIP). This review is focused on the role in the CD95-mediated signaling of the death effector domain-containing proteins procaspase-8 and c-FLIP. We discuss how dynamic cross-talk between procaspase-8 and c-FLIP at the DISC regulates life/death decisions at CD95.

A modular model of the apoptosis machinery.

UNLABELLED: Using a modular principle of computer hardware as a metaphor, we defined and implemented in the BioUML platform a module concept for biological pathways. BioUML provides a user interface to create modular models and convert them automatically into plain models for further simulations. Using this approach, we created the apoptosis model including 13 modules: death stimuli (TRAIL, CD95L, and TNF-α)-induced activation of caspase-8; survival stimuli (p53, EGF, and NF-κB) regulation; the mitochondria level; cytochrome C- and Smac-induced activation of caspase-3; direct activation of effector caspases by caspase-8 and - 12; PARP and apoptosis execution phase modules. Each module is based on earlier published models and extended by data from the Reactome and TRANSPATH databases. The model ability to simulate the apoptosis-related processes was checked; the modules were validated using experimental data. AVAILABILITY: http://www.biouml.org/apoptosis.shtml .

Caspase-8 activity has an essential role in CD95/Fas-mediated MAPK activation.

Stimulation of CD95/Fas/APO-1 results in the induction of both apoptotic and non-apoptotic signaling pathways. The processes regulating these two opposing pathways have not been thoroughly elucidated to date. In this study, using quantitative immunoblots, imaging, and mathematical modeling, we addressed the dynamics of the DED proteins of the death-inducing signaling complex (DISC), procaspase-8, and cellular FLICE inhibitory proteins (c-FLIPs) to the onset of CD95-mediated ERK1/2 and p38 mitogen-activated protein kinase (MAPK) activation. We found that CD95 DISC-induced caspase-8 activity is important for the initiation of ERK1/2 and p38 MAPK activation. The long c-FLIP isoform, c-FLIP(L), and the short c-FLIP isoform, c-FLIP(R), inhibited MAPK induction by blocking caspase-8 processing at the DISC. Furthermore, we built a mathematical model describing CD95 DISC-mediated MAPK activation and apoptosis. The model quantitatively defined the dynamics of DED proteins, procaspase-8, and c-FLIP, which lead to caspase-8 activation and induction of apoptotic and non-apoptotic signaling pathways. In conclusion, the combination of biochemical analysis with mathematical modeling provides evidence for an important role of caspase-8 in CD95-mediated activation of MAPKs, while c-FLIP exerts a regulatory function in this process.

[Regulation of death receptor-induced apoptosis induced via CD95/FAS and other death receptors].

Apoptosis (programmed cell death) is common to all multicellular organisms. Apoptosis can be triggered by the extrinsic (death receptor (DR)) or the intrinsic (mitochondrial) death pathways. Apoptosis plays the central role for the cell differentiation, removal of the damaged cells and the homeostasis of the immune system. CD95 (APO-1/Fas) is a member of the DR family, which was discovered more than 20 years ago. This review is focused on the mechanisms of DR-induced apoptosis focusing on CD95 (APO-1/Fas)-mediated apoptosis and the role of the anti-apoptotic protein c-FLIP in the extrinsic apoptosis. Regulation of apoptosis plays the central role in the immune system and apoptosis deregulation leads to a number of diseases. Gaining insights into these processes will improve our understanding of the pathogenesis of diseases such as cancer, autoimmunity and AIDS, and will open new approaches to rational treatment strategies.

The role of CAP3 in CD95 signaling: new insights into the mechanism of procaspase-8 activation.

Formation of the CD95 (APO-1/Fas) death inducing signaling complex (DISC) plays a central role in CD95 signaling. Previously, CD95 DISC composition was analyzed by two-dimensional gel electrophoresis and four major cytotoxicity-associated proteins (CAP1-4) were found. CAP1 and CAP2 were defined to be unmodified and phosphorylated FADD, respectively. CAP4 was identified as procaspase-8a. CAP3, however, has remained elusive. In this study, we demonstrate that CAP3 is an intermediate of procaspase-8 processing. CAP3 is generated within seconds of DISC formation and subsequently processed to the prodomain of procaspase-8a that is known as p26 (CAP5). These findings lead to new insights into the mechanism of procaspase-8 processing and apoptosis initiation.