Hepatocellular carcinoma (HCC) represents 80% of the primary hepatic neoplasms. It is the sixth most frequent neoplasm, the fourth cause of cancer-related death, and 7% of registered malignancies. Sorafenib is the first line molecular targeted therapy for patients in advanced stage of HCC. The present study shows that Sorafenib exerts free radical scavenging properties associated with the downregulation of nuclear factor E2-related factor 2 (Nrf2)-regulated thioredoxin 1 (Trx1) expression in liver cancer cells. The experimental downregulation and/or overexpression strategies showed that Trx1 induced activation of nitric oxide synthase (NOS) type 3 (NOS3) and S-nitrosation (SNO) of CD95 receptor leading to an increase of caspase-8 activity and cell proliferation, as well as reduction of caspase-3 activity in liver cancer cells. In addition, Sorafenib transiently increased mRNA expression and activity of S-nitrosoglutathione reductase (GSNOR) in HepG2 cells. Different experimental models of hepatocarcinogenesis based on the subcutaneous implantation of HepG2 cells in nude mice, as well as the induction of HCC by diethylnitrosamine (DEN) confirmed the relevance of Trx1 downregulation during the proapoptotic and antiproliferative properties induced by Sorafenib. In conclusion, the induction of apoptosis and antiproliferative properties by Sorafenib were related to Trx1 downregulation that appeared to play a relevant role on SNO of NOS3 and CD95 in HepG2 cells. The transient increase of GSNOR might also participate in the deactivation of CD95-dependent proliferative signaling in liver cancer cells.

Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL/TNFSF10) and Fas Ligand (FasL/TNFSF6), two major cytokines of the TNF (Tumor Necrosis Factor) superfamily, exert their main functions from the immune system compartment. Mice model studies revealed that TRAIL and FasL-mediated signalling both control the homeostasis of the immune cells, mainly from the lymphoid lineage, and function on cytotoxic cells as effector proteins to eliminate the compromised cells. The first clues in the physiological functions of TRAIL arose from the analysis of TRAIL deficient mice, which, even though they are viable and fertile, are prone to cancer and autoimmune diseases development, revealing TRAIL as an important safeguard against autoimmunity and cancer. The naturally occurring gld (generalized lymphoproliferative disease) and lpr (lymphoproliferation) mutant mice develop lymphadenopathy and lupus-like autoimmune disease. The discovery that they are mutated in the fasl and the fas receptor gene, respectively, demonstrates the critical role of the FasL/Fas system in lymphocyte homeostasis and autoimmunity. This review summarizes the state of current knowledge regarding the key death and non-death immune functions that TRAIL and FasL play in the initiation and progression of cancer and autoimmune diseases.

Finely tuned regulation of epithelial cell death maintains tissue integrity and homeostasis. At the cellular level, life and death decisions are controlled by environmental stimuli such as the activation of death receptors. We show that cell polarity and adherens junction formation prevent proapoptotic signals emanating from the Fas death receptor. Fas is sequestered in E-cadherin actin-based adhesion structures that are less able to induce downstream apoptosis signaling. Using a proteomic-based approach, we find that the polarity molecule Dlg1 interacts with the C-terminal PDZ-binding site in Fas and that this interaction decreases formation of the death-inducing complex upon engagement with Fas ligand (FasL), thus acting as an additional cell death protection mechanism. We propose that E-cadherin and Dlg1 inhibit FasL-induced cell death by two complementary but partially independent mechanisms that help to maintain epithelial homeostasis by protecting normal polarized epithelia from apoptosis. When polarity is lost, the Fas-cadherin-Dlg1 antiapoptotic complex is disrupted, and FasL can promote the elimination of compromised nonpolarized cells.

Sorafenib is the first line molecular targeted therapy for patients in advanced hepatocarcinoma stage. The aim of the study was the identification of the relationship between redox and antitumoral properties of Sorafenib in liver cancer cells. Sorafenib induces mitochondrial depolarization and superoxide anion production. However, this effect was counteracted by relevant drug-related scavenging activity, determined using fluorescent and EPR approaches, which were associated with a reduced Nrf2-dependent Thioredoxin 1 (Trx1) signaling. The downregulation of Trx1 was associated with NOS3-denitrosylation, low nitric oxide generation and reduction of caspase-8 activity in HepG2. The reduction of S-nitrosylation of Cys199 and Cys304 residues in CD95 was related to low caspase-8 activation in Sorafenib-treated HepG2 cells. Different overexpression and interfering in vitro designs showed that Trx1 regulated S-transnitrosylation between NOS3 and CD95, caspase-8 activation, EGFR tyrosine phosphorylation and cell proliferation. The two in vivo models, thioacetamide-induced HCC and the subcutaneous HepG2-implantation in nude mice, showed that the antitumoral properties of Sorafenib were related to reduced Trx1 expression, CD95 S-nitrosylation and caspase-8 activity in tumors.

The Fas/FasL system plays a critical role in death by apoptosis and immune escape of cancer cells. The Fas receptor being ubiquitously expressed in tissues, its apoptotic-inducing function, initiated upon FasL binding, is tightly regulated by several negative regulatory mechanisms to prevent inappropriate cell death. One of them, involving the non-receptor tyrosine kinase Btk, was reported mainly in B cells and only poorly described. We report here that Btk negatively regulates, through its tyrosine kinase activity, the FasL-mediated cell death in epithelial cell lines from colon cancer origin. More importantly, we show that Btk interacts not only with Fas but also with the phosphatidylinositol-4-phosphate 5-kinase, PIP5K1γ, which, upon stimulation by Fas ligand, is responsible of a rapid and transient synthesis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). This production requires both the presence and the tyrosine kinase activity of Btk, and participates in the negative regulation of FasL-mediated cell death since knocking down PIP5K1γ expression significantly strengthens the apoptotic signal upon FasL engagement. Altogether, our data demonstrate the cooperative role of Btk and PIP5K1γ in a FasL-induced PI(4,5)P2 production, both proteins participating to the threshold setting of FasL-induced apoptotic commitment in colorectal cell lines.

S-acylation is the covalent addition of a fatty acid, most generally palmitate onto cysteine residues of proteins through a labile thioester linkage. The death receptor CD95 is S-palmitoylated and this post-translational modification plays a crucial role on CD95 organization in cellular membranes and thus on CD95-mediated signaling. Here, we describe the nonradioactive detection of CD95 S-acylation by acyl-biotin exchange chemistry in which a biotin is substituted for the CD95-linked fatty acid. This sensitive technique, which depends on the ability of hydroxylamine to specifically cleave the thioester linkage between fatty acids and proteins, relies on three chemical steps: (1) blockage of free thiols of non-modified cysteine residues, (2) hydroxylamine-mediated cleavage of thioester-linked fatty acids to restore free thiols and (3) biotinylation of free thiols with a thiol reactive biotinylation agent. Resulting biotinylated proteins can be easily purified by an avidin capture and analyzed by SDS-PAGE and immunoblotting.

Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors.

The death receptor Fas undergoes a variety of post-translational modifications including S-palmitoylation. This protein acylation has been reported essential for an optimal cell death signaling by allowing both a proper Fas localization in cholesterol and sphingolipid-enriched membrane nanodomains, as well as Fas high-molecular weight complexes. In human, S-palmitoylation is controlled by 23 members of the DHHC family through their palmitoyl acyltransferase activity. In order to better understand the role of this post-translational modification in the regulation of the Fas-mediated apoptosis pathway, we performed a screen that allowed the identification of DHHC7 as a Fas-palmitoylating enzyme. Indeed, modifying DHHC7 expression by specific silencing or overexpression, respectively, reduces or enhances Fas palmitoylation and DHHC7 co-immunoprecipitates with Fas. At a functional level, DHHC7-mediated palmitoylation of Fas allows a proper Fas expression level by preventing its degradation through the lysosomes. Indeed, the decrease of Fas expression obtained upon loss of Fas palmitoylation can be restored by inhibiting the lysosomal degradation pathway. We describe the modification of Fas by palmitoylation as a novel mechanism for the regulation of Fas expression through its ability to circumvent its degradation by lysosomal proteolysis.

Fas interaction at the plasma membrane with its lipid and protein environment plays a crucial role in the early steps of Fas signalling induced by Fas ligand binding. Particularly, Fas localisation in the raft nanodomains, ezrin-mediated interaction with the actin cytoskeleton and subsequent internalization are critical steps in Fas-mediated cell death. We identified a lysine-rich region (LRR) in the cytoplasmic, membrane-proximal region of Fas as a key determinant modulating these initial events. Through a genetic approach, we demonstrate that Fas LRR represents another signal additional to palmitoylation targeting Fas to the raft nanodomains, and modulates Fas interaction with the cytoskeleton.

Fas ligand (FasL) is a transmembrane protein that regulates cell death in Fas-bearing cells. FasL-mediated cell death is essential for immune system homeostasis and the elimination of viral or transformed cells. Because of its potent cytotoxic activity, FasL expression at the cell surface is tightly regulated, for example, via processing by ADAM10 and SPPL2a generating soluble FasL and the intracellular fragments APL (ADAM10-processed FasL form) and SPA (SPPL2a-processed APL). In this study, we report that FasL processing by ADAM10 counteracts Fas-mediated cell death and is strictly regulated by membrane localization, interactions and modifications of FasL. According to our observations, FasL processing occurs preferentially within cholesterol and sphingolipid-rich nanodomains (rafts) where efficient Fas–FasL contact occurs, Fas receptor and FasL interaction is also required for efficient FasL processing, and FasL palmitoylation, which occurs within its transmembrane domain, is critical for efficient FasL-mediated killing and FasL processing.

Growth factor (GF) deprivation and/or blocking of cognate signaling can induce apoptosis and is the basis of several cancer treatment paradigms. We observed that RXR agonists (rexinoids) induce apoptosis of tumor cells when GF support is abrogated. This "rexinoid apoptosis" involves activation of both iNOS and eNOS by RXR-PPARgamma and results in production of apoptogenic NO. IGF/EGF-induced IGF receptor 1-mediated MAP kinase blocks rexinoid apoptosis by RXR phosphorylation. Combining rexinoids with the MAPK inhibitor U0126 induced apoptosis in human cancer cells in vitro and ex vivo and blocked xenograft growth in vivo. Our results suggest a regulatory mechanism in which GF signaling antagonizes RXR-PPARgamma-mediated default apoptosis to sustain cell life.

S-palmitoylation is a lipid modification that regulates membrane-protein association and influences protein trafficking, stability or aggregation, thus playing an important role in protein signalling. We previously demonstrated that the palmitoylation of Fas, one of the DD (death domain)-containing members of the TNFR [TNF (tumour necrosis factor) receptor] superfamily, is essential for the redistribution of this receptor into lipid rafts, an obligatory step for the death signal transmission. Here we investigate the requirement of protein palmitoylation in the activities of other DD-containing death receptors. We show that DR4 is palmitoylated, whereas DR5 and TNFR1 are not. Furthermore, DR4 palmitoylation is required for its raft localization and its ability to oligomerize, two essential features in TRAIL (TNF-related apoptosis-inducing ligand)-induced death signal transmission.

UNC5H receptors (UNC5H1, UNC5H2, UNC5H3) are putative tumor suppressors whose expression is lost in numerous cancers. These receptors have been shown to belong to the so-called family of dependence receptors. Such receptors induce apoptosis when their ligand netrin-1 is absent, thus conferring a state of cellular dependence towards ligand presence. Along this line, these receptors may limit tumor progression because they induce the death of tumor cells that grow in settings of ligand unavailability. We show here that UNC5H receptors are localized to cholesterol-and sphingolipid-enriched membrane domains called lipid rafts. We then demonstrate that the lipid raft localization of UNC5H2 is required for the pro-apoptotic activity of unbound UNC5H2. We also propose that this lipid raft localization is probably mediated via the recruitment of adaptor protein(s) within the death domain of UNC5H2 but is not dependent on the post-translational modification by palmitoylation of UNC5H2 even though this palmitoylation is required for UNC5H2 pro-apoptotic activity. Moreover we show that the interaction of UNC5H2 with the downstream pro-apoptotic serine threonine kinase DAPk is dependent on both UNC5H2 lipid raft localization and palmitoylation. Thus, we propose that the UNC5H dependence receptors require lipid raft localization and palmitoylation to trigger apoptosis.

The protein Daxx promotes Fas-mediated cell death through activation of apoptosis signal-regulating kinase 1, leading to the activation of the MAPKs JNK and p38. Owing to the in utero lethality of daxx-deficient mice, the in vivo role of Daxx has been so far difficult to analyze. We have generated transgenic mice expressing a dominant-negative form of Daxx (Daxx-DN) in the T-cell lineage. We show that Daxx is recruited to the Fas receptor upon FasL engagement and that Daxx-DN expression protects activated T cells from Fas-induced cell death, by preventing the death-inducing signal complex to be properly formed. Normal lymphocyte development and homeostasis are nevertheless observed. Interestingly, we report that both in vitro and in vivo stimulation of Daxx-DN T-lymphocytes leads to increased proliferative T-cell responses. This increased proliferation is associated with a marked increase in tyrosine phosphorylation of LAT and ZAP70 as Daxx-DN favor their recruitment to the T-cell receptor (TCR) complex. These findings identify Daxx as a critical regulator of T-lymphocyte homeostasis by decreasing TCR-induced cell proliferation and by promoting Fas-mediated cell death.

Apart from PML-RARα acute promyelocytic leukemia all other acute myeloid leukemias (AML) are unresponsive to retinoid differentiation therapy. However, in our study we show that elevating the levels of cyclic AMP (cAMP) confers onto retinoid X receptor (RXR)-selective agonists (“rexinoids”) the ability to induce terminal granulocyte differentiation and apoptosis of all-trans retinoic acid-resistant and insensitive AML cell lines and patients’ AML blasts. Protein kinase A activation leads to co-repressor release from the RAR subunit of the RAR-RXR heterodimer, resulting in “de-subordination” of otherwise silent RXR, which acquires transcriptional competence in response to cognate ligands. Rexinoid-cAMP induction of endogenous RARβ is blunted in mouse embryo fibroblasts lacking RARs, but re-introduction of exogenous RARα re-establishes responsiveness, thus confirming that the RARα-RXR heterodimer is the rexinoid mediator. The apoptogenic effect of this treatment involves enhanced expression of the death receptor DR5 and its cognate ligand, the tumor necrosis factor-related apoptosis inducing ligand (TRAIL), both of which are known to induce apoptosis in a tumor cell-selective manner and lead to the activation of initiator caspases. Immunohistochemistry confirmed induction of TRAIL and DR5 in AML patient blasts cultured “ex vivo”. AML patients’ blasts responded to rexinoid-cAMP combination treatment with induction of maturation and apoptosis, independent of karyotype, immunophenotype, and FAB classification. Clonogenic assays revealed complete inhibition of blast clonogenicity in four out of five tested samples. Indeed, it is known that cAMP levels can be elevated also by treating cells with 3′, 5′-cAMP phosphodiesterase inhibitors (PDEi’s). These observation and the clinical availability of the corresponding drugs provide a rationale for initiating clinical studies addressing the efficacy of combinatorial PDEi-rexinoid therapy in AML patients. Together with our recent finding that a very promising class of epigenetic anti-tumor drugs operates through activation of TRAIL expression (Nat Med2005, 11(1):7784), the possibility to target both the ligand (TRAIL) by HDAC inhibitors and the cognate receptors (DR4, DR5) by the above described rexinoid crosstalk may represent a promising therapeutic option which might lead, despite the genetic, morphologic, and clinical variability of AML, to a novel therapeutic option for AML patients by inducing a tumor-selective death pathway.

Retinoic acid (RA), used as first-line therapy for acute promyelocytic leukemia (APL), exerts its antileukemic activity by inducing blast differentiation and activating tumor-selective TNF-related apoptosis-inducing ligand (TRAIL) signaling. To identify downstream mediators of RA signaling, we used retrovirus-mediated insertion mutagenesis in PLB985 leukemia cells and established the RA-resistant cell line WY-1. In PLB985, but not WY-1 cells, RA induced TRAIL and its DR4 and DR5 receptors. Knocking down TRAIL expression by RNA interference blocked RA-induced apoptosis. WY-1 cells are defective for RA-induced differentiation, G1 arrest and exhibit co-resistance to TRAIL. In WY-1 cells, a single virus copy is integrated into a novel RA-regulated gene termed RAM (retinoic acid modulator). RAM is expressed in the myelomonocytic lineage and extinguished by RA in PLB985, but not WY-1 cells. Whereas knocking down RAM expression by RNA interference promoted RA-induced differentiation and TRAIL-triggered apoptosis of PLB985 and WY-1 cells, overexpression of the predicted 109 amino-acid RAM open reading frame did not alter RA signaling in PLB985 cells. This indicates that, apart from encoding the putative RAM protein, RAM RNA may exert additional functions that are impaired by the retrovirus insertion. Our study demonstrates that RA induction of the TRAIL pathway is also operative in leukemia cells lacking an RARα oncofusion protein and identifies RAM as a novel RA-dependent modulator of myeloid differentiation and death.