Therapeutic approaches targeting CD95L/CD95 signaling in cancer and autoimmune diseases

Cell death plays a pivotal role in the maintenance of tissue homeostasis. Key players in the controlled induction of cell death are the Death Receptors (DR). CD95 is a prototypic DR activated by its cognate ligand CD95L triggering programmed cell death. As a consequence, alterations in the CD95/CD95L pathway have been involved in several disease conditions ranging from autoimmune diseases to inflammation and cancer. CD95L-induced cell death has multiple roles in the immune response since it constitutes one of the mechanisms by which cytotoxic lymphocytes kill their targets, but it is also involved in the process of turning off the immune response. Furthermore, beyond the canonical pro-death signals, CD95L, which can be membrane-bound or soluble, also induces non-apoptotic signaling that contributes to its tumor-promoting and pro-inflammatory roles. The intent of this review is to describe the role of CD95/CD95L in the pathophysiology of cancers, autoimmune diseases and chronic inflammation and to discuss recently patented and emerging therapeutic strategies that exploit/block the CD95/CD95L system in these diseases.Subject terms: Drug development, Apoptosis     

The CD95/CD95L signaling pathway: A role in carcinogenesis

Apoptosis is a fundamental process that contributes to tissue homeostasis, immune responses, and development. The receptor CD95, also called Fas, is a member of the tumor necrosis factor receptor (TNF-R) superfamily. Its cognate ligand, CD95L, is implicated in immune homeostasis and immune surveillance, and various lineages of malignant cells exhibit loss-of-function mutations in this pathway; therefore, CD95 was initially classified as a tumor suppressor gene. However, more recent data indicate that in different pathophysiological contexts, this receptor can transmit non-apoptotic signals, promote inflammation, and contribute to carcinogenesis. A comparison with the initial molecular events of the TNF-R signaling pathway leading to non-apoptotic, apoptotic, and necrotic pathways reveals that CD95 is probably using different molecular mechanisms to transmit its non-apoptotic signals (NF-κB, MAPK, and PI3K). As discussed in this review, the molecular process by which the receptor switches from an apoptotic function to an inflammatory role is unknown. More importantly, the biological functions of these signals remain elusive.     

Multiple interactions of the cytosolic polyproline region of the CD95 ligand: hints for the reverse signal transduction capacity of a death factor.

The CD95/Fas/Apo-1 ligand is expressed on activated lymphocytes, NK cells, platelets, certain immune-privileged cells and some tumor cells and induces apoptosis through the death receptor CD95/Fas/Apo-1. In murine T cells, membrane-bound CD95L (Fas ligand) also acts as a costimulatory receptor to coordinate activation and function in vivo. The molecular basis for this reverse signal transduction is yet unknown. In the present report, we identify individual interaction domains of enzymes and adapter molecules that selectively interact with full-length CD95L from transfectants and human T cells. These results may help to explain the costimulatory capacity of CD95L.     

Differential calcium response in HeLa and HeLa-Fas cells by cytotoxic T lymphocytes

We constructed a CD95 overexpressing HeLa cell line which was extremely sensitive towards CD95 mediated apoptosis. In these CD95 overexpressing cells, CD95 blocks the nuclear calcium signal induced by perforin positive and CD95 ligand positive killer cells. This phenomenon is highly relevant in states of inflammatory syndromes such as systemic inflammatory response syndrome (SIRS) and sepsis which are associated with a high probability to reactivate latent viruses due to a functional deficiency of cytotoxic effectors.     

Neutralization of CD95 ligand promotes regeneration and functional recovery after spinal cord injury

The clinical outcome of spinal cord injury (SCI) depends in part on the extent of secondary damage, to which apoptosis contributes. The CD95 and tumor necrosis factor (TNF) ligand/receptor systems play an essential role in various apoptotic mechanisms. To determine the involvement of these ligands in SCI-induced damage, we neutralized the activity of CD95 ligand (CD95L) and/or TNF in spinal cord-injured mice. Therapeutic neutralization of CD95L, but not of TNF, significantly decreased apoptotic cell death after SCI. Mice treated with CD95L-specific antibodies were capable of initiating active hind-limb movements several weeks after injury. The improvement in locomotor performance was mirrored by an increase in regenerating fibers and upregulation of growth-associated protein-43 (GAP-43). Thus, neutralization of CD95L promoted axonal regeneration and functional improvement in injured adult animals. This therapeutic strategy may constitute a potent future treatment for human spinal injury.     

The role of CD95 and CD95 ligand in cancer

CD95 (Fas/APO-1) and its ligand, CD95L, have long been viewed as a death receptor/death ligand system that mediates apoptosis induction to maintain immune homeostasis. In addition, these molecules are important in the immune elimination of virus-infected cells and cancer cells. CD95L was, therefore, considered to be useful for cancer therapy. However, major side effects have precluded its systemic use. During the last 10 years, it has been recognized that CD95 and CD95L have multiple cancer-relevant nonapoptotic and tumor-promoting activities. CD95 and CD95L were discovered to be critical survival factors for cancer cells, and were found to protect and promote cancer stem cells. We now discuss five different ways in which inhibiting or eliminating CD95L, rather than augmenting, may be beneficial for cancer therapy alone or in combination with standard chemotherapy or immune therapy.     

Diva/Boo is a negative regulator of cell death in human glioma cells

Diva is a novel proapoptotic member of the Bcl-2 protein family which binds apoptosis activating factor-1 (APAF-1). Diva is identical with Boo which was identified as a novel antiapoptotic Bcl-2 family protein. Here, we report that Diva promotes the cell cycle exit of human glioma cells in response to serum deprivation and inhibits apoptosis of these cells induced by CD95 ligand or chemotherapeutic drugs. In glioma cells, Diva interferes with apoptotic signaling downstream of cytochrome c release, but upstream of caspase activation, consistent with an inhibitory effect on the mitochondrial amplification step involving the apoptosome and APAF-1.     

CCR3 expression induced by IL-2 and IL-4 functioning as a death receptor for B cells

We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (approximately 98% CCR3(+)). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19(+) (approximately 40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.     

CD95 signaling in colorectal cancer

CD95 and its ligand (CD95L) are widely expressed in colorectal tumors, but their role in shaping tumor behavior is unclear. CD95 activation on tumor cells can lead to apoptosis, while CD95L attracts neutrophils, suggesting a function in tumor suppression. However, CD95 can also promote tumorigenesis, at least in part by activating non-apoptotic signaling pathways that stimulate tumor cell proliferation, invasion and survival. In addition, CD95 signaling in stromal cells and tumor-infiltrating inflammatory cells has to be taken into account when addressing the function of CD95 and its ligand in colorectal tumor biology. We present a model in which the tumor-suppressing and tumor-promoting activities of CD95/CD95L together determine colorectal tumor behavior. We also discuss how these multiple activities are changing our view of CD95 and CD95L as potential therapeutic targets in the treatment of colorectal cancer. We conclude that locking CD95 in apoptosis-mode may be a more promising anti-cancer strategy than simply inhibiting or stimulating CD95.     

CD14 and apoptosis

In addition to its role as a mediator of innate pro-inflammatory responses following bacterial lipopolysaccharide (LPS) binding, the 55kDa glycosyl-phosphatidylinositol-linked macrophage plasma membrane glycoprotein CD14 is now also known to play a role in phagocytic clearance of apoptotic cells. Although apoptotic cell-associated ligand(s) for CD14 await definition, initial findings suggest that ligand binding occurs close to, or at the same site as, LPS binding. Significantly, in contrast to LPS clearance and in keeping with the non-phlogistic nature of apoptosis, CD14-dependent engulfment of apoptotic cells fails to elicit pro-inflammatory cytokine release from macrophages. Therefore CD14 may be regarded as an innate immune receptor both for microbial products—after binding which activates inflammatory responses—and for self components, which either fail to induce, or alternatively actively suppress, inflammatory responses. Here we review current knowledge of the structure and functions of CD14, its ligands, its possible modes of signal transduction and its place in the panoply of macrophage molecules implicated in apoptotic-cell clearance.     

CD40 ligand (CD40L) does not stimulate proliferation of vascular smooth muscle cells

The present study investigates the effects of CD40 ligand (CD40L) on mitogenic signalling, proliferation, and migration of cultured bovine coronary artery smooth muscle cells (SMC). A time- and concentration-dependent phosphorylation of the extracellular signal-regulated kinases-1/2 (ERK-1/2) and the mitogen-activated protein kinase p38 (p38-MAPK) was observed upon stimulation with soluble CD40L (sCD40L). This phosphorylation was inhibited by neutralizing antibodies against the CD40 and CD40L, respectively. Activation of the phosphatidylinositol-3-phosphate (PI-3) kinase pathway by sCD40L, as determined by the measurement of Akt phosphorylation, was not detected. However, there was evidence for direct activation of the NFkappaB system (degradation of IkappaBalpha and nuclear translocation of the p65 NFkappaB subunit) by sCD40L. Accordingly, sCD40L caused a small but significant increase in DNA synthesis. However, sCD40L-induced DNA synthesis was not followed by proliferation (increase in cell number). Furthermore, sCD40L did not potentiate SMC mitogenesis induced by known mitogens such as platelet-derived growth factor-BB, thrombin or serum. The lack of cell proliferation was not caused by a concomitant induction of SMC apoptosis by sCD40L. The possible role of membrane-bound CD40L in SMC mitogenesis was also studied using different membrane preparations (platelets, lymphocytes). However, no mitogenic effects of membrane-bound CD40L were detected. Finally, sCD40L did not induce SMC migration. From these data it is concluded that CD40L activates mitogenic signalling and DNA synthesis but does not contribute to proliferation or migration of vascular SMC.     

Interleukin 1β-converting Enzyme Related Proteases/Caspases Are Involved in TRAIL-induced Apoptosis of Myeloma and Leukemia Cells

The Fas/APO-1/CD95 ligand (CD95L) and the recently cloned TRAIL ligand belong to the TNFfamily and share the ability to induce apoptosis in sensitive target cells. Little information is available on the degree of functional redundancy between these two ligands in terms of target selectivity and intracellular signalling pathway(s). To address these issues, we have expressed and characterized recombinant mouse TRAIL. Specific detection with newly developed rabbit anti-TRAIL antibodies showed that the functional TRAIL molecule released into the supernatant of recombinant baculovirus-infected Sf9 cells is very similar to that associated with the membrane fraction of Sf9 cells. CD95L resistant myeloma cells were found to be sensitive to TRAIL, displaying apoptotic features similar to those of the CD95L- and TRAIL-sensitive T leukemia cells Jurkat. To assess if IL-1β-converting enzyme (ICE) and/or ICE-related proteases (IRPs) (caspases) are involved in TRAIL-induced apoptosis of both cell types, peptide inhibition experiments were performed. The irreversible IRP/caspase-inhibitor AcYVAD-cmk and the reversible IRP/caspase-inhibitor Ac-DEVD-CHO blocked the morphological changes, disorganization of plasma membrane phospholipids, DNA fragmentation, and loss of cell viability associated with TRAIL-induced apoptosis. In addition, cells undergoing TRAIL-mediated apoptosis displayed cleavage of poly(ADP)-ribose polymerase (PARP) that was completely blocked by Ac-DEVD-CHO.

These results indicate that TRAIL seems to complement the activity of the CD95 system as it allows cells, otherwise resistant, to undergo apoptosis triggered by specific extracellular ligands. Conversely, however, induction of apoptosis in sensitive cells by TRAIL involves IRPs/caspases in a fashion similar to CD95L. Thus, differential sensitivity to CD95L and TRAIL seems to map to the proximal signaling events associated with receptor triggering.

     

CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells

The apoptosis-inducing death receptor CD95 (APO-1/Fas) controls the homeostasis of many tissues. Despite its apoptotic potential, most human tumors are refractory to the cytotoxic effects of CD95 ligand. We now show that CD95 stimulation of multiple apoptosis-resistant tumor cells by CD95 ligand induces increased motility and invasiveness, a response much less efficiently triggered by TNFalpha or TRAIL. Three signaling pathways resulting in activation of NF-kappaB, Erk1/2 and caspase-8 were found to be important to this novel activity of CD95. Gene chip analyses of a CD95-stimulated tumor cell line identified a number of potential survival genes and genes that are known to regulate increased motility and invasiveness of tumor cells to be induced. Among these genes, urokinase plasminogen activator was found to be required for the CD95 ligand-induced motility and invasiveness. Our data suggest that CD95L, which is found elevated in many human cancer patients, has tumorigenic activities on human cancer cells. This could become highly relevant during chemotherapy, which can cause upregulation of CD95 ligand by both tumor and nontumor cells.     

Rocaglamide breaks TRAIL resistance in HTLV-1-associated adult T-cell leukemia/lymphoma by translational suppression of c-FLIP expression

The human T-cell leukemia virus type-1 (HTLV-1)-associated adult T-cell leukemia/lymphoma (ATL) is incurable by currently known therapies. ATL samples and cell lines derived from ATL patients show restricted sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 ligand (CD95L). We have recently shown that HTLV-1-infected cells express elevated levels of cellular caspase-8 FLICE-inhibitory protein (c-FLIP) conferring resistance to receptor-mediated apoptosis. This finding underscores the demand to develop new strategies for treatment of ATL. In this study, we show that the naturally occurring herbal compound Rocaglamide (Roc) sensitizes CD95L- and TRAIL-induced apoptosis in HTLV-1-infected cells by downregulation of c-FLIP expression. Investigation of the molecular mechanism of Roc-mediated downregulation of c-FLIP revealed that it inhibits phosphorylation of the translation initiation factor 4E (eIF4E), a key factor that controls the rate-limiting step of translation, through inhibition of the MEK–ERK–MNK1 signaling pathway. This event prevents de novo synthesis of short-lived proteins such as c-FLIP in HTLV-1-infected cells. Our data suggest that Roc may serve as an adjuvant for TRAIL-based anticancer therapy.     

CD95 tyrosine phosphorylation is required for CD95 oligomerization

Proapoptotic stimuli, such as CD95 ligand and hydrophobic bile acids induce an epidermal growth factor receptor (EGFR)-catalyzed tyrosine phosphorylation of CD95-death receptor in hepatocytes, as a prerequisite for CD95-translocation to the plasma membrane, formation of the death-inducing signalling complex and execution of apoptotic cell death. However, the molecular role played by CD95 tyrosine phosphorylation remained unclear. The present study shows that CD95-tyrosine phosphorylation is required for CD95-oligomerization. Fluorescence resonance energy transfer (FRET)-analysis in Huh7 hepatoma cells, which were cotransfected with CD95-YFP/CD95-CFP revealed that stimulation of these cells with CD95 ligand, proapoptotic bile acids or hyperosmolarity resulted within 30 min in an intracellular FRET-signal, suggestive for CD95/CD95-oligomerization. After 120 min the FRET-signal was detected in the plasma membrane, indicating translocation of the CD95/CD95-oligomer to the plasma membrane. CD95/CD95-oligomerization was abolished in presence of AG1478 or a JNK-inhibitory peptide, i.e. maneuvers known to prevent EGFR-catalyzed CD95-tyrosine phosphorylation. Transfection studies with YFP/CFP-coupled CD95-mutants, which contain tyrosine/phenylalanine-exchanges in positions 232 and 291 (CD95^Y232,291F), revealed that at least one tyrosine (Y^232,291)-phosphorylated CD95 is required for CD95/CD95-oligomerization. FRET-studies in mouse embryonic fibroblasts, which in contrast to Huh7 express endogenous CD95, revealed that EGF, but not CD95L induced EGFR-homomerization, whereas CD95 ligand, but not EGF resulted in EGFR/CD95-heteromerization. These findings suggest that EGFR-catalyzed CD95-tyrosine phosphorylation is involved in the CD95/CD95-oligomerization process, which is induced by proapoptotic stimuli and is required for apoptosis induction.     

APRIL, a new member of the tumor necrosis factor family, modulates death ligand-induced apoptosis

APRIL (a proliferation-inducing ligand) is a newly identified member of the tumor necrosis factor (TNF) family. Tumor growth-promoting as well as apoptosis-inducing effects of APRIL have been described. Here, we report that five of 12 human malignant glioma cell lines express APRIL. APRIL gene transfer experiments revealed that malignant glioma cells are refractory to growth-promoting activity of APRIL in vitro and in vivo. Interestingly, ectopic expression of APRIL confers minor protection from apoptotic cell death induced by the death ligands, CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand (Apo2L). This antiapoptotic activity is specific for death ligand/receptor-mediated apoptosis since APRIL does not protect glioma cells from the cytotoxicity of the drugs, teniposide, vincristine, lomustine or cisplatin. Ectopic expression of APRIL is associated with the upregulation of X-linked inhibitor of apoptosis protein (XIAP), providing a possible explanation for the antiapoptotic activity observed here. In contrast, APRIL does not regulate the expression levels of the antiapoptotic proteins FLICE-inhibitory protein (FLIP), Bcl-2 or Bcl-X(L). These findings suggest that APRIL is involved in the regulation of death ligand-induced apoptotic signaling in malignant glioma cells.     

Hypoxia sensitizes human malignant glioma cells towards CD95L-induced cell death

Death ligands such as CD95 ligand (CD95L) have limited activity against glioma cells under normoxic conditions. Hypoxia is a critical aspect of the microenvironment of gliomas in vivo. We investigated the effect of co-exposure to acute hypoxia and CD95 ligand in three human malignant glioma cell lines with different susceptibility to CD95L under normoxic conditions. Hypoxia sensitized all three cell lines towards CD95L-induced cell death. Co-exposure resulted in apoptotic changes in the early phase, with gradual conversion to secondary necrosis with increasing length of hypoxia. The mitochondrial injury induced by hypoxia was enhanced by co-treatment, and caspase cleavage became prominent. Inhibition of the epidermal growth factor receptor (EGFR), although sensitizing glioma cells to CD95L under normoxia, protects glioma cells from hypoxia by reducing energy consumption. However, the opposing effects of EGFR signalling on death induced by CD95L or hypoxia were neutralized by co-exposure to hypoxia and CD95L. Furthermore, inhibition of protein synthesis by cycloheximide also reduced glucose consumption and conferred protection from hypoxia, but did not modulate CD95L-induced cell death under hypoxic conditions. These results suggest that death ligands may be useful to target hypoxic tumour cells resistant to conventional therapies or to complement strategies aiming at the induction of tumour hypoxia.     

Fas/Fas ligand interactions promote activation-induced cell death of NK T lymphocytes

NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demonstrate herein that in addition to their IFN-gamma production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, alpha-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism.