Expression of tumor necrosis factor-alpha-related apoptosis-inducing ligand and its receptors in rat testis during development

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor-alpha family of cytokines that is known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, the presence of TRAIL and its receptors was investigated in the rat testis during development. TRAIL and its receptors were immunolocalized to the different testicular cell types. TRAIL and its receptors were also identified in the rat testis in terms of protein and mRNA. Our immunohistochemical studies indicate that TRAIL, DR5/TRAIL-R2, and DcR2-TRAIL-R4 are detected in Leydig cells, whereas ligand and all receptors are localized in germ cells. TRAIL was permanently immunodetected in germ cells from the fetal stage to adulthood, whereas its receptors were immunolocalized exclusively in postmeiotic germ cells. The expression of TRAIL and receptor mRNAs was consistent with the immunodetection of TRAIL and receptor proteins. Indeed, TRAIL ligand mRNA was also identified in the rat testis from the fetal stage to adulthood. The mRNAs of the death receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2, were weakly detected during the perinatal period and increased from the pubertal stage to adulthood. The mRNAs of the decoy receptors, DcR1 and DcR2, were present in the rat testis at all ages studied, but the DcR2/TRAIL-R4 mRNa level was higher from the pubertal period to adulthood. Together, the present findings demonstrate that 1) TRAIL and its receptors are expressed in the testis during normal development, and 2) TRAIL protein is present in the different germ cell types, whereas its receptors were predominantly detected in the postmeiotic germ cells.     

Restricted expression of tumor necrosis factor-related apoptosis-inducing ligand receptor 4 in human peripheral blood lymphocytes

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor but not normal cells, thus providing therapeutic possibilities for human cancers. However, it is not fully clear how widespread TRAIL receptors are, or how TRAIL signaling is modulated in normal cells. We characterized cell surface expression of TRAIL receptors in normal healthy donor peripheral blood and report that each of the TRAIL receptors are characteristically expressed on restricted cell populations. TRAIL-R1 is distinctively expressed on B-lymphocytes, TRAIL-R2 on monocytes, TRAIL-R3 on neutrophils and most impressively, CD8+ lymphocytes and NKT lymphocytes but not CD4+ lymphocytes express TRAIL-R4.     

TRAIL pathway components and their putative role in granulosa cell apoptosis in the human ovary

Extensive apoptotic oocyte reduction occurs during fetal ovarian development. The regulatory pathways responsible for oocyte selection to programmed cell death are, however, poorly understood. The aim of this study was to investigate the potential involvement of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 and decoy receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in the apoptotic process characterizing human fetal and adult ovaries. For this purpose, in situ hybridization and immunohistochemistry were applied to human fetal and adult ovarian samples to study the mRNA and protein expression of TRAIL pathway components, and a human granulosa cell tumor-derived cell line (KGN) was used to elucidate functional effects of TRAIL on apoptosis. TRAIL was expressed in human fetal ovary from the 11th week until term. The pro-apoptotic TRAIL-R2/DR5 and the anti-apoptotic TRAIL-R4/DcR2 were also expressed in human ovaries throughout the fetal period. Among the different ovarian cell types, these TRAIL pathway components were mainly localized in the oocytes, and their expression increased towards term. Expression of TRAIL-R1/DR4 and TRAIL-R3/DcR1 was negligible in all of the fetal ovaries studied. Adult ovaries expressed TRAIL, TRAIL-R2/DR5, TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in granulosa cells and oocytes of small primary/secondary follicles as well as in granulosa and theca cells of more developed antral follicles. In KGN cells, TRAIL efficiently induced apoptosis in a dose-dependent manner, and this was blocked by a caspase inhibitor. The results indicate a role of the TRAIL pathway components in the regulation of granulosa cell apoptosis in in vitro and suggest that these factors may have a role in regulating ovarian apoptosis also in vivo.     

TNF-related apoptosis-inducing ligand: signalling of a 'smart' molecule

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor super-family and signals via two death receptors, TRAIL-R1 and TRAIL-R2, and two decoy receptors, TRAIL-R3 and TRAIL-R4, differently expressed in normal and cancer cells. TRAIL is mainly studied for its capacity to induce apoptosis preferentially in cancer cells. TRAIL is expressed in a variety of human tissues, in particular in the lymphoid system, suggesting a strong physiological role in the innate immunity. This review will focus on TRAIL gene structure and regulation, protein folding, tissue expression and molecular signalling. Finally, the potential use of TRAIL as anticancer treatment alone or in combination therapy as well as the use of drugs which signal via TRAIL and its receptors will be analyzed.     

Involvement of TRAIL/TRAIL-receptors in human intestinal cell differentiation

Despite the fact that tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptors (TRAIL-Rs) are expressed in intestinal mucosa, little is known about the biological role of this system in intestinal cell physiology. The expression of surface TRAIL and TRAIL-R1, -R2, -R3, -R4 were examined by flow cytometry in the immortalized human cell line tsFHI under culture conditions promoting growth or growth arrest and expression of differentiated traits. A progressive increase of surface TRAIL expression paralleled tsFHI differentiation, consistently with immunohistochemistry analysis showing an increase of TRAIL immunostaining along the crypt-villus axis in normal jejuneal mucosa. In spite of the presence of TRAIL-R1 and TRAIL-R2 "death receptors," recombinant TRAIL was not cytotoxic for tsFHI cells. Exposure of tsFHI to recombinant TRAIL rather increased/anticipated the expression levels of the cyclin-dependent kinase inhibitors p21 and p27, which mediate the induction of growth arrest and the stabilization of differentiated traits, respectively, as well as of the canonical differentiation marker DPPIV. The differentiation inducing activity of TRAIL was abolished by pre-incubation with a Fc-TRAIL-R2 chimera. On the other hand, TRAIL did not significantly modulate the levels of osteoprotegerin (OPG), CXCL8/IL-8, CXCL9/MIG, and CXCL10/IP10 spontaneously released or induced by inflammatory cytokines. Taken together, these data suggest that TRAIL might act as a paracrine trophic cytokine on intestinal epithelium, promoting intestinal cell differentiation.     

Differential localization and regulation of death and decoy receptors for TNF-related apoptosis-inducing ligand (TRAIL) in human melanoma cells

Induction of apoptosis in cells by TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF family, is believed to be regulated by expression of two death-inducing and two inhibitory (decoy) receptors on the cell surface. In previous studies we found no correlation between expression of decoy receptors and susceptibility of human melanoma cells to TRAIL-induced apoptosis. In view of this, we studied the localization of the receptors in melanoma cells by confocal microscopy to better understand their function. We show that the death receptors TRAIL-R1 and R2 are located in the trans-Golgi network, whereas the inhibitory receptors TRAIL-R3 and -R4 are located in the nucleus. After exposure to TRAIL, TRAIL-R1 and -R2 are internalized into endosomes, whereas TRAIL-R3 and -R4 undergo relocation from the nucleus to the cytoplasm and cell membranes. This movement of decoy receptors was dependent on signals from TRAIL-R1 and -R2, as shown by blocking experiments with Abs to TRAIL-R1 and -R2. The location of TRAIL-R1, -R3, and -R4 in melanoma cells transfected with cDNA for these receptors was similar to that in nontransfected cells. Transfection of TRAIL-R3 and -R4 increased resistance of the melanoma lines to TRAIL-induced apoptosis even in melanoma lines that naturally expressed these receptors. These results indicate that abnormalities in "decoy" receptor location or function may contribute to sensitivity of melanoma to TRAIL-induced apoptosis and suggest that further studies are needed on the functional significance of their nuclear location and TRAIL-induced movement within cells.     

Expression of TRAIL receptors in human autoreactive and foreign antigen-specific T cells

Deletion of T cells due to apoptosis induction is a regulatory mechanism in the human immune system that may be impaired in autoimmune diseases such as multiple sclerosis (MS). Involvement of the apoptosis-mediating CD95/CD95 ligand system in MS has been demonstrated. Here, we report that (auto)antigen-specific human T cells are not killed in vitro by soluble TNF-related apoptosis-inducing ligand (TRAIL) although expressing death-inducing receptors, TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2. Apoptosis was assessed by caspase activation and DNA fragmentation, receptor expression was detected by RT - PCR and flow cytometry. The (auto)antigen-specific T cells were also resistant to specific TRAIL-R1/TRAIL-R2-directed induction of apoptosis, indicating that coexpression of the truncated TRAIL-R3 and TRAIL-R4 in these T cells is not responsible for the observed resistance. Upon stimulation, levels of death-inducing TRAIL receptors decreased whereas TRAIL was up-regulated on the cell surface. In contrast to CD95, the role of TRAIL receptors in MS might not involve regulation of T cell vulnerability.     

TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2-Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family.     

Functional analysis of TRAIL receptors using monoclonal antibodies

mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.     

Chemotherapy overcomes TRAIL-R4-mediated TRAIL resistance at the DISC level

TNF-related apoptosis-inducing ligand or Apo2L (Apo2L/TRAIL) is a promising anti-cancer drug owing to its ability to trigger apoptosis by binding to TRAIL-R1 or TRAIL-R2, two membrane-bound receptors that are often expressed by tumor cells. TRAIL can also bind non-functional receptors such as TRAIL-R4, but controversies still exist regarding their potential to inhibit TRAIL-induced apoptosis. We show here that TRAIL-R4, expressed either endogenously or ectopically, inhibits TRAIL-induced apoptosis. Interestingly, the combination of chemotherapeutic drugs with TRAIL restores tumor cell sensitivity to apoptosis in TRAIL-R4-expressing cells. This sensitization, which mainly occurs at the death-inducing signaling complex (DISC) level, through enhanced caspase-8 recruitment and activation, is compromised by c-FLIP expression and is independent of the mitochondria. Importantly, TRAIL-R4 expression prevents TRAIL-induced tumor regression in nude mice, but tumor regression induced by TRAIL can be restored with chemotherapy. Our results clearly support a negative regulatory function for TRAIL-R4 in controlling TRAIL signaling, and unveil the ability of TRAIL-R4 to cooperate with c-FLIP to inhibit TRAIL-induced cell death.     

TRAIL death receptors as tumor suppressors and drug targets

Various ways of targeting TRAIL-death receptors for the treatment of a diverse set of malignancies are being explored in ongoing clinical trials. Recent data of ours and others suggest that loss of the only death signaling receptor in mice (TRAIL-R) is associated with susceptibility to various stages of lymphomagenesis and carcinogenesis, perhaps in a complex cell- and model-specific manner (1,2). Myc-overexpressing B cell lymphomas with an intact TRAIL-R locus displayed a number of gene expression changes indicating resistance to TRAIL-R signaling. Herein we show some data on the use of recombinant human TRAIL (rhTRAIL) and γ-radiation (10 Gy) in combination in an autochthonous mouse model for hepatocellular carcinoma. As cell death signaling through the death receptors is evolutionary conserved from zebra fish to man, novel genetically engineered mouse tumor models may prove useful in establishing in vivo models that excel our fundamental understanding of resistance to TRAIL-death receptor signaling, off-target effects from TRAIL-death receptor targeting compounds and help in identifying a clinically cogent rationale for efficient targeting of TRAIL death receptors in patients. Once established, mouse tumor models may prove to be a useful tool in understanding TRAIL-death receptor signaling.     

Effect of H. pylori on the expression of TRAIL, FasL and their receptor subtypes in human gastric epithelial cells and their role in apoptosis

BACKGROUND AND AIMS: In the human stomach expression of TNF-related apoptosis inducing ligand (TRAIL) and its receptors and the modulatory role of Helicobacter pylori are not well described. Therefore, we investigated the effect of H. pylori on the expression of TRAIL, FasL and their receptors (TRAIL-R1-R4, Fas) in gastric epithelial cells and examined their role in apoptosis. MATERIALS AND METHODS: mRNA and protein expression of TRAIL, FasL and their receptors were analyzed in human gastric epithelial cells using RT-PCR, Western blot, and immunohistochemistry. Gastric epithelial cells were incubated with FasL, TRAIL and/or H. pylori, and effects on expression, cell viability and epithelial apoptosis were monitored. Apoptosis was analyzed by histone ELISA, DAPI staining and immunohistochemistry. RESULTS: TRAIL, FasL and their receptor subtypes were expressed in human gastric mucosa, gastric epithelial cell primary cultures and gastric cancer cells. TRAIL, FasL and H. pylori caused a time- and concentration-dependent induction of DNA fragmentation in gastric cancer cells with synergistic effects. In addition, H. pylori caused a selective up-regulation of TRAIL, TRAIL-R1 and Fas mRNA and protein expression in gastric cancer cells. CONCLUSIONS: Next to FasL and Fas, TRAIL and all of its receptor subtypes are expressed in the human stomach and differentially modulated by H. pylori. TRAIL, FasL and H. pylori show complex interaction mediating apoptosis in human gastric epithelial cells. These findings might be important for the understanding of gastric epithelial cell kinetics in patients with H. pylori infection.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) decoy receptor TRAIL-R3 is up-regulated by p53 in breast tumor cells through a mechanism involving an intronic p53-binding site

Tumor necrosis factor-related apoptosis-inducing ligand receptor 3 (TRAIL-R3) is a decoy receptor for TRAIL, a member of the tumor necrosis factor family. In several cell types decoy receptors inhibit TRAIL-induced apoptosis by binding TRAIL and thus preventing its binding to proapoptotic TRAIL receptors. We studied the regulation of TRAIL-R3 gene expression in breast tumor cells treated with the genotoxic drug doxorubicin (DXR). The breast tumor cell line MCF-7 (p53 wild type) responded to DXR with a marked elevation of TRAIL-R3 expression at the mRNA, total protein, and cell surface levels. In contrast, in EVSA-T cells (p53 mutant) DXR did not induce increased expression of TRAIL-R3. In MCF-7 cells overexpressing the human papillomavirus protein E6, which causes p53 degradation, DXR-induced TRAIL-R3 expression was notably reduced. Furthermore, in MCF-7 cells overexpressing a temperature-sensitive p53 mutant (Val135), shifting the cultures to the permissive temperature was sufficient to induce the expression of TRAIL-R3. We also cloned and characterized a p53 consensus element located within the first intron of the human TRAIL-R3 gene. This element binds p53 and confers responsiveness to genotoxic damage to constructs of the TRAIL-R3 promoter in transient transfection experiments. Our results indicate that genotoxic treatments such as DXR, frequently used in cancer therapy, may also induce genes such as TRAIL-R3 that potentially have antiapoptotic actions and thus interfere with the TRAIL signaling system. This is particularly important in view of the proposed use of TRAIL in antitumor therapy.     

Functional expression of TRAIL and TRAIL-R2 during human megakaryocytic development

The expression and function of surface TRAIL and TRAIL receptors were investigated in primary megakaryocytic cells, generated in serum-free liquid phase from peripheral human CD34(+) cells. The surface expression of both TRAIL and "death receptor" TRAIL-R2 became detectable starting from the early phase of megakaryocytic differentiation (day 6 of culture) and persisted at later (days10-14) culture times. On the other hand, "death receptor" TRAIL-R1, "decoy receptors" TRAIL-R3, and TRAIL-R4 were barely detectable or undetectable at any time point examined. Addition of recombinant TRAIL at day 6 of culture increased the rate of spontaneous apoptosis of CD34(+)/CD41(dim) megakaryoblasts and it significantly decreased the total output of mature megakaryocytic cells evaluated after additional 4-8 days of culture. Conversely, addition in culture of TRAIL-R2-Fc chimera, which blocked the interaction between endogenous TRAIL and TRAIL-R2 on the surface of cultured megakaryocytic cells, increased the total megakaryocytic cell count. In addition, recombinant TRAIL promoted a small but reproducible increase of maturation in the surviving megakaryocytic cell population, evaluated by both phenotypic analysis and morphology. A similar pro-maturation effect was observed when TRAIL was added to bone marrow-derived CD61(+) megakaryocytic cells. Thus, our data suggest a role of TRAIL as a regulator of megakaryocytopoiesis.     

Acceleration of human neutrophil apoptosis by TRAIL

Neutrophil granulocytes have a short lifespan, with their survival limited by a constitutive program of apoptosis. Acceleration of neutrophil apoptosis following ligation of the Fas death receptor is well-documented and TNF-alpha also has a transient proapoptotic effect. We have studied the role of the death receptor ligand TRAIL in human neutrophils. We identified the presence of mRNAs for TRAIL, TRAIL-R2, and TRAIL-R3, and cell surface expression of TRAIL-R2 and -R3 in neutrophil populations. Neutrophil apoptosis is specifically accelerated by exposure to a leucine zipper-tagged form of TRAIL, which mimics cell surface TRAIL. Using blocking Abs to TRAIL receptors, specifically TRAIL-R2, and a TRAIL-R1:FcR fusion protein, we have excluded a role for TRAIL in regulating constitutive neutrophil apoptosis. No additional proapoptotic effect of leucine zipper TRAIL was identified following TRAIL treatment of neutrophils in the presence of gliotoxin, an inhibitor of NF-kappaB, suggesting TRAIL does not activate NF-kappaB in human neutrophils. TRAIL treatment of human neutrophils did not induce a chemotactic response. The susceptibility of neutrophils to TRAIL-mediated apoptosis suggests a role for TRAIL in the regulation of inflammation and may provide a mechanism for clearance of neutrophils from sites of inflammation.     

TNF-alpha-related apoptosis-inducing ligand decoy receptor DcR2 is targeted by androgen action in the rat ventral prostate

The apoptotic cell death process in the prostate is known to be under the control of androgens. Tumor necrosis factor-alpha (TNF-alpha)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF-alpha family of cytokines, known to induce apoptosis upon binding to its death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. Two additional TRAIL receptors, DcR1/TRAIL-R3 and DcR2/TRAIL-R4, lack functional death domains and act as decoy receptors for TRAIL. In this study, we examined whether TRAIL and cellular receptors expression was targeted by androgens during the apoptotic cell death process in the hormone sensitive ventral prostate. The role of androgens was investigated using two sets of experiment. (1) Androgen deprivation associated with an apoptotic process resulted in a decrease in DcR2 mRNA and protein expression in the ventral prostate 3 days after castration. Testosterone administration to castrated adult rats prevented the decrease in DcR2 mRNA and protein levels in the ventral prostate. In contrast, DcR2 expression was modified, neither in the dorsolateral nor in the anterior prostate following castration. No changes were observed in DR4, DR5, DcR1, and TRAIL mRNA and protein levels in prostate after castration. (2) A specific decrease in DcR2 expression was observed in the ventral prostate after treatment of rats with the anti-androgen flutamide. Together, the present results suggest that testosterone specifically controls DcR2 expression in the adult rat ventral prostate. Androgen withdrawal, by reducing DcR2 expression, might leave the cells vulnerable to cell death signals generated by TRAIL via its functional receptors.     

Expression of osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, tumor necrosis factor-related apoptosis-inducing ligand, stromal cell-derived factor-1 and their receptors in epithelial metastatic breast cancer cell lines

ABSTRACT: BACKGROUND: While breast cancer (BC) is the major cause of death among women worldwide, there is no guarantee of better patient survival because many of these patients develop primarily metastases, despite efforts to detect it in its early stages. Bone metastasis is a common complication that occurs in 65-80 % of patients with disseminated disease, but the molecular basis underlying dormancy, dissemination and establishment of metastasis is not understood. Our objective has been to evaluate simultaneously osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), stromal cell-derived factor-1 (SDF-1), and their receptors (R) in 2 human BC cell lines, MDA-MB-231 and MCF-7. METHODS: OPG, RANKL, TRAIL and SDF-1 expression and release, in addition to the expression of their receptors has been investigated using immunofluorescence, imunocytochemistry and ELISA analyses. RESULTS: MCF-7 cells released higher levels of OPG in conditioned media (CM) than MDA-MB-231 cells; 100 % of both types of cell expressed OPG, RANKL, TRAIL and SDF-1. Moreover, 100 % in both lines expressed membrane RANKL and RANK, whereas only 50 % expressed CXCR4. Furthermore, 100 % expressed TRAIL-R1 and R4, 30-50 % TRAIL-R2, and 40-55 % TRAIL-R3. CONCLUSIONS: MCF-7 and MDA-MB-231 cells not only released OPG, but expressed RANKL, TRAIL and SDF-1. The majority of the cells also expressed RANK, CXCR4 and TRAIL-R. Since these ligands and their receptors are implicated in the regulation of proliferation, survival, migration and future bone metastasis during breast tumor progression, assessment of these molecules in tumor biopsies of BC patients could be useful in identifying patients with more aggressive tumors that are also at risk of bone metastasis, which may thus improve the available options for therapeutic intervention.