Cellular Dynamics of Fas-Associated Death Domain in the Regulation of Cancer and Inflammation.

Fas-associated death domain (FADD) is an adaptor protein that predominantly transduces the apoptosis signal from the death receptor (DR) to activate caspases, leading to the initiation of apoptotic signaling and the coordinated removal of damaged, infected, or unwanted cells. In addition to its apoptotic functions, FADD is involved in signaling pathways related to autophagy, cell proliferation, necroptosis, and cellular senescence, indicating its versatile role in cell survival and proliferation. The subcellular localization and intracellular expression of FADD play a crucial role in determining its functional outcomes, thereby highlighting the importance of spatiotemporal mechanisms and regulation. Furthermore, FADD has emerged as a key regulator of inflammatory signaling, contributing to immune responses and cellular homeostasis. This review provides a comprehensive summary and analysis of the cellular dynamics of FADD in regulating programmed cell death and inflammation through distinct molecular mechanisms associated with various signaling pathways.

Synthesis of N-Glycosylated Soluble Fas Ligand.

Controlled cell death is essential for the regulation of the immune system and plays a role in pathogen defense. It is often altered in pathogenic conditions such as cancer, viral infections and autoimmune diseases. The Fas receptor and its corresponding membrane-bound ligand (FasL) are part of the extrinsic apoptosis pathway activated in these cases. A soluble form of FasL (sFasL), produced by ectodomain shedding, displays a diverse but still elusive set of non-apoptotic functions and sometimes even serves as a pro-survival factor. To gather more knowledge about the characteristics of this protein and the impact N-glycosylations may have, access to homogeneous posttranslationally modified variants of sFasL is needed. Therefore, we developed a flexible strategy to obtain such homogeneously N-glycosylated variants of sFasL by applying chemical protein synthesis. This strategy can be flexibly combined with enzymatic methods to introduce more complex, site selective glycosylations.

NGAL Mediates Anaplastic Thyroid Carcinoma Cells Survival Through FAS/CD95 Inhibition.

Neutrophil gelatinase-associated lipocalin (NGAL), a siderophore-mediated iron binding protein, is highly expressed in human anaplastic thyroid carcinomas (ATCs) where it plays pleiotropic protumorigenic roles including that of a prosurvival protein. Here we show that NGAL inhibits FAS/CD95 death receptor to control ATC cell survival. FAS/CD95 expression in human specimens from patients with ATC and in ATC-derived cell lines negatively correlate with NGAL expression. Silencing of NGAL in ATC cells leads to FAS/CD95 upregulation, whereas NGAL overexpression determines the opposite effect. As a result, an agonist anti-FAS/CD95 antibody induces cell death in NGAL-silenced cells while it is ineffective on NGAL-overexpressing cells. Interestingly, the inhibitory activity of NGAL on FAS/CD95 is due to its iron carrier property given that perturbing iron homeostasis of NGAL-proficient and -deficient ATC cells directly influences FAS/CD95 expression. Accordingly, conditioned media containing a mutant form of NGAL unable to bind siderophores cannot rescue cells from FAS/CD95-dependent death, whereas NGAL wild type-containing conditioned media abolish the effects of the agonist antibody. We also find that downregulation of FAS/CD95 expression is mediated by iron-dependent NGAL suppression of p53 transcriptional activity. Our results indicate that NGAL contributes to ATC cell survival by iron-mediated inhibition of p53-dependent FAS/CD95 expression and suggest that restoring FAS/CD95 by NGAL suppression could be a helpful strategy to kill ATC cells.

Synergistic toxicity of some food additives used in non-alcoholic beverages on renal tubular epithelial cells.

Evidences supported many food additives (FAs) possess toxicity to human health due to chronic excessive exposure. Global hygienic standards strictly limit the dosage of each FA and mixture of the same functional FAs. However, the synergetic effects caused by the combination of FAs with different functions require careful evaluation. In the present study, the content of each FA in beverages was determined by HPLC-UV-Vis detection. The cytotoxic effects of selected typical FAs alone or their combination were evaluated in human renal tubular epithelial cells. Mathematical Modeling and bioinformatics methods were employed to evaluate the toxicity of FAs and to predict the key target proteins of FAs on renal tubular cell toxicity, which were verified by western blot. The results indicated above 5 FAs were used in each surveyed beverage. The content of each FA and the respective ratios of the same functional FAs in each beverage did not exceed the maximum permitted level. But it was intensively shown that the significant synergistic cytotoxicity for the combination of FAs with lower concentration. The intercellular signaling transduction pathways including JNK/STAT, PI3P/AKT, and MAPK pathways, which could also be activated by PDGF signaling, were predicted to be involved in Fas-induced cytotoxicity. The increased expression of p-STAT3, p-JNK and p-AKT was associated with renal tubular injury. The current study implied the synergistic cytotoxic effect caused by multiple FAs at no toxic dosages via activated cellular transduction pathways regulating cell survival and apoptosis function, which warning of the synergistic toxic effects of different types of FAs.

CD95 (Fas) and CD95L (FasL)-mediated non-canonical signaling pathways.

Although the interaction of CD95L (also known as FasL) with its so-called death receptor CD95 (Fas) induces an apoptotic signal responsible for the elimination of infected and cancer cells and maintenance of tissue homeostasis, this receptor can also implement non apoptotic signaling pathways. This latter signaling is involved in metastatic dissemination in certain cancers and the severity of auto-immune disorders. The signaling complexity of this pair is increased by the fact that CD95 expression itself seems to contribute to oncogenesis via a CD95L-independent manner and, that both ligand and receptor might interact with other partners modulating their pathophysiological functions. Finally, CD95L itself can trigger cell signaling in immune cells rendering complex the interpretation of mouse models in which CD95 or CD95L are knocked out. Herein, we discuss these non-canonical responses and their biological functions.

Characterizing the regulatory Fas (CD95) epitope critical for agonist antibody targeting and CAR-T bystander function in ovarian cancer.

Receptor clustering is the most critical step to activate extrinsic apoptosis by death receptors belonging to the TNF superfamily. Although clinically unsuccessful, using agonist antibodies, the death receptors-5 remains extensively studied from a cancer therapeutics perspective. However, despite its regulatory role and elevated function in ovarian and other solid tumors, another tumor-enriched death receptor called Fas (CD95) remained undervalued in cancer immunotherapy until recently, when its role in off-target tumor killing by CAR-T therapies was imperative. By comprehensively analyzing structure studies in the context of the binding epitope of FasL and various preclinical Fas agonist antibodies, we characterize a highly significant patch of positively charged residue epitope (PPCR) in its cysteine-rich domain 2 of Fas. PPCR engagement is indispensable for superior Fas agonist signaling and CAR-T bystander function in ovarian tumor models. A single-point mutation in FasL or Fas that interferes with the PPCR engagement inhibited apoptotic signaling in tumor cells and T cells. Furthermore, considering that clinical and immunological features of the autoimmune lymphoproliferative syndrome (ALPS) are directly attributed to homozygous mutations in FasL, we reveal differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations. As Fas-mediated bystander killing remains vital to the success of CAR-T therapies in tumors, our findings highlight the therapeutic analytical design for potentially effective Fas-targeting strategies using death agonism to improve cancer immunotherapy in ovarian and other solid tumors.

The Role of FAS Receptor Methylation in Osteosarcoma Metastasis.

Osteosarcoma is the most frequent primary malignant bone tumor with an annual incidence of about 400 cases in the United States. Osteosarcoma primarily metastasizes to the lungs, where FAS ligand (FASL) is constitutively expressed. The interaction of FASL and its cell surface receptor, FAS, triggers apoptosis in normal cells; however, this function is altered in cancer cells. DNA methylation has previously been explored as a mechanism for altering FAS expression, but no variability was identified in the CpG island (CGI) overlapping the promoter. Analysis of an expanded region, including CGI shores and shelves, revealed high variability in the methylation of certain CpG sites that correlated significantly with FAS mRNA expression in a negative manner. Bisulfite sequencing revealed additional CpG sites, which were highly methylated in the metastatic LM7 cell line but unmethylated in its parental non-metastatic SaOS-2 cell line. Treatment with the demethylating agent, 5-azacytidine, resulted in a loss of methylation in CpG sites located within the FAS promoter and restored FAS protein expression in LM7 cells, resulting in reduced migration. Orthotopic implantation of 5-azacytidine treated LM7 cells into severe combined immunodeficient mice led to decreased lung metastases. These results suggest that DNA methylation of CGI shore sites may regulate FAS expression and constitute a potential target for osteosarcoma therapy, utilizing demethylating agents currently approved for the treatment of other cancers.

The role of death receptor signaling pathways in mouse Sertoli cell avoidance of apoptosis during LPS- and IL-18-induced inflammatory conditions.

Lipopolysaccharide (LPS) triggers infectious acute inflammation, and interleukin (IL)-18 is an inflammasome-mediated cytokine. We previously demonstrated that endogenous IL-18 induces testicular germ cell apoptosis during acute inflammation when plasma IL-18 levels are high. Additionally, high-dose recombinant IL-18 (rIL-18) induced Leydig cell apoptosis. The blood-testis barrier formed by Sertoli cells protects testicular germ cells from both exogenous and endogenous harmful substances. However, the impact of LPS and IL-18 on Sertoli cells remained unclear. We stimulated TM4 cells, a mouse Sertoli cell line, with LPS (200 or 1000 ng/mL) or rIL-18 (0.1-100 ng/mL) at levels that induced Leydig cell apoptosis in our previous study and assessed caspase 3 cleavage and the mRNA expression of inflammatory cytokines and markers of apoptotic pathways (Tnfr1, Fasl, Fas, Fadd) after stimulation. Il6 mRNA was increased by LPS stimulation. Tnfα mRNA was increased by 200 ng/mL LPS but not 1000 ng/mL LPS. Fas was increased, but Fasl was decreased, by LPS. LPS had little influence on Tnfr1 or Fadd mRNA expression and did not induce apoptosis. Il18 mRNA was not increased, and Il18r1 was significantly decreased following LPS treatment. Treatment with rIL-18 increased Il18r1 mRNA and induced inflammation, but decreased Tnfr1 and had little influence on apoptosis, as indicated by Tnfα, Fasl, Fas, Fadd and cleaved caspase 3. These results suggested that Sertoli cells do not easily undergo apoptosis despite strong inflammatory stimuli. Additionally, Sertoli cells may resist inflammation and play a larger role in protecting testicular homeostasis than other component cells of the testis.

Unsolved mystery of Fas: mononuclear cells may have trouble dying in patients with Sjögren's syndrome.

BACKGROUND: Patients with Sjögren's syndrome, like other patients with autoimmune disorders, display dysregulation in the function of their immune system. Fas and Fas Ligand (FasL) are among the dysregulated proteins. METHODS: We studied Fas and FasL on IL-2Rα+ cells and in serum of patients with Sjögren's syndrome (n = 16) and healthy individuals (n = 16); both from same ethnic and geographical background. We used flow cytometry and enzyme-linked immunosorbent for this purpose. We also measured the expression of Bcl-2 and Bax by reverse transcription quantitative real-time PCR (RT-qPCR) and percentage of apoptotic and dead cells using Annexin V and 7-AAD staining in lymphocytes. RESULTS: FasL was increased in patients' T and B cells while Fas was increased in patients' monocytes, T and B cells. No signs of increased apoptosis were found. sFas and sFasL in patients' serum were increased, although the increase in sFasL was not significant. We suspect an effect of non-steroidal anti-inflammatory therapy on B cells, explaining the decrease of the percentage Fas+ B cells found within our samples. In healthy individuals, there was a noticeable pattern in the expression of FasL which mutually correlated to populations of mononuclear cells; this correlation was absent in the patients with Sjögren's syndrome. CONCLUSIONS: Mononuclear cells expressing IL-2Rα+ had upregulated Fas in Sjögren's syndrome. However, the rate of apoptosis based on Annexin V staining and the Bcl-2/Bax expression was not observed in mononuclear cells. We suspect a functional role of abnormal levels of Fas and FasL which has not been cleared yet.

Insights into Canine Infertility: Apoptosis in Chronic Asymptomatic Orchitis.

Chronic asymptomatic orchitis (CAO) is a common cause of acquired non-obstructive azoospermia in dogs. To understand the impact and mode of action of apoptosis, we investigated TUNEL, Bax, Bcl-2, Fas/Fas ligand, and caspase 3/8/9 in testicular biopsies of CAO-affected dogs and compared the results to undisturbed spermatogenesis in healthy males (CG). TUNEL+ cells were significantly increased in CAO, correlating with the disturbance of spermatogenesis. Bcl-2, Bax (p < 0.01 each), caspase 9 (p < 0.05), Fas, caspase 8 (p < 0.01 each), and caspase 3 (p < 0.05) were significantly increased at the mRNA level, whereas FasL expression was downregulated. Cleaved caspase 3 staining was sporadic in CAO but not in CG. Sertoli cells, some peritubular (CAO/CG) and interstitial immune cells (CAO) stained Bcl-2+, with significantly more immunopositive cells in both compartments in CAO compared to CG. Bcl-2 and CD20 co-expressing B lymphocytes were encountered interstitially and in CAO occasionally also found intratubally, underlining their contribution to the maintenance of CAO. Our results support the crucial role of the intrinsic and extrinsic apoptotic pathways in the pathophysiology of canine CAO. Autoprotective Bcl-2 expression in Sertoli cells and B lymphocytes seems to be functional, however, thereby also maintaining and promoting the disease by immune cell activation.

Evaluation of FAS and eNOS expression in COVID-19 placenta: histopathological and immunohistochemical study.

OBJECTIVE: In this study, the effects of cell adhesion, inflammation and apoptotic changes on fetal development in cases of COVID-19 placenta were investigated. PATIENTS AND METHODS: Placenta tissue samples from 15 COVID-19 and 15 healthy pregnant women were taken after delivery. Tissue samples were fixed in formaldehyde, then blocked with paraffin wax and 4-6 µm thick sections were cut and stained with Harris Hematoxylene-Eosin. Sections were stained with FAS antibody and endothelial nitric oxide synthase (eNOS) antibody. RESULTS: In COVID-19 placenta section, deterioration of the root villus basement membrane structure in the maternal region, decidua cells and syncytial cell degeneration, significant increase in fibrinoid tissue, endothelial dysfunction in free villi and intense congestion in blood vessels, increase in syncytial nodes and bridges were observed. In terms of inflammation, eNOS expression was increased in Hoffbauer cells, dilated blood vessels endothelial cells in chorionic villi, and surrounding inflammatory cells. Positive FAS expression was also increased in the basement membranes of root and free villi, syncytial bridge and nodes, and endothelial cells. CONCLUSIONS: The effect of COVID-19 caused an increase in eNOS activity and acceleration of the proapoptotic process and the deterioration of cell-membrane adhesion.

CD95/Fas ligand induced toxicity.

The role of CD95/Fas ligand (CD95L/FasL) in the induction of CD95-mediated extrinsic apoptosis is well characterized. Trimerized, membrane-bound CD95L ligates the CD95 receptor activating downstream signaling resulting in the execution of cells by caspase proteins. However, the expression of CD95L has been reported to induce cell death in contexts in which this pathway is unlikely to be activated, such as in cell autonomous activation induced cell death (AICD) and in CD95-resistant cancer cell lines. Recent data suggests that the CD95L mRNA exerts toxicity through death induced by survival gene elimination (DISE). DISE results from the targeting of networks of survival genes by toxic short RNA (sRNA)s in the RNA-induced silencing complex (RISC). CD95L mRNA contributes to this death directly, through the processing of its mRNA into toxic sRNAs that are loaded into the RISC, and indirectly, by promoting the loading of other toxic sRNAs. Interestingly, CD95L is not the only mRNA that is processed and loaded into the RISC. Protein-coding mRNAs involved in protein translation are also selectively loaded. We propose a model in which networks of mRNA-derived sRNAs modulate DISE, with networks of genes providing non-toxic RISC substrate sRNAs that protect against DISE, and opposing networks of stress-activated genes that produce toxic RISC substrate sRNAs that promote DISE.

Bifidobacterium animalis sup F1-7 Acts as an Effective Activator to Regulate Immune Response Via Casepase-3 and Bak of FAS/CD95 Pathway.

Intestinal microecology was closely related to immune regulation, but the related mechanism was still unclear. This study aimed to reveal how microorganisms improved immune response via casepase-3 and Bak of FAS/CD95 pathway. Bifidobacterium animalis F1-7 inhibited the melanoma B16-F10 cells in vitro effectively; had a potent anticancer effect of lung cancer mice; effectively improved the spleen immune index and CD3+ (75.8%) and CD8+ (19.8%) expression level; strengthened the phagocytosis of macrophages; inhibited the overexpression of inflammatory factors IL-6 (319.10 ± 2.46 pg/mL), IL-8 (383.05 ± 9.87 pg/mL), and TNF-α (2003.40 ± 11.42 pg/mL); and promoted the expression of anti-inflammatory factor IL-10 (406.00 ± 3.59 pg/mL). This process was achieved by promoting caspase-8/3 and BH3-interacting domain death agonist (Bid), Bak genes, and protein expression. This study confirmed the B. animalis F1-7 could act as an effective activator to regulate immune response by promoting the expression of caspase-8/3, Bid and Bak genes, and proteins and by activating the FAS/CD95 pathway. Our study provided a data support for the application of potentially beneficial microorganisms of B. animalis F1-7 as an effective activator to improve immunity.

CRISPR/Cas9-mediated abrogation of CD95L/CD95 signaling-induced glioma cell growth and immunosuppression increases survival in murine glioma models.

PURPOSE: Glioblastoma is the most common brain tumor in adults and is virtually incurable. Therefore, new therapeutic strategies are urgently needed. Over the last decade, multiple growth-promoting functions have been attributed to CD95, a prototypic death receptor well characterized as an apoptosis mediator upon CD95L engagement. Strategic targeting of non-apoptotic or apoptotic CD95 signaling may hold anti-glioblastoma potential. Due to its antithetic nature, understanding the constitutive role of CD95 signaling in glioblastoma is indispensable. METHODS: We abrogated constitutive Cd95 and Cd95l gene expression by CRISPR/Cas9 in murine glioma models and characterized the consequences of gene deletion in vitro and in vivo. RESULTS: Expression of canonical CD95 but not CD95L was identified in mouse glioma cells in vitro. Instead, a soluble isoform-encoding non-canonical Cd95l transcript variant was detected. In vivo, an upregulation of the membrane-bound canonical CD95L form was revealed. Cd95 or Cd95l gene deletion decreased cell growth in vitro. The growth-supporting role of constitutive CD95 signaling was validated by Cd95 re-transfection, which rescued growth. In vivo, Cd95 or Cd95l gene deletion prolonged survival involving tumor-intrinsic and immunological mechanisms in the SMA-497 model. In the GL-261 model, that expresses no CD95, only CD95L gene deletion prolonged survival, involving a tumor-intrinsic mechanism. CONCLUSION: Non-canonical CD95L/CD95 interactions are growth-promoting in murine glioma models, and glioma growth and immunosuppression may be simultaneously counteracted by Cd95l gene silencing.

MMP7 cleavage of amino-terminal CD95 death receptor switches signaling toward non-apoptotic pathways.

CD95 is a death receptor that can promote oncogenesis through molecular mechanisms that are not fully elucidated. Although the mature CD95 membrane receptor is considered to start with the arginine at position 17 after elimination of the signal peptide, this receptor can also be cleaved by MMP7 upstream of its leucine at position 37. This post-translational modification occurs in cancer cells but also in normal cells such as peripheral blood leukocytes. The non-cleaved CD95 amino-terminal region consists in a disordered domain and its in silico reconstitution suggests that it might contribute to receptor aggregation and thereby, regulate the downstream death signaling pathways. In agreement with this molecular modeling analysis, the comparison of CD95-deficient cells reconstituted with full-length or N-terminally truncated CD95 reveals that the loss of the amino-terminal region of CD95 impairs the initial steps of the apoptotic signal while favoring the induction of pro-survival signals, including the PI3K and MAPK pathways.

Amiodarone-drove XBP1s aggravates endoplasmic reticulum stress and apoptosis in Hashimoto's thyroiditis through regulating LINC00842/miR-214/FASL axis.

Hashimoto's thyroiditis (HT) is an organ-specific autoimmune disease, that eventually lead to hypothyroidism. XBP1s is an endoplasmic reticulum stress related protein and participates in the pathogenesis of several diseases. Nevertheless, the potential role of XBP1s in amiodarone (AMIO)-treated HT patients remains unknown. In this study, AMIO aggravated the endoplasmic reticulum stress responses in HT patients and thyroid epithelial follicular cells. Moreover, MTT assay and flow cytometry analysis revealed that knockdown of XBP1s suppressed AMIO-induced thyroid epithelial follicular cells apoptosis. Mechanically, the Chromatin Immunoprecipitation (ChIP) and luciferase activity assay proved that XBP1s enhanced LINC00842 expression in HT patients and thyroid epithelial follicular cells via binding to LINC00842 promoter. LINC00842 functioned as a miR-214 sponge in HT patients and thyroid epithelial follicular cells. Besides, LINC00842 up-regulated Fas ligand (FASL) expression via inhibition of miR-214. In rescue experiments, overexpression of FASL reversed shXBP1s-induced suppression of cell apoptosis in AMIO-treated thyroid epithelial follicular cells. These findings concluded that AMIO-drove XBP1s aggravated endoplasmic reticulum stress and apoptosis in HT via modulating LINC00842/miR-214/FASL axis, providing a new sight for the therapeutic strategy of AMIO-induced HT.

Serum soluble Fas ligand is a severity and mortality prognostic marker for COVID-19 patients.

Finding cytokine storm initiator factors associated with uncontrolled inflammatory immune response is necessary in COVID-19 patients. The aim was the identification of Fas/Fas Ligand (FasL) role in lung involvement and mortality of COVID-19 patients. In this case-control study, mild (outpatient), moderate (hospitalized), and severe (ICU) COVID-19 patients and healthy subjects were investigated. RNA isolated from PBMCs for cDNA synthesis and expression of mFas/mFasL mRNA was evaluated by RT-PCR. Serum sFas/sFasL protein by ELISA and severity of lung involvement by CT-scan were evaluated. Also, we docked Fas and FasL via Bioinformatics software (in silico) to predict the best-fit Fas/FasL complex and performed molecular dynamics simulation (MDS) in hyponatremia and fever (COVID-19 patients), and healthy conditions. mFasL expression was increased in moderate and severe COVID-19 patients compared to the control group. Moreover, mFas expression showed an inverse correlation with myalgia symptom in COVID-19 patients. Elevation of sFasL protein in serum was associated with reduced lung injury and mortality. Bioinformatics analysis confirmed that blood profile alterations of COVID-19 patients, such as fever and hyponatremia could affect Fas/FasL complex interactions. Our translational findings showed that decreased sFasL is associated with lung involvement; severity and mortality in COVID-19 patients. We think that sFasL is a mediator of neutrophilia and lymphopenia in COVID-19. However, additional investigation is suggested. This is the first report describing that the serum sFasL protein is a severity and mortality prognostic marker for the clinical management of COVID-19 patients.

Gap junction intercellular communication mediates cadmium-induced apoptosis in hepatocytes via the Fas/FasL pathway.

As a common environmental pollutant, cadmium (Cd) causes damage to many organs of the body. Gap junction intercellular communication (GJIC) represents one of the most important routes of rapid signaling between cells. However, the mechanisms underlying GJIC's role in hepatotoxicity induced by Cd remain unknown. We established a Cd poisoning model in vitro by co-culturing Cd-exposed and unexposed hepatocytes and found that 18ß-glycyrrhetinic acid (GA), a GJIC inhibitor, can effectively reduce the apoptosis rate of healthy cells co-cultured with apoptotic cells treated with Cd. We also found that anti-FasL antibody had the same effect. However, in mono-cultured cells, GA treatment in combination with Cd was found to aggravate the damage induced by Cd exposure, increase the level of oxidative stress and protein expression of HO-1, decrease the mitochondrial membrane potential, incur more serious morphological damage to mitochondria than Cd treatment alone. Moreover, compared with Cd-only exposure, GA and Cd co-treatment further increased the expression levels of the apoptosis-related proteins Fas, FasL, FADD and the ratio of Bax/Bcl-2, inhibited the protein expression of ASK1 and Daxx. We also found that the protein expression of Daxx in siFADD + Cd hepatocytes was significantly higher than in Cd-treated cells. Thus, our study suggests that gap junction inhibition may play a dual role in Cd-induced cell damage by inhibiting the transmission of death signals from damaged cells to healthy cells but also aggravating the transmission of death signals between damaged cells, and that the Fas/FasL-mediated death receptor pathway may play an important role in this process.

Lipid Nanoparticle Delivery of Fas Plasmid Restores Fas Expression to Suppress Melanoma Growth In Vivo.

Fas ligand (FasL), expressed on the surface of activated cytotoxic T lymphocytes (CTLs), is the physiological ligand for the cell surface death receptor, Fas. The Fas-FasL engagement initiates diverse signaling pathways, including the extrinsic cell death signaling pathway, which is one of the effector mechanisms that CTLs use to kill tumor cells. Emerging clinical and experimental data indicate that Fas is essential for the efficacy of CAR-T cell immunotherapy. Furthermore, loss of Fas expression is a hallmark of human melanoma. We hypothesize that restoring Fas expression in tumor cells reverses human melanoma resistance to T cell cytotoxicity. DNA hypermethylation, at the FAS promoter, down-regulates FAS expression and confers melanoma cell resistance to FasL-induced cell death. Forced expression of Fas in tumor cells overcomes melanoma resistance to FasL-induced cell death in vitro. Lipid nanoparticle-encapsulated mouse Fas-encoding plasmid therapy eliminates Fas+ tumor cells and suppresses established melanoma growth in immune-competent syngeneic mice. Similarly, lipid nanoparticle-encapsulated human FAS-encoding plasmid (hCOFAS01) therapy significantly increases Fas protein levels on tumor cells of human melanoma patient-derived xenograft (PDX) and suppresses the established human melanoma PDX growth in humanized NSG mice. In human melanoma patients, FasL is expressed in activated and exhausted T cells, Fas mRNA level positively correlates with melanoma patient survival, and nivolumab immunotherapy increases FAS expression in tumor cells. Our data demonstrate that hCOFAS01 is an effective immunotherapeutic agent for human melanoma therapy with dual efficacy in increasing tumor cell FAS expression and in enhancing CTL tumor infiltration.

Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment.

Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas-Fas and Fas-FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD.