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The development of targeted therapy such as tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-based therapy has gained increasing attention as a promising new approach in cancer therapy. TRAIL specifically targets cancer cells while sparing the normal cells, thus, limiting the known side effects of the majority anti-cancer therapies. As more extensive research and clinical trials are conducted, resistance to TRAIL molecule has become one of the significant issues associated with the failure of TRAIL in treating colorectal cancer (CRC). To date, the exact mechanism by which TRAIL resistance may have occurred remains unknown. Interestingly, recent studies have revealed the critical role of the TRAIL decoy receptor family; consisting of decoy receptor 1 (DcR1; also known as TRAIL-R3), decoy receptor 2 (DcR2; also known as TRAIL-R4), and osteoprotegerin (OPG) in driving TRAIL resistance. This review highlights the expression of the decoy receptors in CRC and its possible association with the reduction in sensitivity towards TRAIL treatment based on the currently available in vitro, in vivo, and human studies. Additionally, discrepancies between the outcomes from different research groups are discussed, and essential areas are highlighted for future investigation of the roles of decoy receptors in modulating TRAIL-induced apoptosis. Overcoming TRAIL resistance through modulating the expression(s) and elucidating the role(s) of TRAIL decoy receptors hold great promise for TRAIL-based therapies to be extensively explored in treating human cancers including CRC.

The TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer drug candidate because it selectively binds to the proapoptotic death receptors, which are frequently overexpressed in a wide range of cancer cells, subsequently inducing strong apoptosis in these cells. However, the therapeutic benefit of TRAIL has not been clearly proven, mainly because of its poor pharmacokinetic characteristics and frequent resistance to its application caused by the activation of a survival signal via the EGF/epidermal growth factor receptor (EGFR) signaling pathway. Here, a lumazine synthase protein cage nanoparticle isolated from Aquifex aeolicus (AaLS) was used as a multiple ligand-displaying nanoplatform to display polyvalently both TRAIL and EGFR binding affibody molecules (EGFRAfb) via a SpyTag/SpyCatcher protein-ligation system, to form AaLS/TRAIL/EGFRAfb. The dual-ligand-displaying AaLS/TRAIL/EGFRAfb exhibited a dramatically enhanced cytotoxicity on TRAIL-resistant and EGFR-overexpressing A431 cancer cells in vitro, effectively disrupting the EGF-mediated EGFR survival signaling pathway by blocking EGF/EGFR binding as well as strongly activating both the extrinsic and intrinsic apoptotic pathways synergistically. The AaLS/TRAIL/EGFRAfb selectively targeted A431 cancer cells in vitro and actively reached the tumor sites in vivo. The A431 tumor-bearing mice treated with AaLS/TRAIL/EGFRAfb exhibited a significant suppression of the tumor growth without any significant side effects. Collectively, these findings showed that the AaLS/TRAIL/EGFRAfb could be used as an effective protein-based therapeutic for treating EGFR-positive cancers, which are difficult to manage using mono-therapeutic approaches.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) combined with sensitizer is a potential method to reverse TRAIL-resistance in tumor cells. Rhein (RH) is a monomer extracted from Chinese herbs that has been reported to show anti-tumor effects in a variety of tumor cells, but the role of RH in TRAIL-induced anti-tumor effects in bladder cancer cells has not been reported. In this study, we found that the combined treatment of a non-toxic concentration of RH with TRAIL significantly inhibited the proliferation and induced apoptosis in both TRAIL sensitive and resistant bladder cancer cell lines. Furthermore, we found that RH promoted bladder cancer cell apoptosis by up-regulating DR5 expression. Our findings provide potential value in the clinical treatment of bladder cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death and contributes to tumor rejection by cytotoxic lymphocytes in cancer immunosurveillance and immunotherapy. TRAIL and TRAIL receptor agonists have garnered wide popularity as promising agents for cancer therapy. We previously demonstrated that the loss of fucosylation in cancer cells impairs TRAIL sensitivity; however, the precise structures of the fucosylated glycans that regulate TRAIL sensitivity and their carrier molecules remain elusive. Herein, we observed that Lewis glycans among various fucosylated glycans positively regulate TRAIL-induced cell death. Specifically, Lewis glycans on lacto/neolacto glycosphingolipids, but not glycoproteins including TRAIL receptors, enhanced TRAIL-induced formation of the cytosolic caspase 8 complex, without affecting the formation of the membranous receptor complex. Furthermore, type I Lewis glycan expression in colon cancer cell lines and patient-derived cancer organoids was positively correlated with TRAIL sensitivity. These findings provide novel insights into the regulatory mechanism of TRAIL-induced cell death and facilitate the identification of novel predictive biomarkers for TRAIL-related cancer therapies in future.

Mortality in coronavirus disease 2019 (COVID-19) patients has been linked to the presence of a "cytokine storm" induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which involves elevated levels of circulating cytokines and immune-cell hyperactivation. Targeting cytokines during the management of COVID-19 patients has the potential to improve survival rates and reduce mortality. Although cytokine blockers and immune-host modulators are currently being tested in severely ill COVID-19 patients to cope with the overwhelming systemic inflammation, there is not too many successful cases, thus finding new cytokine blockers to attenuate the cytokine storm syndrome is meaningful. In this paper, we significantly attenuated the inflammatory responses induced by mouse hepatitis viruses A59 and SARS-CoV-2 through a soluble DR5-Fc (sDR5-Fc) chimeric protein that blocked the TNF-related apoptosis-inducing ligand-death receptor 5 (TRAIL-DR5) interaction. Our findings indicates that blocking the TRAIL-DR5 pathway through the sDR5-Fc chimeric protein is a promising strategy to treat COVID-19 severe patients requiring intensive care unit admission or with chronic metabolic diseases.

The tumor microenvironment is important in the initiation and progression of lung adenocarcinoma (LUAD). In this study, we aim to analyze the expression profile of immune-related genes in LUADs, examine the differential expression of immune-related genes in epidermal growth factor receptor (EGFR) wild-type and mutant LUADs, and the clinicopathologic significance of these differentially expressed genes. We used the NanoString PanCancer Immune Profiling Panel to examine 34 cases of LUADs (18 EGFR wild-type, 16 EGFR mutant). In EGFR wild-type LUADs, the macrophage and neutrophil signatures are significantly higher, and significantly higher expression of chemokines, interleukins, leukocyte, macrophage, natural killer cell, pathogen defense, Tumor necrosis factor superfamily, and transporter function signatures are also observed. TNFRSF10B mRNA was preferentially expressed in EGFR wild-type LUADs (P = 6.15e-6, adjusted P = .0244). Immunohistochemical staining for TRAIL-R2 (encoded by TNFRSF10B) on 134 tissue microarray LUAD cases demonstrated strong, moderate, and weak staining in 75 (56.0%), 46 (34.3%), and 13 (9.7%) cases, respectively. Strong TRAIL-R2 expression was significantly associated with poor overall survival (OS) in all stages and EGFR wild-type LUADs, but not in EGFR-mutant tumors. Furthermore, strong TRAIL-R2 expression (P = .004) was an independent risk factor for poor OS. In summary, TNFRSF10B mRNA revealed significantly higher expression in EGFR wild-type LUADs, and strong TRAIL-R2 expression predicts an unfavorable prognosis for these tumors. These patients may benefit from additional treatment with TRAIL-R2-targeted therapies.

Multidrug resistance (MDR), which is common in colorectal cancer (CRC), induces high mortality in patients. Due to its robust and selective apoptosis induction in some CRC cells with MDR, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is attractive as a novel tool for CRC therapy. However, TRAIL is limited by its poor tumor-homing ability and inefficient apoptosis induction in CRC cells expressing low levels of death receptor (DR). Here, the tumor-homing RGR peptide (CRGRRST) was fused to TRAIL to produce RGR-TRAIL. Compared with TRAIL, RGR-TRAIL showed greater cell binding and cytotoxicity in CRC cells. In addition, RGR-TRAIL exerted significantly enhanced tumor uptake and growth suppression in mice bearing CRC tumor xenografts. Notably, RGR-TRAIL eradicated all tumor xenografts of DR-overexpressing COLO205 cells. However, TRAIL only showed mild tumor growth suppression under the same conditions, indicating that RGR fusion significantly increased the antitumor effect of TRAIL in DR-overexpressing CRC cells by improving tumor homing. Nevertheless, RGR fusion did not significantly enhance the antitumor effect of TRAIL in HT29 cells expressing low levels of DR. We found that DR expression in HT29 cells was enhanced by epidermal growth factor receptor (EGFR)-targeted photodynamic therapy (PDT). Moreover, both the in vitro and in vivo antitumor effects of RGR-TRAIL were significantly improved by combination with PDT. HT29 tumor xenografts (∼20%) were even eradicated by combination therapy. These results indicate that it is valuable to further evaluate the combination therapy of RGR-TRAIL and tumor-targeted PDT for clinical therapy of CRC with MDR.

Antibody-based therapeutics, which induce apoptosis of malignant cells by selectively binding to their receptors, hold tremendous promise for clinical cancer therapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received considerable interest due to its favorable capability of activating apoptosis in cancer cells by interacting with death receptors (DRs). However, cancer stem-like cells (CSCs) show deficient or lower DR and are highly resistant to TRAIL-mediated apoptosis limiting the therapeutic efficacy. Here, we report a liposome-mediated acclimatization strategy to overcome the CSC-emanated TRAIL resistance. The liposomal assemblies coencapsulating plasmid DNA encoding TRAIL and salinomycin enable cancer cells as protein generators to express TRAIL, and more importantly, can acclimatize resistant CSCs to be sensitized to the TRAIL-triggered apoptosis by salinomycin-induced upregulation of DR expression on CSCs. This programmable liposome-based drug codelivery system shows the potential to efficiently eliminate CSCs and inhibit CSC-enriched tumor growth in the orthotopic colon tumor mouse model.

Targeting cyclin-dependent kinases 4 and 6 (CDK4/6) is a successful therapeutic approach against breast and other solid tumors. Inhibition of CDK4/6 halts cell cycle progression and promotes antitumor immunity. However, the mechanisms underlying the antitumor activity of CDK4/6 inhibitors are not fully understood. We found that CDK4/6 bind and phosphorylate the p53 family member p73 at threonine 86, which sequesters p73 in the cytoplasm. Inhibition of CDK4/6 led to dephosphorylation and nuclear translocation of p73, which transcriptionally activated death receptor 5 (DR5), a cytokine receptor and key component of the extrinsic apoptotic pathway. p73-mediated induction of DR5 by CDK4/6 inhibitors promoted immunogenic cell death of cancer cells. Deletion of DR5 in cancer cells in vitro and in vivo abrogated the potentiating effects of CDK4/6 inhibitors on immune cytokine TRAIL, 5-fluorouracil chemotherapy, and anti–PD-1 immunotherapy. Together, these results reveal a previously unrecognized consequence of CDK4/6 inhibition, which may be critical for potentiating the killing and immunogenic effects on cancer cells. Significance: This work demonstrates how inhibition of CDK4/6 sensitizes cancer cells to chemotherapy and immune checkpoint blockade and may provide a new molecular marker for improving CDK4/6-targeted cancer therapies. See related commentary by Frank, p. 1170

Age-related macular degeneration (AMD) and central serous chorioretinopathy (CSC) are common diseases that can cause vision loss in older and younger populations. These diseases share pathophysiological conditions derived from retinal pigment epithelium (RPE) dysfunction. Tumor necrosis factor receptor superfamily 10A (TNFRSF10A)-LOC389641 with the same lead single-nucleotide polymorphism (SNP) (rs13278062) is the only overlapped susceptibility locus found in both AMD and CSC through genome-wide association studies. This lead SNP has been reported to alter the transcriptional activity of TNFRSF10A. This study aimed to elucidate the function of TNFRSF10A in RPE degeneration using human primary RPE cells and Tnfrsf10 knockout (Tnfrsf10−/−) mice. TNFRSF10A was found to be localized in human RPE. In vitro assays revealed that a T allele of rs13278062, the risk allele for AMD and CSC, downregulated TNFRSF10A transcription in RPE, leading to decreased cell viability and increased apoptosis through protein kinase C-α (PKCA) downregulation. Treatment with phorbol 12-myristate 13-acetate, a PKC activator, rescued the cell viability. Morphological RPE abnormality was found in the retina of Tnfrsf10−/− mice. Our data suggest that downregulation of TNFRSF10A expression inactivates PKCA signaling and causes cellular vulnerability of the RPE, which may contribute to the pathogenesis of AMD and CSC.

Due to its tumor-specificity and limited side effects, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown great potential in cancer treatments. However, the short half-life of TRAIL protein and the poor death receptor (DR) expression of cancer cells severely compromise the therapeutic outcomes of TRAIL in clinical studies. Herein, a novel ROS-dependent TRAIL-sensitizing nanoplatform, CPT MV, with a Ce6-PLGA core and a TRAIL-modified cell membrane shell was explored to improve the

Endothelial cells (ECs) grant access of disseminated cancer cells to distant organs. However, the molecular players regulating the activation of quiescent ECs at the premetastatic niche (PMN) remain elusive. Here, we find that ECs at the PMN coexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate death receptor 5 (DR5). Unexpectedly, endothelial TRAIL interacts intracellularly with DR5 to prevent its signaling and preserve a quiescent vascular phenotype. In absence of endothelial TRAIL, DR5 activation induces EC death and nuclear factor κB/p38-dependent EC stickiness, compromising vascular integrity and promoting myeloid cell infiltration, breast cancer cell adhesion, and metastasis. Consistently, both down-regulation of endothelial TRAIL at the PMN by proangiogenic tumor-secreted factors and the presence of the endogenous TRAIL inhibitors decoy receptor 1 (DcR1) and DcR2 favor metastasis. This study discloses an intracrine mechanism whereby TRAIL blocks DR5 signaling in quiescent endothelia, acting as gatekeeper of the vascular barrier that is corrupted by the tumor during cancer cell dissemination. ispartof: SCIENCE ADVANCES vol:9 issue:12 ispartof: location:United States status: published

The five-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has remained a dismal 9% for approximately 40 years with an urgent need for novel therapeutic interventions. ONC201 is the founding member of the imipridone class, comprised of orally bioavailable small molecules that have shown efficacy in multiple tumor types both in animal models and in Phase I/II clinical trials. ONC201 is a potent inducer of the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. TRAIL is an innate immune mechanism which induces programmed cell death of cancer cells. We observed that PDAC cells upregulated ATF4, CHOP, and DR5 after treatment with ONC201. This occurred in cell lines that are susceptible to ONC201-induced apoptosis and in ones that are not. In response to ONC201, PDAC cells downregulated anti-apoptotic proteins including c-FLIP, BclXL, XIAP, cIAP1, and survivin. We hypothesized that TRAIL receptor agonists might induce selective, synergistic apoptosis in pancreatic cancer cell lines treated with ONC201. We screened 7 pancreatic cancer cell lines and found synergy with ONC201 and rhTRAIL or the novel TRAIL receptor agonist TLY012 in 6 of the 7 cell lines tested. In vivo experiments using BxPC3 and HPAFII xenograft models showed that the combination of ONC201 plus TLY012 significantly delays tumor growth as compared to controls. Immunohistochemical analysis of the tumors after three doses of the combination showed significantly increased cleavage of caspase 3 in vivo as compared to controls. Taken together, the preclinical efficacy of ONC201 and TLY012 represents a novel therapeutic option for further testing in pancreatic cancer patients. This combination showed marked efficacy in tumor cells that are both sensitive and resistant to the pro-apoptotic effects of ONC201, providing rationale to further investigate the combination of ONC201 plus TLY012 in patients with pancreatic cancer.

Anti-death receptor 5 (DR5) antibody is a potential therapeutic agent for liver fibrosis because it exhibits anti-fibrotic effects by inducing the apoptosis of activated hepatic stellate cells (HSCs), which are responsible for hepatic fibrogenesis. However, the clinical applications of anti-DR5 antibodies have been limited by their low agonistic activity against DR5. In this study, an anti-DR5 antibody-curcumin conjugate (DCC) was prepared to investigate its effect on the clearance of activated HSCs. The DCC was synthesized through a coupling reaction between a maleimide-functionalized curcumin derivative and a thiolated anti-DR5 antibody. No significant differences were observed in the uptake behaviors of activated HSCs between the bare anti-DR5 antibodies and DCC. Owing to the antioxidant and anti-inflammatory effects of curcumin, DCC-treated HSCs produced much lower levels of reactive oxygen species and inducible nitric oxide synthase than the bare anti-DR5 antibody-treated HSCs. Additionally, the anti-fibrotic effects of DCC on activated HSCs were more prominent than those of the bare anti-DR5 antibodies, as demonstrated by the immunocytochemical analysis of α-smooth muscle actin. DCC preferentially accumulated in the liver after its systemic administration to mice with liver fibrosis. Thus, DCC may serve as a potential therapeutic agent for treating liver fibrosis.

ONC201 demonstrated promising activity in patients with advanced endometrial cancer in a Phase I clinical trial. ONC201 activates the integrated stress response (ISR) and upregulates TRAIL and its receptor DR5. We hypothesized ONC201 upregulation of DR5 could sensitize tumors to TRAIL and combination of ONC201 and TRAIL would lead to enhanced cell death in endometrial cancer models. Five endometrial cancer cell lines AN3CA, HEC1A, Ishikawa, RL952, and KLE as well as a murine xenograft model were treated with ONC201 alone or in combination with TRAIL. ONC201 decreased the cell viability of all five endometrial cancer cell lines at clinically achievable low micro-molar concentrations (2–4 μM). ONC201 activated the ISR and induced protein expression of TRAIL and DR5 at the cell surface. Pretreatment with ONC201 sensitized endometrial cancer cell lines to TRAIL, leading to increased cell death induction compared to either agent alone. Tumor growth was reduced in vivo by the ONC201/TRAIL combination treatment in the xenograft model of endometrial cancer (p = .014). Mice treated with combination treatment survived significantly longer than mice from the three control groups (p = .018). ONC201 decreased cell viability in endometrial cancer cells lines primarily through growth arrest while the combination of ONC201 and TRAIL promoted cell death in vitro and in vivo. Our results suggest a novel cancer therapeutic strategy that can be further investigated in the clinic.

Long-term medication with dexamethasone – a synthetic glucocorticoid (GC) drug – results in hyperglycemia, or steroid-induced diabetes. Although recent studies revealed that dexamethasone directly induces pancreatic β-cell apoptosis, its molecular mechanisms remain unclear. In our initial analysis of mRNA transcripts, we discovered the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway may be involved in dexamethasone-induced pancreatic β-cell apoptosis. In the present study, a mechanism of dexamethasone-induced pancreatic β-cell apoptosis through the TRAIL pathway was investigated in cultured cells and isolated mouse islets. INS-1 cells were cultured with and without dexamethasone in the presence or absence of a glucocorticoid receptor (GR) inhibitor, RU486. We found that dexamethasone induced pancreatic β-cell apoptosis in association with the upregulation of TNSF10 (TRAIL) mRNA and protein expression. Moreover, dexamethasone upregulated the TRAIL death receptor (DR5) protein but suppressed the decoy receptor (DcR1) protein. Similar findings were observed in mouse isolated islets: dexamethasone increased TRAIL and DR5 compared to that of control mice. Furthermore, dexamethasone stimulated pro-apoptotic signaling including superoxide production, caspase-8, -9, and -3 activities, NF-κB, and Bax but repressed the anti-apoptotic protein, Bcl-2. All these effects were inhibited by the GR-inhibitor, RU486. Furthermore, knock-down DR5 decreased dexamethasone-induced caspase 3 activity. Caspase-8 and caspase-9 inhibitors protected pancreatic β-cells from dexamethasone-induced apoptosis. Taken together, dexamethasone induced pancreatic β-cell apoptosis by binding to the GR and inducing DR5 and TRAIL pathway.

Age-related disorders, such as Alzheimer’s disease (AD) and age-related macular degeneration (AMD) share common features such as amyloid-β (Aβ) protein accumulation. Retinal deposition of Aβ aggregates in AMD patients has suggested a potential link between AMD and AD. In the present study, we analyzed the expression pattern of a focused set of miRNAs, previously found to be involved in both AD and AMD, in the retina of a triple transgenic mouse model of AD (3xTg-AD) at different time-points. Several miRNAs were differentially expressed in the retina of 3xTg-AD mice, compared to the retina of age-matched wild-type (WT) mice. In particular, bioinformatic analysis revealed that miR-155 had a central role in miRNA-gene network stability, regulating several pathways, including apoptotic and inflammatory signaling pathways modulated by TNF-related apoptosis-inducing ligand (TNFSF10). We showed that chronic treatment of 3xTg-AD mice with an anti-TNFSF10 monoclonal antibody was able to inhibit the retinal expression of miR-155, which inversely correlated with the expression of its molecular target SOCS-1. Moreover, the fine-tuned mechanism related to TNFSF10 immunoneutralization was tightly linked to modulation of TNFSF10 itself and its death receptor TNFRSF10B, along with cytokine production by microglia, reactive gliosis, and specific AD-related neuropathological hallmarks (i.e., Aβ deposition and Tau phosphorylation) in the retina of 3xTg-AD mice. In conclusion, immunoneutralization of TNFSF10 significantly preserved the retinal tissue in 3xTg-AD mice, suggesting its potential therapeutic application in retinal degenerative disorders.

A lucanthone, one of the family of thioxanthenones, has been reported for its inhibitory effects of apurinic endonuclease-1 and autophagy. In this study, we investigated whether lucanthone could enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in various cancer cells. Combined treatment with lucanthone and TRAIL significantly induced apoptosis in human renal carcinoma (Caki and ACHN), prostate carcinoma (PC3), and lung carcinoma (A549) cells. However, combined treatment did not induce apoptosis in normal mouse kidney cells (TCMK-1) and normal human skin fibroblast (HSF). Lucanthone downregulated protein expression of deubiquitinase DUB3, and a decreased expression level of DUB3 markedly led to enhance TRAIL-induced apoptosis. Ectopic expression of DUB3 inhibited combined treatment with lucanthone and TRAIL-induced apoptosis. Moreover, lucanthone increased expression level of DR5 mRNA via downregulation of miR-216a-5p. Transfection of miR-216a-5p mimics suppressed the lucanthone-induced DR5 upregulation. Taken together, these results provide the first evidence that lucanthone enhances TRAIL-induced apoptosis through DR5 upregulation by downregulation of miR-216a-5p and DUB3-dependent Mcl-1 downregulation in human renal carcinoma cells.

Oncogenic transformation leads to changes in glutamine metabolism that make transformed cells highly dependent on glutamine for anabolic growth and survival. Herein, we investigated the cell death mechanism activated in glutamine-addicted tumor cells in response to the limitation of glutamine metabolism. We show that glutamine starvation triggers a FADD and caspase-8-dependent and mitochondria-operated apoptotic program in tumor cells that involves the pro-apoptotic TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2), but is independent of its cognate ligand TRAIL. In glutamine-depleted tumor cells, activation of the amino acid-sensing general control nonderepressible-2 kinase (GCN2) is responsible for TRAIL-R2 upregulation, caspase-8 activation, and apoptotic cell death. Interestingly, GCN2-dependent ISR signaling induced by methionine starvation also leads to TRAIL-R2 upregulation and apoptosis. Moreover, pharmacological inhibition of transaminases activates a GCN2 and TRAIL-R2-dependent apoptotic mechanism that is inhibited by non-essential amino acids (NEAA). In addition, metabolic stress upon glutamine deprivation also results in GCN2-independent FLICE-inhibitory protein (FLIP) downregulation facilitating caspase-8 activation and apoptosis. Importantly, downregulation of the long FLIP splice form (FLIP) and apoptosis upon glutamine deprivation are inhibited in the presence of a membrane-permeable α-ketoglutarate. Collectively, our data support a model in which limiting glutamine utilization in glutamine-addicted tumor cells triggers a previously unknown cell death mechanism regulated by GCN2 that involves the TRAIL-R2-mediated activation of the extrinsic apoptotic pathway., This work was supported by grants from Ministerio de Economía y Competitividad (SAF2015-64383-P), Ministerio de Ciencia, Innovación y Universidades (PGC2018-093960-B-I00), Junta de Andalucía Excellence Program (PY20-00754), CIBERONC ISCIII CB16/12/00421 and the European Community through the regional development funding program (FEDER) to ALR.

Myeloid-derived suppressor cells (MDSCs) have been demonstrated to

The tumor necrosis factor (TNF) superfamily member TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in cancer cells via death receptor (DR) activation with little toxicity to normal cells or tissues. The selectivity for activating apoptosis in cancer cells confers an ideal therapeutic characteristic to TRAIL, which has led to the development and clinical testing of many DR agonists. However, TRAIL/DR targeting therapies have been widely ineffective in clinical trials of various malignancies for reasons that remain poorly understood. Triple negative breast cancer (TNBC) has the worst prognosis among breast cancers. Targeting the TRAIL DR pathway has shown notable efficacy in a subset of TNBC in preclinical models but again has not shown appreciable activity in clinical trials. In this review, we will discuss the signaling components and mechanisms governing TRAIL pathway activation and clinical trial findings discussed with a focus on TNBC. Challenges and potential solutions for using DR agonists in the clinic are also discussed, including consideration of the pharmacokinetic and pharmacodynamic properties of DR agonists, patient selection by predictive biomarkers, and potential combination therapies. Moreover, recent findings on the impact of TRAIL treatment on the immune response, as well as novel strategies to address those challenges, are discussed.

TRAIL (TNF-related apoptosis-inducing ligand) and its derivatives are potentials for anticancer therapy due to the selective induction of apoptosis in tumor cells upon binding to death receptors DR4 or DR5. Previously, we generated a DR5-selective TRAIL mutant variant DR5-B overcoming receptor-dependent resistance of tumor cells to TRAIL. In the current study, we improved the antitumor activity of DR5-B by fusion with a tumor-homing iRGD peptide, which is known to enhance the drug penetration into tumor tissues. The obtained bispecific fusion protein DR5-B-iRGD exhibited dual affinity for DR5 and integrin αvβ3 receptors. DR5-B-iRGD penetrated into U-87 tumor spheroids faster than DR5-B and demonstrated an enhanced antitumor effect in human glioblastoma cell lines T98G and U-87, as well as in primary patient-derived glioblastoma neurospheres in vitro. Additionally, DR5-B-iRGD was highly effective in a xenograft mouse model of the U-87 human glioblastoma cell line in vivo. We suggest that DR5-B-iRGD may become a promising candidate for targeted therapy for glioblastoma.

Objective: Cancer treatment using a targeted inducer of apoptosis like tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) faced the obstacle of resistance, thus providing a plus drug like Thymoquinone (TQ) could be of great interest to tackle breast cancer cells. The aim of the present work is to examine the genetic modulation impacts of the TRAIL receptors and apoptotic markers upon the combinatorial remedy of TRAIL plus TQ on human breast cancer cell lines. Methods: To achieve this rationale, the protein content-based cytotoxicity using SRB assay, as well as the genetic expressions of the TRAIL receptors (DR4 and DR5) and apoptotic markers (Bcl-2, Cas-8, and FADD) using real time qRT-PCR technique were preceded against breast cancer MCF-7 and MDA-MB-231 cancerous cell lines. Results: The current study showed that the combination therapy of TQ+TRAIL significantly inhibited the protein content-based proliferation of MDA-MB-231 cells more than MCF-7 cells. The synergistic effect of them significantly up-regulated the genetic expressions of DR4, DR5, Cas-8, and FADD genes and inhibited the genetic expression of the Bcl-2 gene in the proposed cell lines treated for 24 h. The induction of the apoptotic genes using the combined therapy was stimulated by the elevation of the reactive oxygen species (ROS); nitric oxide (NO) and malondialdehyde (MDA) levels. Conclusions: The synergistic influence between TQ which induced the DR5 and TRAIL, facilitating the connection between TRAIL and its receptors on the cancerous cell membrane. Hence, the proposed combination therapy induced the ROS-mediated apoptotic stimulus.

Cancer cells that are resistant to Bax/Bak-dependent intrinsic apoptosis can be eliminated by proteasome inhibition. Here, we show that proteasome inhibition induces the formation of high molecular weight platforms in the cytosol that serve to activate caspase-8. The activation complexes contain Fas-associated death domain (FADD) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Furthermore, the complexes contain TRAIL-receptor 2 (TRAIL-R2) but not TRAIL-receptor 1 (TRAIL-R1). While RIPK1 inhibition or depletion did not affect proteasome inhibitor-induced cell death, TRAIL-R2 was found essential for efficient caspase-8 activation, since the loss of TRAIL-R2 expression abrogated caspase processing, significantly reduced cell death, and promoted cell re-growth after drug washout. Overall, our study provides novel insight into the mechanisms by which proteasome inhibition eliminates otherwise apoptosis-resistant cells, and highlights the crucial role of a ligand-independent but TRAIL-R2-dependent activation mechanism for caspase-8 in this scenario., Irish Research Council, European Molecular Biology Organization (EMBO), Deutsche Forschungsgemeinschaft, Projekt DEAL

Death receptor 4 (DR4) is a cell surface protein that is generally thought to mediate apoptosis upon binding to its ligand named TRAIL. However, its contribution to apoptosis resistance has also been reported. MET (or c-MET) gene amplification represents an important mechanism for acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) against EGFR mutant non-small cell lung cancer (NSCLC). This study focuses on demonstrating the impact of MET inhibition on DR4 modulation in MET-amplified EGFR mutant NSCLC cell lines and the underlying mechanisms. Several MET inhibitors decreased DR4 levels in MET-amplified HCC827 cell lines resistant to EGFR-TKIs with no or limited effects on modulating DR5 levels, while increasing DR4 levels in HCC827 parental cells and other NSCLC cell lines. MET inhibitors did not affect DR4 stability, but decreased DR4 mRNA levels with suppression of AP-1-dependent DR4 promoter transactivation. Moreover, these inhibitors suppressed ERK and c-Jun phosphorylation accompanied with decreasing c-Jun levels. Hence, it is likely that MET inhibition downregulates DR4 expression in MET-amplified EGFR mutant NSCLC cells through suppressing AP-1-mediated DR4 transcription. Osimertinib combined with MET inhibition synergistically induces apoptosis in the MET-amplified EGFR mutant NSCLC cells accompanied with augmented DR4 reduction both in vitro and in vivo. Furthermore, MET inhibition combined with TRAIL enhanced killing of MET-amplified EGFR mutant HCC827/AR cells, but not HCC827 parental cells. These data collectively suggest that DR4 may possess an unrecognized anti-apoptotic function, contributing to apoptosis resistance under given conditions.

ONC201/TIC10 activates TRAIL signaling through ATF4 and the integrated stress response (ISR). ONC201 demonstrated tumor regressions and disease stability in patients with histone H3K27M-mutated midline-glioma. H3K27M-mutation prevents H3K27-methylation on the mutated allele. EZH2 inhibitors (EZH2i) reduce H3K27 methylation and have anti-tumor effects. We hypothesized ONC201 sensitivity and tumor apoptosis may increase by reducing H3K27-methylation with EZH2i or HDACi as mimics of H3K27M-mutation. EZH2i EPZ-6438 (tazemetostat) or PF-06821497 and HDACi vorinostat were combined with ONC201 to treat multiple cancer cell lines and cell viability and histone modifications were analyzed. We observed synergistic effects towards cell viability in multiple cancers by EPZ-6438 or PF-06821497 plus ONC201 or triple therapy with vorinostat, EPZ-6438, and ONC201. EPZ-6438 and vorinostat synergized with ONC201 to enhance apoptosis. Activation of the ISR and TRAIL-DR5 were observed in cells treated with ONC201 -/+ epigenetic modulators. Knockdown of ATF4 reduced DR5 induction and apoptosis following EZH2i and ONC201 treatment of U251 glioma cells. mRNA expression of dopamine-receptors did not correlate with ONC201 sensitivity in the tumor cell lines tested (N = 12), including changes after epigenetic drugs. Dopamine did not rescue apoptosis by ONC201 in different tumor cell lines (N = 10) including 2 GBM, 3 DIPG and did not prevent DR5 activation or apoptosis. DRD2 agonist sumanirole did not protect brain tumor cells (N = 6 including 4 DIPG cell lines) from ONC201 reduction in viability. Although synergy was observed with ONC201 and vorinostat, there was no significant increase in H3K27 acetylation in cell lines including DIPG as compared to vorinostat alone, and in some cases the acetylation was less than vorinostat alone at 72 H. H3K27 methylation reduction correlated with synergy from combinations of either EPZ-6438 or vorinostat with ONC201 or triple combination. Our findings provide a rationale for combination of ONC201 and epigenetic modulators including triple therapy for in vivo and clinical testing in treatment of human malignancies including brain tumors and DIPG.

TNF-related apoptosis-inducing ligand (TRAIL) receptor 2 (TRAIL-R2) can induce apoptosis in cancer cells upon crosslinking by TRAIL. However, TRAIL-R2 is highly expressed by many cancers suggesting pro-tumor functions. Indeed, TRAIL/TRAIL-R2 also activate pro-inflammatory pathways enhancing tumor cell invasion, migration, and proliferation. In addition, nuclear TRAIL-R2 (nTRAIL-R2) promotes malignancy by inhibiting miRNA let-7-maturation. Here, we show that TRAIL-R2 interacts with the tumor suppressor protein p53 in the nucleus, assigning a novel pro-tumor function to TRAIL-R2. Knockdown of TRAIL-R2 in p53 wild-type cells increases the half-life of p53 and the expression of its target genes, whereas its re-expression decreases p53 protein levels. Interestingly, TRAIL-R2 also interacts with promyelocytic leukemia protein (PML), a major regulator of p53 stability. PML-nuclear bodies are also the main sites of TRAIL-R2/p53 co-localization. Notably, knockdown or destruction of PML abolishes the TRAIL-R2-mediated regulation of p53 levels. In summary, our finding that nTRAIL-R2 facilitates p53 degradation and thereby negatively regulates p53 target gene expression provides insight into an oncogenic role of TRAIL-R2 in tumorigenesis that particularly manifests in p53 wild-type tumors.

Colorectal cancer (CRC) remains a leading causes of cancer-related death in the world, mainly due to the lack of effective treatment of advanced disease. TNF-related apoptosis-inducing ligand (TRAIL)-driven cell death, a crucial event in the control of tumor growth, selectively targets malignant rather than non-transformed cells. However, the fact that cancer cells, including CRC cells, are either intrinsically resistant or acquire resistance to TRAIL, represents a major hurdle to the use of TRAIL-based strategies in the clinic. Agents able to overcome CRC cell resistance to TRAIL have thus great therapeutic potential and many researchers are making efforts to identify TRAIL sensitizers. The anthelmintic drug rafoxanide has recently emerged as a potent anti-tumor molecule for different cancer types and we recently reported that rafoxanide restrained the proliferation of CRC cells, but not of normal colonic epithelial cells, both in vitro and in a preclinical model mimicking sporadic CRC. As these findings were linked with the induction of endoplasmic reticulum stress, a phenomenon involved in the regulation of various components of the TRAIL-driven apoptotic pathway, we sought to determine whether rafoxanide could restore the sensitivity of CRC cells to TRAIL. Our data show that rafoxanide acts as a selective TRAIL sensitizer in vitro and in a syngeneic experimental model of CRC, by decreasing the levels of c-FLIP and survivin, two key molecules conferring TRAIL resistance. Collectively, our data suggest that rafoxanide could potentially be deployed as an anti-cancer drug in the combinatorial approaches aimed at overcoming CRC cell resistance to TRAIL-based therapies.

TRAIL, a member of TNF superfamily, is a potent inducer of neuronal death. Neurotoxic effects of TRAIL appear mediated by its death receptor TRAIL-R2/DR5. To assess the role of TRAIL/TRAIL-R2 pathway in AD-related neurodegeneration, we studied the impact of the treatment with amyloid-β (Aβ) upon cell viability and inflammation in TRAIL-R-deficient mice (TRAIL-R-/-). Here, we demonstrate that the lack of TRAIL-R2 protects from death cultured TRAIL-R-/- mouse embryonic hippocampal cells undergone treatment with either Aβ1–42 or TRAIL. Consistently, stereotaxic injection of Aβ1–42 resulted in blunted caspase activation, as well as in reduction of JNK phosphorylation and increased AKT phosphorylation in TRAIL-R-/- mice. Moreover, the lack of TRAIL-R2 was associated with blunted constitutive p53 expression in mice undergone Aβ1–42 treatment, as well as in decrease of phosphorylated forms of tau and GSK3β proteins. Likewise, TRAIL-R2 appears essential to both TRAIL and Aβ-mediated neurotoxicity and inflammation. Indeed, hippocampi of TRAIL-R-/- mice challenged with Aβ1–42, showed a scanty expression of microglial (Iba-1) and astrocytic (GFAP) markers along with attenuated levels of IL-1β, TNF-α, NOS2 and COX2. In conclusion, the bulk of these results demonstrate that the constitutive lack of TRAIL-R2 is associated with a substantial reduction of noxious effects of Aβ1–42, providing further evidence on the prominent role played by TRAIL in course of Aβ-related neurodegeneration and confirming that the TRAIL system represents a potential target for innovative AD therapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is selectively lethal to cancer cells and harmless to normal cells, making it a potential agent for cancer therapy. However, some breast cancer cells are resistant to TRAIL. This study revealed that andrographolide (Andro), an extract from Andrographis paniculate, a natural compound, sensitized breast cancer cells to TRAIL-induced tumor suppression; it identified apoptosis-associated protein regulation, reactive oxygen species accumulation, mitochondria membrane potential disruption, caspase cascade activation, and gasdermin-E cleavage to be involved in the tumor lethality mediated by Andro combined with TRAIL treatment. The flow cytometry results showed the combination of Andro and TRAIL repressed breast cancer cells by cell death induction, and the assessment of combined index indicated that the combined treatment with Andro and TRAIL repressed breast cancer cells synergistically. Blotting results displayed Andro and TRAIL combination elevated TRAIL-associated receptors, death receptors 4 and 5, at protein levels. These results provided critical insight into breast cancer patients' therapy and exploration direction for TRAIL clinical application.

First-line therapy with interferon beta (IFN-β), involved in gene expression modulation in immune response, is widely used for multiple sclerosis. However, 30-50% of patients do not respond optimally. Variants in CBLB, CTSS, GRIA3, OAS1 and TNFRSF10A genes have been proposed to contribute to the variation in the individual response. The purpose of this study was to evaluate the influence of gene polymorphisms on the IFN-β response in relapsing-remitting multiple sclerosis (RRMS) patients. CBLB (rs12487066), GRIA3 (rs12557782), CTSS (rs1136774), OAS1 (rs10774671) and TNFRSF10A (rs20576) polymorphisms were analysed by Taqman in 137 RRMS patients. Response to IFN-β and change in the Expanded Disability Status Scale (EDSS) after 24 months were evaluated using multivariable logistic regression analysis. Carriers of at least one copy of the C allele of CTSS-rs1136774 had a better response to IFN-β (p = 0.0423; OR = 2.94; CI95% = 1.03, 8.40). Carriers of TT genotype of TNFRSF10A-rs20576 had a higher probability of maintaining their EDSS stable after 24 months of IFN-β treatment (p = 0.0251; OR = 5.71; CI95% = 1.39, 31.75). No influence of CBLB (rs12487066), OAS1 (rs10774671) and GRIA3 (rs12557782) gene polymorphisms in the variation of the individual response to IFN-β was shown. Our results suggest that the TNFRSF10A-rs20576 and CTSS-rs1136774 gene polymorphisms influence the response to IFN-β after 24 months, while the CBLB (rs12487066), OAS1 (rs10774671) or GRIA3 (rs12557782) gene polymorphisms had no effect on the variation of the individual response to IFN-β.

An extensive body of research has demonstrated that pulmonary toxicity induced by fluoride is related to cell apoptosis. Although induction of death receptor-initiated extrinsic apoptosis by sodium fluoride (NaF) has been reported, its mechanism of action is still not clearly defined. Herein, we found that NaF treatment induced activation of caspase-8 in BEAS-2B cells, resulting in apoptosis, which was markedly reduced by blocking caspase-8 using small interfering RNA (siRNA). In this study, we report that death receptor 5 (DR5), a major component of the extrinsic apoptotic pathway, is markedly induced upon NaF stimulation. Enhanced DR5 induction was necessary for the apoptotic effects of NaF, inasmuch as transfected BEAS-2B cells with DR5 siRNA attenuated NaF-induced caspase-8 activation in lung cells. Mechanism investigation indicated that the induction of DR5, following NaF exposure, was mediated by tumor protein 53 (p53)-dependent transcriptional activation. Notably, we demonstrated that NaF could induce a significant increase in intracellular reactive oxygen species (ROS) level derived from nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4). Specifically, NOX4 knockdown inhibited NaF-induced the activation of p53/DR5 axis by reducing NOX4-derived ROS production. Further in vivo investigation demonstrated that NOX4 deficiency markedly attenuates NaF-induced lung injury, apoptosis, and ROS levels in the lung. Moreover, the expressions of p53 and DR5 were significantly reduced after NaF treatment in NOX4 knockout mice compared with the wild type mice. Taken together, our findings provide a novel insight into for the pulmonary apoptosis in response to NaF exposure.

Due to the absence of curative treatments for glioblastoma (GBM), we assessed the efficacy of single and combination treatments with a translationally relevant 2nd generation TRAIL-receptor agonist (IZI1551) and the blood–brain barrier (BBB) permeant proteasome inhibitor marizomib in a panel of patient-derived glioblastoma cell lines. These cells were cultured using protocols that maintain the characteristics of primary tumor cells. IZI1551+marizomib combination treatments synergistically induced apoptotic cell death in the majority of cases, both in 2D, as well as in 3D spheroid cultures. In contrast, single-drug treatments largely failed to induce noticeable amounts of cell death. Kinetic analyses suggested that time-shifted drug exposure might further increase responsiveness, with marizomib pre-treatments indeed strongly enhancing cell death. Cell death responses upon the addition of IZI1551 could also be observed in GBM cells that were kept in a medium collected from the basolateral side of a human hCMEC/D3 BBB model that had been exposed to marizomib. Interestingly, the subset of GBM cell lines resistant to IZI1551+marizomib treatments expressed lower surface amounts of TRAIL death receptors, substantially lower amounts of procaspase-8, and increased amounts of cFLIP, suggesting that apoptosis initiation was likely too weak to initiate downstream apoptosis execution. Indeed, experiments in which the mitochondrial apoptosis threshold was lowered by antagonizing Mcl-1 re-established sensitivity to IZI1551+marizomib in otherwise resistant cells. Overall, our study demonstrates a high efficacy of combination treatments with a latest-generation TRAIL receptor agonist and the BBB permeant proteasome inhibitor marizomib in relevant GBM cell models, as well as strategies to further enhance responsiveness and to sensitize subgroups of otherwise resistant GBM cases., European Union’s Horizon 2020 research and innovation program, Deutsche Forschungsgemeinschaft, Projekt DEAL

AimsTo identify single-nucleotide polymorphisms (SNPs) associated with central serous chorioretinopathy (CSCR) by a systematic review and meta-analysis, and to compare the association profiles between CSCR, neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV).MethodsWe searched the EMBASE, PubMed and Web of Science for genetic studies of CSCR from the starting dates of the databases to 12 September 2020. We then performed meta-analyses on all SNPs reported by more than two studies and calculated the pooled OR and 95% CIs. We also conducted sensitivity analysis and adopted the funnel plot to assess potential publication bias.ResultsTotally 415 publications were reviewed, among them 10 were eligible for meta-analysis. We found 10 SNPs that have been reported at least twice. Meta-analysis and sensitivity analysis confirmed significant associations between CSCR and six SNPs in three genes, namely age-related maculopathy susceptibility 2 (ARMS2) (rs10490924, OR=1.37; p=0.00064), complement factor H (CFH) (rs800292, OR=1.44; p=7.80×10−5; rs1061170, OR=1.34; p=0.0028; rs1329428, OR=1.40; p=0.012; and rs2284664, OR=1.36; p=0.0089) and tumour necrosis factor receptor superfamily, member 10a (TNFRSF10A) (rs13278062, OR=1.34; p=1.44×10−15). Among them, onlyTNFRSF10Ars13278062 showed the same trend of effect on CSCR, nAMD and PCV, while the SNPs inARMS2andCFHshowed opposite trends in the SNP associations.ConclusionsThis study confirmed the associations ofARMS2,CFHandTNFRSF10Awith CSCR, and revealed thatARMS2,CFHandTNFRSF10Amay affect different phenotypic expressions of CSCR, nAMD and PCV.

The nanoscale spatial organization of transmembrane tumor necrosis factor (TNF) receptors has been implied as a regulator of cellular fate. Accordingly, molecular tools that can induce specific arrangements of these receptors on cell surfaces would give us an opportunity to study these effects in detail. To achieve this, we introduce DNA origami nanostructures, that precisely scaffold the patterning of TNF-related apoptosis-inducing ligand (TRAIL)-mimicking peptides at nanoscale level. Stimulating human breast cancer cells with these patterns, we find that around 5 nm is the critical inter-ligand distance of hexagonally patterned peptides to induce death receptor clustering and a resulting apoptosis. We thus offer a strategy to reverse the non-efficacy of current ligand- and antibody-based methods for TNF superfamily (TNFRSF) activation.

TRAIL can activate cell surface death receptors, resulting in potent tumor cell death via induction of the extrinsic apoptosis pathway. Eftozanermin alfa (ABBV-621) is a second generation TRAIL receptor agonist engineered as an IgG1-Fc mutant backbone linked to two sets of trimeric native single-chain TRAIL receptor binding domain monomers. This hexavalent agonistic fusion protein binds to the death-inducing DR4 and DR5 receptors with nanomolar affinity to drive on-target biological activity with enhanced caspase-8 aggregation and death-inducing signaling complex formation independent of FcγR-mediated cross-linking, and without clinical signs or pathologic evidence of toxicity in nonrodent species. ABBV-621 induced cell death in approximately 36% (45/126) of solid cancer cell lines in vitro at subnanomolar concentrations. An in vivo patient-derived xenograft (PDX) screen of ABBV-621 activity across 15 different tumor indications resulted in an overall response (OR) of 29% (47/162). Although DR4 (TNFSFR10A) and/or DR5 (TNFSFR10B) expression levels did not predict the level of response to ABBV-621 activity in vivo, KRAS mutations were associated with elevated TNFSFR10A and TNFSFR10B and were enriched in ABBV-621–responsive colorectal carcinoma PDX models. To build upon the OR of ABBV-621 monotherapy in colorectal cancer (45%; 10/22) and pancreatic cancer (35%; 7/20), we subsequently demonstrated that inherent resistance to ABBV-621 treatment could be overcome in combination with chemotherapeutics or with selective inhibitors of BCL-XL. In summary, these data provide a preclinical rationale for the ongoing phase 1 clinical trial (NCT03082209) evaluating the activity of ABBV-621 in patients with cancer. Significance: This study describes the activity of a hexavalent TRAIL-receptor agonistic fusion protein in preclinical models of solid tumors that mechanistically distinguishes this molecular entity from other TRAIL-based therapeutics.

Head and neck squamous cell carcinomas (HNSCC) induced by human papillomavirus (HPV) have increased recently in the US. However, the distinct alterations of molecules involved in the death pathways and drug effects targeting inhibitor of apoptosis proteins (IAPs) have not been extensively characterized in HPV(+) HNSCC cells. In this study, we observed the distinct genomic and expression alterations of nine genes involved in cell death in 55% HNSCC tissues, which were associated with HPV status, tumor staging, and anatomic locations. Expression of four genes was statistically correlated with copy number variation. A panel of HPV(+) HNSCC lines showed abundant TRAILR2 and IAP1 protein expression, but were not sensitive to IAP inhibitor birinapant alone, while combinatory treatment with TNFα or especially TRAIL enhanced this drug sensitivity. The death agonistic TRAILR2 antibody alone showed no cell inhibitory effects, whereas its combination with birinapant and/or TRAIL protein demonstrated additive or synergistic effects. We observed predominantly late apoptosis mode of cell death after combinatorial treatments, and pan-caspase (ZVAD) and caspase-8 (ZIETD) inhibitors attenuated treatment-induced cell death. Our genomic and expression data-driven study provides a framework for identifying relevant combinatorial therapies targeting death pathways in HPV(+) HNSCC and other squamous cancer types.

TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, can induce apoptosis in cancer cells, sparing normal cells when bound to its associated death receptors (DR4/DR5). This unique mechanism makes TRAIL a potential anticancer therapeutic agent. However, clinical trials of recombinant TRAIL protein and TRAIL receptor agonist monoclonal antibodies have shown disappointing results due to its short half-life, poor pharmacokinetics and the resistance of the cancer cells. This review summarizes TRAIL-induced apoptotic and survival pathways as well as mechanisms leading to apoptotic resistance. Recent development of methods to overcome cancer cell resistance to TRAIL-induced apoptosis, such as protein modification, combination therapy and TRAIL-based gene therapy, appear promising. We also discuss the challenges and opportunities in the development of TRAIL-based therapies for the treatment of human cancers.

Death receptor 4 (DR4), a cell surface receptor, mediates apoptosis or induces inflammatory cytokine secretion upon binding to its ligand depending on cell contexts. Its prognostic impact in lung cancer and connection between EGFR-targeted therapy and DR4 modulation has not been reported and thus was the focus of this study. Methods: Intracellular protein alterations were measured by Western blotting. Cell surface protein was detected with antibody staining and flow cytometry. mRNA expression was monitored with qRT-PCR. Gene transactivation was analyzed with promoter reporter assay. Drug dynamic effects in vivo were evaluated using xenografts. Gene modulations were achieved with gene overexpression and knockdown. Proteins in human archived tissues were stained with immunohistochemistry. Results: EGFR inhibitors (e.g., osimertinib) decreased DR4 levels only in EGFR mutant NSCLC cells and tumors, being tightly associated with induction of apoptosis. This modulation was lost once cells became resistant to these inhibitors. Increased levels of DR4 were detected in cell lines with acquired osimertinib resistance and in NSCLC tissues relapsed from EGFR-targeted therapy. DR4 knockdown induced apoptosis and augmented apoptosis when combined with osimertinib in both sensitive and resistant cell lines, whereas enforced DR4 expression significantly attenuated osimertinib-induced apoptosis. Mechanistically, osimertinib induced MARCH8-mediated DR4 proteasomal degradation and suppressed MEK/ERK/AP-1-dependent DR4 transcription, resulting in DR4 downregulation. Moreover, we found that DR4 positive expression in human lung adenocarcinoma was significantly associated with poor patient survival. Conclusions: Collectively, we suggest that DR4 downregulation is coupled to therapeutic efficacy of EGFR-targeted therapy and predicts improved prognosis, revealing a previously undiscovered connection between EGFR-targeted therapy and DR4 modulation.

BackgroundAdoptive cell therapy (ACT) using genetically modified T cells has evolved into a promising treatment option for patients with cancer. However, even for the best-studied and clinically validated CD19-targeted chimeric antigen receptor (CAR) T-cell therapy, many patients face the challenge of lack of response or occurrence of relapse. There is increasing need to improve the efficacy of ACT so that durable, curative outcomes can be achieved in a broad patient population.MethodsHere, we investigated the impact of indomethacin (indo), a non-steroidal anti-inflammatory drug (NSAID), on the efficacy of ACT in multiple preclinical models. Mice with established B-cell lymphoma received various combinations of preconditioning chemotherapy, infusion of suboptimal dose of tumor-reactive T cells, and indo administration. Donor T cells used in the ACT models included CD4+ T cells expressing a tumor-specific T cell receptor (TCR) and T cells engineered to express CD19CAR. Mice were monitored for tumor growth and survival. The effects of indo on donor T cell phenotype and function were evaluated. The molecular mechanisms by which indo may influence the outcome of ACT were investigated.ResultsACT coupled with indo administration led to improved tumor growth control and prolonged mouse survival. Indo did not affect the activation status and tumor infiltration of the donor T cells. Moreover, the beneficial effect of indo in ACT did not rely on its inhibitory effect on the immunosuppressive cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis. Instead, indo-induced oxidative stress boosted the expression of death receptor 5 (DR5) in tumor cells, rendering them susceptible to donor T cells expressing TNF-related apoptosis-inducing ligand (TRAIL). Furthermore, the ACT-potentiating effect of indo was diminished against DR5-deficient tumors, but was amplified by donor T cells engineered to overexpress TRAIL.ConclusionOur results demonstrate that the pro-oxidative property of indo can be exploited to enhance death receptor signaling in cancer cells, providing rationale for combining indo with genetically modified T cells to intensify tumor cell killing through the TRAIL-DR5 axis. These findings implicate indo administration, and potentially similar use of other NSAIDs, as a readily applicable and cost-effective approach to augment the efficacy of ACT.

Ubiquitin-specific protease 1 (USP1) is a deubiquitinase involved in DNA damage repair by modulating the ubiquitination of major regulators, such as PCNA and FANCD2. Because USP1 is highly expressed in many cancers, dysregulation of USP1 contributes to cancer therapy. However, the role of USP1 and the mechanisms underlying chemotherapy remain unclear. In this study, we found high USP1 expression in tumor tissues and that it correlated with poor prognosis in RCC. Mechanistically, USP1 enhanced survivin stabilization by removing ubiquitin. Pharmacological inhibitors (ML23 and pimozide) and siRNA targeting USP1 induced downregulation of survivin expression. In addition, ML323 upregulated DR5 expression by decreasing miR-216a-5p expression at the post-transcriptional level, and miR-216a-5p mimics suppressed the upregulation of DR5 by ML323. Inhibition of USP1 sensitized cancer cells. Overexpression of survivin or knockdown of DR5 markedly prevented the co-treatment with ML323 and TRAIL-induced apoptosis. These results of in vitro were proved in a mouse xenograft model, in which combined treatment significantly reduced tumor size and induced survivin downregulation and DR5 upregulation. Furthermore, USP1 and survivin protein expression showed a positive correlation, whereas miR-216a-5p and DR5 were inversely correlated in RCC tumor tissues. Taken together, our results suggest two target substrates of USP1 and demonstrate the involvement of survivin and DR5 in USP1-targeted chemotherapy.

NT157, a small-molecule tyrosine kinase inhibitor, exhibits broad-spectrum anti-tumor activity. However, NT157-mediated inhibition against glioma has not been explored yet. Herein, the anticancer effects and underlying mechanism of NT157 against human giloma growth were evaluated. The results showed that NT157 alone significantly inhibited glioma cells growth in vitro by lunching cell cycle arrest through up-regulating p21 and p27, and down-regulating cell cycle-related factors. NT157 alone also induced significant glioma cells apoptosis, followed by PARP cleavage and caspase-3 activation. Our findings further revealed that NT157 triggered significant DNA damage and dysfunction of PI3K/AKT, MAPKs and EGFR-STAT3 signaling pathways. Addition of several kinases inhibitors effectively abrogated NT157-induced DR5 up-regulation, which further confirmed the significant role of DR5 pathway. Moreover, combined treatment of NT157 and TRAIL showed enhanced apoptosis against U251 and U87 cells. However, Knockdown of DR5 expression significantly attenuated combined treatment-induced PARP cleavage and caspase-3 activation. Importantly, combined administration of NT157 and TRAIL in vivo effectively inhibited glioma xenograft growth of nude mice by inhibiting cell proliferation and angiogenesis, and inducing DNA damage and apoptosis. Taken together, our findings validated the rational design that combined strategy of NT157 and TRAIL to trigger DNA damage and apoptosis by up-regulating DR5 could be a high efficient way to combat human glioma.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces cancer cell death by binding to TRAIL receptors. Because of its selective cytotoxicity toward cancer cells, TRAIL therapeutics, such as recombinant TRAIL and agonistic antibodies targeting TRAIL receptors, have garnered attention as promising cancer treatment agents. However, many cancer cells acquire resistance to TRAIL-induced cell death. To overcome this issue, we searched for agents to sensitize cancer cells to TRAIL-induced cell death by screening a small-molecule chemical library consisting of diverse compounds. We identified a cardiac glycoside, proscillaridin A, as the most effective TRAIL sensitizer in colon cancer cells. Proscillaridin A synergistically enhanced TRAIL-induced cell death in TRAIL-sensitive and -resistant colon cancer cells. Additionally, proscillaridin A enhanced cell death in cells treated with TRAIL and TRAIL sensitizer, the second mitochondria-derived activator of caspase mimetic. Proscillaridin A upregulated TRAIL receptor expression, while downregulating the levels of the anti-cell death molecules, cellular FADD-like IL-1β converting enzyme-like inhibitor protein and Mcl1, in a cell type-dependent manner. Furthermore, proscillaridin A enhanced TRAIL-induced cell death partly via O-glycosylation. Taken together, our findings suggest that proscillaridin A is a promising agent that enhances the anti-cancer efficacy of TRAIL therapeutics.

Prostaglandin (PG) A

In the last two decades, bifunctional proteins have been created by genetic and protein engineering methods to increase therapeutic effects in various diseases, including cancer. Unlike conventional small molecule or monotargeted drugs, bifunctional proteins have increased biological activity while maintaining low systemic toxicity. The recombinant anti-cancer cytokine TRAIL has shown a limited therapeutic effect in clinical trials. To enhance the efficacy of TRAIL, we designed the HRH–DR5-B fusion protein based on the DR5-selective mutant variant of TRAIL fused to the anti-angiogenic synthetic peptide HRHTKQRHTALH. Initially low expression of HRH–DR5-B was enhanced by the substitution of E. coli-optimized codons with AT-rich codons in the DNA sequence encoding the first 7 amino acid residues of the HRH peptide. However, the HRH–DR5-B degraded during purification to form two adjacent protein bands on the SDS-PAGE gel. The replacement of His by Ser at position P2 immediately after the initiator Met dramatically minimized degradation, allowing more than 20 mg of protein to be obtained from 200 mL of cell culture. The resulting SRH–DR5-B fusion bound the VEGFR2 and DR5 receptors with high affinity and showed increased cytotoxic activity in 3D multicellular tumor spheroids. SRH–DR5-B can be considered as a promising candidate for therapeutic applications.

The ethanol extract from the wood ofTaxus Yunnanensis(TY) induced apoptosis in all cancer cell lines tested, which was mainly due to activation of an extrinsic pathway in human colon cancer DLD-1 cells. The extrinsic pathway was activated by the upregulation of the expression levels of Fas and TRAIL/DR5, which led to the activation of caspase-8. Of note, the machinery of this increase in expression was promoted by the upregulation of MIR32a expression, which silenced MIR34a-targeting E2F3 transcription factor. Furthermore, ectopic expression of MIR32a or siR-E2F3 silencingE2F3increased Fas and TRAIL/DR5 expression. Thus, the extract activated the extrinsic pathway through the MIR34a/E2F3 axis, resulting in the autocrine and paracrine release of TRAIL, and upregulated expression of death receptors Fas and DR5 in the treated DLD-1 cells, which were functionally validated by Fas immunocytochemistry, and using anti-Fas and anti-TRAIL antibodies, respectively. In vivo, TY showed significant anti-tumor effects on xenografted and syngeneic model mice. The extract may also aid in chemoprevention by selectively making marked tumor cells susceptible to the tumor immunosurveillance system.

Costimulatory signals potently promote T‐cell proliferation and effector function. Agonistic antibodies targeting costimulatory receptors of the TNFR family, such as 4‐1BB and CD27, have entered clinical trials in cancer patients. Currently there is limited information how costimulatory signals regulate antigen‐specific but also bystander activation of human CD8 T cells. Engineered antigen presenting cells (eAPC) efficiently presenting several common viral epitopes on HLA‐A2 in combination with MHC class I tetramer staining were used to investigate the impact of costimulatory signals on human CD8 T‐cell responses. CD28 costimulation potently augmented the percentage and number of antigen‐reactive CD8 T cells, whereas eAPC expressing 4‐1BB‐ligand induced bystander proliferation of CD8 T cells and massive expansion of NK cells. Moreover, the 4‐1BB agonist urelumab similarly induced bystander proliferation of CD8 T cells and NK cells in a dose‐dependent manner. However, the promotion of bystander CD8 T‐cell responses is not a general attribute of costimulatory TNF receptor superfamily (TNFRSF) members, since CD27 signals enhanced antigen‐specific CD8 T cells responses without promoting significant bystander activation. Thus, the differential effects of costimulatory signals on the activation of human bystander CD8 T cells should be taken into account when costimulatory pathways are harnessed for cancer immunotherapy., Using a novel system of engineered antigen presenting cells (eAPC), we demonstrate that 4‐1BB signals generated by 4‐1BBL or agonistic 4‐1BB antibodies promote bystander activation of human CD8 T cells