Design and Synthesis of DNA Origami Nanostructures to Control TNF Receptor Activation.

Clustering of type II tumor necrosis factor (TNF) receptors (TNFRs) is essential for their activation, yet currently available drugs fail to activate signaling. Some strategies aim to cluster TNFR by using multivalent streptavidin or scaffolds based on dextran or graphene. However, these strategies do not allow for control of the valency or spatial organization of the ligands, and consequently control of the TNFR activation is not optimal. DNA origami nanostructures allow nanometer-precise control of the spatial organization of molecules and complexes, with defined spacing, number and valency. Here, we demonstrate the design and characterization of a DNA origami nanostructure that can be decorated with engineered single-chain TNF-related apoptosis-inducing ligand (SC-TRAIL) complexes, which show increased cell killing compared to SC-TRAIL alone on Jurkat cells. The information in this chapter can be used as a basis to decorate DNA origami nanostructures with various proteins, complexes, or other biomolecules.

Targeting Integrin α3 Blocks ß1 Maturation, Triggers Endoplasmic Reticulum Stress, and Sensitizes Glioblastoma Cells to TRAIL-Mediated Apoptosis.

Glioblastoma (GBM) is a devastating brain cancer for which new effective therapies are urgently needed. GBM, after an initial response to current treatment regimens, develops therapeutic resistance, leading to rapid patient demise. Cancer cells exhibit an inherent elevation of endoplasmic reticulum (ER) stress due to uncontrolled growth and an unfavorable microenvironment, including hypoxia and nutrient deprivation. Cancer cells utilize the unfolded protein response (UPR) to maintain ER homeostasis, and failure of this response promotes cell death. In this study, as integrins are upregulated in cancer, we have evaluated the therapeutic potential of individually targeting all αß1 integrin subunits using RNA interference. We found that GBM cells are uniquely susceptible to silencing of integrin α3. Knockdown of α3-induced proapoptotic markers such as PARP cleavage and caspase 3 and 8 activation. Remarkably, we discovered a non-canonical function for α3 in mediating the maturation of integrin ß1. In its absence, generation of full length ß1 was reduced, immature ß1 accumulated, and the cells underwent elevated ER stress with upregulation of death receptor 5 (DR5) expression. Targeting α3 sensitized TRAIL-resistant GBM cancer cells to TRAIL-mediated apoptosis and led to growth inhibition. Our findings offer key new insights into integrin α3's role in GBM survival via the regulation of ER homeostasis and its value as a therapeutic target.

Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

TRAIL and IP-10 dynamics in pregnant women post COVID-19 vaccination: associations with neutralizing antibody potency.

Introduction: The aim of this study is to investigate changes in TNF-related apoptosis-inducing ligand (TRAIL) and gamma interferon-induced protein 10 (IP-10) after COVID-19 vaccination in pregnant women and to explore their association with neutralizing antibody (Nab) inhibition. Methods: The study evaluated 93 pregnant women who had previously received two (n=21), three (n=55) or four (n=17) doses of COVID-19 vaccine. Also we evaluated maternal blood samples that were collected during childbirth. The levels of TRAIL, IP-10 and Nab inhibition were measured using enzyme-linked immunosorbent assays (ELISA). Results and discussion: Our study revealed four-dose group resulted in lower TRAIL levels when compared to the two-dose and three-dose groups (4.78 vs. 16.07 vs. 21.61 pg/ml, p = 0.014). The two-dose group had reduced IP-10 levels than the three-dose cohort (111.49 vs. 147.89 pg/ml, p=0.013), with no significant variation compared to the four-dose group. In addition, the four-dose group showed stronger Nab inhibition against specific strains (BA.2 and BA.5) than the three-dose group. A positive correlation was observed between TRAIL and IP-10 in the two-dose group, while this relationship was not found in other dose groups or between TRAIL/IP-10 and Nab inhibition. As the doses of the COVID-19 vaccine increase, the levels of TRAIL and IP-10 generally increase, only by the fourth dose, the group previously vaccinated with AZD1222 showed lower TRAIL but higher IP-10. Despite these changes, more doses of the vaccine consistently reinforced Nab inhibition, apparently without any relation to TRAIL and IP-10 levels. The variation may indicate the induction of immunological memory in vaccinated mothers, which justifies further research in the future.

Pro-Inflammatory Activation Suppresses TRAIL-induced Apoptosis of Acute Myeloid Leukemia Cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a promising agent for treatment of AML due to its specific apoptosis-inducing effect on tumor cells but not normal cells. However, emergence of resistance to TRAIL in the AML cells limits its potential as an antileukemic agent. Previously, we revealed increase in the resistance of the human AML THP-1 cells to the TRAIL-induced death during their LPS-dependent proinflammatory activation and in the in vitro model of LPS-independent proinflammatory activation - in a long-term high-density cell culture. In this study, we investigated mechanisms of this phenomenon using Western blot analysis, caspase 3 enzymatic activity analysis, quantitative reverse transcription-PCR, and flow cytometry. The results showed that the increased resistance to the TRAIL-induced cell death of AML THP-1 cells during their pro-inflammatory activation is associated with the decrease in the surface expression of the proapoptotic receptors TRAIL-R1/DR4 and TRAIL-R2/DR5, as well as with the increased content of members of the IAPs family - Livin and cIAP2. The results of this article open up new insights into the role of inflammation in formation of the resistance of AML cells to the action of mediators of antitumor immunity, in particular TRAIL.

Glutamine-mediated epigenetic regulation of cFLIP underlies resistance to TRAIL in pancreatic cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it kills cancer cells while sparing normal cells. However, many cancers, including pancreatic ductal adenocarcinoma (PDAC), exhibit intrinsic or acquired resistance to TRAIL, and the molecular mechanisms underlying TRAIL resistance in cancers, particularly in PDAC, remain unclear. In this study, we demonstrated that glutamine (Gln) endows PDAC cells with resistance to TRAIL through KDM4C-mediated epigenetic regulation of cFLIP. Inhibition of glutaminolysis significantly reduced the cFLIP level, leading to TRAIL-mediated formation of death-inducing signaling complexes. Overexpression of cFLIP dramatically rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Alpha-Ketoglutarate (aKG) supplementation significantly reversed the decrease in the cFLIP level induced by glutaminolysis inhibition and rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Knockdown of glutamic-oxaloacetic transaminase 2, which facilitates the conversion of oxaloacetate and glutamate into aspartate and aKG, decreased aKG production and the cFLIP level and activated TRAIL-induced apoptosis. AKG-mediated epigenetic regulation was necessary for maintaining a high level of cFLIP. Glutaminolysis inhibition increased the abundance of H3K9me3 in the cFLIP promoter, indicating that Gln-derived aKG production is important for Jumonji-domain histone demethylase (JHDM)-mediated cFLIP regulation. The JHDM KDM4C regulated cFLIP expression by binding to its promoter, and KDM4C knockdown sensitized PDAC cells to TRAIL-induced apoptosis. The present findings suggest that Gln-derived aKG production is required for KDM4C-mediated epigenetic regulation of cFLIP, which leads to resistance to TRAIL.

DMC-siERCC2 hybrid nanoparticle enhances TRAIL sensitivity by inducing cell cycle arrest for glioblastoma treatment.

ERCC2 plays a pivotal role in DNA damage repair, however, its specific function in cancer remains elusive. In this study, we made a significant breakthrough by discovering a substantial upregulation of ERCC2 expression in glioblastoma (GBM) tumor tissue. Moreover, elevated levels of ERCC2 expression were closely associated with poor prognosis. Further investigation into the effects of ERCC2 on GBM revealed that suppressing its expression significantly inhibited malignant growth and migration of GBM cells, while overexpression of ERCC2 promoted tumor cell growth. Through mechanistic studies, we elucidated that inhibiting ERCC2 led to cell cycle arrest in the G0/G1 phase by blocking the CDK2/CDK4/CDK6/Cyclin D1/Cyclin D3 pathway. Notably, we also discovered a direct link between ERCC2 and CDK4, a critical protein in cell cycle regulation. Additionally, we explored the potential of TRAIL, a low-toxicity death ligand cytokine with anticancer properties. Despite the typical resistance of GBM cells to TRAIL, tumor cells undergoing cell cycle arrest exhibited significantly enhanced sensitivity to TRAIL. Therefore, we devised a combination strategy, employing TRAIL with the nanoparticle DMC-siERCC2, which effectively suppressed the GBM cell proliferation and induced apoptosis. In summary, our study suggests that targeting ERCC2 holds promise as a therapeutic approach to GBM treatment.

Intratumoural delivery of TRAIL mRNA induces colon cancer cell apoptosis.

Novel strategies in intratumoral injection and emerging immunotherapies have heralded a new era of precise cancer treatments. The affinity of SARS-CoV-2 to ACE2 receptors, a feature which facilitates virulent human infection, is leveraged in this research. Colon cancer cells, with their high ACE2 expression, provide a potentially strategic target for using this SARS-CoV-2 feature. While the highly expression of ACE2 is observed in several cancer types, the idea of using the viral spike protein for targeting colon cancer cells offers a novel approach in cancer treatment. Intratumoral delivery of nucleic acid-based drugs is a promising alternative to overcoming the limitations of existing therapies. The increasing importance of nucleic acids in this realm, and the use of Lipid Nanoparticles (LNPs) for local delivery of nucleic acid therapeutics, are important breakthroughs. LNPs protect nucleic acid drugs from degradation and enhance cellular uptake, making them a rapidly evolving nano delivery system with high precision and adaptability. Our study leveraged a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) combined with a receptor-binding domain from the SARS-CoV-2 spike protein, encapsulated in LNPs, to target colon cancer cells. Our results indicated that the TRAIL fusion-mRNA induced apoptosis in vitro and in vivo. Collectively, our findings highlight LNP-encapsulated TRAIL fusion-mRNA as a potential colon cancer therapy.

Aponermin: First Approval.

Aponermin () is a recombinant circularly permuted human tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) developed by Beijing Sunbio Biotech (a wholly owned subsidiary of Wuhan Hiteck Biological Pharma CO., LTD) for the treatment of multiple myeloma. Aponermin binds to and activates the death receptors 4 and/or 5 on tumour cells, triggering intracellular caspase reactions and inducing apoptosis, thereby exerting antitumor effects. In November 2023, aponermin in combination with thalidomide and dexamethasone received its first approval in China for the treatment of patients with relapsed or refractory multiple myeloma who have received at least two prior therapies. This article summarizes the milestones in the development of aponermin leading to this first approval for relapsed or refractory multiple myeloma.

Bacteriophage-based particles carrying the TNF-related apoptosis-inducing ligand (TRAIL) gene for targeted delivery in hepatocellular carcinoma.

The TRAIL (Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand) is a promising candidate for cancer treatment due to its unique ability to selectively induce programmed cell death, or apoptosis, in cancer cells while sparing healthy ones. This selectivity arises from the preferential binding of the TRAIL to death receptors on cancer cells, triggering a cascade of events that lead to their demise. However, significant limitations in using the TRAIL for cancer treatment are the administration of the TRAIL protein that can potentially lead to tissue toxicity (off-target) and the short half-life of the TRAIL in the body which may necessitate frequent and sustained administration; these can pose logistical challenges for long-term treatment regimens. We have devised a novel approach for surmounting these limitations by introducing the TRAIL gene directly into cancer cells, enabling them to produce the TRAIL locally and subsequently trigger apoptosis. A novel gene delivery system such as a bacteriophage-based particle TPA (transmorphic phage/AAV) was utilized to address these limitations. TPA is a hybrid M13 filamentous bacteriophage particle encapsulating a therapeutic gene cassette with inverted terminal repeats (ITRs) from adeno-associated viruses (AAVs). The particle also showed a tumour targeting ligand, CDCRGDCFC (RGD4C), on its capsid (RGD4C.TPA) to target the particle to cancer cells. RGD4C selectively binds to αvß3 and αvß5 integrins overexpressed on the surface of most of the cancer cells but is barely present on normal cells. Hepatocellular carcinoma (HCC) was chosen as a model because it has one of the lowest survival rates among cancers. We demonstrated that human HCC cell lines (Huh-7 and HepG2) express αvß5 integrin receptors on their surface. These HCC cells also express death receptors and TRAIL-binding receptors. We showed that the targeted TPA particle carrying the transmembrane TRAIL gene (RGD4C.TPA-tmTRAIL) selectively and efficiently delivered the tmTRAIL gene to HCC cells resulting in the production of tmTRAIL from transduced cells and subsequently induced apoptotic death of HCC cells. This tumour-targeted particle can be an excellent candidate for the targeted gene therapy of HCC.

Rolapitant treats lung cancer by targeting deubiquitinase OTUD3.

BACKGROUND: Lung cancer is cancer with the highest morbidity and mortality in the world and poses a serious threat to human health. Therefore, discovering new treatments is urgently needed to improve lung cancer prognosis. Small molecule inhibitors targeting the ubiquitin-proteasome system have achieved great success, in which deubiquitinase inhibitors have broad clinical applications. The deubiquitylase OTUD3 was reported to promote lung tumorigenesis by stabilizing oncoprotein GRP78, implying that inhibition of OTUD3 may be a therapeutic strategy for lung cancer. RESULTS: In this study, we identified a small molecule inhibitor of OTUD3, Rolapitant, by computer-aided virtual screening and biological experimental verification from FDA-approved drugs library. Rolapitant inhibited the proliferation of lung cancer cells by inhibiting deubiquitinating activity of OTUD3. Quantitative proteomic profiling indicated that Rolapitant significantly upregulated the expression of death receptor 5 (DR5). Rolapitant also promoted lung cancer cell apoptosis through upregulating cell surface expression of DR5 and enhanced TRAIL-induced apoptosis. Mechanistically, Rolapitant directly targeted the OTUD3-GRP78 axis to trigger endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP)-DR5 signaling, sensitizing lung cancer cells to TRAIL-induced apoptosis. In the vivo assays, Rolapitant suppressed the growth of lung cancer xenografts in immunocompromised mice at suitable dosages without apparent toxicity. CONCLUSION: In summary, the present study identifies Rolapitant as a novel inhibitor of deubiquitinase OTUD3 and establishes that the OTUD3-GRP78 axis is a potential therapeutic target for lung cancer.

Ionizable Lipid Nanoparticle-Mediated TRAIL mRNA Delivery in the Tumor Microenvironment to Inhibit Colon Cancer Progression.

Introduction: Immunotherapy has revolutionized cancer treatment by harnessing the immune system to enhance antitumor responses while minimizing off-target effects. Among the promising cancer-specific therapies, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted significant attention. Methods: Here, we developed an ionizable lipid nanoparticle (LNP) platform to deliver TRAIL mRNA (LNP-TRAIL) directly to the tumor microenvironment (TME) to induce tumor cell death. Our LNP-TRAIL was formulated via microfluidic mixing and the induction of tumor cell death was assessed in vitro. Next, we investigated the ability of LNP-TRAIL to inhibit colon cancer progression in vivo in combination with a TME normalization approach using Losartan (Los) or angiotensin 1-7 (Ang(1-7)) to reduce vascular compression and deposition of extracellular matrix in mice. Results: Our results demonstrated that LNP-TRAIL induced tumor cell death in vitro and effectively inhibited colon cancer progression in vivo, particularly when combined with TME normalization induced by treatment Los or Ang(1-7). In addition, potent tumor cell death as well as enhanced apoptosis and necrosis was found in the tumor tissue of a group treated with LNP-TRAIL combined with TME normalization. Discussion: Together, our data demonstrate the potential of the LNP to deliver TRAIL mRNA to the TME and to induce tumor cell death, especially when combined with TME normalization. Therefore, these findings provide important insights for the development of novel therapeutic strategies for the immunotherapy of solid tumors.

TNF-Related Apoptosis-Inducing Ligand: Non-Apoptotic Signalling.

TNF-related apoptosis-inducing ligand (TRAIL or Apo2 or TNFSF10) belongs to the TNF superfamily. When bound to its agonistic receptors, TRAIL can induce apoptosis in tumour cells, while sparing healthy cells. Over the last three decades, this tumour selectivity has prompted many studies aiming at evaluating the anti-tumoral potential of TRAIL or its derivatives. Although most of these attempts have failed, so far, novel formulations are still being evaluated. However, emerging evidence indicates that TRAIL can also trigger a non-canonical signal transduction pathway that is likely to be detrimental for its use in oncology. Likewise, an increasing number of studies suggest that in some circumstances TRAIL can induce, via Death receptor 5 (DR5), tumour cell motility, potentially leading to and contributing to tumour metastasis. While the pro-apoptotic signal transduction machinery of TRAIL is well known from a mechanistic point of view, that of the non-canonical pathway is less understood. In this study, we the current state of knowledge of TRAIL non-canonical signalling.

TRAIL-induced apoptosis and proteasomal activity - Mechanisms, signalling and interplay.

Programmed cell death, in particular apoptosis, is essential during development and tissue homeostasis, and also is the primary strategy to induce cancer cell death by cytotoxic therapies. Precision therapeutics targeting TRAIL death receptors are being evaluated as novel anti-cancer agents, while in parallel highly specific proteasome inhibitors have gained approval as drugs. TRAIL-dependent signalling and proteasomal control of cellular proteostasis are intricate processes, and their interplay can be exploited to enhance therapeutic killing of cancer cells in combination therapies. This review provides detailed insights into the complex signalling of TRAIL-induced pathways and the activities of the proteasome. It explores their core mechanisms of action, pharmaceutical druggability, and describes how their interplay can be strategically leveraged to enhance cell death responses in cancer cells. Offering this comprehensive and timely overview will allow to navigate the complexity of the processes governing cell death mechanisms in TRAIL- and proteasome inhibitor-based treatment conditions.

Significance of TRAIL/Apo-2 ligand and its death receptors in apoptosis and necroptosis signalling: Implications for cancer-targeted therapeutics.

The human immune defensesystem routinely expresses the tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), which is the most prevalent element for antitumor immunity. TRAIL associates with its death receptors (DRs), DR4 (TRAIL-R1), and DR5 (TRAIL-R2), in cancer cells to initiate the intracellular apoptosis cascade. Accordingly, numerous academic institutions and pharmaceutical companies havetried to exploreTRAIL's capacity to kill tumourcells by producing recombinant versions of it (rhTRAIL) or TRAIL receptor agonists (TRAs) [monoclonal antibody (mAb), synthetic and natural compounds, etc.] and molecules that sensitize TRAIL signalling pathway for therapeutic applications. Recently, several microRNAs (miRs) have been found to activate or inhibit death receptor signalling. Therefore, pharmacological regulation of these miRs may activate or resensitize the TRAIL DRs signal, and this is a novel approach for developing anticancer therapeutics. In this article, we will discuss TRAIL and its receptors and molecular pathways by which it induces various cell death events. We will unravel potential innovative applications of TRAIL-based therapeutics, and other investigated therapeutics targeting TRAIL-DRs and summarize the current preclinical pharmacological studies and clinical trials. Moreover, we will also emphasizea few situations where future efforts may be addressed to modulate the TRAIL signalling pathway.

ASB3 promotes hepatocellular carcinoma progression by mediating DR5 ubiquitination in TRAIL resistance.

Ankyrin-repeat proteins with a suppressor of cytokine signaling box (ASB) proteins belong to the E3 ubiquitin ligase family. 18 ASB members have been identified whose biological functions are mostly unexplored. Here, we discovered that ASB3 was essential for hepatocellular carcinoma (HCC) development and high ASB3 expression predicted poor clinical outcomes. ASB3 silencing induced HCC cell growth arrest and apoptosis in vitro and in vivo. Liver-specific deletion of Asb3 gene suppressed diethylnitrosamine (DEN)-induced liver cancer development. Mechanistically, ASB3 interacted with death receptor 5 (DR5), which promoted ubiquitination and degradation of DR5. We further showed that ASB3 knockdown stabilized DR5 and increased the sensitivity of liver cancer cells to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a DR5-dependent manner in cellular and in animal models. In summary, we demonstrated that ASB3 promoted ubiquitination and degradation of DR5 in HCC, suggesting the potential of targeting ASB3 to HCC treatment and overcome TRAIL resistance.

Strophanthidin Induces Apoptosis of Human Lung Adenocarcinoma Cells by Promoting TRAIL-DR5 Signaling.

Strophanthidin (SPTD), one of the cardiac glycosides, is refined from traditional Chinese medicines such as Semen Lepidii and Antiaris toxicaria, and was initially used for the treatment of heart failure disease in clinic. Recently, SPTD has been shown to be a potential anticancer agent, but the underlying mechanism of action is poorly understood. Herein, we explored the molecular mechanism by which SPTD exerts anticancer effects in A549 human lung adenocarcinoma cells by means of mass spectrometry-based quantitative proteomics in combination with bioinformatics analysis. We revealed that SPTD promoted the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2, or DR5) in A549 cells to activate caspase 3/6/8, in particular caspase 3. Consequently, the activated caspases elevated the expression level of apoptotic chromatin condensation inducer in the nucleus (ACIN1) and prelamin-A/C (LMNA), ultimately inducing apoptosis via cooperation with the SPTD-induced overexpressed barrier-to-autointegration factor 1 (Banf1). Moreover, the SPTD-induced DEPs interacted with each other to downregulate the p38 MAPK/ERK signaling, contributing to the SPTD inhibition of the growth of A549 cells. Additionally, the downregulation of collagen COL1A5 by SPTD was another anticancer benefit of SPTD through the modulation of the cell microenvironment.

E-cadherin re-expression: Its potential in combating TRAIL resistance and reversing epithelial-to-mesenchymal transition.

The major limitation of conventional chemotherapy drugs is their lack of specificity for cancer cells. As a selective apoptosis-inducing agent, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has emerged as an attractive alternative. However, most of the cancer cells are found to be either intrinsically resistant to the TRAIL protein or may develop resistance after multiple treatments, and TRAIL resistance can induce epithelial-to-mesenchymal transition (EMT) at a later stage, promoting cancer invasion and migration. Interestingly, E-cadherin loss has been linked to TRAIL resistance and initiation of EMT, making E-cadherin re-expression a potential target to overcome these obstacles. Recent research suggests that re-expressing E-cadherin may reduce TRAIL resistance by enhancing TRAIL-induced apoptosis and preventing EMT by modulating EMT signalling factors. This reversal of EMT, can also aid in improving TRAIL-induced apoptosis. Therefore, this review provides remarkable insights into the mechanisms underlying E-cadherin re-expression, clinical implications, and potentiation, as well as the research gaps of E-cadherin re-expression in the current cancer treatment.

Heparan sulfate promotes TRAIL-induced tumor cell apoptosis.

TRAIL (TNF-related apoptosis-inducing ligand) is a potent inducer of tumor cell apoptosis through TRAIL receptors. While it has been previously pursued as a potential anti-tumor therapy, the enthusiasm subsided due to unsuccessful clinical trials and the fact that many tumors are resistant to TRAIL. In this report, we identified heparan sulfate (HS) as an important regulator of TRAIL-induced apoptosis. TRAIL binds HS with high affinity (KD = 73 nM) and HS induces TRAIL to form higher-order oligomers. The HS-binding site of TRAIL is located at the N-terminus of soluble TRAIL, which includes three basic residues. Binding to cell surface HS plays an essential role in promoting the apoptotic activity of TRAIL in both breast cancer and myeloma cells, and this promoting effect can be blocked by heparin, which is commonly administered to cancer patients. We also quantified HS content in several lines of myeloma cells and found that the cell line showing the most resistance to TRAIL has the least expression of HS, which suggests that HS expression in tumor cells could play a role in regulating sensitivity towards TRAIL. We also discovered that death receptor 5 (DR5), TRAIL, and HS can form a ternary complex and that cell surface HS plays an active role in promoting TRAIL-induced cellular internalization of DR5. Combined, our study suggests that TRAIL-HS interactions could play multiple roles in regulating the apoptotic potency of TRAIL and might be an important point of consideration when designing future TRAIL-based anti-tumor therapy.

The E3 ubiquitin ligase Itch regulates death receptor and cholesterol trafficking to affect TRAIL-mediated apoptosis.

The activation of apoptosis signalling by TRAIL (TNF-related apoptosis-inducing ligand) through receptor binding is a fundamental mechanism of cell death induction and is often perturbed in cancer cells to enhance their cell survival and treatment resistance. Ubiquitination plays an important role in the regulation of TRAIL-mediated apoptosis, and here we investigate the role of the E3 ubiquitin ligase Itch in TRAIL-mediated apoptosis in oesophageal cancer cells. Knockdown of Itch expression results in resistance to TRAIL-induced apoptosis, caspase-8 activation, Bid cleavage and also promotes cisplatin resistance. Whilst the assembly of the death-inducing signalling complex (DISC) at the plasma membrane is not perturbed relative to the control, TRAIL-R2 is mis-localised in the Itch-knockdown cells. Further, we observe significant changes to mitochondrial morphology alongside an increased cholesterol content. Mitochondrial cholesterol is recognised as an important anti-apoptotic agent in cancer. Cells treated with a drug that increases mitochondrial cholesterol levels, U18666A, shows a protection from TRAIL-induced apoptosis, reduced caspase-8 activation, Bid cleavage and cisplatin resistance. We demonstrate that Itch knockdown cells are less sensitive to a Bcl-2 inhibitor, show impaired activation of Bax, cytochrome c release and an enhanced stability of the cholesterol transfer protein STARD1. We identify a novel protein complex composed of Itch, the mitochondrial protein VDAC2 and STARD1. We propose a mechanism where Itch regulates the stability of STARD1. An increase in STARD1 expression enhances cholesterol import to mitochondria, which inhibits Bax activation and cytochrome c release. Many cancer types display high mitochondrial cholesterol levels, and oesophageal adenocarcinoma tumours show a correlation between chemotherapy resistance and STARD1 expression which is supported by our findings. This establishes an important role for Itch in regulation of extrinsic and intrinsic apoptosis, mitochondrial cholesterol levels and provides insight to mechanisms that contribute to TRAIL, Bcl-2 inhibitor and cisplatin resistance in cancer cells.