Proteome and phosphoproteome profiling of non-small cell lung cancer cell line A549 treated with TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is recognized for its promising therapeutic effects against cancer. However, mechanisms underlying the effect of TRAIL on protein expression, signal transduction, and apoptosis induction remain unclear. We surmised that a systematic analysis of the proteome and phosphoproteome associated with TRAIL signaling may help elucidate the mechanisms involved and facilitate the development of therapeutics. Therefore, we investigated the proteome and phosphoproteome of non-small cell lung cancer cell line A549 treated with TRAIL. Our results indicated that 126 proteins and 1684 phosphosites were markedly differentially expressed between the phosphate-buffered saline- and TRAIL-treated groups. The expression at protein and phosphosite levels were not completely consistent. Gene ontology functional analysis revealed that metal ion (zinc) binding was highly affected by TRAIL treatment. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that almost all pathways that involved differentially expressed phosphosites were associated with apoptosis. We also identified an important kinase, AKT1, and its series of substrates in TRAIL signaling. The results of this study may provide guidance for future research on tumor therapy using TRAIL.

Regulation of death receptor signaling by the autophagy protein TP53INP2.

TP53INP2 positively regulates autophagy by binding to Atg8 proteins. Here, we uncover a novel role of TP53INP2 in death-receptor signaling. TP53INP2 sensitizes cells to apoptosis induced by death receptor ligands. In keeping with this, TP53INP2 deficiency in cultured cells or mouse livers protects against death receptor-induced apoptosis. TP53INP2 binds caspase-8 and the ubiquitin ligase TRAF6, thereby promoting the ubiquitination and activation of caspase-8 by TRAF6. We have defined a TRAF6-interacting motif (TIM) and a ubiquitin-interacting motif in TP53INP2, enabling it to function as a scaffold bridging already ubiquitinated caspase-8 to TRAF6 for further polyubiquitination of caspase-8. Mutations of key TIM residues in TP53INP2 abrogate its interaction with TRAF6 and caspase-8, and subsequently reduce levels of death receptor-induced apoptosis. A screen of cancer cell lines showed that those with higher protein levels of TP53INP2 are more prone to TRAIL-induced apoptosis, making TP53INP2 a potential predictive marker of cancer cell responsiveness to TRAIL treatment. These findings uncover a novel mechanism for the regulation of caspase-8 ubiquitination and reveal TP53INP2 as an important regulator of the death receptor pathway.

The Prognostic, Diagnostic, and Therapeutic Potential of TRAIL Signalling in Cardiovascular Diseases.

TNF-related apoptosis-inducing ligand (TRAIL) was originally discovered, almost 20 years ago, for its ability to kill cancer cells. More recent evidence has described pleiotropic functions, particularly in the cardiovascular system. There is potential for TRAIL concentrations in the circulation to act as prognostic and/or diagnostic factors for cardiovascular diseases (CVD). Pre-clinical studies also describe the therapeutic capacity for TRAIL signals, particularly in the context of atherosclerotic disease and diseases of the myocardium. Because diabetes mellitus significantly contributes to the progression and pathogenesis of CVDs, in this review we highlight recent evidence for the prognostic, diagnostic, and therapeutic potential of TRAIL signals in CVDs, and where relevant, the impact of diabetes mellitus. A greater understanding of how TRAIL signals regulate cardiovascular protection and pathology may offer new diagnostic and therapeutic avenues for patients suffering from CVDs.

Escaping cell death via TRAIL decoy receptors: a systematic review of their roles and expressions in colorectal cancer.

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.

Improving Targeted Delivery and Antitumor Efficacy with Engineered Tumor Necrosis Factor-Related Apoptosis Ligand-Affibody Fusion Protein.

Tumor necrosis factor-related apoptosis ligand (TRAIL) is a promising protein candidate for selective apoptosis of a variety of cancer cells. However, the short half-life and a lack of targeted delivery are major obstacles for its application in cancer therapy. Here, we propose a simple strategy to solve the targeting problem by genetically fusing an anti-HER2 affibody to the C-terminus of the TRAIL. The fusion protein TRAIL-affibody was produced as a soluble form with high yield in recombinant Escherichia coli. In vitro studies proved that the affibody domain promoted the cellular uptake of the fusion protein in the HER2 overexpressed SKOV-3 cells and improved its apoptosis-inducing ability. In addition, the fusion protein exhibited higher accumulation at the tumor site and greater antitumor effect than those of TRAIL in vivo, indicating that the affibody promoted the tumor homing of the TRAIL and then improved the therapeutic efficacy. Importantly, repeated injection of high-dose TRAIL-affibody showed no obvious toxicity in mice. These results demonstrated that the engineered TRAIL-affibody is promising to be a highly tumor-specific and targeted cancer therapeutic agent.

Revisiting the role of TRAIL/TRAIL-R in cancer biology and therapy.

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.

OSMI-1 Enhances TRAIL-Induced Apoptosis through ER Stress and NF-κB Signaling in Colon Cancer Cells.

Levels of O-GlcNAc transferase (OGT) and hyper-O-GlcNAcylation expression levels are associated with cancer pathogenesis. This study aimed to find conditions that maximize the therapeutic effect of cancer and minimize tissue damage by combining an OGT inhibitor (OSMI-1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). We found that OSMI-1 treatment in HCT116 human colon cancer cells has a potent synergistic effect on TRAIL-induced apoptosis signaling. Interestingly, OSMI-1 significantly increased TRAIL-mediated apoptosis by increasing the expression of the cell surface receptor DR5. ROS-induced endoplasmic reticulum (ER) stress by OSMI-1 not only upregulated CHOP-DR5 signaling but also activated Jun-N-terminal kinase (JNK), resulting in a decrease in Bcl2 and the release of cytochrome c from mitochondria. TRAIL induced the activation of NF-κB and played a role in resistance as an antiapoptotic factor. During this process, O-GlcNAcylation of IκB kinase (IKK) and IκBα degradation occurred, followed by translocation of p65 into the nucleus. However, combination treatment with OSMI-1 counteracted the effect of TRAIL-mediated NF-κB signaling, resulting in a more synergistic effect on apoptosis. Therefore, the combined treatment of OSMI-1 and TRAIL synergistically increased TRAIL-induced apoptosis through caspase-8 activation. Conclusively, OSMI-1 potentially sensitizes TRAIL-induced cell death in HCT116 cells through the blockade of NF-κB signaling and activation of apoptosis through ER stress response.

Repression of Death Receptor-Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen.

Hepatitis B virus (HBV) e antigen (HBeAg) is associated with viral persistence and pathogenesis. Resistance of HBV-infected hepatocytes to apoptosis is seen as one of the primary promotors for HBV chronicity and malignancy. Fas receptor/ligand (Fas/FasL) and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) system plays a key role in hepatic death during HBV infection. We found that HBeAg mediates resistance of hepatocytes to FasL or TRAIL-induced apoptosis. Introduction of HBeAg into human hepatocytes rendered resistance to FasL or TRAIL cytotoxicity in a p53-dependent manner. HBeAg further inhibited the expression of p53, total Fas, membrane-bound Fas, TNF receptor superfamily member 10a, and TNF receptor superfamily member 10b at both mRNA and protein levels. In contrast, HBeAg enhanced the expression of soluble forms of Fas through facilitation of Fas alternative mRNA splicing. In a mouse model, expression of HBeAg in mice injected with recombinant adenovirus-associated virus 8 inhibited agonistic anti-Fas antibody-induced hepatic apoptosis. Xenograft tumorigenicity assay also found that HBeAg-induced carcinogenesis was resistant to the proapoptotic effect of TRAIL and chemotherapeutic drugs. These results indicate that HBeAg may prevent hepatocytes from FasL and TRAIL-induced apoptosis by regulating the expression of the proapoptotic and antiapoptotic forms of death receptors, which may contribute to the survival and persistence of infected hepatocytes during HBV infection.

Glioma progression is suppressed by Naringenin and APO2L combination therapy via the activation of apoptosis in vitro and in vivo.

Naringenin (NG) is a natural antioxidant flavonoid which is isolated from citrus fruits, and has been reported to inhibit colon cancer proliferation. However, the effects of NG treatment on glioma remain to be elucidated. The present study aimed to explore the effects of NG on glioma in vitro and in vivo. Also, the interactions between NG and APO2 ligand (APO2L; also known as tumor necrosis factor-related apoptosis-inducing ligand) were investigated in glioma. A synergistic effect of NG and APO2L combination on apoptotic induction was observed, though glioma cells were insensitive to APO2L alone. After NG treatment, glioma cells resumed the sensitivity to APO2L and cell apoptosis was induced via the activation of caspases, elevation of decoy receptors 4 and 5 (DR4 and DR5) and induction of p53. Coadministration of NG and APO2L decreased levels of anti-apoptotic B cell lymphoma 2 (Bcl-2) family members Bcl-2 and Bcl-extra large (Bcl-xL), while increased levels of proapoptotic factors Bcl-2-associated agonist of cell death (Bad) and Bcl-2 antagonist/killer 1 (Bak). Furthermore, an in vivo mouse xenograft model demonstrated that NG and APO2L cotreatment markedly suppressed glioma growth by activating apoptosis in tumor tissues when compared with NG or APO2L monotherapy. The present study provides a novel therapeutic strategy for glioma by potentiating APO2L-induced apoptosis via the combination with NG in glioma tumor cells.

Designing combination therapies with modeling chaperoned machine learning.

Chemotherapy resistance is a major challenge to the effective treatment of cancer. Thus, a systematic pipeline for the efficient identification of effective combination treatments could bring huge biomedical benefit. In order to facilitate rational design of combination therapies, we developed a comprehensive computational model that incorporates the available biological knowledge and relevant experimental data on the life-and-death response of individual cancer cells to cisplatin or cisplatin combined with the TNF-related apoptosis-inducing ligand (TRAIL). The model's predictions, that a combination treatment of cisplatin and TRAIL would enhance cancer cell death and exhibit a "two-wave killing" temporal pattern, was validated by measuring the dynamics of p53 accumulation, cell fate, and cell death in single cells. The validated model was then subjected to a systematic analysis with an ensemble of diverse machine learning methods. Though each method is characterized by a different algorithm, they collectively identified several molecular players that can sensitize tumor cells to cisplatin-induced apoptosis (sensitizers). The identified sensitizers are consistent with previous experimental observations. Overall, we have illustrated that machine learning analysis of an experimentally validated mechanistic model can convert our available knowledge into the identity of biologically meaningful sensitizers. This knowledge can then be leveraged to design treatment strategies that could improve the efficacy of chemotherapy.

Sequential Delivery and Cascade Targeting of Peptide Therapeutics for Triplexed Synergistic Therapy with Real-Time Monitoring Shuttled by Magnetic Gold Nanostars.

Due to the outstanding synergistic effects and low-toxicity, combination therapy exhibits more considerable potential in antitumor activity than monotherapy. Herein, a core-shell magnetic gold nanostar (Fe3O4@GNS, MGNS)-based system for codelivery of a mitochondrial targeting amphipathic tail-anchoring peptide (ATAP) and a membrane-associated cytokine (tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) was constructed. The magnetic core can facilitate delivery of the drug vehicle by external magnetic field, which results in accurate accumulation and enhances tumor cellular uptake for preliminary targeting. TRAIL and ATAP could sequentially target and be released toward the plasma membrane and mitochondria, initiating the extrinsic and intrinsic apoptosis pathways, respectively. The gold shell of MGNS can cause local tumor hyperthermia due to broad-band plasmon resonances in the near-infrared region, which can act as a complement with the peptide drug to further enhance apoptosis. Both in vitro and in vivo experiments revealed that rationally integrating extrinsic apoptosis, intrinsic apoptosis and hyperthermia for triplexed synergistic therapy, enabled the smart drug vehicle with pinpoint peptide drug delivery capabilities, and minimized side effects, enhancing the antitumor efficiency.

Non-viral nucleic acid delivery to the central nervous system and brain tumors.

Gene therapy is a rapidly emerging remedial route for many serious incurable diseases, such as central nervous system (CNS) diseases. Currently, nucleic acid medicines, including DNAs encoding therapeutic or destructive proteins, small interfering RNAs or microRNAs, have been successfully delivered to the CNS with gene delivery vectors using various routes of administration and have subsequently exhibited remarkable therapeutic efficiency. Among these vectors, non-viral vectors are favorable for delivering genes into the CNS as a result of their many special characteristics, such as low toxicity and pre-existing immunogenicity, high gene loading efficiency and easy surface modification. In this review, we highlight the main types of therapeutic genes that have been applied in the therapy of CNS diseases and then outline non-viral gene delivery vectors.

Combination therapy with c-met inhibitor and TRAIL enhances apoptosis in dedifferentiated liposarcoma patient-derived cells.

BACKGROUND: Liposarcoma (LPS) is a tumor derived from adipose tissue, and has the highest incidence among soft tissue sarcomas. Dedifferentiated liposarcoma (DDLPS) is a malignant tumor with poor prognosis. Recurrence and metastasis rates in LPS remain high even after chemotherapy and radiotherapy following complete resection. Therefore, the development of advanced treatment strategies for LPS is required. In the present study, we investigated the effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment, and of combination treatment using TRAIL and a c-Met inhibitor on cell viability and apoptosis in LPS and DDLPS cell lines of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment, and of combination treatment using TRAIL and a c-Met inhibitor. METHODS: We analyzed cell viability after treatment with TRAIL and a c-Met inhibitor by measuring CCK8 and death receptor 5 (DR5) expression levels via fluorescence activated cell sorting (FACS) in both sarcoma cell lines and DDLPS patient-derived cells (PDCs). Moreover, we validated the effects of TRAIL alone and in combination with c-Met inhibitor on apoptosis in LPS cell lines and DDLPS PDCs via FACS. RESULTS: Our results revealed that combination treatment with a c-Met inhibitor and human recombinant TRAIL (rhTRAIL) suppressed cell viability and induced cell death in both sarcoma cell lines and DDLPS PDCs, which showed varying sensitivities to rhTRAIL alone. Also, we confirmed that treatment with a c-Met inhibitor upregulated DR5 levels in sarcoma cell lines and DDLPS PDCs. In both TRAIL-susceptible and TRAIL-resistant cells subjected to combination treatment, promotion of apoptosis was dependent on DR5 upregulation. CONCLUSION: From these results, our findings validated that DR5 up-regulation caused by combination therapy with a c-Met inhibitor and rhTRAIL enhanced TRAIL sensitization and promoted apoptosis. We propose the use of this approach to overcome TRAIL resistance and serve as a novel treatment strategy for clinical trials.

Identification of miRNA-7 by genome-wide analysis as a critical sensitizer for TRAIL-induced apoptosis in glioblastoma cells.

Glioblastoma (GBM) is still one of the most lethal forms of brain tumor despite of the improvements in treatments. TRAIL (TNF-related apoptosis-inducing ligand) is a promising anticancer agent that can be potentially used as an alternative or complementary therapy because of its specific antitumor activity. To define the novel pathways that regulate susceptibility to TRAIL in GBM cells, we performed a genome-wide expression profiling of microRNAs in GBM cell lines with the distinct sensitivity to TRAIL-induced apoptosis. We found that the expression pattern of miR-7 is closely correlated with sensitivity of GBM cells to TRAIL. Furthermore, our gain and loss of function experiments showed that miR-7 is a potential sensitizer for TRAIL-induced apoptosis in GBM cells. In the mechanistic study, we identified XIAP is a direct downstream gene of miR-7. Additionally, this regulatory axis could also exert in other types of tumor cells like hepatocellular carcinoma cells. More importantly, in the xenograft model, enforced expression of miR-7 in TRAIL-overexpressed mesenchymal stem cells increased apoptosis and suppressed tumor growth in an exosome dependent manner. In conclusion, we identify that miR-7 is a critical sensitizer for TRAIL-induced apoptosis, thus making it as a promising therapeutic candidate for TRAIL resistance in GBM cells.

Role of CCL20 mediated immune cell recruitment in NF-κB mediated TRAIL resistance of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers. From a clinical view, the transcription factor NF-κB is of particular importance, since this pathway confers apoptosis resistance and limits drug efficacy. Whereas the role of the most abundant NF-κB subunit p65/RelA in therapeutic resistance is well documented, only little knowledge of the RelA downstream targets and their functional relevance in TRAIL mediated apoptosis in PDAC is available. In the present study TRAIL resistant and sensitive PDAC cell lines were analyzed for differentially expressed RelA target genes, to define RelA downstream targets mediating TRAIL resistance. The most upregulated target gene was then further functionally characterized. Unbiased genome-wide expression analysis demonstrated that the chemokine CCL20 represents the strongest TRAIL inducible direct RelA target gene in resistant PDAC cells. Unexpectedly, targeting CCL20 by siRNA, blocking antibodies or by downregulation of the sole CCL20 receptor CCR6 had no effect on PDAC cell death or cancer cell migration, arguing against an autocrine role of CCL20 in PDAC. However, by using an ex vivo indirect co-culture system we were able to show that CCL20 acts paracrine to recruit immune cells. Importantly, CCL20-recruited immune cells further increase TRAIL resistance of CCL20-producing PDAC cells. In conclusion, our data show a functional role of a RelA-CCL20 pathway in PDAC TRAIL resistance. We demonstrate how the therapy-induced cross-talk of cancer cells with immune cells affects treatment responses, knowledge needed to tailor novel bi-specific treatments, which target tumor cell as well as immune cells.

miR-494 Sensitizes Gastric Cancer Cells to TRAIL Treatment Through Downregulation of Survivin.

BACKGROUND/AIMS: TNF-related apoptosis-inducing ligand (TRAIL) is a novel and low-toxic anti-tumor drug used for various cancers. However, cancer cells usually develop mechanisms to acquire the resistance against TRAIL. Among these changes, dysregulation of microRNAs (miRNAs) usually occurs in cancer cells and is responsible for induction of drug resistance. METHODS: Expression of miR-494 in gastric cancer tissues and cell lines was detected by quantitative reverse transcriptase real time PCR (qRT-PCR) analysis. Effect of miR-494 on regulating the TRAIL sensitivity to gastric cancer cell lines was evaluated by MTT assays. Bioinformatics and luciferase reporter assays were used to confirm the regulation of miR-494 on survivin. Mitochondrial apoptosis pathway in gastric cancer cells was tested by western blot and flow cytometry analysis. RESULTS: Obvious downregulation of miR-494 was observed in gastric cancer cells. Furthermore, we found that expression profile of miR-494 was associated with TRAIL-sensitivity in gastric cancer. Enforced expression of miR-494 was found to sensitize the gastric cancer cells to TRAIL-induced cytotoxicity. Mechanically, Luciferase reporter assays proved that survivin was the target of miR-494 in gastric cancer cells. Enforced expression of miR-494 decreased the expression of survivin, and thus promoted the TRAIL-induced mitochondria collapse and apoptosis pathway. CONCLUSION: MiR-494/survivin axis represents a potential mechanism which is responsible for TRAIL resistance in gastric cancer cells. Increasing the miR-494 expression may serve as a novel therapeutic strategy to sensitize gastric cancer cells to TRAIL treatment.

Aldehyde Dehydrogenase 1 (ALDH1) Promotes the Toxicity of TRAIL in Non-Small Cell Lung Cancer Cells via Post-Transcriptional Regulation of MEK-1 Expression.

BACKGROUND/AIM: Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL)-based therapies have been used in many human cancers. However, some tumors are resistant to TRAIL-induced cell death. Aldehyde dehydrogenase 1 (ALDH1) is a functional marker for identification of CSCs. METHODS: In this study, we used the colony formation assay, AnnexinV/ PI double staining and PI staining to detect proliferation, apoptosis and cell cycle in ALDH1+ non-small cell lung cancer (NSCLC) cells with TRAIL treatment. In addition, we established xenograft mouse models to confirm the anti-tumor roles of TRAIL in vivo. Finally, gene array and western blot were used to detect the deeper mechanism of the susceptibility of ALDH1+ NSCLC cells to TRAIL. RESULTS: We confirmed that TRAIL could inhibit proliferation, and induce apoptosis and G1 arrest in ALDH1+ NSCLC cells. Correspondingly, TRAIL was associated with decreased tumor size and the favorable survival rate of ALDH1+ cells established xenograft mouse models. ALDH1 could increase the death receptors (DR) 4 and DR5 expression in ALDH1+ NSCLC cells via activating MEK/ERK signaling pathway. CONCLUSION: ALDH1 protein induced MEK-1 mRNA stability and promoted its translation via its 3'UTR.

N-Acetyl-Glucosamine Sensitizes Non-Small Cell Lung Cancer Cells to TRAIL-Induced Apoptosis by Activating Death Receptor 5.

BACKGROUND/AIMS: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anti-cancer agent due to its selective toxicity. However, many human non-small cell lung cancer (NSCLC) cells are partially resistant to TRAIL, thereby limiting its clinical application. Therefore, there is a need for the development of novel adjuvant therapeutic agents to be used in combination with TRAIL. METHODS: In this study, the effect of N-acetyl-glucosamine (GlcNAc), a type of monosaccharide derived from chitosan, combined with TRAIL was evaluated in vitro and in vivo. Thirty NSCLC clinical samples were used to detect the expression of death receptor (DR) 4 and 5. After GlcNAc and TRAIL co-treatment, DR expression was determined by real-time PCR and western blotting. Cycloheximide was used to detect the protein half-life to further understand the correlation between GlcNAc and the metabolic rate of DR. Non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to detect receptor clustering, and the localization of DR was visualized by immunofluorescence under a confocal microscope. Furthermore, a co-immunoprecipitation assay was performed to analyze the formation of death-inducing signaling complex (DISC). O-linked glycan expression levels were evaluated following DR5 overexpression and RNA interference mediated knockdown. RESULTS: We found that the clinical samples expressed higher levels of DR5 than DR4, and GlcNAc co-treatment improved the effect of TRAIL-induced apoptosis by activating DR5 accumulation and clustering, which in turn recruited the apoptosis-initiating protease caspase-8 to form DISC, and initiated apoptosis. Furthermore, GlcNAc promoted DR5 clustering by improving its O-glycosylation. CONCLUSION: These results uncovered the molecular mechanism by which GlcNAc sensitizes cancer cells to TRAIL-induced apoptosis, thereby highlighting a novel effective agent for TRAIL-mediated NSCLC-targeted therapy.

Phase III study of dulanermin (recombinant human tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand) combined with vinorelbine and cisplatin in patients with advanced non-small-cell lung cancer.

Background Dulanermin is a recombinant soluble human Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that activates apoptotic pathways by binding to proapoptotic death receptor (DR) 4 and DR5. The purpose of this study was to evaluate the efficacy and safety of dulanermin combined with vinorelbine and cisplatin (NP) as the first-line treatment for patients with advanced non-small-cell lung cancer (NSCLC). Experimental design Patients were randomly assigned to receive NP chemotherapy (vinorelbine 25 mg/m2 on days 1 and 8 and cisplatin 30 mg/m2 on days 2 to 4) for up to six cycles plus dulanermin (75 µg/kg on days 1 to 14) or placebo every three weeks until disease progression, intolerable toxicity, or withdrawal of consent. The primary end point was progression-free survival (PFS), and the secondary end points included objective response rate (ORR), overall survival (OS), and safety evaluation. Results Between October 2009 and June 2012, 452 untreated patients with stage IIIB to IV NSCLC were randomly assigned to receive dulanermin plus NP (n = 342) and placebo plus NP (n = 110). Median PFS was 6.4 months in the dulanermin arm versus 3.5 months in the placebo arm (hazard ratio (HR), 0.4034; 95% CI, 0.3181 to 0.5117, p < 0.0001). ORR was 46.78% in the dulanermin arm versus 30.00% in the placebo arm (p = 0.0019). Median OS was 14.6 months in the dulanermin arm versus 13.9 months in the placebo arm (HR, 0.94; 95% CI, 0.74 to 1.21, p = 0.64). The most common grade ≥ 3 adverse events (AEs) were oligochromemia, leukopenia, neutropenia, and oligocythemia. Overall incidence of AEs, grade ≥ 3 AEs, and serious AEs were similar across the two arms. Conclusion Addition of dulanermin to the NP regimen significantly improved PFS and ORR. However, our results showed that the combination of dulanermin with chemotherapy had a synergic activity and favorable toxic profile in the treatment of patients with advanced NSCLC.

Development of acquired resistance to lapatinib may sensitise HER2-positive breast cancer cells to apoptosis induction by obatoclax and TRAIL.

BACKGROUND: Lapatinib has clinical efficacy in the treatment of trastuzumab-refractory HER2-positive breast cancer. However, a significant proportion of patients develop progressive disease due to acquired resistance to the drug. Induction of apoptotic cell death is a key mechanism of action of lapatinib in HER2-positive breast cancer cells. METHODS: We examined alterations in regulation of the intrinsic and extrinsic apoptosis pathways in cell line models of acquired lapatinib resistance both in vitro and in patient samples from the NCT01485926 clinical trial, and investigated potential strategies to exploit alterations in apoptosis signalling to overcome lapatinib resistance in HER2-positive breast cancer. RESULTS: In this study, we examined two cell lines models of acquired lapatinib resistance (SKBR3-L and HCC1954-L) and showed that lapatinib does not induce apoptosis in these cells. We identified alterations in members of the BCL-2 family of proteins, in particular MCL-1 and BAX, which may play a role in resistance to lapatinib. We tested the therapeutic inhibitor obatoclax, which targets MCL-1. Both SKBR3-L and HCC1954-L cells showed greater sensitivity to obatoclax-induced apoptosis than parental cells. Interestingly, we also found that the development of acquired resistance to lapatinib resulted in acquired sensitivity to TRAIL in SKBR3-L cells. Sensitivity to TRAIL in the SKBR3-L cells was associated with reduced phosphorylation of AKT, increased expression of FOXO3a and decreased expression of c-FLIP. In SKBR3-L cells, TRAIL treatment caused activation of caspase 8, caspase 9 and caspase 3/7. In a second resistant model, HCC1954-L cells, p-AKT levels were not decreased and these cells did not show enhanced sensitivity to TRAIL. Furthermore, combining obatoclax with TRAIL improved response in SKBR3-L cells but not in HCC1954-L cells. CONCLUSIONS: Our findings highlight the possibility of targeting altered apoptotic signalling to overcome acquired lapatinib resistance, and identify potential novel treatment strategies, with potential biomarkers, for HER2-positive breast cancer that is resistant to HER2 targeted therapies.