Death Receptors DR5 Research Articles

LDL accelerates monocyte to macrophage differentiation: Effects on adhesion and anoikis

Background and aimsHigh LDL triggers dyslipidemia and atherosclerosis, a chronic inflammatory disease with participation of the innate immunity system. Monocytes are recruited to areas of LDL-induced endothelial damage and initiate differentiation. This study was aimed to investigate the effects of LDL on the early transitional stages of monocyte differentiation into macrophages. MethodsBlood monocytes, isolated from healthy donors by their adhesion properties, were exposed to native-LDL (1.80 mg/mL) for 48-h. Monocyte phenotype was assessed at transcript and miRNA levels by real-time PCR. Protein-expression was determined by western-blot and flow-cytometry. ResultsCD14 time-dependently decreased in adhered monocytes, reaching a >4fold decrease at transcript- and protein-levels after 7-days in culture when cells were already differentiated into macrophages. At 4-days differentiation, monocytes exposed to LDL reduced CD14-transcrition >1.5fold in mRNA (p = 0.002) and 34% CD14-protein (p = 0.039), whereas increased in CD16-expression (p = 0.019). Besides, LDL induced a significant increase in integrin CD49c (α3-subunit) at mRNA (>2fold, p = 0.008) and protein (>3fold, p = 0.045) level and a decrease in the apoptosis-effectors CASP8 and CASP3 (p = 0.002 and p = 0.035, respectively) as well as in the precursor form of the death-receptor DR5 (p = 0.045) without affecting its mRNA-expression level, suggesting a LDL-dependent post-transcriptional regulation of DR5. In silico prediction analysis indicated miR-126-3p as a candidate to regulate DR5-expression and miR-126-3p was shown affected by LDL reaching a significant increase (p = 0.033). ConclusionsIn differentiating human monocytes, LDL stimulates expression of cell-adhesion molecules and downregulates apoptosis-effectors, regulating anoikis and survival programs in the early stage macrophages.

Dual Agonist Surrobody Simultaneously Activates Death Receptors DR4 and DR5 to Induce Cancer Cell Death.

Death receptors of the TNF family are found on the surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors 4 and 5 (DR4 and DR5) is TNF-related apoptosis-inducing ligand, TRAIL (Apo2L). As most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing proapoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 monospecific antibodies. Taken together, Surrobody shows promising preclinical proapoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent.

Abstract LB-A16: Novel Small Molecule ONC201 Induces Cell Death in Triple Negative and Non-triple Negative Breast Cancer Cells

Abstract Breast cancer is a major cause of cancer related death in United States women, and there is an urgent need for novel targeted therapies with low toxicity and increased efficacy. The TRAIL pathway has been of interest in the field of oncology, but resistance has been identified in cancer cell lines and primary tumors including those of the breast. Only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL, with other breast cancer molecular subtypes being resistant (Rahman et al., Breast Cancer Res. Treat., 2008). Clinical translation of these findings has been limited, as TRAIL receptor agonistic antibodies have shown minimal effects. We identified a small molecule inducer of the TRAIL pathway, ONC201/TIC10 (Allen et al., Sci. Trans. Med., 2013) that functions through dual inhibition of AKT/ERK, induction of the ATF4/CHOP pathway (Kline et al., Submitted, 2015) and cytotoxic effects dependent on TRAIL/DR5 upregulation. Recently, ONC201 completed its first-in-man phase 1 clinical trial in advanced solid tumors that defined its safety, pharmacokinetics and recommended phase II dose (Stein et al., Abstract C138, 2015 AACR-EORTC meeting). The objective of this work was to investigate ONC201 efficacy in TNBC (TRAIL-sensitive) and non-TNBC (TRAIL-resistant) cells. Using cell viability assays, we demonstrate IC50 values for ONC201 in the low micromolar range for both TNBC (n = 6) and non-TNBC cells (n = 5). These doses are achievable based on human PK. The evaluated non-TNBC cell lines, including those with estrogen receptor positivity and HER2 amplifications, have been shown to be highly resistant to TRAIL therapy. Propidium iodide staining and analysis of SubG1 DNA content indicates that ONC201 induces apoptosis in both TNBC and non-TNBC cells. We also show that a single dose of ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 triple negative human breast cancer xenograft model. The effects of ONC201 on well-known mechanisms of TRAIL-resistance, such as increases in inhibitor of apoptosis (IAP) family proteins, were investigated to explain sensitivity of TRAIL-resistant breast cancer cells to ONC201. We observed that ONC201 mediates a decrease in expression of IAP family proteins XIAP, c-IAP1, and c-IAP2 across TNBC and non-TNBC cell lines. We have shown that XIAP levels correlate with sensitivity to ONC201-induced apoptosis (Kline et al., Submitted, 2015). We also examined levels of death receptors DR4 and DR5 using flow cytometry, as a known mechanism of TRAIL resistance involves constitutive death receptor endocytosis from the cell surface (Zhang et al., Mol. Cancer Res., 2008). Interestingly, increases in cell surface DR4 and DR5 levels are greater in TNBC cell lines when compared with non-TNBC cell lines. Overall, our findings suggest that ONC201 exerts cytotoxic effects against a broad range of breast cancer cells, including TNBC and non-TNBC subtypes. These effects are observed regardless of the TRAIL sensitivity of the cells, and may be mediated through mechanisms involving downregulation of anti-apoptotic proteins and upregulation of cell surface death receptors. Our work will further understanding of these mechanisms and contribute to development of a preclinical rationale for the use of ONC201 as a treatment for breast cancers. Citation Format: Marie D. Baumeister, Jessica Wagner, Christina L.B. Kline, Joshua E. Allen, David T. Dicker, Wafik S. El-Deiry. Novel Small Molecule ONC201 Induces Cell Death in Triple Negative and Non-triple Negative Breast Cancer Cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A16.

Goniothalamin enhances TRAIL-induced apoptosis in colorectal cancer cells through DR5 upregulation and cFLIP downregulation.

The combination of TNF-related apoptosis-inducing ligand (TRAIL) and bioactive compound to enhance apoptosis in TRAIL-resistant cancer is one of cancer treatment strategies. TRAIL possesses the unique capacity to selectively induce apoptosis in cancer cells both in vitro and in vivo with little effect on normal cells. Recent studies have reported that there are many TRAIL-resistant cancers. Thus, bioactive compounds that enhance cytotoxicity of TRAIL would be potential candidates for cancer therapeutic application. This study evaluated the cytotoxic and apoptosis induction upon combined treatment of TRAIL and goniothalamin, the natural styryl-lactone compound extracted from plant Goniothalamus spp., in LoVo cells. The results showed that a combination of goniothalamin and TRAIL enhanced caspase-dependent apoptosis induction in LoVo cells via both death receptor- and mitochondrial-mediated apoptosis pathways. In addition, goniothalamin enhanced TRAIL-induced apoptosis through increased death receptor DR5 expression and decreased anti-apoptotic regulator cFLIP. Interestingly, goniothalamin increased translocation of DR5 to cell surface and consequently contributed to the enhancement of TRAIL-induced apoptosis. In conclusion, this is the first report showing the combined treatment of goniothalamin and TRAIL was able to effectively enhance TRAIL-mediated apoptosis induction in TRAIL-refractory colorectal cancer, LoVo cells. Therefore, this study may offer a strategic cancer treatment against TRAIL-resistant cancers.

Associations of Crohn's disease with DR4 and DR5 gene polymorphisms

To assess the associations of death receptor DR4 and DR5 gene polymorphisms with Crohn's disease (CD). A total of 295 CD patients and 490 healthy controls were recruited. Three single nucleotide polymorphisms (SNPs) of the DR4 (rs13278062, rs20575) and DR5 (rs1047266) genes were determined with a SNaPshot method. Unconditional logistic regression analysis was carried out for determining the allelic and genotypic differences of the three SNPs between CD patients and the controls, as well as the influence of the DR4 and DR5 gene polymorphisms on the clinical features of CD patients. Linkage disequilibrium and haplotype analysis were calculated by haplotype 4.2 and R language software. A gene-gene interaction model was established to analyze whether the three SNPs can exert a synergistic effect on the susceptibility to CD. The mutant allele (T) and genotype (GT+TT) of DR4 (rs13278062) were increased among CD patients compared to the controls (37.12% vs. 32.04%, P = 0.040, 95%CI: 1.010-1.550; 62.71% vs. 54.90%, P = 0.032, 95%CI: 1.028-1.855, respectively). However, the allelic and genotypic frequencies of DR4 (rs20575) and DR5 (rs1047266) did not differ between the two groups (all P > 0.05). Based on the Montreal Classification Standards, the CD patients were stratified by locations and behaviors of the disease. After multiple comparison correction (P < 0.0125), compared to ileocolonic CD patients respectively, the mutant allele (T) and genotype (GT+TT) of the rs13278062 polymorphism were significantly increased in colonic CD patients (41.04% vs. 25.64%, P = 0.002, 95%CI: 0.315-0.778; 66.04% vs. 41.03%, P = 0.001, 95%CI: 0.196-0.655, respectively) and terminal ileum CD patients (41.44% vs. 25.64%, P = 0.002, 95%CI: 0.311-0.762; 74.77% vs. 41.03%, P < 0.001, 95%CI: 0.126-0.437, respectively). In comparison to penetrating CD patients, the mutant allele (T) and genotype (GT+TT) of DR4 (rs13278062) were significantly decreased in stricturing CD patients (32.29% vs. 48.91%, P = 0.007, 95%CI: 0.300-0.828; 57.29% vs. 86.96%, P = 0.001, 95%CI: 0.078-0.520, respectively). A similar conclusion was drawn for the mutant genotype (GT+TT) of DR4 (rs13278062) in non-stricturing, non-penetrating CD patients (58.82% vs. 86.96%, P = 0.001, 95%CI: 0.086-0.536). Haplotype analysis indicated that the CT haplotype formed by rs20575 and rs13278062 was increased in CD patients compared to the controls (37.1% vs. 31.8%, P = 0.029, OR=1.279, 95%CI: 1.022-1.600). The outcome of a gene-gene interaction model indicated that the mutant genotype (GT+TT) of DR4 (rs13278062) and mutant genotype (CT+TT) of DR5 (rs1047266) may play a negatively synergistic role in CD patients (B = - 0.483, OR = 0.617, P = 0.030). The rs13278062 polymorphism of the DR4 gene not only can confer an increased risk for CD, but may also influence the location of the lesions and the disease behaviors. The CT haplotype formed by rs20575 and rs13278062 may be an independent risk factor for CD. Furthermore, the mutant genotype (GT+TT) of DR4 (rs13278062) and mutant genotype (CT+TT) of DR5 (rs1047266) may exert a negative synergistic effect on CD.

Caspase-8 expression is predictive of tumour response to death receptor 5 agonist antibody in Ewing's sarcoma.

Background:Despite good initial response to chemotherapy, 30% of Ewing's sarcoma (EWS) patients with localised tumours develop recurrent disease, associated with poor prognosis. The aim of this study was to address this challenge by conducting preclinical evaluation of a death receptor targeted agent in vitro and in vivo, and by identifying predictive biomarkers.Methods:Cell viability assays, drug dose responses, immunoblots, rescue with gene transfer, mice tumour models, and statistical comparisons of tumour growth and Kaplan–Meier survival curves.Results:This study shows that many EWS cell lines are selectively sensitive to a death receptor DR5 antibody and are more resistant to a DR4 antibody. Preclinical evaluation of these cell lines indicates their sensitivity to human DR5 agonist antibody conatumumab in vitro, which induces rapid activation of caspase-8 and apoptosis. We also found that sensitivity to conatumumab correlates with expression of caspase-8. Furthermore, the catalytic activity of caspase-8 is both necessary and sufficient to confer this sensitivity. In vivo, conatumumab is active against an EWS cell line and a patient-derived xenograft with higher caspase-8 expression, but is not effective against another with lower caspase-8 expression.Conclusions:These studies suggest the potential of conatumumab as a therapeutic agent against EWS and caspase-8 as a predictive biomarker for sensitivity.

Role Of S-Nitrosylation In The Extrinsic Apoptotic Signalling Pathway In Cancer

One of the key features of tumour cells is the acquisition of resistance to apoptosis. Thus, determining therapeutic strategies that circumvent apoptotic resistance and result in tumor regression is a challenge. One strategy to induce apoptosis is to activate death receptor signalling pathways. Members of the Tumor Necrosis Factor TNF-family death receptors ligand (TRAIL, FasL and TNF-α) can originate from immune and non-immune cells. Death receptors, engaged by cognate ligands, can initiate multiple signaling pathways, which can generate diverse outcomes, including non-apoptosis-related signal. Knowledge on the molecular mechanisms (that determine death or survival of tumour cells) following exposure to the TNF-family death receptors ligands have demonstrated that post-translational modifications of the signaling pathway components play a critical role in determining cell fate. Cell death can be sensed by nitric oxide (NO) in a wide variety of tumour cells. S-nitrosylation, the covalent modification of a protein cysteine thiol by an NO moiety, has emerged as an important post-translational regulation for the TNF-family death receptor signaling pathways. It has been demonstrated that death receptor DR4 (TRAIL-R1) becomes S-nitrosylated and promotes apoptosis following a specific NO donor treatment (Tang et al., 2006). Then, our group has shown that S-nitrosylation of Fas, following glyceryl trinitrate (GTN) exposure, promotes redistribution of the receptor to lipid rafts, formation of the death-inducing signal complex (DISC), and induction of cell death. Finally, I will discuss our recent efforts to decipher regulatory mechanism of the TNF-α/TNFR1 signalling cell death pathway by S-nitrosylation following GTN treatment.

Mutations Enhancing Selectivity of Antitumor Cytokine TRAIL to DR5 Receptor Increase Its Cytotoxicity against Tumor Cells.

Tumor necrosis factor superfamily cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces apoptosis in tumor cells by binding to death receptors DR4 and DR5 without affecting normal cells. However, the therapeutic use of TRAIL is limited, because many tumor cells are resistant to it. The resistance is partially related to interaction of TRAIL with the decoy receptors DcR1 and DcR2, which do not trigger the apoptotic signal and inhibit signaling of death receptors. Previously, we designed a unique DR5-specific TRAIL mutant variant DR5-B, which binds to DR5 receptor as effectively as the original cytokine, but has practically no interaction with DR4 and DcR1 receptors, and its affinity for DcR2 is reduced 400-fold. In the present work, the cytotoxity of TRAIL and DR5-B was analyzed on 12 different tumor cell lines and two types of normal cells. In nine of 12 tumor cell lines, DR5-B killed 1.5-5.0 times more tumor cells than TRAIL, and it did not exhibit toxicity towards normal cells. Chemotherapeutic drugs such as doxorubicin, paclitaxel, and bortezomib augmented the effect of both TRAIL variants, and the enhancing effect was more pronounced for DR5-B. Half-maximal effective concentrations (EC50) for DR5-B in combination with chemotherapeutic agents were 1.5-10.0 times lower than for wild-type TRAIL. Thus, DR5-B is a promising candidate both for monotherapy and in combination with chemotherapy for treatment of TRAIL-resistant tumors.

Abstract 2917: Caspase-8 expression is predictive of tumor response to death receptor 5 agonist antibody conatumumab in Ewing's sarcoma

Abstract Despite good initial response to cytotoxic chemotherapy, approximately 30% of Ewing's sarcoma (EWS) patients who present with localized tumors develop recurrent disease, which is associated with poor prognosis. Addressing this clinical challenge requires the development of targeted therapies. This study shows that many EWS cell lines are selectively sensitive to a death receptor DR5 antibody and are more resistant to a DR4 antibody. Preclinical evaluation of these cell lines indicates their sensitivity to human DR5 agonist antibody conatumumab in vitro, which induces rapid activation of caspase-8 and apoptosis. Further analysis reveals the correlation of sensitivity to conatumumab with the expression of caspase-8, with its catalytic activity both necessary and sufficient to confer such sensitivity. In vivo, conatumumab has active against both EWS cell lines and a patient-derived xenograft with higher caspase-8 expression, but is not effective against another patient-derived xenograft with lower caspase-8 expression. These studies suggest the potential of conatumumab as a therapeutic agent against EWS and caspase-8 as a biomarker for correlative studies. Citation Format: Zhigang Kang, D Seth Goldstein, Yunkai Yu, Paul S. Meltzer, David M. Loeb, Liang Cao. Caspase-8 expression is predictive of tumor response to death receptor 5 agonist antibody conatumumab in Ewing's sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2917. doi:10.1158/1538-7445.AM2015-2917

Abstract B34: Sensitization of TRAIL-resistant malignant melanomas by ellagic acid

Abstract The incidence of malignant melanoma has been on the rise for the past 30 years with a lifetime risk of 1 in 75. In its early stage, melanoma is easily cured, but the prognosis associated with metastatic malignant melanoma remains very poor and is one of the most treatment-refractory malignancies. FDA-approved melanoma therapeutics, Dacarbazine (DTIC) and Interleukin-2 (IL-2) are associated with response rates of only 10-20% and do not yield long-lasting remissions. A direct and selective p53-independent cancer therapy is the application of Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL). TRAIL can induce apoptosis in a broad range of transformed human cells while showing minimal toxicity towards non-malignant cells. However, some cancers are resistant to rhTRAIL-induced apoptosis as a direct result of overexpressed antiapoptotic proteins. One key driver mediating the expression of such antiapoptotic proteins is Casein Kinase 2 (CK2). Upregulated in the nucleus of all cancer cells, CK2 serves as an oncoprotein, promoting the expression of inhibitors of apoptosis resulting in resistance to cell death. By inhibiting CK2 and, therefore, downregulating its antiapoptotic products, TRAIL-resistant cells may become more sensitive to TRAIL-induced apoptosis. To date, Ellagic Acid (EA) is the most potent, naturally-occurring CK2 inhibitor, possessing a Ki of 20 nM. To test whether the addition of EA could sensitize cancer cells to the effects of TRAIL, we utilized cotreatment with TRAIL and EA against the V600E BRAF-mutated melanoma cell lines A375 and WM164, which have previously been reported as TRAIL-resistant. Our in vitro analyses showed that the cotreatment of TRAIL plus EA does not augment the ability of TRAIL to induce apoptosis in A375 as determined by Annexin-V Apoptosis assays and Western Blot analysis. TRAIL-alone was able to induce apoptosis in A375 characterized by the formation of Annexin-V positive cells, PARP fragmentation and the activation of caspase 3. TRAIL initiated both the extrinsic and intrinsic pathways of apoptosis marked by caspase 8-activation and the mitochondrial release of cytochrome c, respectively. However, EA treatment alone did not induce apoptosis marked by the absence of Annexin-V positive cells and PARP fragmentation. Additionally, both TRAIL and EA, as single-agents, inhibited the growth of A375 in a dose-dependent manner, and the combination treatment of TRAIL plus EA enhanced the cytostatic effects of the single-agent treatments as determined by Sulforhodamine-B (SRB) colorimetric assays. Cell cycle analysis through Propidium Iodide (PI) staining and FACS analysis revealed that EA impeded cell growth through an EA-mediated S-phase arrest in A375. Microarray analysis showed that EA downregulates antiapoptotic proteins survivin, XIAP, cIAP-2 and cIAP-1 and upregulates the death receptor DR5. In vivo, we showed that the combination treatment of TRAIL plus EA did not potentiate the ability of TRAIL to inhibit the growth of established A375 tumors. TRAIL alone inhibited tumor growth by ∼80% and H&amp;E staining of TRAIL-treated tumors presented evidence of apoptotic cells. EA was not able to stop the growth of established A375 tumors and did not augment the in vivo antiproliferative effects of TRAIL. Lastly, we showed that TRAIL is cytotoxic towards WM164 cells and can reduce the viability of A375 as determined by Trypan Blue exclusion assays. Whereas EA, although it can inhibit the growth of WM164, does not reduce the viability of the cells. Additional analysis of the potential of EA to sensitize WM164 to the effects of TRAIL is ongoing. These data demonstrate that EA can enhance the cytostatic but not the cytotoxic or proapoptotic effects of TRAIL in vitro. In vivo, TRAIL can inhibit the growth of established tumors as a single-agent but is not augmented by cotreatment with EA. Citation Format: Katherine A. Turner, Daniel Lindner, Michael Kalafatis. Sensitization of TRAIL-resistant malignant melanomas by ellagic acid. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B34.

Decoy receptors block TRAIL sensitivity at a supracellular level: the role of stromal cells in controlling tumour TRAIL sensitivity.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand cytokine known for its cytotoxic activity against malignantly transformed cells. TRAIL induces cell death through binding to death receptors DR4 and DR5. The inhibitory decoy receptors (DcR1 and DcR2) co-expressed with death receptor 4 (DR4)/DR5 on the same cell can block the transmission of the apoptotic signal. Here, we show that DcRs also regulate TRAIL sensitivity at a supracellular level and thus represent a mechanism by which the microenvironment can diminish tumour TRAIL sensitivity. Mathematical modelling and layered or spheroid stroma-extracellular matrix-tumour cultures were used to model the tumour microenvironment. By engineering TRAIL to escape binding by DcRs, we found that DcRs do not only act in a cell-autonomous or cis-regulatory manner, but also exert trans-cellular regulation originating from stromal cells and affect tumour cells, highlighting the potent inhibitory effect of DcRs in the tumour tissue and the necessity of selective targeting of the two death-inducing TRAIL receptors to maximise efficacy.

Comprehensive identification of genes driven by ERV9-LTRs reveals TNFRSF10B as a re-activatable mediator of testicular cancer cell death.

The long terminal repeat (LTR) of human endogenous retrovirus type 9 (ERV9) acts as a germline-specific promoter that induces the expression of a proapoptotic isoform of the tumor suppressor homologue p63, GTAp63, in male germline cells. Testicular cancer cells silence this promoter, but inhibitors of histone deacetylases (HDACs) restore GTAp63 expression and give rise to apoptosis. We show here that numerous additional transcripts throughout the genome are driven by related ERV9-LTRs. 3' Rapid amplification of cDNA ends (3'RACE) was combined with next-generation sequencing to establish a large set of such mRNAs. HDAC inhibitors induce these ERV9-LTR-driven genes but not the LTRs from other ERVs. In particular, a transcript encoding the death receptor DR5 originates from an ERV9-LTR inserted upstream of the protein coding regions of the TNFRSF10B gene, and it shows an expression pattern similar to GTAp63. When treating testicular cancer cells with HDAC inhibitors as well as the death ligand TNF-related apoptosis-inducing ligand (TRAIL), rapid cell death was observed, which depended on TNFRSF10B expression. HDAC inhibitors also cooperate with cisplatin (cDDP) to promote apoptosis in testicular cancer cells. ERV9-LTRs not only drive a large set of human transcripts, but a subset of them acts in a proapoptotic manner. We propose that this avoids the survival of damaged germ cells. HDAC inhibition represents a strategy of restoring the expression of a class of ERV9-LTR-mediated genes in testicular cancer cells, thereby re-enabling tumor suppression.

Identification of TRAIL-inducing compounds highlights small molecule ONC201/TIC10 as a unique anti-cancer agent that activates the TRAIL pathway.

BackgroundWe previously reported the identification of ONC201/TIC10, a novel small molecule inducer of the human TRAIL gene that improves efficacy-limiting properties of recombinant TRAIL and is in clinical trials in advanced cancers based on its promising safety and antitumor efficacy in several preclinical models.MethodsWe performed a high throughput luciferase reporter screen using the NCI Diversity Set II to identify TRAIL-inducing compounds.ResultsSmall molecule-mediated induction of TRAIL reporter activity was relatively modest and the majority of the hit compounds induced low levels of TRAIL upregulation. Among the candidate TRAIL-inducing compounds, TIC9 and ONC201/TIC10 induced sustained TRAIL upregulation and apoptosis in tumor cells in vitro and in vivo. However, ONC201/TIC10 potentiated tumor cell death while sparing normal cells, unlike TIC9, and lacked genotoxicity in normal fibroblasts. Investigating the effects of TRAIL-inducing compounds on cell signaling pathways revealed that TIC9 and ONC201/TIC10, which are the most potent inducers of cell death, exclusively activate Foxo3a through inactivation of Akt/ERK to upregulate TRAIL and its pro-apoptotic death receptor DR5.ConclusionThese studies reveal the selective activity of ONC201/TIC10 that led to its selection as a lead compound for this novel class of antitumor agents and suggest that ONC201/TIC10 is a unique inducer of the TRAIL pathway through its concomitant regulation of the TRAIL ligand and its death receptor DR5.

α-Hispanolol sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis via death receptor up-regulation

Hispanolone derivatives have been previously described as anti-inflammatory and antitumoral agents. However, their effects on overcoming Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance remain to be elucidated. In this study, we analyzed the cytotoxic effects of the synthetic hispanolone derivative α-hispanolol (α-H) in several tumor cell lines, and we evaluated the induction of apoptosis, as well as the TRAIL-sensitizing potential of α-H in the hepatocellular carcinoma cell line HepG2. Our data show that α-H decreased cell viability in a dose-dependent manner in HeLa, MDA-MB231, U87 and HepG2 cell lines, with a more prominent effect in HepG2 cells. Interestingly, α-H had no effect on non-tumoral cells. α-H induced activation of caspase-8 and caspase-9 and also increased levels of the proapoptotic protein Bax, decreasing antiapoptotic proteins (Bcl-2, X-IAP and IAP-1) in HepG2 cells. Specific inhibition of caspase-8 abrogated the cascade of caspase activation, suggesting that the extrinsic pathway has a critical role in the apoptotic events induced by α-H. Furthermore, combined treatment of α-H with TRAIL enhanced apoptosis in HepG2 cells, activating caspase-8 and caspase-9. This correlated with up-regulation of both the TRAIL death receptor DR4 and DR5. DR4 or DR5 neutralizing antibodies abolished the effect of α-H on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the death receptor pathway. Our results demonstrate that α-H induced apoptosis in the human hepatocellular carcinoma cell line HepG2 through activation of caspases and induction of the death receptor pathway. In addition, we describe a novel function of α-H as a sensitizer on TRAIL-induced apoptotic cell death in HepG2 cells.

Trailing TRAIL Resistance: Novel Targets for TRAIL Sensitization in Cancer Cells.

Resistance to chemotherapeutic drugs is the major hindrance in the successful cancer therapy. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family of ligands, which initiates apoptosis in cancer cells through interaction with the death receptors DR4 and DR5. TRAIL is perceived as an attractive chemotherapeutic agent as it specifically targets cancer cells while sparing the normal cells. However, TRAIL therapy has a major limitation as a large number of the cancer develop resistance toward TRAIL and escape from the destruction by the immune system. Therefore, elucidation of the molecular targets and signaling pathways responsible for TRAIL resistance is imperative for devising effective therapeutic strategies for TRAIL resistant cancers. Although, various molecular targets leading to TRAIL resistance are well-studied, recent studies have implicated that the contribution of some key cellular processes toward TRAIL resistance need to be fully elucidated. These processes primarily include aberrant protein synthesis, protein misfolding, ubiquitin regulated death receptor expression, metabolic pathways, epigenetic deregulation, and metastasis. Novel synthetic/natural compounds that could inhibit these defective cellular processes may restore the TRAIL sensitivity and combination therapies with such compounds may resensitize TRAIL resistant cancer cells toward TRAIL-induced apoptosis. In this review, we have summarized the key cellular processes associated with TRAIL resistance and their status as therapeutic targets for novel TRAIL-sensitizing agents.

Cisplatin-mediated c-myc overexpression and cytochrome c (cyt c) release result in the up-regulation of the death receptors DR4 and DR5 and the activation of caspase 3 and caspase 9, likely responsible for the TRAIL-sensitizing effect of cisplatin.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) reverses multidrug resistance (MDR) and induces apoptosis in MDR gastric carcinoma cells. In our previous study, cisplatin proved to be a sensitizing agent for TRAIL. To study the synergistic effects of cisplatin and TRAIL, we investigated the mechanism by which TRAIL reverses multidrug resistance, the role of c-myc in modulating the death receptors DR4 and DR5 and the relationship between cisplatin and cytochrome c (cyt c) release in SGC7901/VCR and SGC7901/DDP cells. We found that after treatment with TRAIL, the DNA-PKcs/Akt/GSK-3β pathway, which is positively correlated with the levels of MDR1 and MRP1, was significantly inhibited and that this tendency can be abolished by Z-DEVD-FMK (a specific caspase 3 inhibitor). We also found that suppression of c-myc by siRNA reduced the expression of DR4 and DR5 and that transfection with a pAVV-c-myc expression vector increased the expression of DR4 and DR5. Moreover, cisplatin increased the expression of c-myc in the presence of TRAIL, and there is a clear increase in cyt c release from mitochondria with the increasing concentrations of cisplatin. Meanwhile, the intrinsic death receptor pathway of caspase 9, as well as the common intrinsic and extrinsic downstream target, caspase 3, was potently activated by the release of cyt c. Together, we conclude that in TRAIL-treated MDR gastric carcinoma cells, cisplatin induces the death receptors DR4 and DR5 through the up-regulation of c-myc and strengthens the activation of caspases via promoting the release of cyt c. These effects would then be responsible for the TRAIL sensitization effect of cisplatin.

Downregulation of external death receptor genes FAS and DR5 in colorectal cancer samples positive for human papillomavirus infection

AimHuman papillomaviruses (HPV) have frequently been detected in colorectal cancer tumor samples, and may play a role in the pathogenesis of colorectal cancer. This study was designed to investigate the presence of DNA and RNA for the high-risk HPV genotypes 16 and 18 in samples of colorectal cancer tumors and adjacent normal tissues. We also investigated the expression of proapoptotic genes in HPV-positive colorectal tumors compared to normal tissue samples. MethodsSamples of tumoral and adjacent normal tissues were fresh-frozen, and HPV DNA was identified by nested and semiquantitative PCR. Real time PCR was used to quantitatively compare the expression of HPV-18 E6 and nine proapoptotic genes in HPV-positive tumors and samples of adjacent normal tissue. ResultsHPV-16 DNA was found in 10.5% of the tumor samples, and HPV-18 DNA was found in 23.6% of the samples. Real time PCR results showed lower expression of the E6 gene in HPV-positive tumors than in adjacent normal tissue. The expression of two proapoptotic genes, FAS and DR5, was significantly lower in tumor samples than in adjacent normal tissues. ConclusionsHPV infection, especially HPV-18, may play a role in colorectal cancer tumorigenesis by downregulating death receptor genes and interfering with the extrinsic pathway of apoptosis.

Abstract T P243: Death Receptors and Mitochondria as Crucial Targets for Drug Discovery in Cerebral Amyloid Angiopathy

The vascular deposition of amyloid, known as Cerebral Amyloid Angiopathy (CAA) is an age-associated condition featured in about 90% of Alzheimer’s disease cases and in the aging brain. Amyloid beta (Abeta) deposition in CAA compromises cerebral blood flow and can cause cerebral hemorrhage and cognitive impairment, by mechanisms that are still poorly understood. Our goal was to identify the molecular events underlying the apoptotic cascade generated by Abeta in cerebrovascular cells and to pinpoint new targets for drug discovery. Human brain microvascular endothelial cells were challenged with vasculotropic Abeta variants associated with cerebrovascular deposition and hemorrhagic outcome, and the resulting signaling pathways were analysed. The in vitro findings were validated in vivo in mice subjected to intrahippocampal Abeta injections and confirmed in human CAA cases. Our findings highlighted an activation of caspase-8 and -9, together with mitochondrial dysfunction and release of cytochrome C, suggesting death receptor mediated apoptosis, which was confirmed by an overexpression of the TRAIL (TNF-related apoptosis inducing ligand) death receptors DR4 and DR5. Signaling cascades typical of TRAIL death receptor-mediated pathways were activated. The same receptors colocalized with Abeta on the cell membrane after amyloid challenge, and immunoprecipitated in vitro with Abeta oligomers. SiRNAs against DR4 and DR5 and the prevention of mitochondrial dysfunction through Carbonic Anhydrase Inhibitors, significantly diminished Abeta mediated apoptosis in endothelial cells. In vivo experiments in mice injected with vasculotropic Abeta peptide and in human CAA cases confirmed the upregulation of the receptors, their colocalization with Abeta on the cerebral vasculature, and the activation of caspases, which could be prevented by carbonic anhydrase inhibitors. Our data strongly suggests that TRAIL death-receptors and mitochondrial functioning are key cellular targets for therapeutic intervention against Abeta-induced vascular cell death in CAA.

Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future.

Screen of Small Molecule ONC201/TIC10 Identifies Single Agent Activity and Combinatorial Efficacy with Bortezomib, Rituximab or Dexamethasone in Killing of Acute Lymphoblastic Leukemia Cells

The antitumor protein recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anticancer therapy although it has notable shortcomings that have limited its clinical development. Our lab previously identified a novel use for TRAIL-inducing compound 10 (ONC201/TIC10) as an efficacious antitumor therapeutic that induces TRAIL pathway activation through dual blockade of Akt and ERK that releases Foxo3a to transcriptionally activate the TRAIL and its death receptor DR5 genes. ONC201/TIC10 is being developed by Oncoceutics for the clinical therapy of cancer. Acute lymphoblastic leukemia (ALL) is a heterogeneous disease with an increasing rate of refractory/relapsed population of patients, making it a challenging disease to treat. Therefore, new therapeutic options as single dose or combinational treatments are required. Therapeutic agonist TRAIL antibodies have been investigated as a therapy for solid tumors; however, the biologic agents have not been explored extensively as a potential single or combinational therapy for the treatment of ALL. The aim of this study was to begin to characterize ONC201/TIC10 as a single agent for ALL therapy and in combination with bortezomib to increase induction of tumor cell death.Single agent ONC201/TIC10 decreased ALL cell viability at IC50 values ranging from 2.5-5 µM after a 48 hour treatment. ONC201/TIC10 was combined with various accepted clinical treatments of ALL to determine if a synergistic or additive effect exists. Treatments of compounds at doses below their IC50, including the proteasome inhibitor bortezomib and monoclonal antibody-based rituximab therapy, showed synergistic or additive anti-leukemic effects when treated mutually with ONC201/TIC10 in ALL and T-cell ALL. TRAIL and Foxo3a expression was monitored by western blot analysis and apoptosis was indicated by both subG1 and caspase activity to investigate the effect of these combination treatments.Bortezomib-ONC201/TIC10 combinational treatments decreased cell viability by approximately 30% in MALT-4 cells and 20% in Jurkat compared to single dose bortezomib treatments. Rituximab and the steroid compound dexamethasone were administered both below their IC50 in combination with ONC201/TIC10 and exhibited 20-40% cell viability decrease in ALL in vitro beyond the monoagent effects. Pharmacological enhancement by the addition of ONC201/TIC10 increased apoptotic efficacy and significantly decreased cell viability during combination treatments as compared to single agent therapy. The formation of the TRAIL death-inducing signaling complex could provide a rationale for further investigation into ONC201/TIC10 combinational treatment in ALL. These preclinical results suggest that there may be a clinical therapeutic advantage to modifying current ALL patient therapies to include the addition of ONC201/TIC10. DisclosuresAllen:Oncoceutics, Inc.: Employment, Equity Ownership, Patents & Royalties. El-Deiry:Oncoceutics, Inc.: Equity Ownership, Patents & Royalties.