Upregulation Of Death Receptor Research Articles

3‑Bromopyruvate sensitizes human breast cancer cells to TRAIL‑induced apoptosis via the phosphorylated AMPK‑mediated upregulation of DR5.

Previous studies have indicated that the sensitivity of breast cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is associated with the expression of death receptors on the cell membrane. However, drug resistance limits the use of TRAIL in cancer therapy. Numerous studies have indicated that death receptors, which induce apoptosis, are upregulated by the endoplasmic reticulum (ER) stress response. 3-Bromopyruvate (3-BP), an anticancer agent, inhibits cell growth and induces apoptosis through interfering with glycolysis. In the present study, it was demonstrated that 3-BP synergistically sensitized breast cancer cells to TRAIL-induced apoptosis via the upregulation of death receptor 5 (DR5). Furthermore, we found that the protein levels of glucose-related protein 78 (GRP78) and CCAAT-enhancer-binding protein homologous protein (CHOP) increased following treatment with 3-BP. The expression of Bax (in MCF-7 cells) and caspase-3 (in MDA-MB-231 cells) increased following co-treatment with 3-BP and TRAIL, whereas the expression of the anti-apoptotic protein Bcl-2 decreased. In order to investigate the molecular mechanism regulating this effect, the expression of adenosine monophosphate-activated protein kinase (AMPK), activated by 3-BP, was determined. It was demonstrated that phosphorylated-AMPK was upregulated following treatment with 3-BP. Notably, Compound C, an AMPK inhibitor, reversed the effects of 3-BP. Finally, a synergistic antitumor effect of 3-BP and TRAIL was observed in MCF-7 cell xenografts in nude mice. In conclusion, these results indicated that 3-BP sensitized breast cancer cells to TRAIL via the AMPK-mediated upregulation of DR5.

Inhibition of euchromatin histone-lysine N-methyltransferase 2 sensitizes breast cancer cells to tumor necrosis factor-related apoptosis-inducing ligand through reactive oxygen species-mediated activating transcription factor 4-C/EBP homologous protein-death receptor 5 pathway activation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been characterized as an anti-cancer therapeutic agent with prominent cancer cell selectivity over normal cells. However, breast cancer cells are generally resistant to TRAIL, thus limiting its therapeutic potential. In this study, we found that BIX-01294, a selective inhibitor of euchromatin histone methyltransferase 2/G9a, is a strong TRAIL sensitizer in breast cancer cells. The combination of BIX-01294 and TRAIL decreased cell viability and led to an increase in the annexin V/propidium iodide-positive cell population, DNA fragmentation, and caspase activation. BIX-01294 markedly increased death receptor 5 (DR5) expression, while silencing of DR5 using small interfering RNAs abolished the TRAIL-sensitizing effect of BIX-01294. Specifically, BIX-01294 induced C/EBP homologous protein (CHOP)-mediated DR5 gene transcriptional activation and DR5 promoter activation was induced by upregulation of the protein kinase R-like endoplasmic reticulum kinase-mediated activating transcription factor 4 (ATF4). Moreover, inhibition of reactive oxygen species by N-acetyl-L-cysteine efficiently blocked BIX-01294-induced DR5 upregulation by inhibiting ATF4/CHOP expression, leading to diminished sensitization to TRAIL. These findings suggest that BIX-01294 sensitizes breast cancer cells to TRAIL by upregulating ATF4/CHOP-dependent DR5 expression with a reactive oxygen species-dependent manner. Furthermore, combination treatment with BIX-01294 and TRAIL suppressed tumor growth and induced apoptosis in vivo. In conclusion, we found that epigenetic regulation can contribute to the development of resistance to cancer therapeutics such as TRAIL, and further studies of unfolded protein responses and the associated epigenetic regulatory mechanisms may lead to the discovery of new molecular targets for effective cancer therapy.

Abstract 1351: Inhibition of autophagy enhances dihydroartemisinin (DHA)-induced cytotoxicity through DR5 upregulation

Abstract Artemisinin (Qinghaosu) comes originally from Qinghao has been used against Plasmodium falciparum malaria for a long time. Dihydroartemisinin, an analogue of artemisinin, is also be listed by WHO to exhibit antimalarial and anticancer properties. In our study, DHA exhibits antiproliferative activity in several solid tumors, especially against colorectal cancer (CRC) HCT116 cells. Our current results show DHA suppresses cell proliferation and induces caspase-dependent apoptosis without inducing necrosis-like cell death in HCT116 cells. In addition, DHA interferes autophagy pathway by upregulating of LC3 II formation in HCT116 cells. Inhibition of autophagy with either autophagy inhibitor 3-methyladenine (3MA) or KD of ATG5 potentiated DHA-induced cell death. We found DHA increased expression level of DR5 while had no effect on DR4 expression level on surface of tumor cells. Further, cell-surface DR5 expression was significantly elevated in DHA-treated ATG5-KD cells. These findings suggest that inhibition of the autophagy enhances DHA-induced cytotoxicity through DR5 upregulation. Key words: Apooptsis, DR5 (Death Receptor 5), autophagy, colorectal cancer Citation Format: Chen Mei-Chuan. Inhibition of autophagy enhances dihydroartemisinin (DHA)-induced cytotoxicity through DR5 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1351.

Abstract LB-082: ONC201 sensitizes resistant breast cancer cells to TRAIL through death receptor 5 upregulation

Abstract ONC201 is a potent inducer of cancer cell death through the tumor necrosis factor related apoptosis inducing ligand (TRAIL) pathway. ONC201 and its structural analogs comprise a novel class known as the imipridones. The compound is being tested clinically against a range of solid tumors and hematological malignancies. We found that both triple negative and non-triple negative breast cancer cells are sensitive to ONC201. This was initially surprising, as the majority of non-triple negative breast cancer cells are resistant to TRAIL. In TRAIL-sensitive cells, ONC201 induced cell death in a TRAIL-dependent manner. In TRAIL-resistant cells, the effects of ONC201 are not pro-apoptotic but are rather anti-proliferative. Here we further investigate the effects of ONC201 in TRAIL-resistant breast cancers from multiple molecular subtypes. ONC201 has been previously shown to induce transcriptional upregulation of TRAIL receptor death receptor 5 (DR5). A known mechanism of TRAIL resistance in breast cancer is low cell surface DR5 expression. We hypothesized that pre-treatment with ONC201 would upregulate DR5 and lead to TRAIL sensitization in resistant cells. Our results confirm our hypothesis and show that treatment with ONC201 transcriptionally upregulates DR5 and increases its expression at the cell surface. In addition, pre-treatment with ONC201 sensitizes TRAIL-resistant breast cancer cells to the pro-apoptotic effects of TRAIL. Caspase-8 and PARP are cleaved when cells are treated with the combination of ONC201 and TRAIL, but not when treated with either compound alone. Annexin-V PI staining and quantitation of subG1 DNA content via flow cytometry further confirmed these results. Knockdown of DR5 abrogated TRAIL sensitization by ONC201. Natural killer (NK) cells are known to use TRAIL to kill targets like tumor cells. We are exploring the hypothesis that pre-treatment of tumor cells with ONC201 would increase their killing by NK cells. We are currently investigating the importance of the TRAIL pathway and DR5 upregulation by imipridone compounds in this sensitization to NK cell induced apoptosis. In summary, these results describe a novel mechanism by which ONC201 sensitizes breast cancer cells to the effects of apoptosis inducing ligand TRAIL. They also provide preclinical rationale for the testing of the compound against breast cancers in the clinic. Citation Format: Marie D. Ralff, Jessica Wagner, Wafik S. El-Deiry. ONC201 sensitizes resistant breast cancer cells to TRAIL through death receptor 5 upregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-082.

Telmisartan generates ROS-dependent upregulation of death receptor 5 to sensitize TRAIL in lung cancer via inhibition of autophagy flux.

Telmisartan broadly used for the treatment of hypertension that is also known for its anticancer properties. TRAIL has the potential to kill tumor cells with minimal toxicity in normal cells by binding to death receptors, DR4 and DR5. Unfortunately, these TRAIL-death receptors have failed as most human cancers are resistant to TRAIL-mediated apoptosis. In this study, we evaluated telmisartan as a novel TRAIL-DR5-targeting agent with the aim of rendering TRAIL-based cancer therapies more active. Herein, we demonstrated that telmisartan could sensitize TRAIL and enhance NSCLC tumor cell death. The molecular mechanism includes the blocking of AMPK phosphorylation causes inhibition of autophagy flux by telmisartan resulting in ROS generation leading to death receptor (DR5) upregulation and subsequent activation of the caspase cascade by TRAIL treatment. Furthermore, using chloroquine and siATG5 significantly enhances ROS production and application of the ROS scavenger N-acetyl-cysteine (NAC) rescues the cells undergoing apoptosis by abrogating the expression of DR5 and finally the caspase cascade. Additionally, NAC treatment also maintains autophagy flux and makes the cells unresponsive to TRAIL. In summary, telmisartan in combination with TRAIL exhibits enhanced cytotoxic capacity toward lung cancer cells, thereby providing the potential for effective and novel therapeutic approaches to treat lung cancer.

Isoalantolactone induces apoptosis through reactive oxygen species-dependent upregulation of death receptor 5 in human esophageal cancer cells

Esophageal cancer is the eighth most prevalent cancer and has high mortality in our society. Isoalantolactone, extracted from Inula helenium L, has shown potent anticancer effects on a variety of cancers. However, its effect on human esophageal cancer, and the underlying molecular mechanism, remain to be investigated. In the present study, we demonstrated that isoalantolactone induced apoptosis in esophageal cancer cells. Treatment with isoalantolactone activated caspases-3, -7, and -10, and upregulated death receptor (DR)5. Furthermore, DR5 knockdown partially reversed the effect of isoalantolactone. These results indicated the extrinsic apoptosis was induced by isoalantolactone. In addition, intracellular reactive oxygen species (ROS) were significantly elevated after treatment with isoalantolactone. N-Acetylcysteine, an ROS scavenger, blocked both the apoptosis and decreased cell viability caused by isoalantolactone. In vivo, significant suppression of tumor growth by isoalantolactone was observed in an ECA109 cell xenograft mouse model. Isoalantolactone showed no obvious adverse effects on mouse weight and histology of heart, liver, spleen, lung, and kidney. In conclusion, our results revealed that isoalantolactone induced apoptosis through the extrinsic pathway via upregulation of DR5 and elevation of ROS in human esophageal cancer cells. Isoalantolactone, therefore, could be a potential candidate in developing anticancer agents for esophageal cancer patients.

Calmodulin antagonist enhances DR5-mediated apoptotic signaling in TRA-8 resistant triple negative breast cancer cells.

Patients with triple negative breast cancer (TNBC) have no successful "targeted" treatment modality, which represents a priority for novel therapy strategies. Upregulated death receptor 5 (DR5) expression levels in breast cancer cells compared to normal cells enable TRA-8, a DR5 specific agonistic antibody, to specifically target malignant cells for apoptosis without inducing normal hepatocyte apoptosis. Drug resistance is a common obstacle in TRAIL-based therapy for TNBC. Calmodulin (CaM) is overexpressed in breast cancer. In this study, we characterized the novel function of CaM antagonist in enhancing TRA-8 induced cytotoxicity in TRA-8 resistant TNBC cells and its underlying molecular mechanisms. Results demonstrated that CaM antagonist(s) enhanced TRA-8 induced cytotoxicity in a concentration and time-dependent manner for TRA-8 resistant TNBC cells. CaM directly bound to DR5 in a Ca2+ dependent manner, and CaM siRNA promoted DR5 recruitment of FADD and caspase-8 for DISC formation and TRA-8 activated caspase cleavage for apoptosis in TRA-8 resistant TNBC cells. CaM antagonist, trifluoperazine, enhanced TRA-8 activated DR5 oligomerization, DR5-mediated DISC formation, and TRA-8 activated caspase cleavage for apoptosis, and decreased anti-apoptotic pERK, pAKT, XIAP, and cIAP-1 expression in TRA-8 resistant TNBC cells. These results suggest that CaM could be a key regulator to mediate DR5-mediated apoptotic signaling, and suggests a potential strategy for using CaM antagonists to overcome drug resistance of TRAIL-based therapy for TRA-8 resistant TNBC.

Interleukin-4 Enhances the Sensitivity of Human Monocytes to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Through Upregulation of Death Receptor 4.

Interleukin (IL)-4 is generally thought to promote tumor cell growth and inhibit apoptosis. However, its role in characteristics of monocytic leukemia cells was rarely reported. In this study, we assessed the role of IL-4 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity of human monocytes. After incubation with IL-4 for 24 h, death receptor 4 (DR4) was significantly increased without downregulation of TRAIL decoy receptors and antiapoptotic proteins in THP-1 monocytes, and human primary monocytes and U-937 cells also exhibited increased TRAIL-induced apoptosis compared with control. Enhancement of TRAIL-mediated apoptosis by IL-4 was blocked by anti-DR4-neutralizing antibodies. Both upregulation of DR4 and enhancement of TRAIL-mediated apoptosis by IL-4 could be blocked by inhibitors of Janus kinase (JAK)/signal transducer and activator of transcription (STAT), phosphoinositol 3-kinase (PI3K)/Akt, and extracellular signal-regulated kinase to varying degrees. Thus, our data demonstrated a novel effect on TRAIL sensitivity on monocytes and monocytic leukemia cells of IL-4 and suggested that it may be necessary to reconsider the impact of current therapies against IL-4, JAK/STAT, and PI3K/Akt pathways with regard to TRAIL sensitivity.

The poly(ADP-ribose) polymerase inhibitor olaparib induces up-regulation of death receptors in primary acute myeloid leukemia blasts by NF-κB activation

Olaparib is a potent orally bioavailable poly(ADP-ribose) polymerase inhibitor (PARPi), approved for BRCA-mutated ovarian and breast cancers. We recently showed that olaparib at clinically achievable concentrations exerts anti-proliferative and pro-apoptotic effects in vitro as monotherapy against primary acute myeloid leukemia (AML) blasts, while sparing normal bone marrow (BM) hematopoietic cells. Since AML expresses low levels of death receptors that may contribute to apoptosis resistance, in this study we investigated whether the anti-leukemia activity of olaparib involves modulation of FAS and TRAIL receptors DR5 and DR4. Our data show that the primary AML samples tested express FAS and DR5 transcripts at levels lower than normal BM. In this context, apoptosis triggered by olaparib is associated with a dose-dependent up-regulation of death receptors expression and caspase 8 activation. Olaparib-mediated FAS up-regulation requires NF-κB activation, as indicated by the increase of p65 phosphorylation and decrease of IκBα. Moreover, FAS up-regulation is abrogated by pretreatment of AML cells with two different NF-κB inhibitors. These results indicate that NF-κB activation and consequent induction of death receptor expression contribute to the anti-leukemia effect of olaparib in AML.

Silibinin sensitizes TRAIL-mediated apoptosis by upregulating DR5 through ROS-induced endoplasmic reticulum stress-Ca2+-CaMKII-Sp1 pathway.

In this study, we addressed how silibinin enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in various cancer cells. Combined treatment with silibinin and TRAIL (silibinin/TRAIL) induced apoptosis accompanied by the activation of caspase-3, caspase-8, caspase-9, and Bax, and cytosolic accumulation of cytochrome c. Anti-apoptotic proteins such as Bcl-2, IAP-1, and IAP-2 were inhibited as well. Silibinin also triggered TRAIL-induced apoptosis in A549 cells through upregulation of death receptor 5 (DR5). Pretreatment with DR5/Fc chimeric protein and DR5-targeted small interfering RNA (siRNA) significantly blocked silibinin/TRAIL-mediated apoptosis in A549 cells. Furthermore, silibinin increased the production of reactive oxygen species (ROS), which led to the induction of TRAIL-mediated apoptosis through DR5 upregulation. Antioxidants such as N-acetyl-L-cysteine and glutathione reversed the apoptosis-inducing effects of TRAIL. Silibinin further induced endoplasmic reticulum (ER) stress as was indicated by the increase in ER marker proteins such as PERK, eIF2α, and ATF-4, which stimulate the expression of CCAAT/enhancer binding protein homologous protein (CHOP). CHOP-targeted siRNA eliminated the induction of DR5 and resulted in a significant decrease in silibinin/TRAIL-mediated apoptosis. We also found that silibinin/TRAIL-induced apoptosis was accompanied with intracellular influx of Ca2+, which was stimulated by ER stress and the Ca2+ chelator, ethylene glycol tetraacetic acid (EGTA). Ca2+/calmodulin-dependent protein kinase (CaMKII) inhibitor, K252a, blocked silibinin/TRAIL-induced DR5 expression along with TRAIL-mediated apoptosis. Accordingly, we showed that ROS/ER stress-induced CaMKII activated Sp1, which is an important transcription factor for DR5 expression. Our results showed that silibinin enhanced TRAIL-induced apoptosis by upregulating DR5 expression through the ROS-ER stress-CaMKII-Sp1 axis.

Sensitization of rhTRAIL-resistant Triple-negative Breast Carcinoma Through Silibinin Co-Treatment.

Triple-negative breast cancer (TNBC) is the most fatal form of breast cancer due to the shortcomings of therapies. However, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is a promising anticancer therapeutic that possesses the capability to promote the induction of apoptosis in cancer cells, but some TNBCs are resistant to rhTRAIL's pro-apoptotic effects. Therefore, a combinatorial treatment approach with silibinin and rhTRAIL was considered in order to sensitize rhTRAIL-resistant TNBCs. The co-treatment of rhTRAIL and silibinin's impact on apoptosis induction in rhTRAIL-resistant TNBC BT-20 and HCC1937 cells was inspected via application of Annexin V/PI assays and western blot analysis. Silibinin possessed the ability to sensitize the examined rhTRAIL-resistant TNBC cells to rhTRAIL-induced apoptosis through the up-regulation of death receptors 4 and 5 and the down-regulation of survivin transcriptionally. Silibinin is a good sensitizing agent for rhTRAIL-resistant TNBCs.

Cell death and restoration of TRAIL-sensitivity by ciglitazone in resistant cervical cancer cells.

Known activators of the Peroxisome Proliferator-Activated Receptor γ (PPARγ), thiazolidinediones (TZD) induce apoptosis in a variety of cancer cells through dependent and/or independent mechanisms of the receptor. We tested a panel of TZD (Rosiglitazone, Pioglitazone, Ciglitazone) to shed light on their potential therapeutic effects on three cervical cancer cell lines (HeLa, Ca Ski, C-33 A). In these cells, only ciglitazone triggered apoptosis through PPARγ-independent mechanisms and in particular via both extrinsic and intrinsic pathways in Ca Ski cells containing Human PapillomaVirus (HPV) type 16. It also inhibits cervical cancer xenograft development in nude mice. Ciglitazone kills cervical cancer cells by activating death receptor signalling pathway, caspase cascade and BH3 interacting-domain death agonist (Bid) cleavage through the up-regulation of Death Receptor 4 (DR4)/DR5 and soluble and membrane-bound TNF related apoptosis inducing ligand (TRAIL). Importantly, the drug let TRAIL-resistant Ca Ski cells to respond to TRAIL through the downregulation of cellular FLICE-Like Inhibitory Protein (c-FLIP) level. For the first time, we revealed that ciglitazone is able to decrease E6 viral oncoprotein expression known to block TRAIL pathway and this was associated with cell death. Our results highlight the capacity of ciglitazone to restore TRAIL sensitivity and to prevent E6 blocking action to induce apoptosis in cervical cancer cells.

Kaempferol Sensitizes Human Ovarian Cancer Cells-OVCAR-3 and SKOV-3 to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis via JNK/ERK-CHOP Pathway and Up-Regulation of Death Receptors 4 and 5.

BackgroundOvarian cancer is the most common gynecological malignancies in women, with high mortality rates worldwide. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily which preferentially induces apoptosis of cancer cells. However, acquired resistance to TRAIL hampers its therapeutic application. Identification of compounds that sensitize cancer cells to TRAIL is vital in combating resistance to TRAIL. The effect of kaempferol, a flavonoid enhancing TRAIL-induced apoptosis in ovarian cancer cells, was investigated in this study.Material/MethodsThe cytotoxic effects of TRAIL (25 ng/mL) and kaempferol (20–100 μM) on human ovarian cancer cells OVCAR-3 and SKOV-3 were assessed. Effect of kaempferol on the expression patterns of cell survival proteins (Bcl-xL, Bcl-2, survivin, XIAP, c-FLIP) and apoptotic proteins (caspase-3, caspase-8, caspase-9, Bax) were studied. The influence of kaempferol on expression of DR4 and DR5 death receptors on the cell surface and protein and mRNA levels was also analyzed. Apoptosis following silencing of DR5 and CHOP by small interfering RNA (siRNA), and activation of MAP kinases were analyzed as well.ResultsKaempferol enhanced apoptosis and drastically up-regulated DR4, DR5, CHOP, JNK, ERK1/2, p38 and apoptotic protein expression with decline in the expression of anti-apoptotic proteins. Further transfection with siRNA specific to CHOP and DR5 indicated the involvement of CHOP in DR5 up-regulation and also the contribution of DR5 in kaempferol-enhanced TRAIL-induced apoptosis.ConclusionsKaempferol sensitized ovarian cancer cells to TRAIL-induced apoptosis via up-regulation of DR4 and DR5 through ERK/JNK/CHOP pathways.

A study on the expression of apoptotic molecules related to death receptor and endoplasmic reticulum pathways in the jejunum of AFB1-intoxicated chickens.

Aflatoxin B1 (AFB1) is a common contaminant of poultry feeds in tropical and subtropical climates. Early researches have well established the hepatotoxic, carcinogenic, and immunotoxic effects of AFB1 on humans and animals. Recently, it has been shown that AFB1 could cause the up- or down-alteration of mitochondrial pathway molecule expression. However, the information on the expression of death receptor and endoplasmic reticulum molecules in the jejunal apoptosis induced by AFB1 were unavailable. So the present study was conducted to explore the expression of apoptotic molecules related to death receptor and endoplasmic reticulum in the jejunal cells of chickens exposed to AFB1 diet for 3 weeks. Total of 144 one-day-old chickens was randomly divided into two groups, namely control group (containing 0 mg/kg AFB1) and AFB1 group (containing 0.6 mg/kg AFB1). Histopathological observation and microscopic quantitative analysis revealed morphological changes in the jejunum such as the shedding of the mucosal epithelial cells in the apical region of villi along with the decrease of villus height, villus area and villus/crypt ratio in the AFB1 group. Both TUNEL and flow cytometry assays showed that AFB1 intake induced excessive apoptosis of jejunal cells. Quantitative real-time PCR test displayed the general upregulation of death receptors (FAS, FASL, TNF-α and TNF-R1), endoplasmic reticulum signals (GRP78 and GRP94) as well as initiator and executioner caspases (CASPASE-10, CASPASE-8 and CASPASE-3) in the jejunum of AFB1-intoxicated chickens. It's the first study demonstrating that AFB1 induced apoptosis of chickens’ jejunum accompanied by the alteration of death receptor and endoplasmic reticulum molecule expression.

N-myc downstream-regulated gene 1 promotes apoptosis in colorectal cancer via up-regulating death receptor 4.

The aim of this study was to evaluate the clinical significance of N-myc downstream-regulated gene 1 (NDRG1) in colorectal cancer (CRC) patients and to explore the mechanisms governing the role of NDRG1 in apoptosis of CRC cells. In the current study, we found that NDRG1 was a prognostic marker of CRC patients. Moreover, NDRG1 expression negatively correlated to tumor size and clinical TNM stage, suggesting that NDRG1 might act as a tumor suppressor by inhibiting proliferation or inducing apoptosis in CRC. Consistently, substantial apoptosis was observed in vitro and in vivo in the presence of NDRG1. From a mechanistic standpoint, we discovered that NDRG1 was able to prevent death receptor 4 from degradation induced by MARCH-8, a member of the membrane-associated RING-CH (MARCH) ubiquitin ligase family. As a consequence, CRC cells expressing NDRG1 were more sensitive to reagents targeting death receptors such as tumor necrosis factor-related apoptosis-inducing ligands (TRAIL). Additionally, the pro-apoptotic effect of NDRG1 was also validated in mouse xenograft model. In conclusion, our results provided further insights of the pivotal role of NDRG1 in apoptosis initiated by death receptors and demonstrated a novel marker to predict the sensitivity of CRC to TRAIL treatment in future clinical study.

MicroRNA-7 upregulates death receptor 5 and primes resistant brain tumors to caspase-mediated apoptosis.

MicroRNAs (miRs) are known to play a pivotal role in tumorigenesis, controlling cell proliferation and apoptosis. In this study, we investigated the potential of miR-7 to prime resistant tumor cells to apoptosis in glioblastoma (GBM). We created constitutive and regulatable miR-7 expression vectors and utilized pharmacological inhibition of caspases and genetic loss of function to study the effect of forced expression of miR-7 on death receptor (DR) pathways in a cohort of GBM with established resistance to tumor necrosis factor apoptosis inducing ligand (TRAIL) and in patient-derived primary GBM stem cell (GSC) lines. We engineered adeno-associated virus (AAV)-miR-7 and stem cell (SC) releasing secretable (S)-TRAIL and utilized real time in vivo imaging and neuropathology to understand the effect of the combined treatment of AAV-miR-7 and SC-S-TRAIL in vitro and in mouse models of GBM from TRAIL-resistant GSC. We show that expression of miR-7 in GBM cells results in downregulation of epidermal growth factor receptor and phosphorylated Akt and activation of nuclear factor-kappaB signaling. This leads to an upregulation of DR5, ultimately priming resistant GBM cells to DR-ligand, TRAIL-induced apoptotic cell death. In vivo, a single administration of AAV-miR-7 significantly decreases tumor volumes, upregulates DR5, and enables SC-delivered S-TRAIL to eradicate GBM xenografts generated from patient-derived TRAIL-resistant GSC, significantly improving survival of mice. This study identifies the unique role of miR-7 in linking cell proliferation to death pathways that can be targeted simultaneously to effectively eliminate GBM, thus presenting a promising strategy for treating GBM.

Abstract 2117: TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma

Abstract Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), the optimized form of the endogenous death ligand TRAIL, shows therapeutic potential for cancer due to its ability to induce apoptosis in cancer cells independent of p53, while exhibiting minimal toxicity to normal cells. Despite this, a majority of breast cancers display resistance to rhTRAIL treatment due to up-regulation of pro-apoptotic proteins, down-regulation of anti-apoptotic proteins, and/or up-regulation of death receptors (DR) 4 and 5. To overcome rhTRAIL resistance, natural compounds have been investigated as sensitizing agents. This study considered the application of the naturally occurring flavonol Quercetin (Q). Q has been shown to have the ability to up-regulate DR5 and down-regulate anti-apoptotic proteins in cancer cells, and thereby, making Q a favorable choice to be employed as a sensitizing agent. The intention of this study was to ascertain the capacity of Q to sensitize rhTRAIL-resistant triple negative breast cancer BT-20 cells and hormone-dependent breast cancer MCF-7 cells to rhTRAIL-induced apoptosis and elucidate the underlying mechanism for Q’s sensitization. Q demonstrated the ability to intensify rhTRAIL’s pro-apoptotic effects in the breast cancer BT-20 and MCF-7 cell lines as detected through Annexin V/PI assays followed by FACS analysis. In comparison to single agent treatments, the cotreatment of Q and rhTRAIL enhanced the induction of the extrinsic pathway of apoptosis as marked by PARP cleavage (a hallmark of apoptosis), activation of caspase 8, and activation of the executioner caspases 3 and 7. The mechanism for Q’s augmentation in breast cancer was determined to be through the down-regulation of c-FLIPL (caspase 8 inhibitor) in a dose-dependent manner. Furthermore, Q promoted the ubiquitination of c-FLIPL facilitating the proteasome-mediated degradation of c-FLIPL in breast cancer. An additional mechanism for Q’s sensitization was displayed in breast cancer BT-20 cells. Q up-regulated DR5 membrane and protein expression in breast cancer BT-20 cells in a dose-dependent manner. RT-PCR analysis revealed that Q’s influence on DR5 expression occurred at the transcriptional level in those breast cancer cells. Thus, these data suggest that the cotreatment of Q and rhTRAIL possesses the therapeutic potential to be an effective anti-breast carcinoma regimen. Citation Format: Jasmine M. Manouchehri, Michael Kalafatis. TRAIL-induced apoptosis in TRAIL-resistant breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2117. doi:10.1158/1538-7445.AM2017-2117

Abstract 124: Imipridone ONC201 promotes intra-tumoral accumulation of CD3+/NK+ cells that contribute to its anti-tumor efficacy

Abstract ONC201, a first-in-class oral anti-tumor agent, upregulates the pro-apoptotic immune cytokine TRAIL and activates the integrated stress response leading to upregulation of death receptor 5 in bulk tumor and cancer stem cells. We previously demonstrated that ONC201 exerts a dose- and schedule-dependent effect on tumor progression in vivo while suppressing Akt/ERK signaling in tumors in a dose/frequency-dependent manner (Wagner et al., AACR, 2016). We also provided evidence that ONC201 exhibits a potent anti-metastatic effect (Wagner et al., AACR, 2016). We observe accumulation and activation of TRAIL-secreting NK+ cells within ONC201-treated tumors in C57/BL6, Balb/c, and athymic nude tumor-bearing mice. Importantly, ONC201 exerts in vivo anti-tumor efficacy on tumor cell lines that are ONC201-resistant in vitro, including acquired stable resistance. Using the NK-depleting antibody GM1, we demonstrate that the activation and TRAIL secretion of NK cells by ONC201 significantly contributes to in vivo anti-tumor efficacy, including TRAIL/ONC201-resistant tumors. We are currently investigating how ONC201 recruits NK cells to the tumor by examining NK-recruiting chemokine factors within the tumor site. We have also demonstrated upregulation of CD3+ T cells by ONC201 in syngeneic mice. Finally, we observed an increase in activated TRAIL-secreting NK cells in the peripheral blood of patients upon ONC201 administration in the clinic. Our results demonstrate novel and potentially significant increases in cytotoxic NK cell recruitment to tumors. The results offer a unique pathway of immune stimulation for cancer therapy that may be combined with immune checkpoint or targeted cancer therapy strategies. We are currently investigating the role of NK cells and CD3+ cells in ONC201’s ability to inhibit metastasis by using a metastatic model that involves surgically removing the primary tumor and allowing metastases to grow in vivo before treatment. These findings indicate that ONC201 possess immunomodulatory activity and provide a rationale for combining ONC201 with PD-1/PDL-1 inhibitors, a combination we are currently testing in syngeneic immunocompetent mouse models. Citation Format: Jessica Wagner, C. Leah Kline, Lanlan Zhou, Andrew Zloza, Charles Chesson, Jenna Newman, Howard Kaufman, Joseph Bertino, Mark Stein, Wafik El-Deiry. Imipridone ONC201 promotes intra-tumoral accumulation of CD3+/NK+ cells that contribute to its anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 124. doi:10.1158/1538-7445.AM2017-124

Abstract 4047: NF-kB inhibitor DMAPT enhances cisplatin efficacy and reduces its toxicity in a carcinogen-induced model of muscle-invasive bladder cancer

Abstract BACKGROUND: Nuclear factor kappaB (NF-κB) blocks apoptosis and promotes chemoresistance to drugs like cisplatin (CDDP). However, the clinical use of NF-κB inhibitors is restricted to hematological malignancies and the benefit of NF-κB inhibition in solid tumors has yet to be realized. The novel agent dimethylaminoparthenolide (DMAPT) blocks NF-κB in bladder cancer cell lines and can overcome chemoresistance as part of a combination therapy. METHODS AND RESULTS: We tested DMAPT's ability to enhance CDDP's efficacy against muscle-invasive bladder cancer. In vitro, 2.5 µM DMAPT was shown to up-regulate death receptor 4 and 5 expression and induce poly (ADP-ribose) polymerase (PARP) cleavage in UMUC-3 cells. Both 2.5 µM DMAPT and 2.5 µM CDDP reduced UMUC-3 cell viability as single agents, and their effect was enhanced when combined. We next tested CDDP (4.0 mg/kg/week i.p) and DMAPT (100.0 mg/kg/day oral) in combination and as single agents for 6 weeks in an immune-competent mouse model of muscle-invasive (T2) bladder cancer induced by the tobacco-related agent N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN). Procedures followed the 2010/63/EU animal welfare directive. One DMAPT-treated and 1 CDDP-treated mouse died during the experiments. The cohorts were: negative control (no BBN exposure, n=15), positive control (BBN but no therapy, n=19), BBN and DMAPT-treated (n=17), BBN and CDDP-treated (n=18) and BBN and the combination of CDDP plus DMAPT (n=19). Negative control, positive control, DMAPT, CDDP and combination-treated mice showed 0.0%, 42.1%, 35.3%, 11.1% and 0.0% bladder cancer incidence, respectively. T2 (muscle-invasive) lesions were present in positive controls (15.8%), DMAPT (17.6%) and CDDP (5.6%), but not in the combination group (0.0%). High-grade urothelial dysplasia, a pre-malignant lesion, was observed at rates of: 57.9% (positive controls), 29.4% (DMAPT), 16.6% (CDDP) and 0.0% (combination). CDDP induced renal tubular necrosis and interstitial nephritis seen on H&E with increased urea (BUN) and creatinine. The combination therapy reduced interstitial nephritis, BUN (p<0.05, Student's t test) and creatinine levels. CDDP reduced gastrocnemius muscle mass, increased muscular fatigue (grip strength test) and reduced body weight. These effects were ameliorated in combination-treated mice. CDDP and the combination therapy induced anemia and atrophy of bone marrow, thymus and spleen at identical levels. CONCLUSION: The DMAPT-CDDP combination was more effective than CDDP as single agent in vitro and in vivo, and totally eradicated malignant and pre-malignant bladder lesions in an immune-competent mouse model. The combination ameliorated CDDP-induced nephrotoxicity and muscle wasting presumably by blocking CDDP mediated injury from NF-κB activation in normal tissues, without imposing additional hematological toxicity. Citation Format: Rui M. Gil da Costa, Pedro Ferreirinha, Nazaré Pinto da Cunha, Carlos Santos, Tiago Neto, Ana I. Faustino-Rocha, Manuel Vilanova, Margarida M. Bastos, Carlos Lopes, Paula A. Oliveira, Joaquim Gabriel, Peter Nelson, Christopher Sweeney. NF-kB inhibitor DMAPT enhances cisplatin efficacy and reduces its toxicity in a carcinogen-induced model of muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4047. doi:10.1158/1538-7445.AM2017-4047

DATS sensitizes glioma cells to TRAIL-mediated apoptosis by up-regulation of death receptor 5 via ROS

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) is a promising anticancer reagent for antitumor therapy. However, many cancer cells, including malignant glioma cells, tend to be resistant to TRAIL, due to repeat treat to cancer cells, highlighting the need for strategies to overcome TRAIL resistance. Here we present that in combination with diallyl trisulfide (DATS), exposure to TRAIL induced apoptosis in TRAIL-resistant glioma cells. Surprisingly, we found that subtoxic concentrations of DATS significantly potentiated TRAIL-induced cytotoxicity and apoptosis in glioma cells. DATS dramatically upregulated DR5 receptor expression but had no effects on DR4 receptor. In addition, DATS enhances TRAIL-induced apoptosis through the downregulation of anti-apoptotic protein (Mcl-1) and the upregulation of DR5 receptors through actions on the ROS- induced-p53.