Tumor Necrosis Factor-related Apoptosis-inducing Ligand Sensitivity Research Articles

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.

MP55-07 FLAVONOIDS ENHANCE TRAIL SENSITIVITY IN PROSTATE CANCER CELLS BY TARGETING ADENINE NUCLEOTIDE TRANSLOCASE-2

You have accessJournal of UrologyProstate Cancer: Basic Research III1 Apr 2015MP55-07 FLAVONOIDS ENHANCE TRAIL SENSITIVITY IN PROSTATE CANCER CELLS BY TARGETING ADENINE NUCLEOTIDE TRANSLOCASE-2 Masakatsu Oishi, Takashi Ueda, Terukazu Nakamura, Yoshio Naya, Fumiya Hongo, Kazumi Kamoi, Koji Okihara, and Tsuneharu Miki Masakatsu OishiMasakatsu Oishi More articles by this author , Takashi UedaTakashi Ueda More articles by this author , Terukazu NakamuraTerukazu Nakamura More articles by this author , Yoshio NayaYoshio Naya More articles by this author , Fumiya HongoFumiya Hongo More articles by this author , Kazumi KamoiKazumi Kamoi More articles by this author , Koji OkiharaKoji Okihara More articles by this author , and Tsuneharu MikiTsuneharu Miki More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2050AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. Recombinant human TRAIL has been under clinical trials, whereas various kinds of malignant tumors have resistance to TRAIL. It has been shown that several anticancer agents and flavonoids (apigenin, quercetin etc.) overcome resistance to TRAIL by upregulating death receptor 5 (DR5) in malignant tumor cells. However, the mechanisms by which these compounds induce DR5 expression remain unknown. On the other hand, although docetaxel plus prednisone is effective in hormone-refractory prostate cancer, the outcome of this treatment is still insufficient. Therefore, new strategies are needed to treat this cancer. Thus we investigated the mechanism in a hormone-refractory prostate cancer cell line DU145. METHODS To elucidate the mechanisms by which flavonoids induce DR5 expression in DU145 cells, we explored the proteins binding to flavonoids using FG beads with epoxy linkers. We fixed genistein, which enhances TRAIL sensitivity without upregulating DR5, apigenin, and quercetin onto these beads (Figure 1A). The binding proteins of apigenin, quercetin, and genistein were purified from the DU145 whole cell extracts and were identified by MALDI-TOF MS analysis. Then, function of binding proteins was analized. RESULTS Comparing the binding proteins, we discovered that ANT2 was a target of apigenin and quercetin, but not genistein in DU145 cells (Figure 1B). Similarly to treatment of apigenin and quercetin, knockdown of ANT2 enhanced TRAIL-induced apoptosis by upregulating DR5 expression. Moreover, silencing of ANT2 attenuated the enhancement of TRAIL-induced apoptosis by apigenin or quercetin. CONCLUSIONS These results suggest that flavonoids upregulate DR5 and enhance TRAIL-induced apoptosis in hormone-refractory prostate cancer cells by binding and inhibiting ANT2 (Figure 1C). We propose that ANT2 inhibitors may contribute to TRAIL therapy in hormone-refractory prostate cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e675 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Masakatsu Oishi More articles by this author Takashi Ueda More articles by this author Terukazu Nakamura More articles by this author Yoshio Naya More articles by this author Fumiya Hongo More articles by this author Kazumi Kamoi More articles by this author Koji Okihara More articles by this author Tsuneharu Miki More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

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.

Novel TRAIL sensitizer Taraxacum officinale F.H. Wigg enhances TRAIL-induced apoptosis in Huh7 cells.

TRAIL (TNF-related apoptosis inducing ligand) is a promising anti-cancer drug target that selectively induces apoptosis in cancer cells. However, many cancer cells are resistant to TRAIL-induced apoptosis. Therefore, reversing TRAIL resistance is an important step for the development of effective TRAIL-based anti-cancer therapies. We previously reported that knockdown of the TOR signaling pathway regulator-like (TIPRL) protein caused TRAIL-induced apoptosis by activation of the MKK7-c-Jun N-terminal Kinase (JNK) pathway through disruption of the MKK7-TIPRL interaction. Here, we identified Taraxacum officinale F.H. Wigg (TO) as a novel TRAIL sensitizer from a set of 500 natural products using an ELISA system and validated its activity by GST pull-down analysis. Furthermore, combination treatment of Huh7 cells with TRAIL and TO resulted in TRAIL-induced apoptosis mediated through inhibition of the MKK7-TIPRL interaction and subsequent activation of MKK7-JNK phosphorylation. Interestingly, HPLC analysis identified chicoric acid as a major component of the TO extract, and combination treatment with chicoric acid and TRAIL induced TRAIL-induced cell apoptosis via JNK activation due to inhibition of the MKK7-TIPRL interaction. Our results suggest that TO plays an important role in TRAIL-induced apoptosis, and further functional studies are warranted to confirm the importance of TO as a novel TRAIL sensitizer for cancer therapy. © 2015 Wiley Periodicals, Inc.

Icaritin Sensitizes Human Glioblastoma Cells to TRAIL-Induced Apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been considered to be one of the most promising candidates in research on treatments for cancer, because it induces apoptosis in a wide variety of cancer cells but not in most normal human cell types. However, many cells including glioblastoma (GBM) cells are resistant to TRAIL-induced apoptosis, which limits the potential application of TRAIL in cancer therapy. Icaritin, a hydrolytic product of icariin from Epimedium Genus, has been identified as a potential therapeutic and preventive agent in renal cell carcinoma and breast cancer. In this study, we investigated whether Icaritin treatment could modulate TRAIL-induced apoptosis in GBM. The effect of icaritin on TRAIL sensitivity was assessed in human GBM U87 and U373 cells. The underlying regulatory cascades were approached by biochemical and pharmacological strategies. We found that nontoxic concentration of icaritin alone had no significant effect on the level of apoptosis, but a combination treatment of TRAIL and icaritin caused a significantly more profound apoptosis. The sensitization was accompanied by c-FLIP down-regulation and inhibition of NF-κB activity. Studies have further demonstrated that silencing NF-κB alone was sufficient to down-regulate c-FLIP expression and sensitized both tested cells to TRAIL-induced apoptosis. These data suggest that icaritin sensitizes TRAIL-induced tumor cell apoptosis via suppression of NF-κB-dependent c-FLIP expression, providing in vitro evidence supporting the notion that icaritin is a potential sensitizer of TRAIL in anticancer therapy against human GBM.

Novel Molecular Regulators of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis in NSCLC Cells.

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in some non-small cell lung cancer (NSCLC) cells, some NSCLC cells exhibit TRAIL-resistance. The underlying mechanisms that regulate TRAIL sensitivity in NSCLC cells are not well understood. The objective of this study was to investigate molecular regulators of the TRAIL pathway in NSCLC cells. The TRAIL-sensitive NSCLC cell line NCI-H358 and a TRAIL-resistant cell line A549 were treated with rmhTRAIL for 24 hours. Then cell viability were measured by MTT assay, meanwhile cell cycle and apoptosis were measured by flow cytometry. Furthermore, mass spectrometry (LC-MS/MS) was used to identify the difference in the protein expression profiles. Finally, real-time PCR was performed to detect the mRNA expression of TRAIL receptors and apoptotic related proteins. These results confirmed that NCI-H358 cells were sensitive to TRAIL, whereas A549 cells were resistant. Both mRNA and protein levels of voltage-dependent anion-selective channel proteinl (VDAC1), caspase9 (CASP9), and cytochrome c1 (CYC1) were upregulated in H358 cells but downregulated in A549 cells, whereas antiapoptotic protein BAG-2 was downregulated. In addition, TRAIL also causes DR5 low expression in A549 cells. These results indicate that rmhTRAIL had different anti-tumor activity in different NSCLC cell lines. Downregulation of VDAC1, CYC1, CASP9, and upregulation of BAG-2 might be associated with underlying TRAIL-resistance mechanisms. These findings motivated further studies to explore new therapeutic strategy overcoming TRAIL-resistance of NSCLC cells through modulating dysregulation of the proteins above.

Upregulation of Death Receptor 5 and Production of Reactive Oxygen Species Mediate Sensitization of PC-3 Prostate Cancer Cells to TRAIL Induced Apoptosis by Vitisin A

Background/Aims: Although Vitisin A, derived from wine grapes, is known to have cytotoxic, anti-adipogenic, anti-inflammatory and antioxidant effects, the underlying antitumor mechanism has not been investigated in prostate cancer cells to date. In the present study, the apoptotic mechanism of Vitisin A plus TNF-related apoptosis-inducing ligand (TRAIL) in prostate cancer cells was elucidated. Methods: The cytotoxicity of Vitisin A and/or TRAIL against PC-3, DU145 and LNCaP prostate cancer cells was measured by MTT colorimetric assay. Annexin V-FITC Apoptosis Detection kit was used to detect apoptotic cells by flow cytometry. Intracellular levels of ROS were measured by flow cytometry using 2070-diacetyl dichlorofluorescein (DCFDA). Results: Combined treatment with Vitisin A and TRAIL enhanced cytotoxicity and also increased sub-G1 population in PC-3 cells better than DU145 or LNCap prostate cancer cells. Similarly, Annexin V and PI staining revealed that combination increased early and late apoptosis in PC-3 cells compared to untreated control. Consistently, combination attenuated the expression of pro-caspases 7/8, DcR1, Bcl-XL or Bcl-2 and activated caspase 3, FADD, DR5 and DR4 in PC-3 cells. Also, combination increased DR5 promoter activity compared to untreated control. Furthermore, combination increased the production of reactive oxygen species (ROS) and DR5 cell surface expression. The ROS inhibitor NAC and silencing of DR5 by siRNA transfection inhibited the ability of combination to induce PARP cleavage and generate ROS. Conclusion: These findings provide evidence that Vitisin A can be used in conjunction with TRAIL as a potent TRAIL sensitizer for synergistic apoptosis induction via upregulation of DR5 and production of ROS in prostate cancer cells.

XIAP-targeting drugs re-sensitize PIK3CA-mutated colorectal cancer cells for death receptor-induced apoptosis.

Mutations in the oncogenic PIK3CA gene are found in 10–20% of colorectal cancers (CRCs) and are associated with poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic TRAIL death receptor antibodies emerged as promising anti-neoplastic therapeutics, but to date failed to prove their capability in the clinical setting as especially primary tumors exhibit high rates of TRAIL resistance. In our study, we investigated the molecular mechanisms underlying TRAIL resistance in CRC cells with a mutant PIK3CA (PIK3CA-mut) gene. We show that inhibition of the constitutively active phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway only partially overcame TRAIL resistance in PIK3CA-mut-protected HCT116 cells, although synergistic effects of TRAIL plus PI3K, Akt or cyclin-dependent kinase (CDK) inhibitors could be noted. In sharp contrast, TRAIL triggered full-blown cell death induction in HCT116 PIK3CA-mut cells treated with proteasome inhibitors such as bortezomib and MG132. At the molecular level, resistance of HCT116 PIK3CA-mut cells against TRAIL was reflected by impaired caspase-3 activation and we provide evidence for a crucial involvement of the E3-ligase X-linked inhibitor of apoptosis protein (XIAP) therein. Drugs interfering with the activity and/or the expression of XIAP, such as the second mitochondria-derived activator of caspase mimetic BV6 and mithramycin-A, completely restored TRAIL sensitivity in PIK3CA-mut-protected HCT116 cells independent of a functional mitochondrial cell death pathway. Importantly, proteasome inhibitors and XIAP-targeting agents also sensitized other CRC cell lines with mutated PIK3CA for TRAIL-induced cell death. Together, our data suggest that proteasome- or XIAP-targeting drugs offer a novel therapeutic approach to overcome TRAIL resistance in PIK3CA-mutated CRC.

Co-acting gene networks predict TRAIL responsiveness of tumour cells with high accuracy

BackgroundIdentification of differentially expressed genes from transcriptomic studies is one of the most common mechanisms to identify tumor biomarkers. This approach however is not well suited to identify interaction between genes whose protein products potentially influence each other, which limits its power to identify molecular wiring of tumour cells dictating response to a drug. Due to the fact that signal transduction pathways are not linear and highly interlinked, the biological response they drive may be better described by the relative amount of their components and their functional relationships than by their individual, absolute expression.ResultsGene expression microarray data for 109 tumor cell lines with known sensitivity to the death ligand cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was used to identify genes with potential functional relationships determining responsiveness to TRAIL-induced apoptosis. The machine learning technique Random Forest in the statistical environment “R” with backward elimination was used to identify the key predictors of TRAIL sensitivity and differentially expressed genes were identified using the software GeneSpring. Gene co-regulation and statistical interaction was assessed with q-order partial correlation analysis and non-rejection rate. Biological (functional) interactions amongst the co-acting genes were studied with Ingenuity network analysis. Prediction accuracy was assessed by calculating the area under the receiver operator curve using an independent dataset. We show that the gene panel identified could predict TRAIL-sensitivity with a very high degree of sensitivity and specificity (AUC = 0 · 84). The genes in the panel are co-regulated and at least 40% of them functionally interact in signal transduction pathways that regulate cell death and cell survival, cellular differentiation and morphogenesis. Importantly, only 12% of the TRAIL-predictor genes were differentially expressed highlighting the importance of functional interactions in predicting the biological response.ConclusionsThe advantage of co-acting gene clusters is that this analysis does not depend on differential expression and is able to incorporate direct- and indirect gene interactions as well as tissue- and cell-specific characteristics. This approach (1) identified a descriptor of TRAIL sensitivity which performs significantly better as a predictor of TRAIL sensitivity than any previously reported gene signatures, (2) identified potential novel regulators of TRAIL-responsiveness and (3) provided a systematic view highlighting fundamental differences between the molecular wiring of sensitive and resistant cell types.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1144) contains supplementary material, which is available to authorized users.

Bortezomib sensitizes primary meningioma cells to TRAIL-induced apoptosis by enhancing formation of the death-inducing signaling complex.

A meningioma is the most common primary intracranial tumor in adults. Here, we investigated the therapeutic potential of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in 37 meningiomas. Freshly isolated primary meningioma cells were treated with TRAIL with or without different sensitizing protocols, and apoptotic cell death was then quantified. Mechanisms of TRAIL sensitization were determined by a combination of Western blotting, flow cytometry, receptor complex immunoprecipitation, and siRNA-mediated knockdown experiments. Tumor necrosis factor-related apoptosis-inducing ligand receptor expression was analyzed using immunohistochemistry and quantified by an automated software-based algorithm. Primary tumor cells from 11 (29.7%) tumor samples were sensitive to TRAIL-induced apoptosis, 12 (32.4%) were intermediate TRAIL resistant, and 14 (37.8%) were completely TRAIL resistant. We tested synergistic apoptosis-inducing cotreatment strategies and determined that only the proteasome inhibitor bortezomib potently enhanced expression of the TRAIL receptors TRAIL-R1 and/or TRAIL-R2, the formation of the TRAIL death-inducing signaling complex, and activation of caspases; this treatment resulted in sensitization of all TRAIL-resistant meningioma samples to TRAIL-induced apoptosis. Bortezomib pretreatment induced NOXA expression and downregulated c-FLIP, neither of which caused the TRAIL-sensitizing effect. Native TRAIL receptor expression could not predict primary TRAIL sensitivity. This first report on TRAIL sensitivity of primary meningioma cells demonstrates that TRAIL/bortezomib cotreatment may represent a novel therapeutic option for meningiomas.

Sensitization of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant primary prostate cancer cells by isoegomaketone from Perilla frutescens.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently in clinical trials as a cancer treatment due to its ability to induce apoptosis selectively in cancer cells. Nevertheless, the risk of developing resistance warrants the development of sensitizers that can overcome resistance to TRAIL. In this study, isoegomaketone (1) acted as a synergistic TRAIL sensitizer by mediating up-regulation of DR5 expression in primary prostate cancer RC-58T/h/SA#4 cells. Combined with 1, TRAIL exhibited enhanced apoptotic activity in a human prostate cancer cell line designated RC-58T/h/SA#4, as indicated by increases in annexin V-positive and sub-G1 cell populations as well as condensation of chromatin or apoptotic bodies. Combined treatment also activated caspases-8, -9, and -3; increased the protein levels of Bax, AIF, and cytosolic cytochrome c; and induced PARP cleavage while reducing Bcl-2 protein expression. Human recombinant DR5 Fc chimera efficiently attenuated 1-induced apoptosis, thereby demonstrating the critical role of DR5 in 1-mediated apoptotic cell death. Furthermore, DR5 expression induced by 1 was mediated via a ROS-independent pathway that required CHOP and p53. Overall, these findings provide evidence that 1 potentiates TRAIL-mediated apoptosis through up-regulation of DR5 via a ROS-independent pathway. This suggests that 1 has potential for increasing the effectiveness of prostate cancer therapy with TRAIL.

Resveratrol sensitizes HepG2 cells to TRAIL-induced apoptosis.

Resveratrol is a natural polyphenol found in a wide variety of plants, including grapes, berries, and peanuts. Resveratrol can modulate a wide spectrum of molecular targets, including those involved in cancer signaling pathways. Here, we evaluated the role of resveratrol in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and examined the molecular mechanisms in the human hepatocellular carcinoma cell line HepG2. We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay to assess cell viability, flow cytometry to analyze cell cycle and apoptosis, and immunoblotting to detect protein expression. Resveratrol decreased cell viability at a concentration of 100 μmol/l or higher. At a concentration of 50 μmol/l, resveratrol induced S phase arrest of the cell cycle without apoptosis. In addition, phospho-AMPK increased significantly in a dose-dependent manner. Resveratrol was found to synergistically augment TRAIL-induced apoptosis. The rates of early apoptosis were 3.4, 9.6, and 49.6% on treatment with 50 μmol/l resveratrol, 10 ng/ml TRAIL, and both reagents, respectively. Resveratrol significantly downregulated the expression of survivin in a dose-dependent manner. In conclusion, we found that that resveratrol could augment TRAIL sensitivity by downregulating survivin. These results suggest that combination resveratrol with TRAIL may be an effective new strategy for the treatment of hepatocellular carcinoma.

Abstract 5118: Decursin synergistically enhances TRAIL-induced apoptosis through CHOP dependent DR5 signaling in TRAIL resistant lung cancer cells

Abstract Targeting the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which has been clinically established for anticancer therapy, is the attractive strategy to specifically kill cancer cells through the death receptors. Nonetheless, its therapeutic potential is limited due to the resistance in cancer cells. Thus, in the present study, we evaluated the therapeutic potential of Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root as a TRAIL sensitizer in a variety of resistant lung cancer cells. Co-treatment of Decursin with TRAIL synergistically induced apoptosis in TRAIL resistant lung cancer cells, A545, H1299, H596 and Calu-1. This synergistic effect through the induction of DR5 in Decursin treated cells was also confirmed by immunoblotting and immunoanalysis of endogeneous and surface expression of DR5, respectively. In contrast, in DR knockdown cells, combination treatment Decursin and TRAIL did not induce apoptosis at all. Furthermore, we found that DR5 induction by Decursin was mediated by ER-stress and its byproduct CHOP. Depletion of CHOP via siRNA transfection completely blocked Decusin induced apoptosis cell death in TRAIL resistant lung cancer cells. Taken together, induction of the DR5 via upregulation of CHOP and its inducer, ER stress, by Decursin leads to synergistically increase the TRAIL mediated cell death in TRAIL resistant lung cancer cells. Our results indicate that Decursin is a useful therapeutic strategy for overcoming TRAIL resistance in lung tumor treatment. Key words: Decursin, DR-5, ER stress, CHOP, Trail resistant, Lung cancer Conflict of Interests: The authors disclose no potential conflicts of interest. Acknowledgment: This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korea Government (MEST) (no. 2012-0005755). Citation Format: Kim Jae Kwang. Decursin synergistically enhances TRAIL-induced apoptosis through CHOP dependent DR5 signaling in TRAIL resistant lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5118. doi:10.1158/1538-7445.AM2014-5118

Abstract 2286: Regulation of GSK3β axis by combination treatment with TRAIL and Troglitazone in cancer cells

Abstract Prostate cancer is estimated to account for 29% of all new cancers and is the second leading cause of cancer-related death in men in the United States. Hormonal therapy is the only treatment for advanced forms, but androgen-independence eventually develops in most patients. Developing new treatment strategies are urgently needed, which needs a deeper molecular understanding of the events that lead to tumor progression. TNF-related apoptosis inducing ligand (TRAIL) has gained much importance recently due to its ability to preferentially induce cell death in malignant and transformed cells. However, since many tumor cells develop resistance to TRAIL, recent approaches are focused on developing combinatorial therapeutic regimens that can enhance TRAIL sensitivity. There are multiple reasons behind TRAIL resistance, and recent studies (including ours) indicate that, Glycogen Synthase Kinase 3β (GSK3β) might be a key player in mediating this. Thus pathways that can antagonize GSK3β axis are important targets for cancer drug development. GSK3β is a serine/threonine kinase, initially identified as a critical mediator of glycogen metabolism and insulin signaling and is now well accepted to regulate various cellular processes including cell survival. In our earlier studies, combinatorial treatment with Troglitazone (TZD), a synthetic ligand for peroxisome proliferator-activator receptor gamma (PPARγ) and TRAIL induced significant apoptosis in TRAIL-resistant cancer cells. Utilizing this combination, we also observed an increase in GSK3βSer9 phosphorylation that preceded the onset of apoptosis. At a later time, however, TRAIL-TZD combination produced a dramatic reduction of total GSK3β levels, suggesting that GSK3β is being targeted by multiple pathways following this treatment. Similar results were observed in various prostate as well as pancreatic cancer cells, indicating this to be a generalized event. Interestingly, pretreatment of Bx-PC3 pancreatic cancer cells (with a shorter half-life of GSK3β) with protein synthesis inhibitor Cycloheximide (CHX) significantly reduced TRAIL-TZD-induced inhibition of total GSK3β expression, suggesting mostly a transcriptional regulation. Luciferase assays carried out in both pancreatic and prostate cancer cells indicated dramatic reduction of GSK3β promoter activity with TRAIL-TZD. Knockdown of endogenous PPARγ expression by PPARγ- siRNA resulted in a reduction of PPARγ levels with siRNA transfection, which was unable to antagonize TRAIL-TZD-induced reduction of GSK3β transcription or protein expression. These suggested a potential PPARγ-independent regulation of GSK3β in this pathway. Since the mechanisms that regulate GSK3β expression in cancer cells are largely unknown, these studies indicate a novel strategy of targeting GSK3β, elucidation of which might provide newer insights to improve our understanding of TRAIL resistance. Citation Format: Sunipa Majumdar, Sreevidya Santha, Ajay Rana, Basabi Rana. Regulation of GSK3β axis by combination treatment with TRAIL and Troglitazone in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2286. doi:10.1158/1538-7445.AM2014-2286

Aplysin sensitizes cancer cells to TRAIL by suppressing P38 MAPK/survivin pathway.

TNF-related apoptosis-inducing ligand (TRAIL) is a tumor-selective apoptosis inducer and has been shown to be promising for treating various types of cancers. However, the application of TRAIL is greatly impeded by the resistance of cancer cells to its action. Studies show that overexpression of some critical pro-survival proteins, such as survivin, is responsible for TRAIL resistance. In this study, we found that Aplysin, a brominated compound from marine organisms, was able to restore the sensitivity of cancer cells to TRAIL both in vitro and in vivo. Aplysin was found to enhance the tumor-suppressing capacity of TRAIL on several TRAIL-resistant cancer cell lines. TRAIL-induced apoptosis was also potentiated in A549 and MCF7 cells treated with Aplysin. Survivin downregulation was identified as a mechanism by which Aplysin-mediated TRAIL sensitization of cancer cells. Furthermore, the activation of p38 MAPK was revealed in Aplysin-treated cancer cells, and its inhibitor SB203580 was able to abrogate the promoting effect of Aplysin on the response of cancer cells to TRAIL action, as evidenced by restored survivin expression, elevated cell survival and reduced apoptotic rates. In conclusion, we provided evidence that Aplysin acts as a sensitizer for TRAIL and its effect on p38 MAPK/survivin pathway may partially account for this activity. Considering its low cytotoxicity to normal cells, Aplysin may be a promising agent for cancer treatment in combination with TRAIL.

H-Ras regulation of TRAIL death receptor mediated apoptosis.

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists.

Surrounding pancreatic adenocarcinoma by killer mesenchymal stromal/stem cells.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic malignancy still associated with an unacceptably poor prognosis. In addition to neoplastic tissue, PDAC is characterized by a prominent desmoplastic reaction infiltrating and surrounding cancer cells (Chu et al., 2007). This fibrotic microenvironment is generated predominantly by stromal cells known as tumor-associated fibroblasts, the origin of which has been also attributed to mesenchymal stromal/stem cells (MSCs) deriving from either bone marrow (BM) or adipose tissue (AD) (Grisendi et al., 2011). Because MSCs can be induced to produce anticancer molecules, such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) (Grisendi et al., 2010), we here show the impact of gene-modified human MSCs producing TRAIL against PDAC. The BxPC3 cell line (Fig. 1, left) was selected on the basis of its high expression of functional TRAIL receptors associated with recombinant human TRAIL sensitivity confirmed in a dose–response assay (data not shown). FIG. 1. Pancreatic adenocarcinoma cell line BxPC3 interplays with adipose-derived mesenchymal stem cells expressing green fluorescent protein (GFP AD-MSCs) only or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/GFP AD-MSCs). Scale bar, 50 μm. ... Microscopic examination of cocultures (Fig. 1, middle) of BxPC3 cells with adipose-derived MSCs expressing green fluorescent protein (GFP AD-MSCs), as control, revealed a peculiar interaction between MSCs and tumor cells, with BxPC3 cells arranged as islets surrounded by green AD-MSC bundles, in parallel to what is described for the PDAC-associated desmoplastic reaction in vivo (Chu et al., 2007). Having proved the interaction between GFP AD-MSCs and BxPC3 cells, coculture experiments were then performed for both 24 and 48 hr, testing various target-to-effector (T:E) ratios (1:1, 1:2, and 1:5), using TRAIL/GFP AD-MSCs. Tumor cell death was detected in all tested conditions. The interplay between TRAIL/GFP AD-MSCs and BxPC3 cells was followed by a breakdown of tumor islets together with the disruption of adherent BxPC3 cells, resulting in clusters of dead floating elements in culture medium above an established layer of TRAIL/GFP AD-MSCs (Fig. 1, right; T:E ratio, 1:2 at 48 hr; scale bar, 50 μm). These morphological findings were quantified by flow cytometric detection (data not shown) of apoptotic propidium iodide-positive cells, reaching 69±3% (average±SD) for TRAIL/GFP AD-MSCs versus 14±1% for GFP AD-MSCs (p=0.001 by two-tailed t test). Similar features were observed in cocultures of TRAIL/GFP AD-MSCs and an additional PDAC cell line (MIA PaCa-2 cells; data not shown). Depicting the strong interaction with genetically modified AD-MSCs and PDAC lines, we here suggest MSCs as suitable vectors for anticancer agent delivery, opening novel therapeutic opportunities for this still incurable cancer.

Targeting c-Met receptor overcomes TRAIL-resistance in brain tumors.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced apoptosis specifically in tumor cells. However, with approximately half of all known tumor lines being resistant to TRAIL, the identification of TRAIL sensitizers and their mechanism of action become critical to broadly use TRAIL as a therapeutic agent. In this study, we explored whether c-Met protein contributes to TRAIL sensitivity. We found a direct correlation between the c-Met expression level and TRAIL resistance. We show that the knock down c-Met protein, but not inhibition, sensitized brain tumor cells to TRAIL-mediated apoptosis by interrupting the interaction between c-Met and TRAIL cognate death receptor (DR) 5. This interruption greatly induces the formation of death-inducing signaling complex (DISC) and subsequent downstream apoptosis signaling. Using intracranially implanted brain tumor cells and stem cell (SC) lines engineered with different combinations of fluorescent and bioluminescent proteins, we show that SC expressing a potent and secretable TRAIL (S-TRAIL) have a significant anti-tumor effect in mice bearing c-Met knock down of TRAIL-resistant brain tumors. To our best knowledge, this is the first study that demonstrates c-Met contributes to TRAIL sensitivity of brain tumor cells and has implications for developing effective therapies for brain tumor patients.

Quercetin enhances apoptotic effect of tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) in ovarian cancer cells through reactive oxygen species (ROS) mediated CCAAT enhancer‐binding protein homologous protein (CHOP)‐death receptor 5 pathway

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown efficacy in a phase 2 clinical trial, development of resistance to TRAIL by tumor cells is a major roadblock. We investigated whether quercetin, a flavonoid, can sensitize human ovarian cancer cells to TRAIL. Results indicate that quercetin sensitized cancer cells to TRAIL. The quercetin induced expression of death receptor DR5 but did not affect expression of DR4 in cancer cells. The induction of DR5 was mediated through activation of JNK and through upregulation of a transcription factor CCAAT enhancer-binding protein homologous protein (CHOP); as silencing of these signaling molecules abrogated the effect of quercetin. Upregulation of DR5 was mediated through the generation of reactive oxygen species (ROS), as ROS scavengers reduced the effect of quercetin on JNK activation, CHOP upregulation, DR induction, TRAIL sensitization, downregulated the expression of cell survival proteins and upregulated the proapoptotic proteins. Furthermore, quercetin enhances TRAIL mediated inhibition of tumor growth of human SKOV-3 xenograft was associated with induction of apoptosis, activation of caspase-3, CHOP and DR5. Overall, our data suggest that quercetin enhances apoptotic death of ovarian cancer cells to TRAIL through upregulation of CHOP-induced DR5 expression following ROS mediated endoplasmic reticulum-stress.

Identification of novel molecular regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in breast cancer cells by RNAi screening.

IntroductionTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to its receptors, TRAIL-receptor 1 (TRAIL-R1) and TRAIL-receptor 2 (TRAIL-R2), leading to apoptosis by activation of caspase-8 and the downstream executioner caspases, caspase-3 and caspase-7 (caspase-3/7). Triple-negative breast cancer (TNBC) cell lines with a mesenchymal phenotype are sensitive to TRAIL, whereas other breast cancer cell lines are resistant. The underlying mechanisms that control TRAIL sensitivity in breast cancer cells are not well understood. Here, we performed small interfering RNA (siRNA) screens to identify molecular regulators of the TRAIL pathway in breast cancer cells.MethodsWe conducted siRNA screens of the human kinome (691 genes), phosphatome (320 genes), and about 300 additional genes in the mesenchymal TNBC cell line MB231. Forty-eight hours after transfection of siRNA, parallel screens measuring caspase-8 activity, caspase-3/7 activity, or cell viability were conducted in the absence or presence of TRAIL for each siRNA, relative to a negative control siRNA (siNeg). A subset of genes was screened in cell lines representing epithelial TNBC (MB468), HER2-amplified breast cancer (SKBR3), and estrogen receptor-positive breast cancer (T47D). Selected putative negative regulators of the TRAIL pathway were studied by using small-molecule inhibitors.ResultsThe primary screens in MB231 identified 150 genes, including 83 kinases, 4 phosphatases, and 63 nonkinases, as potential negative regulators of TRAIL. The identified genes are involved in many critical cell processes, including apoptosis, growth factor-receptor signaling, cell-cycle regulation, transcriptional regulation, and DNA repair. Gene-network analysis identified four genes (PDPK1, IKBKB, SRC, and BCL2L1) that formed key nodes within the interaction network of negative regulators. A secondary screen of a subset of the genes identified in additional cell lines representing different breast cancer subtypes and sensitivities to TRAIL validated and extended these findings. Further, we confirmed that small-molecule inhibition of SRC or BCL2L1, in combination with TRAIL, sensitizes breast cancer cells to TRAIL-induced apoptosis, including cell lines resistant to TRAIL-induced cytotoxicity.ConclusionsThese data identify novel molecular regulators of TRAIL-induced apoptosis in breast cancer cells and suggest strategies for the enhanced application of TRAIL as a therapy for breast cancer.