Exploring the TRAILs less travelled: TRAIL in cancer biology and therapy.

In an attempt to improve the chemotherapy of acute leukemia in adults, the author has studied the induction therapy on 63 cases with acute leukemia with Neocarzinostatin (NCS; N) in combination with conventional antileukemic agents; Vincristine (V), Daunorubicin (D), Cytosine Arabinoside (A), 6-Mercaptopurine-Riboside (M) and Prednisolone (P). Eighteen cases with acute lymphocytic leukemia (ALL) was treated with protocol A (NVMP, NDMP, NAMP). Out of 45 cases with acute non-lymphocytic leukemia (ANLL), 25 was treated with protocol A and 20 treated with protocol B (NADP, NAVP), and obtained the following results. 1) Of all 63 cases 51 (81.6%) attained complete remission and 9 (14.3%) partial remission. 2) Complete remission was obtained in 15 cases (83.3%) out of 18 with ALL and 36 cases (80.0%) of 45 with ANLL; the latter cases with ANLL consisted of 18 of 25 cases (72.0%) treated with protocol A and 18 of 20 cases (90.0%) with protocol B. 3) Refractory varieties included cases with acute myelogenous leukemia who had passed through atypical hematological findings, cases with monocytic leukemia, cases with WBC count over 50,000 per μl or with nucleated cell count in bone marrow over 500,000 per μl before treatment and cases over 50 years old. 4) In this induction therapy with NCS in combination, antileukemic effects was remarkable, while suppression on normal hematopoiesis, especially on thrombocytes, was very slight. 5) As to the other side effects no noteworthy disturbances other than occasional nausea, anorexia and liver damage in protocol B were observed. From these results it can be concluded that NCS in combination is one of the most potent remission induction therapies in acute leukemia in adults.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.

Because Bcl-2 family members inhibit the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis, we investigated whether ABT-737, a small molecule Bcl-2 inhibitor, enhances TRAIL killing. We demonstrate that a combination of ABT-737 and TRAIL induced significant cell death in multiple cancer types, including renal, prostate, and lung cancers, although each agent individually had little activity in these tumor cells. All of these cell lines expressed the Mcl-1 protein that is known to block the activity of ABT-737 and TRAIL but did not block the synergy between these agents. However, Bax-deficient cell lines, including DU145 and HCT116 cells and those cell lines expressing low levels of TRAIL receptor, were resistant to apoptosis induced by these agents. To understand how ABT-737 functions to markedly increase TRAIL sensitivity, the levels of specific death-inducing signaling complex components were evaluated. Treatment with ABT-737 did not change the levels of c-FLIP, FADD, and caspase-8 but up-regulated the levels of the TRAIL receptor DR5. DR5 up-regulation induced by ABT-737 treatment occurred through a transcriptional mechanism, and mutagenesis studies demonstrated that the NF-kappaB site found in the DR5 promoter was essential for the ability of ABT-737 to increase the levels of this mRNA. Using luciferase reporter plasmids, ABT-737 was shown to stimulate NF-kappaB activity. Together, these results demonstrate that the ability of ABT-737 and TRAIL to induce apoptosis is mediated through activation of both the extrinsic and intrinsic pathways. Combinations of ABT-737 and TRAIL can be exploited therapeutically where antiapoptotic Bcl-2 family members drive tumor cell resistance to current anticancer therapies.

Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable interest as a potential anti-cancer strategy, TRAIL agonists have had very limited anti-cancer activity in humans. TRAIL signaling in T cells may also potentially provide an immune checkpoint function as it can inhibit T cell activation and proliferation by interfering with T cell receptor signaling. However, this potential immune checkpoint function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, provides a model to examine the potential immune checkpoint function of TRAIL. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both TRAIL and TRAIL receptor (TR) were implanted into livers of WT and Tr−/− mice. Hence, in this model the host immune cells express TRAIL but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr−/− mice had a significant reduction in tumor burden compared to WT mice. Moreover, tumor bearing Tr−/− mice had a significant increase in cytotoxic T lymphocytes (CTLs) and enhanced CTL effector function. However, coculture of T cells with SB cells or SB cells deficient in Tr (SB-Tr−/−) did not result in a significant difference in T cell apoptosis or function, implying that TRAIL-TR is not a direct T cell checkpoint. Myeloid derived suppressor cells (MDSCs) were significantly decreased in Tr−/− tumors compared to WT tumors. Furthermore, implantation of SB cells devoid of Trail (SB-Trail−/−) into WT mice resulted in a significant reduction in tumor burden and MDSC infiltration. Coculture of SB cells with MDSCs from Tr−/− mice attenuated MDSC proliferation and immunosuppression compared to WT MDSCs. Moreover, treatment of MDSCs from Tr−/− mice with TRAIL recombinant protein resulted in a reduction in their proliferation and immunosuppressive function compare to MDSCs from WT mice implying that TRAIL-TR fosters MDSC growth and immunosuppressive function. In conclusion, we have demonstrated that Tr−/− mice have a significant reduction in CCA tumor burden and MDSC infiltration. Consequently, Tr−/− mice bearing tumors have enhanced CTL infiltration and function. These data suggest that the TRAIL-TR system mediates tumor immune evasion via MDSCs. Herein, we suggest that TRAIL-TR appears to function as an indirect T cell checkpoint by augmenting the immunosuppressive effects of MDSCs. Citation Format: Emilien Loeuillard, Juan Wang, Jingchun Yang, Haidong Dong, Gregory Gores, Sumera Ilyas. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) foster myeloid-derived suppressor cell-mediated tumor immune evasion in cholangiocarcinoma [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P072.

In the present study, it was found that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-R2 protein expression did not correlate with mRNA expression in melanoma cell lines. In particular, early passage primary cultures from patients had low TRAIL-R2 protein expression compared with later passage cultures although TRAIL-R2 mRNA expression was similar in early and late passages. Similarly, cell lines made resistant to TRAIL by cultures in TRAIL had low TRAIL-R2 protein expression but normal levels of mRNA for TRAIL-R2. Expression from a luciferase reporter gene construct with the 3'-untranslated region (UTR) (but not the 5'-UTR) of TRAIL-R2 was suppressed when transfected into the TRAIL-selected (resistant) melanoma lines compared with that seen in the parental (sensitive) lines. Similar results were seen in early passage (resistant) cultures compared with late passage (sensitive) primary melanoma cultures. RNA gel shift assays demonstrated protein(s) binding to the 3'-UTR of TRAIL-R2 mRNA that were more evident in TRAIL-resistant cultures with low TRAIL-R2 protein expression. A 23-base fragment of the 3'-UTR inhibited binding of the proteins to the 3'-UTR, and a probe using this fragment bound to proteins in TRAIL-selected melanoma lines and early passage isolates of melanoma. Binding of the 3'-UTR probe to the cytosolic protein(s) was induced by exposure to TRAIL and was lost from the TRAIL selected lines 2-3 days after withdrawal of TRAIL from the cultures. These results are consistent with post-transcriptional regulation of TRAIL-R2 expression by cytosolic proteins induced by TRAIL that bind to the 3'-UTR region of TRAIL-R2 mRNA.

Background and Aims: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable interest as a potential anti-cancer strategy, TR agonists have had very limited anti-cancer activity in humans. TRAIL signaling in T cells may also potentially provide an immune checkpoint function as it can inhibit T cell activation and proliferation by interfering with T cell receptor signaling. However, this potential immune checkpoint function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a malignancy of the bile ducts, provides a model to examine the potential immune checkpoint function of TRAIL. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both TRAIL and TRAIL receptor (TR) were implanted into livers of WT C57BL/6J and Tr−/− mice. Hence, in this model the host immune cells express TRAIL but not the receptor; they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to potential TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed and tumor and immune characterization (via flow cytometry) was conducted. Results: Implantation of SB cells into Tr−/− mice result in a significant reduction in tumor volumes compared to WT mice. Tumor-bearing Tr−/− mice had a significant infiltration of cytotoxic T lymphocytes (CTLs) (CD45+CD3+CD8+CD11a+) and enhanced CTL effector function. Moreover, Tr−/− mice tumors had a significant decrease in granulocytic and monocytic myeloid-derived suppressor cell (MDSCs) as well as tumor-associated macrophages (TAMs) (CD45+F4/80+CD11b+CD206+) compared to WT mice tumors. Conclusion: Using a unique syngeneic orthotopic implantation model of murine CCA, we demonstrate that CCA cells expressing TRAIL in a mouse genetically deficient for TR exhibit reduced tumor volumes enhanced CTL infiltration and function, and reduced MDSC and TAM infiltration into the tumors. These data suggest that TRAIL has a potential immune checkpoint function, and targeting TRAIL signaling with consequent augmentation of CTL function maybe a promising therapeutic approach in human cancers. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera Ilyas. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates tumor immune evasion in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2738.

Tumor growth is often associated with insufficient apoptosis. The Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) and its proapoptotic receptors death receptor 4 (DR4) and DR5 agonistic monoclonal antibodies are being developed as targeted therapeutics because they kill cancer cells while sparing normal cells. A challenge to targeted therapeutics is the selection of patients who are most likely to benefit from targeted drugs because of the heterogeneity of cancer. Molecular imaging may be useful in targeted drug development by assessing the target expression and drug-target interaction, and for predicting therapeutic response. We hypothesized that the cell surface expression level of DR4/5 may predict the proapoptotic targeted therapeutic response if the signaling pathway downstream is intact. The goal of this proof-of-concept study was to develop a molecular imaging strategy to predict proapoptotic anti-cancer therapy response at an early stage of treatment. TRAIL and the DR5 agonistic monoclonal antibody HGS-ETR2 (Lexatumumab, TRM-2) were labeled with a near-infrared dye and these were used to image the TRAIL receptors on cultured TRAIL sensitive and TRAIL resistant human tumor cells as well as tumor xenografts. Imaging of cells and tumor-bearing animals was conducted with near infrared fluorescence imagers and apoptosis in cells was assessed by western blots of PARP-cleavage and flow cytometry of sub-G1 content. Apoptosis in tumors was evaluated by imaging near-infrared dye-labeled Annexin V and tumor tissue activated caspase-3 staining. Both in vitro and in vivo studies showed that imaging of death inducing ligand-receptor interaction was consistent with the apoptosis readout. Thus TRAIL sensitive tumors that express TRAIL receptors underwent cell death following treatment whereas tumors lacking TRAIL receptor expression were shown to be TRAIL resistant. In vivo molecular imaging of TRAIL receptor expression correlated with response to TRAIL therapy and an apoptotic response in vivo.

Introduction: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can kill tumor cells while leaving normal cells unharmed. However, many breast and ovarian cancer cells are resistant to TRAIL-mediated apoptosis. Breast cancers can be divided into those which express the estrogen (ER) and progesterone (PR) receptors, those with HER-2 amplification, and those without expression of ER, PR, or HER-2 amplification (referred to as basal or triple-negative breast cancer). Due to the lack of specific target there is no targeted therapy for triple negative breast cancer. Recently, we demonstrated that a set of triple negative breast cancer cell lines with mesenchymal features are sensitive to TRAIL. Still TRAIL has very little or no effect on the triple negative cells with epithelial characteristics. We are investigating if this is true for the cell lines derived from different ethnic group. Method: Both epithelial (viz., MDA-MB-468, from African American and HCC1937 from non Hispanic white) and mesenchymal cell lines (viz., MDA-MB-157 from African American, and MDA-MB-231 from non Hispanic white patient) were selected for this study. All of the cell lines were grown in MEM medium supplemented with 10% FCS with or without 1ug/ml TRAIL for 16h. Percentage of growth inhibition was determined by MTS assay. Correlation between growth inhibition and apoptosis were established by mitochondrial membrane depolarization, caspase assay, and DNA fragmentation. Result: Both of the epithelial triple negative cells were non responsive to TRAIL. Only some (10.5% for MB-468 and 18.6% for HCC1937) growth inhibition was observed even with highest concentration (5ug/ml) of TRAIL irrespective to the ethnicity differences. The effect of TRAIL in these cell lines was not dose dependant. On the other hand, both cell lines with mesenchymal feature from both ethnicities were highly responsive to TRAIL. More than 70% (71.5% for MB-157 and 75.2% for MB-231) of growth inhibition was achieved with TRAIL at 1ug/ml concentration and the effect was dose dependent. In epithelial cells both the TRAIL-receptor-1 (TR-1) and TRAIL receptor-2 (TR-2) expression were very low. In MB-231 silencing of only TR-2 reduces the effect of TRAIL but not TR-1. Conclusions: Though cells from non Hispanic white patients were a little more sensitive to TRAIL than cells from African American patient, the difference was minor and it was not statistically significant. Low level or TR-2 expression in epithelial cell lines may contribute to TRAIL resistant to these cell lines. We think TRAIL could be used either alone or in combination to treat breast cancer with mesenchymal phenotype. Appropriate combination could be searched for TRAIL therapy for breast cancer with epithelial phenotype. We are investigating additional cell lines derived from different racial or ethnic groups to determine if there is any correlation between ethnicity and TRAIL sensitivity and underlying possible molecular mechanism. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):B45.

Colorectal cancer is the third most common malignancy in the United States. Modest advances with therapeutic approaches that include oxaliplatin (L-OHP) have brought the median survival rate to 22 months, with drug resistance remaining a significant barrier. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is undergoing clinical evaluation. Although human colon carcinomas express TRAIL receptors, they can also demonstrate TRAIL resistance. Constitutive NF-kappaB activation has been implicated in resistance to TRAIL and to cytotoxic agents. We have demonstrated constitutive NF-kappaB activation in five of six human colon carcinoma cell lines; this activation is inhibited by quinacrine. Quinacrine induced apoptosis in colon carcinomas and potentiated the cytotoxic activity of TRAIL in RKO and HT29 cells and that of L-OHP in HT29 cells. Similarly, overexpression of IkappaBalpha mutant (IkappaBalphaM) or treatment with the IKK inhibitor, BMS-345541, also sensitized these cells to TRAIL and L-OHP. Importantly, 2 h of quinacrine pretreatment resulted in decreased expression of c-FLIP and Mcl-1, which were determined to be transcriptional targets of NF-kappaB. Extended exposure for 24 h to quinacrine did not further sensitize these cells to TRAIL- or L-OHP-induced cell death; however, exposure caused the down-regulation of additional NF-kappaB-dependent survival factors. Short hairpin RNA-mediated knockdown of c-FLIP or Mcl-1 significantly sensitized these cells to TRAIL and L-OHP. Taken together, data demonstrate that NF-kappaB is constitutively active in colon cancer cell lines and NF-kappaB, and its downstream targets may constitute an important target for the development of therapeutic approaches against this disease.

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.

2A Peptide-based, Lentivirus-mediated Anti-death Receptor 5 Chimeric Antibody Expression Prevents Tumor Growth in Nude Mice

Introduction: Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and accounts for most cancer-related deaths worldwide. Despite progress in the treatment of subgroups of patients with targeted therapies, better treatments are required to improve overall prognosis. Agents that target the tumor-necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) receptors (TRAIL-R1 and -R2) are able to trigger selective apoptosis in tumor cells, however showed discouraging activity in clinical studies. Resistance to TRAIL-induced apoptosis and non-canonical pro-tumorigenic signaling hampers therapeutic efficacy. Previously, we found that TRAIL induces migration and invasion via RIPK1/Src/STAT3 pathway in apoptosis resistant NSCLC cells. Here we aim to analyze in more detail the role of Src in TRAIL non-canonical signaling and apoptosis resistance. Methods: Cytotoxicity to rhTRAIL was assessed by MTT assays. Src was chemically inhibited or genetically ablated by short hairpin(sh)RNA or CRISPR/CAS9. Src phosphorylation was studied by western blotting. Protein interactions were examined by co-immunoprecipitation (IP) and subsequent western blotting. Src interacting proteins were examined by co-IP experiments in conjunction with LC-mass spectrometric (MS) analyses. Results: The function of Src in TRAIL signaling was examined in TRAIL resistant A549 and sensitive H460 NSCLC cells. rhTRAIL treatment revealed distinct Src phosphorylation patterns, indicating that Src is differentially activated by TRAIL. Subsequently, co-IP experiments were performed showing that in A549 cells Src interacts with RIPK1 and Caspase-8 upon TRAIL treatment, but not in H460 cells. Next, we explored the possible role of Src in regulating TRAIL-induced apoptosis by chemical modulation of Src or by gene silencing or genetic knockout of Src. We found no role for Src in regulating sensitivity or resistance to TRAIL-induced apoptosis. To further investigate possible biological consequences of TRAIL-dependent Src activation we performed co-IP coupled with LC-MS analysis. Abundant differences were found in the Src interactome of A549 and H460 cells and in absence and presence of TRAIL. Various proteins known to be involved in tumor signaling were identified to be in complex with Src, including components of the RAF/MEK/ERK, Wnt and SMAD3 signaling pathways. Currently, mechanistic and validation studies are in progress to elucidate the role of these proteins. Conclusions: Src has no role in controlling sensitivity or resistance to TRAIL-induced apoptosis in the examined NSCLC cells. On the contrary, the Src interactome showed the activation of various pro-tumorigenic pathways by TRAIL. We anticipate that a deeper knowledge of TRAIL signaling will lead to novel therapeutic strategies to improve TRAIL-receptor targeted therapy. Supported by a grant from the Dutch Cancer Society (KWF 2011-5211). Citation Format: Margot de Looff, Steven de Jong, Frank A.E. Kruyt. The role of Src in TRAIL signaling in non-small cell lung cancer cells [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 4377.

Mechanisms of tumor necrosis factor-related apoptosis inducing ligandcombined with Triptolide in inducing the apoptosis of pancreatic cancer cells

Non-small cell lung cancer (NSCLC) is a disease with poor prognosis and novel therapeutic approaches are greatly needed. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an interesting agent that is able to trigger apoptosis through interactions with TRAIL receptors. An important feature of TRAIL is its ability to induce apoptosis in a wide variety of tumors without harming normal cells, making it an attractive anti-cancer therapeutic compared to conventional anti-cancer agents. However, TRAIL is also able to activate signaling pathways that are involved in survival, proliferation and migration of tumor cells. Thus, TRAIL-based therapies combined with inhibitors of such pathways are expected to enhance therapeutic benefit. In this study, we aimed to identify and characterize kinases that are activated by recombinant TRAIL in NSCLC cells. We monitored the activation of a number of kinases known to be involved in TRAIL signaling by Western blotting, including p38 MAPK, JNK, ERK and Akt. In addition we employed PepChip kinase arrays. With these arrays 1024 peptide kinase substrates can be screened in one experiment, whereby a comparison of kinase patterns between untreated and treated cells can be obtained. NSCLC, H460 and A549 cell lines, which are sensitive and resistant for TRAIL, respectively, were exposed to 50 ng/ml TRAIL for different periods of time (5 to 240 minutes) to evaluate the kinetics of kinase activation. In H460 cells, TRAIL induced the phosphorylation of p38 MAPK after 2 hours and JNK1/2 after 3 hours. As the activation of these kinases can be both anti- and pro-apoptotic, kinase inhibitors were used to explore this further. In H460 cells the activation of JNK, ERK and Akt had anti-apoptotic activity. Inhibition of these kinases with SP600125, PD098059, and LY294002, respectively, showed a 2-fold increase in apoptosis when combined with TRAIL. The activation of MAPK p38 on the other hand was pro-apoptotic, since its inhibition with SB203580 resulted in a reduction of TRAIL-induced apoptosis in H460 cells. In resistant A549 cells, however, Akt, ERK, p38 MAPK, and JNK1/2 activation appeared to have anti-apoptotic activity. Furthermore, in these cells an increase in IκBα phosphorylation was observed that was not seen in H460 cells, where levels of phosphorylated IκBα decreased after 1 hour that correlated with cleavage of RIP. Thus suppression of NFκB activation could be associated with TRAIL sensitivity. PepChip kinase arrays, revealed the activation of kinases that are involved in cell migration, such as Rho/Rock in A549 cells, and further investigations are ongoing. In conclusion, we observed differential TRAIL-dependent activation of p38 MAPK, JNK1/2, ERK, Akt and IκBα in sensitive and resistant NSCLC cells as well as in pathways that regulate migration. The relationship with TRAIL antitumor activity is currently further explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1941. doi:10.1158/1538-7445.AM2011-1941

The aim of this study is to investigate whether apoptosis in human beta cells can be related to the induction of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. We examined the expression of TRAIL and TRAIL receptors in two human pancreatic beta-cell lines and in human primary islet cells using RT-PCR assays and flow cytometric analyses and tested TRAIL-mediated beta-cell destruction in (51)Cr release cytotoxicity assays, Annexin-V and APO-DIREC assays. Most of the human beta cells express TRAIL receptors-R1, -R2, -R3, -R4 and/or TRAIL. TRAIL induced much stronger cytotoxicity and apoptosis to beta-cell lines CM and HP62 than did FasL, TNF-alpha, LTalpha1beta2, LTalpha2beta1, LIGHT, and IFN-gamma. The cytotoxicity and apoptosis induced by TRAIL to beta-cell lines CM were inhibited competitively by soluble TRAIL receptors, R1, R2, R3 or R4. Treatment of these beta cells with antibodies against TRAIL receptors was able to block the cytotoxicity of TRAIL to these cells. Beta-cell antigen-specific CTL (CD4(+) and CD8(+)) clones express TRAIL, suggesting that these cells are potential sources of TRAIL-inducing beta-cell destruction. Normal primary islet cells from most donors are resistant to the cytotoxicity mediated by TRAIL. However, treatment with an inhibitor of protein synthesis (cycloheximide) or with an enzyme (PI-PLC) that can remove TRAIL-R3 from the islet-cell membrane was able to increase the susceptibility of TRAIL-resistant primary islet cells to the TRAIL death pathway. The TRAIL death pathway is present and can function in human islet beta cells, but unidentified inhibitors of the TRAIL death pathway are present in normal islet cells.