TRAIL Sensitivity Research Articles

Abstract B81: Oncolytic adenovirus YSC-02 enhanced tumor cell killing and immune response with broad spectrums, which resulted in both TGF-β and HSP27 cooperatively becoming silenced

Abstract As combining therapeutic agents with different action mechanisms may enhance efficacy, YSC-02, an oncolytic adenovirus with multi targets was loaded with five different genes, which were designed with the expectation that different action mechanisms would cooperate. In spite of concerns regarding many APIs, YSC-02 was constructed to be an adenovirus-based anti-cancer drug. By using our own established mouse model system suitable for efficient adenoviral infection and replication, immune activity as well as survival potential could be precisely estimated for anti-cancer drug efficacy. YSC-02 was designed to decrease tumor cell survival, metastasis and to increase tumor cell death, and immune activation. It is composed of five different target genes, including one fused form and two shRNAs, but each of these genes functions is closely linked to produce the maximal antitumor effect. YSC-02 is like an organic complex designed to be applied primarily to heterogeneous liver cancer and melanoma. The greatest difficulty in developing anticancer drugs is deeply related to the existence of many genetic variations even in the same tumor tissue. However, in our study, the downregulation of HSP27 greatly increased the death potential of most tumor cell types, irrespective of differential cell death induced by TGF-β1 or TGF-β2 isotypes. The simultaneous silencing of both HSP27 and TGF-β could significantly repress the tumor survival potential of various cancer cells, which leads to most cell types of death in the complex network of cells. These combined shRNAs have also contributed to the increase of TRAIL sensitivity. Genetically modified mouse cancer cells within syngenic mice could make it possible to estimate the superiority of YSC-02 over its derivatives including GMCSF-expressing oncolytic adenovirus or GMCSF-FETZ-expressing oncolytic adenovirus. At the immunological level, NK cells or T cells were shown to be most activated in the treatment of YSC-02, which may be another contributor of anti-tumor activity. Additionally, even the highest dose of YSC-02 did not produce any toxicity in the liver. Citation Format: Zhezhu Han, Suyeon Je, Hye J. Choi, Dongxu Kang, Rong Xu, Hyun S. Kim, Jae J. Song. Oncolytic adenovirus YSC-02 enhanced tumor cell killing and immune response with broad spectrums, which resulted in both TGF-β and HSP27 cooperatively becoming silenced. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B81.

A Microplate-Based Nonradioactive Protein Synthesis Assay: Application to TRAIL Sensitization by Protein Synthesis Inhibitors.

Non-radioactive assays based on incorporation of puromycin into newly synthesized proteins and subsequent detection using anti-puromycin antibodies have been previously reported and well-validated. To develop a moderate- to high-throughput assay, an adaptation is here described wherein cells are puromycin-labeled followed by simultaneously probing puromycin-labeled proteins and a reference protein in situ. Detection using a pair of near IR-labeled secondary antibodies (InCell western, ICW format) allows quantitative analysis of protein synthesis in 384-well plates. After optimization, ICW results were compared to western blot analysis using cycloheximide as a model protein synthesis inhibitor and showed comparable results. The method was then applied to several protein synthesis inhibitors and revealed good correlation between potency as protein synthesis inhibitors to their ability to sensitize TRAIL-resistant renal carcinoma cells to TRAIL-induced apoptosis.

Aspirin induces cell death, TRAIL sensitization, and mitochondrial network abnormalities in human melanoma cells

Aspirin induces cell death, TRAIL sensitization, and mitochondrial network abnormalities in human melanoma cells

Tumor-selective killing and TRAIL sensitization by garlic organosulfur compounds in human melanoma cells: Role of l-type calcium channel

Tumor-selective killing and TRAIL sensitization by garlic organosulfur compounds in human melanoma cells: Role of l-type calcium channel

YM155 sensitizes TRAIL-induced apoptosis through cathepsin S-dependent down-regulation of Mcl-1 and NF-κB-mediated down-regulation of c-FLIP expression in human renal carcinoma Caki cells.

YM155, a small-molecule survivin inhibitor, has been reported for its anti-cancer activity in various cancer cells. In this study, we investigated the effect of YM155 to enhance TRAIL-mediated apoptosis in human renal carcinoma cells. We found that YM155 alone had no effect on apoptosis, however, combined treatment with YM155 and TRAIL markedly induced apoptosis in human renal carcinoma cells (Caki, ACHN, and A498), breast cancer cells (MDA-MB231), and glioma cells (U251MG), but not normal cells [mesangial cell (MC) and human skin fibroblast (HSF)]. YM155 induced down-regulation of Mcl-1 expression at the post-translational levels, and the overexpression of Mcl-1 markedly inhibited YM155 plus TRAIL-induced apoptosis. Furthermore, YM155 induced down-regulation of c-FLIP mRNA expression through inhibition of NF-κB transcriptional activity. Ectopic expression of c-FLIP markedly blocked YM155-induced TRAIL sensitization. Taken together, our results suggested that YM155 sensitizes TRAIL-mediated apoptosis via down-regulation of Mcl-1 and c-FLIP expression in renal carcinoma Caki cells.

Abstract 3513: Promoting TRAIL apoptosis signaling using 17-beta-hydroxywithanolides

Abstract We have previously reported that withanolide E (WE), a steroidal lactone from Physalis peruviana, was highly active in sensitizing various human carcinoma cell lines to TRAIL-mediated apoptosis. Treatment of cancer cells with WE induced a reduction in the levels of the antiapoptotic proteins cFLIPL and cFLIPS, resulting in an increased activation of caspase-8 on subsequent TRAIL binding to its death receptors DR4 or DR5. The reduction in cFLIPL and cFLIPS was due to their destabilization and increased degradation by the proteasome. Interestingly, WE (a 17-beta-hydoxywithanolide, 17-BHW) was a far superior TRAIL sensitizer than more widely studied withaferin A (WFA) and its analogues, which lack the 17-beta-hydroxy group and bear an opposite side chain orientation, exhibit more promiscuous reactivity and are much more directly toxic to cells. Therefore, over 30 natural and semi-synthetic 17-BHWs were evaluated for their ability to promote death ligand-mediated cancer cell death. The 17-BHWs used in this work were obtained by the application of an efficient method of plant biomass production involving our innovative and patented soil-less aeroponic cultivation of P. crassifolia and P. peruviana and by chemical modification of natural withanolides produced by these plants. Our studies identified several 17-BHWs that were 4-8 fold more potent than WE in sensitizing the renal carcinoma cells ACHN to TRAIL-mediated apoptosis. These more active 17-BHWs were also more efficient at reducing cellular levels of cFLIPL and cFLIPS and enhancing caspase-8 activation. Preliminary structure activity relationship (SAR) studies suggested that the enone moiety in ring A was essential for activity. In addition acetoxylation at C-18, an alpha orientation of the lactone group and the double bond at C-24(25) of the lactone ring played important roles in determining the activity of 17-BHWs as TRAIL sensitizers. This suggests that the 17-BHW scaffold is amenable to optimization by a medicinal chemistry approach, which could lead to the identification of highly active natural product-based sensitizers of cancer cells to TRAIL-mediated apoptosis. The cellular molecular target(s) of active 17-BHWs are currently under further investigation. Funded by FNLCR Contract HHSN261200800001E Citation Format: Alan D. Brooks, Ya-ming Xu, E. M. Kithsiri Wijeratne, Poonam Tewary, Curtis J. Henrich, Cheryl L. Thomas, A. A. Leslie Gunatilaka, Thomas J. Sayers. Promoting TRAIL apoptosis signaling using 17-beta-hydroxywithanolides. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3513.

Abstract 3012: ONC201 induces cell death in triple negative, BRCA1-deficient and non-triple negative breast cancer cells

Abstract Breast cancer is a major cause of cancer related death. TRAIL has been of interest as a cancer therapeutic, but resistance has been observed in breast cancer and only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL. Small molecule ONC201 functions through the ATF4/CHOP pathway to upregulate TRAIL receptor DR5 (Kline et. al., Sci. Sig., in press, 2015), and through dual inhibition of Akt/ERK signaling to upregulate TRAIL (Allen et. al Sci. Trans. Med., 2013). We recently reported that ONC201 depletes colorectal cancer stem cell (CSC) markers and prevents formation of colonospheres. ONC201 also prevents colorectal CSCs from initiating growth of xenografted tumors (Prabhu et. al Can. Res., 2015). ONC201 has completed its first-in-human clinical trial in advanced solid tumors (Stein et al., 2015 AACR-NCI-EORTC meeting) and is being tested in multiple phase I/II clinical trials (NCT02250781, NCT02324621, NCT02420795, NCT02392572, NCT02609230, NCT02525692, NCT02038699). We investigated ONC201 efficacy in TNBC (TRAIL-sensitive), BRCA1-deficient, and non-TNBC (TRAIL-resistant) cells. We demonstrate IC50 values for ONC201 in the low μM range for TNBC or BRCA1-deficient (n = 6) and non-TNBC cells (n = 5), doses that are achievable based on human PK. Importantly, ONC201 induces apoptotic death in both TNBC and non-TNBC cells. ONC201 depletes Aldefluor+ putative breast CSCs in vitro whereas paclitaxel does not. ONC201 inhibits growth of TNBC, BRCA1-deficient and non-TNBC CSC-like mammospheres while paclitaxel chemotherapy does not. We show ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 TNBC xenograft model. We investigated effects of ONC201 on mechanisms of TRAIL resistance in breast cancer cells, including expression of inhibitor of apoptosis (IAP) family proteins. We found that ONC201 mediates a decrease in XIAP, c-IAP1, and c-IAP2 expression in the breast cancer cells used. We also examined levels of DR5 using flow cytometry, as a mechanism of TRAIL resistance in breast cancer cells involves receptor endocytosis from the cell surface (Zhang et al., Mol Cancer Res., 2008). Interestingly, we observed greater increases in cell surface DR5 in TNBC cells than non-TNBC cells after ONC201 treatment. Our findings suggest that ONC201 exerts cytotoxic effects against a broad range of breast cancer cells, including TNBC, BRCA1-deficient, and non-TNBC subtypes. ONC201 is efficacious against breast cancer cells regardless of the TRAIL sensitivity of the cells, and this efficacy may be mediated through mechanisms involving downregulation of IAP proteins and upregulation of cell surface death receptors. Furthermore, ONC201 may target paclitaxel-resistant breast CSCs. Our findings suggest unique targeting of multiple non-TNBC, BRCA1-deficient and TNBC cells and develop a preclinical rationale for the use of ONC201 as a treatment for breast cancer. Citation Format: Marie D. Baumeister, Jessica Wagner, Varun V. Prabhu, Christina LB Kline, Bora Lim, Josh E. Allen, David T. Dicker, Wafik S. El-Deiry. ONC201 induces cell death in triple negative, BRCA1-deficient and non-triple negative breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3012.

HuR Contributes to TRAIL Resistance by Restricting Death Receptor 4 Expression in Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal cancers, in part, due to resistance to both conventional and targeted therapeutics. TRAIL directly induces apoptosis through engagement of cell surface Death Receptors (DR4 and DR5), and has been explored as a molecular target for cancer treatment. Clinical trials with recombinant TRAIL and DR-targeting agents, however, have failed to show overall positive outcomes. Herein, we identify a novel TRAIL resistance mechanism governed by Hu antigen R (HuR, ELAV1), a stress-response protein abundant and functional in PDA cells. Exogenous HuR overexpression in TRAIL-sensitive PDA cell lines increases TRAIL resistance whereas silencing HuR in TRAIL-resistant PDA cells, by siRNA oligo-transfection, decreases TRAIL resistance. PDA cell exposure to soluble TRAIL induces HuR translocation from the nucleus to the cytoplasm. Furthermore, it is demonstrated that HuR interacts with the 3'-untranslated region (UTR) of DR4 mRNA. Pre-treatment of PDA cells with MS-444 (Novartis), an established small molecule inhibitor of HuR, substantially increased DR4 and DR5 cell surface levels and enhanced TRAIL sensitivity, further validating HuR's role in affecting TRAIL apoptotic resistance. NanoString analyses on the transcriptome of TRAIL-exposed PDA cells identified global HuR-mediated increases in antiapoptotic processes. Taken together, these data extend HuR's role as a key regulator of TRAIL-induced apoptosis. Discovery of an important new HuR-mediated TRAIL resistance mechanism suggests that tumor-targeted HuR inhibition increases sensitivity to TRAIL-based therapeutics and supports their re-evaluation as an effective treatment for PDA patients. Mol Cancer Res; 14(7); 599-611. ©2016 AACR.

Modulation of TRAIL mediated apoptosis signaling by Helicobacter pylori

Abstract Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. However, the mechanisms by which cause disease remain unclear. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through down-regulation of cellular FLICE-inhibitory protein (FLIP). Our study thus demonstrates H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. We further demonstrated that FLIPS expression was consistent with Akt activation. H. pylori induced reduced Akt phosphorylation and led to decreased FLIPS expression in human gastric epithelial cells. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.

Identification of Mitoxantrone as a TRAIL-sensitizing agent for Glioblastoma Multiforme

ABSTRACTTumor necrosis factor related apoptosis-inducing ligand (TRAIL) has tremendous promise in treating various forms of cancers. However, many cancer cells exhibit or develop resistance to TRAIL. Interestingly, many studies have identified several secondary agents that can overcome TRAIL resistance. To expand on these studies, we conducted an extensive drug-re-profiling screen to identify FDA-approved compounds that can be used clinically as TRAIL-sensitizing agents in a very malignant type of brain cancer, Glioblastoma Multiforme (GBM). Using selected isogenic GBM cell pairs with differential levels of TRAIL sensitivity, we revealed 26 TRAIL-sensitizing compounds, 13 of which were effective as single agents. Cardiac glycosides constituted a large group of TRAIL-sensitizing compounds, and they were also effective on GBM cells as single agents. We then explored a second class of TRAIL-sensitizing drugs, which were enhancers of TRAIL response without any effect on their own. One such drug, Mitoxantrone, a DNA-damaging agent, did not cause toxicity to non-malignant cells at the doses that synergized with TRAIL on tumor cells. We investigated the downstream changes in apoptosis pathway components upon Mitoxantrone treatment, and observed that Death Receptors (DR4 and DR5) expression was upregulated, and pro-apoptotic and anti-apoptotic gene expression patterns were altered in favor of apoptosis. Together, our results suggest that combination of Mitoxantrone and TRAIL can be a promising therapeutic approach for GBM patients.

MiR-424 and miR-27a increase TRAIL sensitivity of acute myeloid leukemia by targeting PLAG1.

Although microRNAs have been elaborated to participate in various physiological and pathological processes, their functions in TRAIL resistance of acute myeloid leukemia (AML) remain obscure. In this study, we detected relatively lower expression levels of miR-424&27a in TRAIL-resistant and semi-resistant AML cell lines as well as newly diagnosed patient samples. Overexpression of miR-424&27a, by targeting the 3′UTR of PLAG1, enhanced TRAIL sensitivity in AML cells. Correspondingly, knockdown of PLAG1 sensitized AML cells to TRAIL-induced apoptosis and proliferation inhibition. We further found that PLAG1 as a transcription factor could reinforce Bcl2 promoter activity, causing its upregulation at the mRNA level. Both downregulated PLAG1 and elevated expression of miR-424&27a led to Bcl2 downregulation and augmented cleavage of Caspase8, Caspase3 and PARP in the presence of TRAIL. Restoration of Bcl2 could eliminate their effects on AML TRAIL sensitization. Overall, we propose that miR-424&27a and/or PLAG1 might serve as novel therapeutic targets in AML TRAIL therapy.

Galangin sensitizes TRAIL-induced apoptosis through down-regulation of anti-apoptotic proteins in renal carcinoma Caki cells.

Galangin, bioflavonoids, has been shown anti-cancer properties in various cancer cells. In this study, we investigated whether galangin could enhance TRAIL-mediated apoptosis in TRAIL resistant renal carcinoma Caki cells. Galangin alone and TRAIL alone had no effect on apoptosis, while combined treatment with galangin and TRAIL significantly induced apoptosis in renal carcinoma (Caki, ACHN and A498) but not normal cells (normal mouse kidney cells and human normal mesangial cells). Galangin induced down-regulation of Bcl-2 protein at the transcriptional level via inhibition of NF-κB activation but not p53 pathway. Furthermore, galangin induced down-regulation of cFLIP, Mcl-1 and survivin expression at the post-translational levels, and the over-expression of Bcl-2, cFLIP, Mcl-1 and survivin markedly reduced galangin-induced TRAIL sensitization. In addition, galangin increased proteasome activity, but galangin had no effect on expression of proteasome subunits (PSMA5 and PSMD4). In conclusion, our investigation suggests that galangin is a potent candidate for sensitizer of TRAIL resistant cancer cell therapy.

Cytoplasmic levels of cFLIP determine a broad susceptibility of breast cancer stem/progenitor-like cells to TRAIL.

BackgroundThe clinical application of TRAIL receptor agonists as a novel cancer therapy has been tempered by heterogeneity in tumour responses. This is illustrated in breast cancer, where TRAIL is cytotoxic in cell lines of mesenchymal origin but refractory in lines with an epithelial-like phenotype. However, it is now evident that intra-tumour heterogeneity includes a minority subpopulation of tumour-initiating stem/progenitor-like cells (CSCs) that possess mesenchymal characteristics. We hypothesised therefore that TRAIL may target these phenotypically distinct CSC-like cells that are common to most - if not all - breast cancers, thus impacting on the source of malignancy in a much broader range of breast tumour subtypes than previously envisaged.MethodsWe used colony formation, tumoursphere, flow cytometry and xenograft tumour initiation assays to observe the TRAIL sensitivity of CSC-like cells in a panel of two mesenchymal-like (TRAIL-sensitive) and four epithelial-like (TRAIL-resistant) breast cancer cell lines. Subcellular levels of the endogenous TRAIL inhibitor, cFLIP, were determined by western blot and immunofluorescence microscopy. The effect of the subcellular redistribution of cFLIP on TRAIL sensitivity and Wnt signalling was determined using cFLIP localisation mutants and the TOPFlash reporter assay respectively.ResultsTRAIL universally suppressed the clonal expansion of stem/progenitors in all six of the breast cancer cell lines tested, irrespective of their phenotype or overall sensitivity to TRAIL. A concomitant reduction in tumour initiation was confirmed in the TRAIL-resistant epithelial cell line, MCF-7, following serial dilution xenotransplantation. Furthermore TRAIL sensitivity of breast CSCs was inversely proportional to the relative cytoplasmic levels of cFLIP while overexpression of cFLIP in the cytosol using subcellular localization mutants of cFLIP protected these cells from cytotoxicity. The accumulation of nuclear cFLIP on the other hand did not influence TRAIL cytotoxicity but instead promoted Wnt-dependent signalling.ConclusionThese data propose a novel role for TRAIL as a selective CSC agent with a broad specificity for both epithelial and mesenchymal breast tumour subtypes. Furthermore we identify a dual role for cFLIP in the maintenance of breast CSC viability, dependent upon its subcellular distribution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0478-y) contains supplementary material, which is available to authorized users.

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

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

EPIG-01THE FUNCTION OF CHROMATIN-MODIFYING ENZYMES IN GBM CELL APOPTOSIS

Emerging evidence suggests that apoptotic resistance in cancers is associated with aberrant expression of the key components of the apoptotic program. However, how these components are regulated at an epigenetic level is not understood. In this study, we aimed to identify novel epigenetic mechanisms regulating the response of Glioblastoma Multiforme (GBM) cells to a pro-apoptotic ligand and an emerging therapeutic candidate, TRAIL. To this end, we conducted a short-hairpin RNA (shRNA) screen to assess the function of chromatin modifying-enzymes in TRAIL-induced apoptosis. We employed 2-3 shRNAs/gene to interrogate the role of 47 genes in DNA and histone modification pathways. Accordingly, we tested shRNAs targeting DNA-methyltransferases (DNMTs), Histone-Methyltransferases (HMTs), Histone demethylases (HDMs), Methyl-DNA binding proteins, and Polycomb complex members (PrC). Upon infection with individual shRNAs, U87MG cells were tested for their TRAIL response using cell viability assays. As a result, we identified KDM2B, an H3K36-specific HDM, as a novel regulator of TRAIL response. Accordingly, silencing of KDM2B significantly enhanced TRAIL sensitivity and accelerated the apoptosis process, as revealed by live cell imaging experiments. In order to decipher the downstream molecular pathways regulated by KDM2B, we examined expression levels of apoptosis-related genes by RNA-sequencing and quantitative PCR, and observed de-repression of pro-apoptotic genes and repression of anti-apoptotic genes upon KDM2B silencing. We also observed attenuated tumor growth upon KDM2B silencing in vivo. Taken together, our findings suggest a novel mechanism where the key apoptosis components are under epigenetic control of KDM2B in GBM cells. Therefore, identifying the direct targets of KDM2B might provide a new understanding of apoptosis response in GBMs and aid in the design of successful pro-apoptotic therapies.

Abstract 2928: Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation

Abstract Withanolide E (WE), a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to TRAIL-mediated apoptosis. WE, the most potent and least toxic of 5 TRAIL sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling via a rapid decline in the levels of both cFLIPL and cFLIPS proteins. Normal non-transformed human renal epithelial cells were completely resistant to the WE plus TRAIL combination. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in various pro-and anti-apoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIPL and cFLIPS was predominantly due to destabilization and/or aggregation of the proteins subsequently leading to their proteasomal degradation. Since cFLIPL and cFLIPS are reported to be HSP90 client proteins, and WE can inhibit HSP90 activity, the effect of WE on various other HSP90 client proteins was assessed. WE treatment of ACHN renal carcinoma cells altered the stability of a limited number of HSP90 client proteins when compared to the well-known HSP90 inhibitor geldanamycin. The destabilization of cFLIP proteins provides a potentially novel mechanism for sensitizing cancer cells to TRAIL mediated apoptosis. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by WE and its lack of toxicity were confirmed in animal studies, in which a combination treatment of WE with an agonist antibody to TRAIL death receptor 5 (DR5) provided a significant therapeutic benefit compared to either agent alone in a renal cancer xenograft model. Due to its novel activity, WE is a promising reagent for analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. Various structural analogs of WE are currently being tested for their TRAIL-sensitizing activity to provide more insight into Structure Activity Relationships (SAR) for these compounds. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, WE's more specific mechanism of action suggests minimal toxic side effects might accompany its combination with TRAIL death receptor agonists for cancer therapy. Citation Format: Brooks D. Alan, Curtis J. Henrich, Karen L. Erickson, Cheryl L. Thomas, Heidi R. Bokesch, Poonam Tewary, Candace R. Thompson, Richard J. Pompei, Kirk R. Gustafson, James B. McMahon, Thomas J. Sayers. Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2928. doi:10.1158/1538-7445.AM2015-2928

Abstract 15: gp78 is a negative regulator of TRAIL-induced apoptosis in breast cancer cells

Abstract TNF-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in transformed cells by activating the extrinsic apoptotic pathway via its cognate receptors on the cell surface, TRAIL receptor 1 and TRAIL receptor 2. Triple negative breast cancer (TNBC) cells (so-called because TNBC lacks estrogen and progesterone receptor expression and Her-2 amplification) have been found to be sensitive to TRAIL while breast cancer cells of other subtypes of disease remain relatively resistant. Unfortunately, the mechanisms that govern sensitivity to TRAIL are not yet understood. The identification and characterization of novel regulators of the TRAIL pathway would provide new insights into the mechanisms that regulate TRAIL and potentially provide therapeutically exploitable molecular targets for the enhancement of TRAIL-based cancer treatments. In order to identify candidate regulators of the TRAIL pathway, our lab carried out a high-throughput RNAi-mediated screen of ∼1300 genes using the TNBC cell line MB231. One hundred fifty candidate regulators were identified. The RING finger ubiquitin ligase gp78 (also known as AMFR) was identified as a candidate negative regulator of TRAIL sensitivity. gp78 is an endoplasmic reticulum (ER)-residing protein that helps facilitate the retrotranslocation of substrates across the ER membrane into the cytosol during ER-associated protein degradation. This process is critical to maintaining cellular homeostasis by promoting the proteasomal elimination of misfolded proteins and is integral to cell survival. Interestingly, gp78 has previously been found to promote metastasis in a mouse sarcoma model, and in this study, we have further characterized gp78 as an inhibitor of apoptosis. The initial findings from the RNAi screen were confirmed by carrying out siRNA-mediated knockdown of gp78 in MB231 cells. The cells with inhibited gp78 expression were found to be significantly sensitive to TRAIL-induced caspase-3/7 activity and loss in viability. Knockdown of gp78 using in total 11 independent siRNAs demonstrated a reduction in gp78 expression is associated with TRAIL sensitization. Furthermore, pan-caspase inhibition with the pharmacologic inhibitor ZVAD-FMK completely abrogated sensitization to TRAIL with gp78 knockdown, demonstrating that loss in viability is caspase dependent. These results were further characterized by knocking down gp78 alone or along with one of the initiator caspases, 2, 8, 9, or 10. Loss of the initiator caspases 2 and 8 reduced sensitivity to TRAIL with gp78 knockdown, suggesting that pathways involved in the activation of these caspases in particular may be conferring sensitivity to TRAIL. In summary, our lab has identified and characterized gp78 as a regulator of TRAIL sensitivity through a caspase-mediated mechanism in breast cancer cells. Continued study is warranted to fully elucidate the molecular mechanisms by which gp78 inhibits TRAIL-induced apoptosis. Citation Format: Jennifer L. Dine, Sireesha V. Garimella, Kristie Gehlhaus, Magda Grandin, Daniel Letwin, Natasha Caplen, Stanley Lipkowitz. gp78 is a negative regulator of TRAIL-induced apoptosis in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 15. doi:10.1158/1538-7445.AM2015-15

Abstract 994: PU.1 inhibition confers resistance to TRAIL- and anthracycline-mediated apoptosis through NF-κB activation and TRAIL receptor downregulation in acute myeloid leukemia cells

Abstract Earlier studies found that acute myeloid leukemia (AML) cells show resistance to TRAIL, but did not reveal the exact mechanism. The myeloid master regulator PU.1 is a key molecule for myeloid and B-cell development. Its inactivation results in a differentiation block and contributes to the pathogenesis AML. On the other hand, several reports directly linked PU.1 to cell death. We now aimed at analyzing the role of PU.1 in TRAIL-mediated apoptosis of AML cells. We found that inhibiting PU.1 significantly increased resistance to TRAIL-induced apoptosis in several AML cell lines (NB4, HL60, MOLM13) as seen by significantly decreased AnnexinV/PI staining and caspase-3/7 activation. Interestingly, this resistance was specific to TRAIL-induced apoptosis since TNFα induced similar levels of cell death in control and PU.1 knockdown AML cells. AML PU.1 knockdown cells were further characterized by increased expression of anti-apoptotic genes such as FLIPShort, XIAP, Bcl-2 and Mcl-1 upon TRAIL treatment. Given that some of these genes are regulated by NF-κB we investigated if knocking down PU.1 activates this transcription factor. Indeed, PU.1 knockdown AML cells showed increased nuclear translocation of the NF-κB subunit p65 and increased activity of NF-κB specific reporters possibly accounting for FLIP, XIAP and Bcl-2 induction. In line with these findings, blocking the NF-κB pathway with the pharmacological NF-κB inhibitors JSH-23 and BAY 11-7085 or co-depleting FLIP restored TRAIL sensitivity in AML PU.1 knockdown cells. As a possible mechanism we found that PU.1 interacts with and inactivates the p65 protein directly. As an alternative mechanism of TRAIL-resistance in AML PU.1 knockdown cells, we observed a significant down-regulation of TRAIL receptor 1 (-R1) and -R2 levels as well as increased expression of the NF-κB-activating TRAIL decoy-receptor 2 (DcR2). To extend our study to a more clinical setting, we tested if AML PU.1 knockdown cells are resistant to anthracyclines currently used in AML therapy. As with TRAIL treatment AML PU.1 knockdown cells displayed significantly less cell death than control cells upon anthracycline treatment. This resistance was paralleled by increased expression of the above-listed anti-apoptotic factors. In summary, our results reveal a novel function of PU.1 that is keeping NF-κB in check and modulating TRAIL receptor expression. Our data suggest that PU.1 functions in resistance mechanisms not only against TRAIL- but also against anthracycline-based therapies in AML. Citation Format: Aladin Haimovici, Thomas Kaufmann, Mario P. Tschan. PU.1 inhibition confers resistance to TRAIL- and anthracycline-mediated apoptosis through NF-κB activation and TRAIL receptor downregulation in acute myeloid leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 994. doi:10.1158/1538-7445.AM2015-994

Tanshinone IIA Facilitates TRAIL Sensitization by Up-regulating DR5 through the ROS-JNK-CHOP Signaling Axis in Human Ovarian Carcinoma Cell Lines.

Tanshinone IIA (TIIA) extracted from Salvia miltiorrhiza has been shown to possess antitumor and TRAIL-sensitizing activity. The involvement of DR5 in the mechanism whereby TIIA exerts its effects is unknown. This study aimed to explore the mechanism underlying TIIA augmentation of TRAIL-induced cell death in ovarian carcinoma cells. Cell viability was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of relating signaling proteins. Transcriptional activation was explored by a dual-luciferase reporter assay. We found that TIIA sensitized human ovarian carcinoma cells to TRAIL-induced extrinsic apoptosis. Combined treatment with subtoxic concentrations of TIIA and TRAIL was more effective than single treatments with respect to cytotoxicity, clonogenic inhibition, and the induction of caspase-8 and PARP activity in ovarian carcinoma cell lines TOV-21G and SKOV3. TIIA induced DR5 protein and mRNA expression in a concentration-dependent manner. DR5/Fc treatment markedly suppressed the TRAIL cytotoxicity enhanced by TIIA. These results indicate that DR5 plays an essential role in TIIA-induced TRAIL sensitization and that induction of DR5 by TIIA is mediated through the up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP). Knockdown of CHOP gene expression by shRNA attenuated DR5 up-regulation and rescued cell viability under the treatment of TIIA-TRAIL combination. TIIA promoted JNK-mediated signaling to up-regulated CHOP and thereby inducing DR5 expression as shown by the ability of a JNK inhibitor to potently suppress the TIIA-mediated activation of CHOP and DR5. In addition, the quenching of ROS using NAC prevented the induction of JNK phosphorylation and CHOP induction. Furthermore, inhibition of ROS by NAC significantly attenuated TRAIL sensitization by TIIA. Taken together, these data suggest that TIIA enhances TRAIL-induced apoptosis by upregulating DR5 receptors through the ROS-JNK-CHOP signaling axis in human ovarian carcinoma cells.

Tanshinone IIA enhances the effects of TRAIL by downregulating survivin in human ovarian carcinoma cells

BackgroundTanshinone IIA (TIIA), a diterpene quinone from the medicinal plant Salvia miltiorrhiza Bunge (Lamiaceae) was shown to possess apoptotic and TRAIL-sensitizing effects. Still, the molecular mechanisms whereby TIIA induces apoptosis remain largely unknown. PurposeThe role of survivin, an inhibitor of apoptosis protein, in TIIA-induced apoptosis has never been addressed before and hence was the primary goal of this study. MethodsIn this study, we explored the anticancer effect of TIIA in TOV-21G, SKOV3, and OVCAR3 ovarian carcinoma cells. Cytotoxicity was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of related signaling proteins. ResultsOur results illustrated that TIIA's cytotoxic effect was caused by apoptosis with the involvement of caspases activity. Moreover, TIIA downregulated survivin in a concentration-dependent manner without affecting the expression of Bcl-2, Bcl-xL, and Bax. TIIA-induced survivin downregulation is regulated by both transcriptional processes and proteasomal degradation. Using TOV-21G cells as our cellular model, we demonstrated that TIIA-induced survivin downregulation requires p38 MAPK activation. Importantly, genetic overexpression of survivin rendered cells more resistant to TIIA, indicating an essential role of survivin downregulation in TIIA-induced apoptosis. This TRAIL sensitization effect of TIIA is ascribed to survivin downregulation because the effect was abrogated in cells that overexpressed survivin. ConclusionOur findings provide new insights into the action modes of TIIA-mediated anticancer effects and further implicate a rational design for cancer therapeutic regimens by combining TIIA-sensitized TRAIL via downregulating survivin to elicit ovarian cancer cell death.