TRAIL-mediated Cell Death Research Articles

MK-2206 induces cell cycle arrest and apoptosis in HepG2 cells and sensitizes TRAIL-mediated cell death

It has become evident that AKT inhibitors have great potential in cancer treatment. In this study, we investigate the anticancer activity of MK-2206, a novel AKT inhibitor, on HepG2 hepatocellular carcinoma cell, and to show whether MK-2206 enhances the apoptosis-inducing potential of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The cell growth inhibition was evaluated by MTT assay and colony formation assay. Cell cycle distribution was assessed by propidium iodide flow cytometry. Apoptosis was determined by AnnexinV-FITC/PI double staining assay and caspase-9, casapse-7, caspase-3, and PARP cleavage. The results of present study showed that MK-2206-induced G1-phase arrest was associated with a marked decrease in the protein expression of cyclin D1 with concomitant induction of p21 and p27. MK-2206-induced apoptosis was characterized by cleavage of a pro-caspase in a concentration-dependent manner. Moreover, the MAP family kinases p38 kinase and JNK were activated by exposure to MK-2206. SB203580, an p38-specific inhibitor, partially blocked MK-2206-induced death of HepG2 cells and caspase activation. A combination of MK-2206 with TRAIL significantly inhibited growth of TRAIL resistant HepG2 cells. Taken together, our findings provide a new insight to better understand anticancer mechanisms of MK-2206, at least in HepG2 cell. Using of MK-2206 as a potent sensitizer to TRAIL-induced apoptotic cell death offers a promising means of enhancing the efficacy of TRAIL-based HCC treatments.

Abstract 3732: Therapeutic targeting of colorectal cancer stem cells by TRAIL-inducing small molecule TIC10.

Abstract Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the USA. A growing body of evidence indicates that CRC tumors contain a small population of stem/progenitor cells (colorectal cancer stem cells, CRCSCs) capable of self-renewal that contribute to tumor maintenance, resistance to therapy and metastasis. Selective therapeutic targeting of CRCSCs in combination with approved therapies could improve treatment response and prolong patient survival. The endogenous protein TNF-related apoptosis-inducing ligand (TRAIL; Apo2L) selectively induces cell death in cancer cells while sparing normal cells. This approach is particularly attractive for targeting CRCSCs potentially avoiding toxic effects in normal stem/progenitor cells in the colonic crypt. TIC10 is a first-in-class anti-tumor agent that transcriptionally induces TRAIL via the transcription factor Foxo3a, independent of tumor suppressor p53 status. We previously demonstrated that TIC10 activates Foxo3a by dual inhibition of the upstream regulators Akt and ERK. TIC10 induces TRAIL-mediated cell death in cancer cells in vitro and in vivo. TIC10 improves the half-life, tissue distribution, route of administration and spectrum of activity of TRAIL. These attributes overcome therapeutic limitations of recombinant TRAIL and TRAIL-agonist antibodies that have been or are in clinical trials to combat human cancer. TIC10 exhibits potent anti-tumor efficacy in a wide range of preclinical mouse models of cancer including subcutaneous and orthotopic colon cancer without observed toxicity. We hypothesize that the potent anti-tumor effect of TIC10 in colon cancer involves targeting of the CRCSC population. In this study we evaluated the effects of TIC10 on the CRCSC population in three human colon cancer cell lines. Flow cytometric analysis revealed that TIC10 significantly depleted CD133-, CD44- and Aldefluor-positive CRCSCs in HCT116 and SW480 cells. TIC10 significantly induced surface TRAIL expression in CD133- and CD44-positive CRCSCs. TIC10 also significantly inhibited colonosphere formation in DLD1 and SW480 cells. A targeted network analysis of gene expression profiles of HCT116 p53-null human colon cancer cells treated with TIC10 revealed that several stem cell-related pathways and proteins are affected by TIC10 and are likely involved in the anti-CRCSC effects of TIC10. In particular, mRNA transcript levels of Id2 (3.2 fold) and Wnt16 (13.5 fold) were significantly downregulated. Ongoing studies will validate these potential anti-CRCSC molecular targets of TIC10 and determine their contribution to the overall anti-CRCSC effect, evaluate the anti-CRCSC efficacy of TIC10 in vivo and examine combinatorial anti-CRCSC effects of TIC10 with chemotherapies used to treat CRC. In conclusion, TIC10 targets bulk tumor cells as well as CRCSCs, making this molecule a promising novel agent for the treatment of CRC. Citation Format: Varun Vijay Prabhu, Joshua E. Allen, David T. Dicker, Yifu Ding, Wafik S. El-Deiry. Therapeutic targeting of colorectal cancer stem cells by TRAIL-inducing small molecule TIC10. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3732. doi:10.1158/1538-7445.AM2013-3732

The Role of c-FLIP in Cisplatin Resistance of Human Bladder Cancer Cells

We investigated the mechanisms underlying cisplatin resistance in human bladder cancer cells to provide novel molecular targets for the treatment of cisplatin resistant bladder cancer. The differential gene expression of cisplatin sensitive (T24) and resistant (T24R2) human bladder cancer cell lines was analyzed and validated by microarray and Western blot analysis. Changes in cisplatin sensitivity by c-FLIP knockdown and related mechanisms in T24R2 cells were assessed using the Cell Counting Kit-8 assay (Dojindo Molecular Technologies, Gaithersburg, Maryland) and Western blot. siRNA oligonucleotides that specifically target c-FLIP were prepared and siRNA transfection was done. Microarray analysis revealed that the expression of 1,086 and 322 genes showed more than twofold and fourfold changes in the T24R2 and T24 cell lines, respectively. Especially genes involved in the c-FLIP related death receptor apoptosis pathway, including caspase 2 and 9, NF-kB, BID, c-FLIP, XIAP, and cIAP1 and 2, showed differential expression in the 2 cell lines. Western blot demonstrated complete cisplatin mediated suppression of c-FLIP expression in T24 cells but no change in c-FLIP expression was observed in T24R2 cells after cisplatin treatment in the same dose range. Suppression of c-FLIP expression in T24R2 cells by siRNA transfection rendered these cells significantly more sensitive to cisplatin treatment than untransfected T24R2 cells (p <0.05). Results reveal that c-FLIP has an important role in the cisplatin resistance of human bladder cancer cells and c-FLIP modulation may at least partially reverse cisplatin resistance in bladder cancer cells.

Ouabain downregulates Mcl-1 and sensitizes lung cancer cells to TRAIL-induced apoptosis

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a prerequisite for cancer progression, and TRAIL resistance is prevalent in lung cancer. Ouabain, a recently identified human hormone, has shown therapeutic promise by potentiating the apoptotic response of metastatic lung cancer cells to TRAIL. Nontoxic concentrations of ouabain are shown to increase caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage, and apoptosis of H292 cells in response to TRAIL. While ouabain had a minimal effect on c-FLIP, Bcl-2, and Bax levels, we show that it possesses an ability to downregulate the antiapoptotic Mcl-1 protein. The present study also reveals that the sensitizing effect of ouabain is associated with its ability to generate reactive oxygen species (ROS), and hydrogen peroxide is identified as the principle ROS triggering proteasomal Mcl-1 degradation. In summary, our results indicate a novel function for ouabain in TRAIL-mediated cancer cell death through Mcl-1 downregulation, thereby providing new insight into a potential lung cancer treatment as well as a better understanding of the physiological activity of ouabain.

Breast Cancer Proteome Takes More Than Two to Tango on TRAIL: Beat Them at Their Own Game

Breast carcinogenesis is a multidimensional disease that has resisted drug-related solutions to date because of heterogeneity, disorganized spatiotemporal behavior of signal transduction cascades, cell cycle checkpoints, cell transition, plasticity, and impaired pro-apoptotic response. These synchronized oncogenic events, including protein-protein interaction, transcriptional-regulatory, and signaling networks, trigger genomic and transcriptional disturbances in TRAIL-mediated signaling network neighborhoods. Therefore, tumor cells often acquire the ability to escape death by suppressing cell death pathways that normally function to eliminate damaged and harmful cells. This review describes the TRAIL-mediated cell death signaling pathways, the interactions between these pathways, and the ways in which these pathways are deregulated in breast cancer.

Role of syndecan-1 in TRAIL-induced apoptosis in myeloma cells (127.18)

Abstract Syndecan-1 is a transmembrane heparin sulfate proteoglycan which functions as co-receptor for cell signaling and extracellular matrix in many types of malignancies. TNF-related apoptosis inducing ligand (TRAIL) is an antitumor agent which initiates apoptosis through the assembly of death receptor-inducing signaling complex (DISC) after ligand binding. However, role of syndecan-1 in TRAIL -mediated cell death is unknown. Here, we elucidated the pro-survival role of syndecan-1 in multiple myeloma cell lines. Knockdown of syndecan-1 in H929 and RPMI8226 cells increased TRAIL-induced apoptosis. The expression level of signaling molecules downstream of TRAIL was normal in knockdown cells. Although the binding and internalization ability of TRAIL were still normal, an enhanced formation of DISC and caspase-8 activation were detected in knockdown cells. In addition, TRAIL-induced oligomerization of DR4 was enhanced in syndecan-1-knockdwon cells. Thus, syndecan-1 may regulate TRAIL-mediated cell death in the clustering of death receptors in multiple myeloma cells

Abstract 1935: Potent anti-tumor effects of TIC10 require Foxo3a and TRAIL gene upregulation

Abstract TNF-related apoptosis-inducing ligand (TRAIL; Apo2L) is an endogenous protein that induces tumor-selective apoptosis and is currently being tested in clinical trials as an anti-tumor agent. Despite promising preclinical activity, recombinant human TRAIL suffers from efficacy-limiting properties such as short serum half-life, stability, cost, and limitations in delivery, e.g. to the central nervous system. We hypothesized that induction of the human TRAIL gene by small molecules may overcome these limitations of TRAIL-based protein therapy. A cell-based luciferase reporter screen using the human TRAIL promoter identified TIC10 as a p53-independent small molecule inducer of the TRAIL gene. TIC10 induces TRAIL on the surface of normal and tumor cells, in sera of non-tumor-bearing mice, and throughout the tumor microenvironment. Co-culture experiments suggest that TRAIL upregulation by normal cells can contribute to the anti-tumor response of TIC10 through a TRAIL-mediated bystander effect. TIC10-induced TRAIL causes tumor-specific and TRAIL-mediated cell death as indicated by inhibition of cytotoxic effects following TRAIL gene knockdown in vitro and in vivo. Expression profiling of TIC10-induced transcriptional changes revealed altered expression levels of Foxo3a target genes, including DR5. We found that TIC10 causes the nuclear translocation of Foxo3a, but not Foxo1, to upregulate TRAIL gene transcription as determined by chromatin-immunoprecipitation assays. Western blot analysis indicated that TIC10 potently inhibits the phosphorylation of Foxo3a at Ser253 and Ser294 that normally docks the transcription factor to 14-3-3 proteins in the cytosol. Dual inhibition of Akt and ERK by TIC10, by other individual small molecule kinase inhibitors in combination or targeted siRNA inhibited the phosphorylation of these critical serine residues and synergistically induced Foxo3a-dependent TRAIL and TRAIL-mediated cell death. TIC10 is a promising first-in-class anti-tumor agent with a broad range of anti-tumor effects in preclinical animal models including othrotopic GBM, immunocompetent lymphoma, xenografted colon, lung, or breast cancers, and exhibits synergy with taxanes as well as bevacizumab among other agents. TIC10 appears to have no measurable toxic effects in mice including evaluation of serum chemistries, liver function studies, LDH, tissue histology, weight of the animals or behavioral changes. We are pursuing the clinical translation of these important findings that indicate a favorable and wide therapeutic index for a novel anti-tumor agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1935. doi:1538-7445.AM2012-1935

Sensitivity of a novel model of mammary cancer stem cell-like cells to TNF-related death pathways.

Cancer stem cells (CSC) are resistant to radiation and chemotherapy and play a significant role in cancer recurrence and metastatic disease. It is therefore important to identify alternative strategies, such as immunotherapies that can be used to control this refractory population. A CD44(+)CD24(-/low) subpopulation of cells within the B6 PyMT-MMTV transgenic mouse-derived AT-3 mammary carcinoma cell line was identified, which had CSC-like characteristics, including pluripotency and a resistance to chemo- and radiotherapy. Therefore, unlike xenograph models that require immunocompromised settings, this novel system may provide a means to study immune-mediated responses against CSC-like cells. The immunobiology of the AT-3 CSC-like cell population was studied by their surface molecule expression profile and their sensitivity to specified cell death pathways. Comparable levels of Rae-1, CD155, CD54 and higher levels of Fas and DR5 were expressed on the AT-3 CSC-like cells compared to non-CSC-like tumor cells. Expression correlated with an in vitro sensitivity to cell death by NK cells or through the ligation of the death receptors (Fas or DR5), by their ligands or anti-Fas and anti-DR5 mAbs. Indeed, compared to the rest of the AT-3 tumor cells, the CD44(+)CD24(-/low) subpopulation of cells were more sensitive to both Fas- and TRAIL-mediated cell death pathways. Therefore, despite the refractory nature of CSC to other conventional therapies, these CSC-like cells were not inherently resistant to specified forms of immune-mediated cell death. These results encourage the continued investigation into immunotherapeutic strategies as a means of controlling breast CSC, particularly through their cell death pathways.

TRAIL and vitamins: opting for keys to castle of cancer proteome instead of open sesame

Cancer is a multifaceted molecular disorder that is modulated by a combination of genetic, metabolic and signal transduction aberrations, which severely impair the normal homeostasis of cell growth and death. Accumulating findings highlight the fact that different genetic alterations, such as mutations in tumor suppressor genes, might be related to distinct and differential sensitivity to targeted therapies. It is becoming increasingly apparent that a multipronged approach that addresses genetic milieu (alterations in upstream and/or parallel pathways) eventually determines the response of individual tumors to therapy. Cancerous cells often acquire the ability to evade death by attenuating cell death pathways that normally function to eliminate damaged and harmful cells. Therefore impaired cell death nanomachinery and withdrawal of death receptors from cell surface are some of major determinants for the development of chemotherapeutic resistance encountered during treatment. It is therefore essential to emphasize underlying factors which predispose cells to refractoriness against TRAIL mediated cell death pathway and the relevant regulatory components involved. We bring to limelight the strategies to re-sensitize TRAIL resistant cells via vitamins to induce apoptosis.

Death Receptor 5 Signaling Promotes Hepatocyte Lipoapoptosis

Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR(-/-)) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor.

Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonucleotides induces autophagy-dependent cell death in glioma cells

Malignant gliomas are the most common and lethal primary central nervous system neoplasms. Several intriguing lines of evidence have recently emerged indicating that the cellular prion protein (PrPC) may exert neuro- and cyto-protective functions: PrPC overexpression protects cultured neurons and also tumor cell lines exposed to various pro-apoptotic stimuli while, on the contrary, PrPC silencing sensitizes Adriamycin-resistant human breast carcinoma cells to TRAIL-mediated cell death. In order to determine if PrPC is involved in the resistance of glial tumors to cell death, the effects of cellular prion protein downregulation by antisense approach were investigated in different human malignant glioma cell lines. PrPC downregulation induced profound morphological changes and significant cell death. In addition, a significant tumor volume reduction was noted after PrPC silencing in a EGFP-GL261 glioma murine model. Investigations of the molecular effects induced by PrPC silencing were carried out on T98G human glioma cells by analysing autophagic as well as typical apoptotic markers (nuclear morphology, caspase-3/7, p53 and PARP-1). The results indicated that apoptosis was not induced after PrPC downregulation while, on the contrary, electron microscopy analysis, and an accumulation of GFP-LC3-II in autophagosomal membranes of GFP-LC3 transfected cells, indicated a predominant activation of autophagy. PrPC silencing also led to induction of LC3-II, increase in Beclin-1 and a concomitant decrease in p62, Bcl-2 and in the phosphorylation of 4E-BP1, a target of mTOR autophagy signaling. In conclusion, our results show for the first time that interfering with the cellular prion protein expression could modulate autophagy-dependent cell death pathways in glial tumor cells.

Bortezomib Primes Glioblastoma, Including Glioblastoma Stem Cells, for TRAIL by Increasing tBid Stability and Mitochondrial Apoptosis

Searching for novel approaches to sensitize glioblastoma for cell death, we investigated the proteasome inhibitor bortezomib. The effect of bortezomib on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis signaling pathways was analyzed in glioblastoma cell lines, primary glioblastoma cultures, and in an in vivo model. Bortezomib and TRAIL synergistically trigger cell death and reduce colony formation of glioblastoma cells (combination index < 0.1). Investigations into the underlying molecular mechanisms reveal that bortezomib and TRAIL act in concert to cause accumulation of tBid, the active cleavage product of Bid. Also, the stability of TRAIL-derived tBid markedly increases on proteasome inhibition. Notably, knockdown of Bid significantly decreases bortezomib- and TRAIL-mediated cell death. By comparison, silencing of Noxa, which is also upregulated by bortezomib, does not confer protection. Coinciding with tBid accumulation, the activation of Bax/Bak and loss of mitochondrial membrane potential are strongly increased in cotreated cells. Overexpression of Bcl-2 significantly reduces mitochondrial perturbations and cell death, underscoring the functional relevance of the mitochondrial pathway. In addition, bortezomib cooperates with TRAIL to reduce colony formation of glioblastoma cells, showing an effect on long-term survival. Of note, bortezomib profoundly enhances TRAIL-triggered cell death in primary cultured glioblastoma cells and in patient-derived glioblastoma stem cells, underlining the clinical relevance. Importantly, bortezomib cooperates with TRAIL to suppress tumor growth in an in vivo glioblastoma model. These findings provide compelling evidence that the combination of bortezomib and TRAIL presents a promising novel strategy to trigger cell death in glioblastoma, including glioblastoma stem cells, which warrants further investigation.

Abstract 4095: Decreased caspase 8 protein expression and incomplete caspase 3 activation in TRAIL-resistant normal human fibroblasts and epithelial cells

Abstract TNF-related apoptosis-inducing ligand (TRAIL)-mediated cell death is currently being utilized in newly developed cancer therapies. The molecular basis of normal cell resistance to TRAIL-mediated apoptosis, and thus therapeutic index, remains largely unexplored. We investigated TRAIL sensitivity in normal fibroblasts (HFF, WI-38, MRC-5) and epithelial cells (FHs 74 Int). We found that MRC-5 cells were moderately sensitive to TRAIL after 18 hours of treatment while WI-38, HFF, and FHs 74 Int cells were not. Normal fibroblasts and epithelial cells expressed both pro-apoptotic TRAIL receptors DR4 and DR5, although normal cell DR4 and DR5 expression was slightly lower when compared to human lung and colon cancer cells. While normal and cancer cells showed comparable DcR2 expression, normal fibroblasts and epithelial cells expressed little DcR1. c-Myc, a major determinant of TRAIL sensitivity in cancer cells, was found to be decreased in all normal cells compared to cancer cells. Expression of initiator caspase 8, required for complete TRAIL signaling, was assessed. Markedly reduced caspase 8 protein expression was detected in WI-38, HFF and FHs 74 Int cells compared to tumor cells that generally show robust caspase 8 expression. MRC-5 caspase 8 protein expression was found to be noticeably increased compared to TRAIL-resistant normal fibroblasts and epithelial cells and may be a contributing factor in MRC-5 TRAIL sensitivity. Expression and activation of effector caspase 3 was also examined. After 4 hours of TRAIL exposure, expression of full length caspase 3 declined in normal fibroblasts, epithelial cells and tumor cells. Yet, appearance of the mature active caspase 3 subunit p17 was only seen in MRC-5 fibroblasts and cancer cells. Interestingly, cleavage of caspase 3 substrate, PARP, was only seen in cancer cells after 4 hours of TRAIL exposure suggesting that MRC-5 fibroblasts have a delayed cytotoxic response to TRAIL. TRAIL-insensitive normal cells were treated with TRAIL and a proteasome inhibitor to increase susceptibility to TRAIL-mediated cell death. TRAIL-induced cell death was observed in WI-38, HFF and FHs 74 Int cells after combined treatment of TRAIL and MG132. Our current studies are focused on further analysis of caspase 3 activation and stabilization after TRAIL treatment in TRAIL-resistant normal cells. Additionally, we are investigating whether treatment of normal fibroblasts and epithelial cells with histone deacetylase inhibitors, and/or DNA methylation inhibitors might result in increased caspase 8 protein expression and susceptibility to TRAIL-mediated apoptosis. 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 4095. doi:10.1158/1538-7445.AM2011-4095

Abstract LB-222: Identifying cFLIP as a marker and also a potentially “druggable” target of SAHA+TRAIL (TNF-Related Apoptosis Inducing Ligand), cytotoxicity in malignant pleural mesothelioma (MPM)

Abstract Background: Despite expressing adequate levels of receptors for TRAIL significant percentages of cancer cells are resistant to TRAIL-induced apoptosis. We have previously reported that histone deacetylase inhibitor SAHA (vorinostat) + TRAIL combination induces profound supra-additive cytotoxicity in MPM in vitro. We observe that only MPM cells with some TRAIL sensitivity are very susceptible to SAHA+TRAIL cytotoxicity. We hypothesize that TRAIL-initiated death signal at the membrane level determine cellular susceptibility to SAHA+TRAIL. The objective of this study is to identify the molecular marker that predicts TRAIL and thus TRAIL+SAHA sensitivity in MPM cells. Materials and methods: Intrinsic sensitivity of 8 MPM and primary normal endothelial cells to TRAIL and SAHA+TRAIL is determined by cell viability assay; basal expression of cFLIP, caspase 8, DR4/DR5, FADD, RIP, TRADD in these cells are evaluated by western blots. Selective knockdown of FLIP is achieved by siRNA. FLIP mRNA levels were determined by quantitative RT-PCR. Results: 5/8 MPM cells are very suceptible to SAHA+TRAIL cytotoxicity (combination-sensitive cells) while 3 others are classified as combination-resistant. Western blot analysis identifies an inverse relationship between cFLIP as well as procaspase 8 expression and sensitivity to SAHA+TRAIL with only the difference in cFLIP levels as quantified by densitometric analysis being distinctive between two groups: 0.87±0.02 for 5 combination-sensitive MPM cells versus 0.15±0.05 for 3 combination-resistant MPM cells. siRNA-mediated partial cFLIP knockdown restores DISC activity in high cFLIP expressing cells as evidenced by caspase 8 and 3 catalytic processing with stronger caspase activation being noted in combination-treated cells. Quantitative RT-PCR demonstrates high level of cFLIP messenger RNA in high cFLIP expressors indicating that cFLIP is transcriptionally upregulated in these cells. Selective cFLIP downregulation in high cFLIP expressing cells restores susceptibility to TRAIL and strongly sensitizes them to SAHA+TRAIL cytotoxicity. Additionally, selective complete cFLIP knockdown abrogates the need for SAHA to achieve profound TRAIL-mediated cell death in MPM cells regardless of their intrinsic cFLIP epxression. Conclusdion: Our study identifies cFLIP expression as a marker of cellular sensitivity to SAHA+TRAIL in MPM in vitro. More importantly, gene knockdown experiments provide the proof of concept that cFLIP is a pontential “druggable” target and downregulation of which sensitizes resistant cancer cells to TRAIL and SAHA+TRAIL. Ongoing works aim to validate this observation in a larger panel of novel MPM cells and to define treatments strategies to downregulate cFLIP in tumor cells expressing high levels of this antiapoptotic protein. 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 LB-222. doi:10.1158/1538-7445.AM2011-LB-222

Sulforaphane blocks hypoxia-mediated resistance to TRAIL-induced tumor cell death

Hypoxia occurs frequently in various solid tumors and elicits a cellular response designed to improve cell survival through adaptive processes, thereby accelerating cancer progression and the development of chemotherapy resistance. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, leads to tumor cell death via both intrinsic and extrinsic apoptotic signaling pathways. Hypoxia inhibits TRAIL-mediated apoptosis and attenuates the therapeutic activity of TRAIL in cancer management. Hypoxia-inducible factor-1α (HIF-1α) plays a central role in tumor hypoxia by up-regulating gene expression related to angiogenesis, cancer invasion and anti-apoptosis. Sulforaphane (SFN), a phenethyl isothiocyanate, elicits HIF-1α inactivation under hypoxia. This study investigated whether hypoxic inhibition of TRAIL-mediated tumor cell death is increased by SFN-mediated HIF-1α instability. SFN induced cell death in various tumor cells, including SK-N-SH, SNU-638, HeLa and A549 cells, and showed cell cytotoxicity in hypoxia-exposed tumor cells. Western blot analysis showed that SFN treatment increased p53 and activated caspase-3 proteins, and decreased HIF-1α activation under hypoxia. Under low-oxygen conditions, TRAIL-treated cells displayed inhibited apoptosis, while SFN-pre-treated cells exhibited stronger sensitization to TRAIL under the hypoxic conditions. SFN treatment enhanced TRAIL-induced activation of proteins, including caspase-3 and p53. SFN dose-dependently decreased HIF-1α protein levels in cancer cells, which was mediated by decreased protein stability. This study demonstrated that SFN recovered hypoxia-mediated resistance to TRAIL via instability of HIF-1α, and also suggests that combination therapy with SFN and TRAIL may provide a novel strategy for treating hypoxic solid tumors.

Melanoma-associated Chondroitin Sulfate Proteoglycan (MCSP)-targeted delivery of soluble TRAIL potently inhibits melanoma outgrowth in vitro and in vivo

BackgroundAdvanced melanoma is characterized by a pronounced resistance to therapy leading to a limited patient survival of ~6 - 9 months. Here, we report on a novel bifunctional therapeutic fusion protein, designated anti-MCSP:TRAIL, that is comprised of a melanoma-associated chondroitin sulfate proteoglycan (MCSP)-specific antibody fragment (scFv) fused to soluble human TRAIL. MCSP is a well-established target for melanoma immunotherapy and has recently been shown to provide important tumorigenic signals to melanoma cells. TRAIL is a highly promising tumoricidal cytokine with no or minimal toxicity towards normal cells. Anti-MCSP:TRAIL was designed to 1. selectively accrete at the cell surface of MCSP-positive melanoma cells and inhibit MCSP tumorigenic signaling and 2. activate apoptotic TRAIL-signaling.ResultsTreatment of a panel of MCSP-positive melanoma cell lines with anti-MCSP:TRAIL induced TRAIL-mediated apoptotic cell death within 16 h. Of note, treatment with anti-MCSP:sTRAIL was also characterized by a rapid dephosphorylation of key proteins, such as FAK, implicated in MCSP-mediated malignant behavior. Importantly, anti-MCSP:TRAIL treatment already inhibited anchorage-independent growth by 50% at low picomolar concentrations, whereas > 100 fold higher concentrations of non-targeted TRAIL failed to reduce colony formation. Daily i.v. treatment with a low dose of anti-MCSP:TRAIL (0.14 mg/kg) resulted in a significant growth retardation of established A375 M xenografts. Anti-MCSP:TRAIL activity was further synergized by co-treatment with rimcazole, a σ-ligand currently in clinical trials for the treatment of various cancers.ConclusionsAnti-MCSP:TRAIL has promising pre-clinical anti-melanoma activity that appears to result from combined inhibition of tumorigenic MCSP-signaling and concordant activation of TRAIL-apoptotic signaling. Anti-MCSP:TRAIL alone, or in combination with rimcazole, may be of potential value for the treatment of malignant melanoma.

Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent with cancer-selective apoptogenic activity. It evokes the canonical caspase-mediated cell death pathway through death-inducing signaling complex (DISC) formation. We identified that Peroxiredoxin 6 (Prx6) interacts with caspase-10 and caspase-8 via the death effector domain (DED). Prx6 suppresses TRAIL-mediated cell death in human cancer cells, but not that induced by intrinsic apoptosis inducers such as etoposide, staurosporine, or A23187. Among Prx1-6 members, only Prx6 binds to DED caspases and is most effective in suppressing TRAIL or DED caspase-induced cell death. The antiapoptotic activity of Prx6 against TRAIL is not likely associated with its peroxidase activity but is associated with its ability to bind to DED caspases. Increased expression of Prx6 enhances the binding of Prx6 to caspase-10 but reduces TRAIL-induced DISC formation and subsequently caspase activation. Interestingly, Prx6 is highly upregulated in metastatic gastric cancer cells, which are relatively resistant to TRAIL as compared with primary cancer cells. Downregulation of Prx6 sensitizes the metastatic cancer cells to TRAIL-induced cell death. Taken together, these results suggest that Prx6 modulates TRAIL signaling as a negative regulator of caspase-8 and caspase-10 in DISC formation of TRAIL-resistant metastatic cancer cells.

Hypoxia inducing factor-1α regulates tumor necrosis factor-related apoptosis-inducing ligand sensitivity in tumor cells exposed to hypoxia

Hypoxia is a common environmental stress. Particularly, the center of rapidly-growing solid tumors is easily exposed to hypoxic conditions. Hypoxia is well known to attenuate the therapeutic response to radio and chemotherapies including tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) protein. HIF-1α is a critical mediator of the hypoxic response. However, little is known about the function of hypoxia-inducible factor-1α (HIF-1α) on hypoxic inhibition of TRAIL-mediated apoptosis. In this study, we investigated whether hypoxic inhibition of TRAIL-mediated apoptosis can be regulated by modulating HIF-1α protein. Hypoxia- and DEF-induced HIF-1α activation inhibited the TRAIL-mediated apoptosis in SK-N-SH, HeLa, A549 and SNU-638 cells. And also, HIF-1α inactivating reagents including DOX increased the sensitivity to TRAIL protein in tumor cells exposed to hypoxia. Furthermore, knock-down of HIF-1α using lentiviral RNA interference sensitized tumor cells to TRAIL-mediated cell death under hypoxic condition. Taken together, these results indicate that HIF-1α inactivation increased TRAIL sensitivity in hypoxia-induced TRAIL-resistant tumor cells and also suggest that HIF-1α inhibitors may have benefits in combination therapy with TRAIL against hypoxic tumor cells.

243 Platinum-resistance in ovarian cancer is mediated by the IL-6-cIAP-2 axis

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) has attracted considerable attention owing to its selective killing of tumor cells but not normal cells. Melanoma shows weak response to TRAIL because of its low level of TRAIL death receptors. Here, we investigated whether indomethacin, a nonsteroidal anti-inflammatory drug, can potentiate TRAIL-induced apoptosis in melanoma cells. We showed that indomethacin was capable of promoting TRAIL-induced cell death and apoptosis in A375 melanoma cells. Mechanistically, indomethacin induced cell surface expression of death receptor 5 (DR5) in melanoma cells and also in various types of cancer cells. DR5 knockdown abolished the enhancing effect of indomethacin on TRAIL responses. Induction of the DR5 by indomethacin was found to be p53 independent but dependent on the induction of CCAAT/enhancer-binding protein homologous protein (CHOP). Knockdown of CHOP abolished indomethacin-induced DR5 expression and the associated potentiation of TRAIL-mediated cell death. In addition, indomethacin-induced reactive oxygen species (ROS) production preceded upregulation of CHOP and DR5, and consequent sensitization of cells to TRAIL. We also found that indomethacin treatment downregulated survivin via ROS and the NF-κB-mediated signaling pathways. Interestingly, indomethacin also converted TRAIL-resistant melanoma MeWo and SK-MEL-5 cells into TRAIL-sensitive cells. Taken together, our results indicate that indomethacin can potentiate TRAIL-induced apoptosis through upregulation of death receptors and downregulation of survivin.

Abstract 1273: Involvement of PKCδ and NOX4 in reactive oxygen species-mediated tumor resistance to TRAIL-induced cell death

Abstract Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF family of cytokines, causes apoptosis by caspase activation in various cell types, particularly in transformed cells. Numerous types of tumors are relatively resistant to TRAIL-induced cytotoxicity; however, the reasons for this are not yet fully understood. We report here a new signal transduction pathway involving protein kinase Cδ (PKCδ), NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS), that inhibits caspase-dependent cell death induced by TRAIL ligation in human malignant astrocytoma cells. In our experiments, TRAIL ligation-induced generation of intracellular ROS through caspase-dependent proteolytic activation of PKCδ and subsequent activation of the NOX4 complex. Suppression of intracellular ROS induction using various pharmacological inhibitors or PKCδ- or NOX4-specific RNA interference enhanced the enzymatic activity of caspase-3 by blocking the oxidative modification of its catalytic cysteine residue, resulting in marked augmentation of TRAIL-mediated cell death. In addition, combined treatment with ROS scavengers enhanced TRAIL-induced cytotoxicity in an experimental tumor model. These results collectively indicate that TRAIL-induced activation of PKCδ and NOX4 can modulate TRAIL-mediated apoptosis by promoting oxidative modification of active caspase-3 in a negative-feedback manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1273.