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Cancer Research 67, 6946-6955, July 15, 2007. doi: 10.1158/0008-5472.CAN-06-3896
© 2007 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Lipid Rafts and Nonrafts Mediate Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptotic and Nonapoptotic Signals in Non–Small Cell Lung Carcinoma Cells

Jin H. Song1, Margaret C.L. Tse1, Anita Bellail1, Surasak Phuphanich2,3, Fadlo Khuri2,3, Norman M. Kneteman4 and Chunhai Hao1,3

Departments of 1 Pathology and Laboratory Medicine and 2 Hematology and Oncology and 3 Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia; and 4 Department of Surgery, University of Alberta, Edmonton, Alberta, Canada

Requests for reprints: Chunhai Hao, Department of Pathology and Laboratory Medicine, Emory University, 1365-C Clifton Road, Atlanta, GA 30322. Phone: 404-778-4776; Fax: 404-778-5550; E-mail: chao{at}emory.edu or Norman M. Kneteman, Department of Surgery, University of Alberta, Edmonton, Alberta, Canada T6G 2B7. E-mail: nkneteman{at}chaa.ab.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is capable of inducing apoptosis in non–small cell lung carcinoma (NSCLC). However, many of the human NSCLC cell lines are resistant to TRAIL, and TRAIL treatment of the resistant cells leads to the activation of nuclear factor-{kappa}B (NF-{kappa}B) and extracellular signal–regulated kinase 1/2 (ERK1/2). TRAIL can induce apoptosis in TRAIL-sensitive NSCLC cells through the induction of death-inducing signaling complex (DISC) assembly in lipid rafts of plasma membrane. In the DISC, caspase-8 is cleaved and initiates TRAIL-induced apoptosis. In contrast, TRAIL-DISC assembly in the nonraft phase of the plasma membrane leads to the inhibition of caspase-8 cleavage and NF-{kappa}B and ERK1/2 activation in TRAIL-resistant NSCLC cells. Receptor-interacting protein (RIP) and cellular Fas-associated death domain–like interleukin-1ß–converting enzyme-inhibitory protein (c-FLIP) mediates the DISC assembly in nonrafts and selective knockdown of either RIP or c-FLIP with interfering RNA redistributes the DISC from nonrafts to lipid rafts, thereby switching the DISC signals from NF-{kappa}B and ERK1/2 activation to caspase-8–initiated apoptosis. Chemotherapeutic agents inhibit c-FLIP expression, thereby enhancing the DISC assembly in lipid rafts for caspase-8–initiated apoptosis. These studies indicate that RIP and c-FLIP–mediated assembly of the DISC in nonrafts is a critical upstream event in TRAIL resistance and thus targeting of either RIP or c-FLIP may lead to the development of novel therapeutic strategies that can overcome TRAIL resistance in human NSCLC. [Cancer Res 2007;67(14):6946–55]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family (1, 2) is expressed by natural killer and dendritic cells, and plays a role in tumor immunosurveillance (3). Recombinant TRAIL and its agonist antibodies are capable of inducing apoptosis in human cancer cells whereas sparing most normal human cells (4, 5) and are therefore currently under clinical development as therapeutic agents for treating human cancers (6). Lung cancer is the leading cause of cancer death for both men and women in the United States, whereas non–small cell lung carcinomas (NSCLC) constitutes 75% of lung cancers. TRAIL can induce apoptosis in NSCLC cell lines (7) and inhibits the growth of NSCLC xenografts (5). However, the majority of human NSCLC cells are resistant to TRAIL. Here, we show that TRAIL treatment of the resistant cells leads to the activation of nuclear factor-{kappa}B (NF-{kappa}B) and extracellular signal–regulated kinase 1/2 (ERK1/2) cell survival signals. We therefore investigate the molecular mechanisms that control TRAIL apoptotic and nonapoptotic signals in NSCLC cells in search of the therapeutic agents that could switch TRAIL signals from cell survival to cell death.

TRAIL-induced apoptosis occurs through receptor-mediated extrinsic and mitochondria-involved intrinsic pathways. TRAIL binds to the cell surface death receptor DR4 and DR5, which in turn recruit intracellular Fas-associated death domain (FADD). Through its death effector domain, FADD recruits caspase-8 to the receptors for the assembly of a death-inducing signaling complex (DISC; ref. 8). In the DISC, caspase-8 is activated and cleaves caspase-3 directly (9) or indirectly through cleavage of Bcl-2–inhibitory BH3 domain protein (Bid; ref. 10). The Bid then induces mitochondrial release of cytochrome c into the cytosol (11), where caspase-9 is activated and cleaves downstream caspase-3 (12). Mitochondria also releases second mitochondria-derived activators of caspase/direct inhibitor of apoptosis-binding protein with low isoelectric point (Smac/DIABLO; refs. 13, 14), which interacts with X-linked inhibitor of apoptosis protein (XIAP) and releases XIAP inhibition of caspase-3 (15). Once activated, caspase-3 cleaves downstream DNA fragmentation factor 45 (DFF45; ref. 16), leading to apoptotic cell death.

TRAIL-DISC is modulated by intracellular adaptor proteins (8). Receptor-interacting protein (RIP; ref. 17) is a death domain adaptor and is recruited by DR4/DR5 to the DISC, leading to the activation of NF-{kappa}B (18). Studies of the TNFR1-DISC have shown that RIP interacts with inhibitor of {kappa}B kinase {gamma} (IKK{gamma}) for the recruitment of IKK{alpha}/ß to the DISC, where IKK{alpha}/ß kinases are activated and then phosphorylate the inhibitors of {kappa}B (I{kappa}B), thus releasing its inhibition of NF-{kappa}B (19). On the other hand, cellular FADD-like interleukin-1ß–converting enzyme-inhibitory protein (c-FLIP) is a death effector domain protein (20), and is recruited by FADD to the DISC, where it inhibits caspase-8 cleavage (21). Recent studies of T lymphocytes have further shown that c-FLIP is involved in RIP-mediated NF-{kappa}B and Raf1-mediated ERK1/2 signaling (22). These studies suggest that both RIP and c-FLIP are required in the DISC for dual functions: inhibition of caspase-8–initiated apoptosis and linkage to NF-{kappa}B and ERK1/2 pathway.

Recent studies suggest that lipid rafts serve as plasma membrane platforms for death receptor–initiated signals (23). These studies, however, have generated conflicting results concerning the receptor distribution in lipid rafts and subsequent intracellular signal transduction. Upon FasL binding, Fas translocates to lipid rafts in which caspase-8 is recruited and thus initiates apoptosis (24). In contrast, however, lipid rafts are required for TNFR1-initiated activation of NF-{kappa}B (25) and ERK1/2 (26). Lipid rafts are rich in cholesterol and sphingolipids and thus more tightly packaged than the surrounding phospholipid-rich nonraft phase of the plasma membrane (27). Here, we report that lipid rafts mediate TRAIL-DISC–initiated intracellular apoptotic signals in TRAIL-sensitive NSCLC cells. In contrast, nonrafts play a role in TRAIL-induced activation of NF-{kappa}B and ERK1/2 in TRAIL-resistant NSCLC cells. RIP and c-FLIP mediate the assembly of the TRAIL-DISC in nonrafts and targeting of either RIP or c-FLIP results in the redistribution of the DISC from nonrafts to lipid rafts, thereby switching the DISC signals from cell survival to cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, antibodies, and reagents. Human NSCLC cell lines (American Type Culture Collection) were cultured in RPMI 1640 with 10 mmol/L of HEPES, 1 mmol/L of sodium pyruvate, 4.5 g/L of glucose, 1.5 g/L of sodium bicarbonate, 10% fetal bovine serum, and 1% antibiotics (Invitrogen). Recombinant human TRAIL (amino acids 114–281) was purchased from PeproTech, Inc. and recombinant Flag-tagged TRAIL (Flag-TRAIL) was from Alexis. The mitogen-activated protein/ERK kinase (MEK) 1/2 inhibitor, PD98059, was obtained from Cell Signaling Technology. The antibodies used in the study included anti-FLAG M2 (Sigma-Aldrich Canada, Ltd.), DR4, DR5 (ProSci), FADD, cytochrome c, and XIAP (Transduction Laboratories), caspase-8 (Medical & Biological Laboratories), c-FLIP (NF6 clone, a kind gift from Dr. Peter Krammer, German Cancer Research Center, Heidelberg, Germany), caspase-3, DFF45, ERK1/2 (StressGen), Smac (Biomol), caspase-9, phosphorylated and unphosphorylated I{kappa}B, ERK1/2, and NF-{kappa}B (Cell Signaling Technology). Phycoerythrin-conjugated antihuman DR4 and DR5 mouse IgG1 were from eBioscience and phycoerythrin-conjugated IgG1 was from BD PharMingen. Horseradish peroxidase (HRP)–conjugated goat anti-mouse IgG1, IgG2a, IgG2b and rabbit anti-goat IgG were from Southern Biotech; HRP-conjugated goat anti-rabbit antibody was from Jackson ImmunoResearch Laboratories. Goat anti-mouse IgG1-agarose, mouse IgG1, protease inhibitor mixture, Triton X-100, Tween 20, and other chemicals of analytic grade were purchased from Sigma-Aldrich.

Cell death, cleavage of caspases, activation NF-{kappa}B and ERK1/2, and Western blots. Human NSCLC cell lines were seeded in 96-well plates or culture dishes and treated with recombinant TRAIL. Cell death was determined by crystal violet cell viability assay and cellular apoptosis was observed under phase contrast microscopy (5). For the cleavage of caspases, DFF45, and c-FLIP and the phosphorylation of the proteins, cells were treated or untreated, lysed, and subjected to SDS-PAGE on 15% gels and transferred to nitrocellulose membranes. The membranes were blotted with the antibodies against c-FLIP, caspase-8, caspase-9, caspase-3, DFF45, and phosphorylated and unphosphorylated I{kappa}B, ERK1/2, and NF-{kappa}B overnight at 4°C. The membranes were washed and incubated for 1 h at room temperature with the antimouse IgG2b-HRP, antimouse IgG1-HRP, or antirabbit IgG-HRP. The blots were washed and developed by chemiluminescence.

Flow cytometry. Cell surface expression of DR4 and DR5 was measured by flow cytometry. In brief, 0.1 µg/mL of phycoerythrin-conjugated antihuman DR4 and DR5 (mouse IgG1) or mouse IgG1 (a negative control) was added to 106 cells in 200 µL of immunofluorescence buffer (PBS containing 2% fetal bovine serum and 0.02% sodium azide). After 1 h of incubation in the dark at 4°C, the cells were washed with immunofluorescence buffer and dispersed in 500 µL of PBS. For all tested cell samples, 10,000 cells were analyzed using a Becton Dickinson FACScan. The results were processed by using Cell Quest software (Becton Dickinson).

DISC immunoprecipitation. The TRAIL-induced DISC was immunoprecipitated with Flag-tagged TRAIL, based on the protocol as reported (21). In brief, the cells were treated with the mixture of Flag-tagged TRAIL and anti-Flag M2 IgG for 15 min at 37°C and then lysed for 30 min on ice with the lysis buffer. The soluble fraction was immunoprecipitated with goat anti-mouse IgG-agarose overnight at 4°C and subjected to Western blot analysis with the following antibodies: DR4, DR5, FADD, caspase-8, RIP, and c-FLIP. In the unstimulated controls, the cells were lysed first and then treated with the mixed Flag-TRAIL/anti-Flag M2 to immunoprecipitated nonstimulated TRAIL receptors.

Generation of RIP and c-FLIP short hairpin RNA–expressing clones. For a vector-based RNAi approach, a double-stranded short hairpin RNA (shRNA) was cloned into the BamHI-XhoI sites of the pRNAT-U6.1/neo-cGFP vector (GenScript Corporation). The specific sequence for the shRNA against c-FLIP was: 5'-GATCCCGTGTCGGGGACTTGGCTGAACTTTGATATCCGAGTTCAGC CAAGTCCCCGACATTTTTTCCAACTCGAG-3'. The sequence for shRNA against RIP was 5'-GGATCCCGTACCACTAGTCTGACGGATAATTGATATCCGTTATCCGTCAGACTAGTGGTATTTTTTCCAACTCGAG-3'. The underlined, boldface, and italic letters denote the hairpin loop, terminal signal, and target sites of the restriction enzymes BamHI and XhoI, respectively. pRNAT-U6.1/Neo-cGFP empty vector, pRNAT-U6.1/Neo-cGFP c-FLIP/small interfering RNA (siRNA), or RIP/siRNA were transfected by LipofectAMINE 2000 (Invitrogen). After fluorescence-activated cell sorting for green fluorescent protein–expressing cells, transfectants were pooled and grown in 1 mg/mL of G418 (Sigma-Aldrich) and monitored under fluorescence microscopy.

Lipid raft and nonraft fractionation and cholesterol and protein analyses. Lipid raft and nonraft–soluble fractions were separated by discontinuous sucrose density gradients of Triton X-100 cell lysates from treated and untreated cells (28). In brief, subconfluent cells from 10 of 15-cm culture dishes (1 x 108 cells) were lysed on ice for 30 min in 2 mL of MNX buffer [1% Triton X-100 in 25 mmol/L MES, 150 mmol/L NaCl (pH 6.5)] supplemented with 1 mmol/L of phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma), and then homogenized. The homogenates were mixed with 2 mL of 90% sucrose made with MNX buffer and placed on the bottom of a centrifuge tube (14 x 89 mm, Beckman). The samples were then overlaid with 4 mL of 35% sucrose and 4 mL of 5% sucrose and centrifuged at 175,000 x g in a SW32Ti rotor with Optima L-80 XP centrifuge (Beckman) for 16 h at 4°C. Ten fractions of 1 mL were collected from the top to the bottom of the gradient and analyzed by Western blot. To identify lipid raft fractions, cholesterol content in the fractions were determined with the cholesterol assay kit (Wako Pure Chemicals). Cholesterol content in each fraction was indicated as micrograms of total cholesterol per milligram of protein. The protein concentration in each fraction was determined by a Bio-Rad protein assay kit. The fractions were also examined by Western blots with antibodies to the lipid rafts markers glycosphingolipid GM1 (Sigma-Aldrich) and caveolin-1 (BD Biosciences). For the DISC analysis, the cells were treated first with Flag-tagged TRAIL for 30 min and then subjected to discontinuous sucrose density gradients for lipid raft and nonraft fractionation. TRAIL-DISC was immunoprecipitated with anti–IgG-agarose from lipid raft fractions 4 and 5, and nonraft fractions 9 and 10 as previously described (21), and subjected to the Western blot analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TRAIL activates apoptotic and nonapoptotic signals in NSCLC cells. TRAIL has been shown to induce apoptosis in TRAIL-sensitive NSCLC cell lines and inhibit the growth of the xenografts generated from the cell lines (5, 7). We have examined a large panel of 21 human NSCLC cell lines and found out that the majority of the cell lines were either partially or completely resistant to TRAIL treatment (data not shown). To examine the molecular mechanisms in TRAIL resistance, we examined TRAIL signaling pathways in TRAIL-resistant cell lines (A549 and H596) as compared with TRAIL-sensitive cell lines (H460 and H1792). Each of the cell lines were treated with a series of concentrations of recombinant human TRAIL for 16 h at 37°C and the cell viability assay confirmed that H460 and H1792 were TRAIL sensitive, whereas A549 and H596 were TRAIL resistant (Fig. 1A ). The cells were then treated with 300 ng/mL of TRAIL, and Western blots detected the cleavage of caspase-8, caspase-9, caspase-3, XIAP, and DFF45 in the sensitive but not the resistant cell lines (Fig. 1B). TRAIL-treated cells were then subjected to subcellular fractionation; Western blots revealed cytochrome c and Smac in the cytosolic fractions free of mitochondria in the sensitive but not in the resistant cells (Fig. 1B). These studies indicate that TRAIL-induced caspase-8–initiated extrinsic and intrinsic pathways, as observed in the TRAIL-sensitive cells, are inhibited in the TRAIL-resistant NSCLC cells.


Figure 1
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  		Figure 1. TRAIL-induced apoptosis and activation of NF-{kappa}B and ERK1/2. A, each of the cell lines was treated with serial dilutions of TRAIL for 16 h and the percentage of cell death was assessed by cell viability assay. Points, means; bars, SE (n = 8). B, TRAIL-induced cleavage of caspases and DFF45 were examined by Western blots in the NSCLC cells treated with 300 ng/mL of TRAIL for the times indicated. TRAIL-trigged mitochondrial release of cytochrome c and Smac was examined by Western blot analysis of subcellular cytosolic fractions (bottom two rows). C, cell surface expression of DR4 and DR5 in the NSCLC cell lines was examined by flow cytometry. D, TRAIL-DISC was immunoprecipitated by treating the cells with mixed Flag-TRAIL and anti-Flag IgG antibody for 15 min in stimulated cells (+) or after cells were lysed in unstimulated controls (–). The DISC samples, together with cell extracts (EX) as a control for the endogenous proteins, were subjected to Western blots. E, phosphorylated and unphosphorylated forms of I{kappa}-B{alpha}, NF-{kappa}B, and ERK1/2 (left) were examined by Western blots in A549 and H1792 cells treated with 300 ng/mL of TRAIL for the times indicated. A549 cells were treated with PH98059, alone or in combination with TRAIL (right), and examined by Western blots for the phosphorylated and unphosphorylated ERK1/2 and cell viability (bottom).

 
To explore the molecular basis of caspase-8 inhibition, we examined the cell surface expression of DR4 and DR5 and the DR4/DR5-mediated DISC in TRAIL-sensitive (H460, H1792) and TRAIL-resistant cell lines (A549, H596). DR4 and DR5 expression on the cell surface was analyzed by flow cytometry and the results showed that DR5 was highly expressed in both the sensitive and resistant cell lines (Fig. 1C). In contrast, DR4 expression was much lower than DR5 on the cell surface of these four cell lines and, of these four cell lines, DR4 was expressed higher in A549 and H1792 than H460 and H596 (Fig. 1C). Next, we examined DR4/DR5-mediated assembly of the DISC in these two sensitive and two resistant cell lines. Each of the four cell lines were treated with mixed Flag-TRAIL and anti-Flag IgG antibody and the DISC was immunoprecipitated with goat anti-mouse IgG-agarose. For unstimulated control, cells were lysed first and then immunoprecipitated with Flag-TRAIL and anti-Flag IgG. Western blots detected DR4 in H1792 and A549 but not in H460 and H596 cell lines (Fig. 1D); the results correlated well to the lower levels of DR4 expression on the cell surface of H460 and H596 (Fig. 1C). Western blot analysis further revealed DR5, FADD, and caspase-8 in the DISC in both TRAIL-sensitive and TRAIL-resistant cell lines (Fig. 1D). In contrast, however, RIP and c-FLIP were detected in the DISC only in the resistant A549 and H596 cell lines (Fig. 1D).

We then showed that TRAIL treatment resulted in the activation of NF-{kappa}B and ERK1/2 in TRAIL-resistant A549 cells, as evident by the phosphorylated I{kappa}B, NF-{kappa}B, and ERK1/2 on Western blots (Fig. 1E). In contrast, the phosphorylated forms of I{kappa}B and ERK1/2 were not detected in TRAIL-sensitive H1792 under TRAIL treatment (Fig. 1E). The phosphorylated NF-{kappa}B was detected in H1792, but its expression levels did not correlate with TRAIL treatment (Fig. 1E). These results establish the correlation between the recruitment of RIP and c-FLIP to the DISC, and the activation of NF-{kappa}B and ERK1/2 in TRAIL-resistant A549 cells. ERK1/2 kinases are activated through dual phosphorylation by MEK (29). Treatment of A549 with the MEK inhibitor PD98059 prevented TRAIL-induced ERK1/2 phosphorylation, but did not affect the cell resistance to TRAIL (Fig. 1E). A recent study showed that A549 remains resistant to TRAIL after transfection with dominant-negative I{kappa}B{alpha} (30). These studies suggest that inhibition of downstream NF-{kappa}B and ERK1/2 does not affect the A549 sensitivity to TRAIL.

Lipid rafts are required for TRAIL-induced apoptosis. To determine if lipid rafts might serve as a platform for TRAIL-induced signals, lipid rafts were extracted from NSCLC cells by a discontinuous sucrose density gradient applied to Triton X-100 cell lysates of H1792 and A549. Ten fractions of 1 mL were collected from the top to the bottom of the gradient, analyzed biochemically for the cholesterol and protein contents, and examined by Western blots for the presence of the lipid raft marker caveolin-1. The results identified fractions 4 and 5 as lipid rafts by the higher content of cholesterol (Fig. 2A ) and the presence of the lipid raft marker caveolin-1 (Fig. 2A). DR4 and DR5 were detected in both the lipid raft and nonraft fractions, whereas FADD and caspase-8 were found in nonraft fractions in both H1792 and A549 cells (Fig. 2B); the results suggest no correlation between DR4/DR5 distribution in lipid rafts and TRAIL sensitivity of the cell lines.


Figure 2
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  		Figure 2. Lipid rafts mediate TRAIL-induced apoptotic signals. A, H1792 and A549 cell lines were subjected to discontinuous sucrose density gradients of Triton X-100 cell lysates for separation of lipid raft and nonraft fractions. One to 10 fractions were examined on Western blots for the presence of DR4 and DR5 (top two rows). Lipid raft fractions 4 and 5 were identified by Western blots by using lipid raft marker caveolin-1 and GM1 (third row). Bottom, concentration of cholesterol and protein levels were measured in each of the fractions of A549 (points, means; bars, SE; n = 4). B, H1792 cells were stimulated with 300 ng/mL of TRAIL for the times indicated and subjected to discontinuous sucrose density gradients for generation of lipid raft and nonraft fractions. One to 10 fractions were subjected to Western blot analysis of the DISC proteins and lipid raft marker caveolin-1. C, H1792 cells were untreated (0 min) or treated for 2 or 10 min with Flag-TRAIL and subjected to discontinuous sucrose density gradients. Triton X-100–resistant (R) lipid raft (pool of fractions 4–5) and Triton X-100–soluble (S) fractions (pool of fractions 7–10) were incubated with anti-Flag mouse IgG antibody; the DISC was immunoprecipitated by anti-mouse IgG-agarose and examined on Western blots. For unstimulated controls (–), the cells were lysed and the DISC was immunoprecipitated from the Triton X-100–resistant (pool of fractions 4–5) and the soluble (pool of fractions 7–10) fractions and subjected to Western blot. For the endogenous protein expression controls (EX), the cells were lysed and Triton X-100–resistant (R) and –soluble (S) fractions were separated and subjected to the Western blots. D, H1792 cells were treated with MßCD for 30 min and then with 100 ng/mL of TRAIL for 16 h for cell death analysis and for 3 h for Western blot analysis of cleavage of caspase-8.

 
To further examine the role of lipid rafts in TRAIL-induced apoptosis, the H1792 cell line was treated with TRAIL and subjected to a discontinuous sucrose density gradient. Western blot analyses showed that the TRAIL treatment resulted in the redistribution of FADD and caspase-8 from the nonraft to the lipid raft fractions (Fig. 2B). To further examine the DISC assembly in lipid raft and nonraft fractions, H1792 cells were treated with mixed Flag-TRAIL, and then subjected to discontinuous sucrose density gradients for separation of lipid raft and nonraft fractions. The Flag-TRAIL–induced DISC was immunoprecipitated with mouse anti-Flag IgG antibody through rabbit anti-mouse IgG-agarose from lipid raft fractions 4 and 5 and nonraft fractions 9 and 10. The lipid raft and nonraft–associated DISC samples were examined on Western blot. DR5, FADD, and caspase-8 were detected in the DISC obtained from both lipid raft and nonraft fractions (Fig. 2C); the results suggest that TRAIL-induced DISC assembly occurs in both lipid raft and nonraft fractions in the TRAIL-sensitive H1792 cells.

To further examine the role of lipid rafts in TRAIL-induced apoptosis, H1792 cells were treated with the cholesterol-depleting agent methyl-ß-cyclodextrin (MßCD) and the cholesterol content was examined for the cholesterol depletion (data not shown). The MßCD-treated cells were further treated with 300 ng/mL of TRAIL for an additional 16 h for cell death assay and 3 h for Western blot analysis. The results showed that cholesterol depletion inhibited TRAIL-induced cell death and caspase-8 cleavage (Fig. 2D). These results suggest that lipid rafts are required for TRAIL-induced apoptosis in TRAIL-sensitive NSCLC cells.

Nonrafts mediate TRAIL-induced NF-{kappa}B and ERK1/2 signals. TRAIL activates NF-{kappa}B and ERK1/2 in A549 cells (Fig. 1E). We therefore examined A549 cells to determine if lipid rafts and nonrafts were involved in TRAIL-induced NF-{kappa}B and ERK1/2 signaling. A549 cells were treated with 300 ng/mL of TRAIL and subjected to discontinuous sucrose density gradients for separation of lipid raft and nonraft fractions. DR4 and DR5 were detected in the lipid rafts prior to TRAIL stimulation (Fig. 3A ). In contrast, however, FADD and caspase-8 were detected only in the nonraft fractions before or after TRAIL treatment (Fig. 3A). These results suggest the role of nonrafts in TRAIL signaling in resistant cells. To further test this, we examined TRAIL-DISC assembly in lipid rafts and nonrafts. A549 cells were treated with the Flag-TRAIL/IgG anti-Flag antibody; lipid raft and nonraft fractions were separated by discontinuous sucrose density gradients; the Flag-TRAIL-DISC was immunoprecipitated from lipid raft fractions 4 and 5 and nonraft fractions 9 and 10. Western blots detected DR5, FADD, and caspase-8 in the DISC in the nonraft, but not the lipid raft fractions (Fig. 3B). A549 cells were further treated with MßCD; the results showed that cholesterol depletion did not affect TRAIL-induced activation of NF-{kappa}B and ERK1/2 (Fig. 3C). Taken together, these studies indicate that nonrafts mediate TRAIL-induced activation of NF-{kappa}B and ERK1/2 in the TRAIL-resistant NSCLC cells.


Figure 3
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  		Figure 3. Nonrafts mediate TRAIL-induced NF-{kappa}B and ERK1/2 signals. A, A549 cell lines were stimulated with 300 ng/mL of TRAIL for the times indicated; lipid raft and nonraft fractions were separated and examined on Western blots. B, A549 cell lines were untreated (0 min) or treated for 2 or 10 min with Flag-TRAIL, subjected to discontinuous sucrose density gradients, immunoprecipitated by incubating Triton X-100–resistant (R) lipid raft (pool of fractions 4–5) and Triton X-100–soluble (S) fractions (pool of fractions 7–10) with anti-Flag mouse IgG antibody, and subjected to Western blot. The unstimulated controls (–) and cell lysate control (EX) were prepared based on the protocol described in Fig. 2. C, A549 cell lines were treated with MßCD for 30 min and then with 300 ng/mL of TRAIL for 16 h for cell death analysis and for 3 h for Western blot analysis of ERK1/2 and I{kappa}B{alpha} and their phosphorylated forms (p-).

 
The recruitment of c-FLIP to the DISC contributes to the DISC localization in nonrafts. The observation that c-FLIP is recruited to the DISC (Fig. 1D) and the DISC assembly in nonrafts in A549 cells (Fig. 3B) suggest the role of c-FLIP in DISC assembly in nonrafts in A549 cells. To test this, we first generated c-FLIP knockdown clones from A549 and then examined the DISC assembly in lipid rafts and nonrafts. A double-stranded siRNA duplex specific to c-FLIP nucleotides 535 to 555 was able to inhibit the expressions of both c-FLIPL and c-FLIPS (31). We therefore synthesized a shRNA specific to the c-FLIP nucleotides and inserted the shRNA in plasmid pRNAt-U6.1 carrying green fluorescent protein and neomycin-resistance marker. The plasmid was transfected in A549 cells and the pooled clone was selected in the presence of G418. Western blots showed a significant inhibition of c-FLIPL and c-FLIPS expression in the shRNA expression A549 clone, as compared with the empty vector expression A549 clone (Fig. 4A ). The c-FLIPL and c-FLIPS knockdown A549 clone became sensitive to TRAIL, as shown by the cell death (data not shown) and cleavage of caspase-8, caspase-9, caspase-3, and DFF45 (Fig. 4A).


Figure 4
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  		Figure 4. Knockdown of c-FLIP leads to DISC redistribution from nonrafts to lipid rafts for TRAIL-induced apoptosis. A, expression of c-FLIP and cleavage of caspases were determined by Western blots in the c-FLIP knockdown (c-FLIP–) and empty vector expression clone (Vector) after being treated or not with 300 ng/mL of TRAIL for 3 h. B, the c-FLIP knockdown A549 clone was treated with 300 ng/mL of TRAIL for 10 min. Triton X-100–resistant (R) lipid raft and –soluble (S) nonraft fractions were separated and subjected to Western blotting. C, the c-FLIP knockdown A549 clone was untreated (0 min) or treated with Flag-TRAIL for 2 or 10 min. TRAIL-DISC was immunoprecipitated from Triton X-100–resistant (R) lipid raft and –soluble (S) nonraft fractions for Western blot analysis. Triton X-100–resistant (R) and the –soluble (S) fractions from unstimulated cells (–) and cell lysates (EX) were included in the analysis as controls.

 
Next, we examined the c-FLIP knockdown A549 clones for the distribution of the DISC proteins in lipid raft and nonraft fractions. Lipid raft and nonraft fractions were generated from the clone, and Western blot detected DR5 in both lipid raft and nonraft fractions, but detected only FADD and caspase-8 in the nonraft fractions (Fig. 4B). The clone was then treated with TRAIL and lipid rafts and nonraft fractions were separated and examined by Western blots. TRAIL treatment led to the redistribution of FADD and caspase-8 to lipid rafts in the c-FLIP knockdown A549 clone (Fig. 4B). The results suggest that c-FLIP is required for DISC assembly in nonrafts. To further test this, the c-FLIP knockdown A549 clone was treated with Flag-TRAIL and subjected to discontinuous sucrose density gradients for the separation of lipid raft and nonraft fractions. The Flag-TRAIL–induced DISC was isolated from lipid raft and nonraft fractions; and Western blots detected FADD and caspase-8 in the DISC obtained from lipid rafts and nonraft fractions (Fig. 4C). Cleavage products of caspase-8 were detected in lipid raft fractions (Fig. 4C). Taken together, these studies indicate that c-FLIP mediates the assembly of TRAIL-DISC in nonrafts in the TRAIL-resistant A549 cells.

RIP is required for c-FLIP inhibition of caspase-8 and DISC localization in nonrafts. Both RIP and c-FLIP were recruited to the DISC (Fig. 1D) in A549 cells, and selective knockdown of c-FLIP eliminates the cell resistance to TRAIL (Fig. 4A). Therefore, regarding the role of RIP and c-FLIP in the DISC, we speculated that c-FLIP might inhibit caspase-8, whereas RIP might couple with the NF-{kappa}B and ERK1/2 pathway. To examine this, a RIP knockdown clone was generated from the A549 cell line. Several siRNA duplexes specific to the RIP gene were first synthesized and examined for the ability to inhibit RIP expression in A549 cells (data not shown). The siRNA duplex specific to RIP nucleotides 837 to 857 was selected for its ability to inhibit RIP expression (data not shown). A shRNA specific to the RIP nucleotides 837 to 857 was synthesized and inserted into pRNAt-U6.1. The RIP shRNA vector was transfected in A549 cells and a RIP knockdown clone was generated in the presence of G418. Western blots showed a marked reduction in RIP expression (Fig. 5A ). The selective knockdown of RIP did not affect the expression of c-FLIP and caspase-8; however, to our surprise, the RIP knockdown A549 clone became sensitive to TRAIL-induced apoptosis, as shown by the cleavage of caspase-8 (Fig. 5A) and cell death (Fig. 5B). In addition, the selective knockdown of RIP from A549 eliminated TRAIL-induced activation of NF-{kappa}B and ERK1/2 (Fig. 5A). These results indicate that RIP plays a role both in TRAIL resistance and TRAIL-induced activation of NF-{kappa}B and ERK1/2.


Figure 5
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  		Figure 5. RIP is required for c-FLIP inhibition of caspase-8 in the DISC and the DISC assembly in nonrafts. A, the expression of RIP and c-FLIP, the cleavage of caspase-8, and the phosphorylation of NF-{kappa}B and ERK1/2 were examined by Western blot in wild-type A549 (–), the empty vector expressing clone (Vector) and the RIP knockdown clone (RIP–) after treatment with 300 ng/mL of TRAIL for 3 h. B, the cell death was examined in wild-type A549 (–), the empty vector expressing clone (Vector) and the RIP knockdown clone (RIP–) after treatment with 300 ng/mL of TRAIL for 16 h (columns, mean; bars, SE; n = 8). C, the RIP knockdown A549 clone was treated with 300 ng/mL of TRAIL for 10 min. Triton X-100–resistant (R) lipid raft and –soluble (S) nonraft fractions were separated and subjected to Western blotting. D, the RIP knockdown A549 clone was untreated (0 min) or treated with Flag-TRAIL for 2 or 10 min. TRAIL-DISC was immunoprecipitated from Triton X-100–resistant (R) lipid raft and –soluble (S) nonraft fractions for Western blot analysis. Triton X-100–resistant (R) and the –soluble (S) fractions from unstimulated cells (–) and cell lysates (EX) were included as controls.

 
The findings that the RIP knockdown sensitizes A549 cells to TRAIL-induced apoptosis (Fig. 5A) further suggest the role of RIP in the DISC localization in nonrafts in A549 cells. To test this, we separated lipid rafts and nonrafts from the RIP-knockdown A549 clone and showed no difference in the DR5, FADD, and caspase-8 distribution in lipid rafts and nonrafts between wild-type A549 (Fig. 3A) and its RIP knockdown clone (Fig. 5C). However, TRAIL treatment resulted in the redistribution of FADD and caspase-8 from nonrafts to lipid rafts (Fig. 5C) in the RIP knockdown A549 clone. Both c-FLIPL and c-FLIPS were localized in the nonraft fractions prior to TRAIL treatment, but was detected in both lipid raft and nonraft fractions in which c-FLIPL was cleaved into the p43 intermediate form (Fig. 5C). Further examination of the DISC immunoprecipitated from the lipid raft and nonraft fractions showed that TRAIL triggered the DISC assembly in both lipid raft and nonraft fractions (Fig. 5D). Taken together, these results indicate that RIP is required for the c-FLIP–mediated inhibition of caspase-8 in the DISC and the DISC localization in nonrafts for the cell survival. Targeting of RIP therefore leads to the redistribution of TRAIL-DISC from nonrafts to lipid rafts, thereby switching the cells from NF-{kappa}B and ERK1/2 cell survival to caspase-8–initiated apoptotic signals.

Chemotherapy treatment redistributes the DISC from nonrafts to lipid rafts. Chemotherapy agents have been shown to inhibit c-FLIP expression and thus enhance TRAIL-induced apoptosis in human cancer cells and may prove to be effective combination therapies that can overcome TRAIL resistance. Chemotherapy treatment may multiply the molecular mechanisms in TRAIL resistance such as up-regulation of DR5 (32) and inhibition of c-FLIP (33). We therefore speculated that chemotherapy treatment might affect TRAIL-DISC distribution in the lipid raft and nonraft fractions. TRAIL-resistant A549 and H596 cell lines were treated with TRAIL, alone or in combination with the chemotherapeutic agent cisplatin, etoposide (VP16), camptothecin, and doxorubicin. Treatment of the resistant cells with each of the chemotherapy agents enhanced TRAIL-induced apoptosis, as shown by the cleavage of caspases and DFF45 (Fig. 6A ) and cell death (Fig. 6B). TRAIL-resistant A549 cells were treated with each of the chemotherapeutic agents and Western blots showed the increase in DR5 expression and decrease in c-FLIP expression in the cells (Fig. 6C). Treatment with chemotherapeutic drugs did not affect the protein levels of DR4 and RIP (Fig. 6C). Finally, A549 cells were treated with cisplatin, alone or in combination with TRAIL, and then subjected to discontinuous sucrose density gradients for the generation of lipid raft and nonraft fractions. Combination treatments with cisplatin and TRAIL resulted in the redistribution of FADD and caspase-8 from nonraft to lipid raft fractions (Fig. 6D). These results indicate that chemotherapeutic agents enhance TRAIL-DISC redistribution in lipid rafts, thereby causing a synergistic apoptotic effect with TRAIL in TRAIL-resistant NSCLC cells.


Figure 6
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  		Figure 6. Chemotherapy treatment redistributes TRAIL-DISC from nonraft to lipid raft fractions. A, cleavage of caspases and DFF45 was examined in TRAIL-resistant A549 and H596 cell lines after being treated with cisplatin (10 µg/mL), etoposide (VP16, 10 µg/mL), camptothecin (CPT, 300 ng/mL), or doxorubicin (DOX, 300 ng/mL) in the presence or absence of 300 ng/mL of TRAIL. B, cell death was determined by cell viability assay (columns, mean; bars, SE; n = 8) in A549 and H596 cell lines after being treated with TRAIL, cisplatin, and doxorubicin, alone or in combination. C, the expression of DR4, DR5, RIP, and c-FLIP was examined in A549 cells after being treated with cisplatin (10 µg/mL), etoposide (VP16, 10 µg/mL), and camptothecin (CPT, 300 ng/mL) for the times indicated. D, Western blot analysis was carried out on Triton X-100–resistant (R) lipid raft and –soluble (S) nonraft fractions isolated from A549 cells after treatment with 300 ng/mL of TRAIL and 10 g/mL of cisplatin, alone or in combination for 5 min.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several lines of evidence suggest that TRAIL could serve as a therapeutic agent in the treatment of NSCLC, particularly when combined with chemotherapy. TRAIL death receptors DR4 and DR5 were detected in human NSCLC tumors (34); recombinant TRAIL is capable of inducing apoptosis in TRAIL-sensitive NSCLC cells in culture (35) and inhibiting the growth of NSCLC xenografts generated from the sensitive cell lines in mice (5, 7). However, analysis of a large panel of established human NSCLC cell lines shows that the majority of NSCLC cells were either partially or completely resistant to TRAIL. RIP and c-FLIP play an important role in TRAIL resistance, by recruitment to the DISC where they inhibit caspase-8 cleavage and couple NF-{kappa}B and ERK1/2 signals for cell survival. Targeting of either RIP or c-FLIP can overcome TRAIL resistance and thus provides an effective therapeutic approach in TRAIL-based combination treatments of NSCLC.

RIP was initially identified as a death domain adaptor in the Fas and TNFR1 signaling complexes for Fas-mediated apoptosis and TNFR1-activated NF-{kappa}B signals (17, 36). Studies of transfectants first identified RIP in the TRAIL-DISC responsible for NF-{kappa}B activation (18). The endogenous RIP protein was then detected in the TRAIL-DISC and involved in TRAIL-induced IKK activation in TRAIL-resistant HEK293 cells (37). In contrast, RIP was not detected in the TRAIL-DISC in the TRAIL-sensitive B lymphoma cell line BJAB (38). Although RIP was reported in the TRAIL-DISC in HeLa cells, it was quickly cleaved during TRAIL-induced apoptosis (39). In this study, we confirm that RIP plays a role in TRAIL-induced activation of NF-{kappa}B in NSCLC cells. To our surprise, however, the studies establish for the first time that endogenous RIP is required for the c-FLIP–mediated inhibition of caspase-8 in the DISC in TRAIL-resistant NSCLC cells.

c-FLIP was originally identified as a death effector domain adaptor that was recruited by FADD to the Fas-DISC, leading to the inhibition of caspase-8 cleavage and activation (20). c-FLIP exists in two isoforms: c-FLIPS and c-FLIPL; both proteins are recruited to the Fas-DISC in which c-FLIPL is cleaved into an intermediate form of p43 c-FLIPL (40). Both c-FLIPS and c-FLIPL were detected in the TRAIL-DISC in TRAIL-resistant cancer cells (21). Studies of c-FLIP–deficient mice establish the inhibitory role of c-FLIP in TNF and FasL-induced apoptosis (41), and knockdown of c-FLIP with siRNA then confirms the inhibitory role of c-FLIP in TRAIL-induced apoptosis in NSCLC cells (42). On the other hand, however, the study of T lymphocytes reports that c-FLIP promotes Fas-mediated NF-{kappa}B and ERK1/2 activation (43). With shRNA knockdown, we establish that endogenous c-FLIP proteins are required for TRAIL-induced NF-{kappa}B and ERK1/2 signals in TRAIL-resistant NSCLC cells.

The finding that c-FLIP knockdown sensitizes A549 cells to TRAIL (42) has led to speculations regarding the roles of c-FLIP and RIP in DISC function: c-FLIP might inhibit caspase-8 where RIP might couple NF-{kappa}B and/or ERK1/2 signaling. Instead, however, we show that RIP knockdown not only eliminates TRAIL-induced NF-{kappa}B and ERK1/2 signaling in the TRAIL-resistant NSCLC cell line, but also sensitizes the resistant cell line to TRAIL-induced apoptosis. RIP-mediated NF-{kappa}B signals may induce c-FLIP expression (44); however, the study shows that RIP knockdown has no effect on the expression of c-FLIP proteins and caspaspe-8 as well. In fact, the endogenous c-FLIP proteins are recruited to the DISC, but they fail to inhibit caspase-8–initiated apoptosis in the absence of RIP protein. These studies indicate that both RIP and c-FLIP are required for the dual functions of DISC: inhibition of caspase-8–initiated apoptotic cascade and activation of NF-{kappa}B and ERK1/2. This hypothesis may explain the findings that c-FLIP, RIP, and caspase-8 are required for death receptor–induced activation of NF-{kappa}B and ERK1/2 (22, 43).

Recent studies suggest that lipid rafts may serve as the platforms for TNFR1 and Fas-DISC–initiated signals (23). However, the lipid raft model fails to explain TRAIL-DISC–initiated cell death signals in sensitive cells and NF-{kappa}B and ERK1/2 signals in resistant cells. In this study, we provide several lines of evidence in support of the molecular models that lipid rafts are required for TRAIL-induced apoptosis, whereas nonrafts are involved in TRAIL-activated NF-{kappa}B and ERK1/2 signals. TRAIL triggers the DISC assembly in lipid rafts and nonrafts in TRAIL-sensitive cells; however, TRAIL-induced DISC assembly is limited to the nonrafts in TRAIL-resistant cells. Disruption of lipid rafts with cholesterol-depleting agent inhibits TRAIL-induced apoptosis in sensitive cells but not TRAIL-induced NF-{kappa}B and ERK1/2 activation in the resistant cells. Knockdown of either RIP or c-FLIP in the TRAIL-resistant cells results in the redistribution of the DISC from nonrafts to lipid rafts, thereby switching the cells from TRAIL-induced NF-{kappa}B and ERK1/2 cell survival signaling to caspase-8–initiated apoptosis. Both RIP and c-FLIP are required for TRAIL-induced assembly of the DISC in nonrafts and therefore targeting of either RIP and c-FLIP results in the DISC assembly in lipid rafts, leading to TRAIL-induced apoptosis.

TRAIL-induced apoptosis occurs through binding on its receptors DR4 and DR5 (8). In addition to DR4 and DR5, TRAIL also interacts with two other receptors, decoy receptor 1 (DcR1/TRAIL-R3/TRID) and DcR2 (TRAIL-R4). DR4 and DR5 have a cytoplasmic death domain that interacts with FADD for the intracellular assembly of the DISC (8). In contrast, DcR1 lacks a cytoplasmic domain and DcR2 has a truncated death domain, and they therefore inhibit TRAIL-induced apoptosis by competing with DR4 and DR5 for binding to TRAIL (45, 46). In this study, we show that DR5 is much more expressed than DR4 in NSCLC cells and thus plays a critical role in DISC assembly; however, it remains to be determined if and how DcR1 and DcR2 modulate DR5-mediated assembly of the DISC in NSCLC cells.

Chemotherapy remains the standard clinical treatment of NSCLC and recent studies suggest that combination treatment with chemotherapy can overcome TRAIL resistance in NSCLC cells (7). This synergistic effect has been reported due to diverse effects such as the up-regulation of DR5 (32, 47, 48) and the down-regulation of c-FLIP (33, 49). Here, we confirm that DR5 is the major death receptor that mediates the DISC assembly for TRAIL-induced apoptosis in TRAIL-sensitive NSCLC cells. In addition, we show that targeting of either RIP or c-FLIP is sufficient enough for TRAIL to induce apoptosis in TRAIL-resistant NSCLC cells. Chemotherapy treatment leads to the up-regulation of DR5 and down-regulation of c-FLIP, and the redistribution of the DISC from nonrafts to lipid rafts, thus causing synergistic apoptotic effects with TRAIL on TRAIL-resistant NSCLC cells. This result is consistent with a recent study of colon carcinoma (50). These studies therefore indicate that targeting of the molecular modulation of the DISC distribution in nonrafts may provide novel therapeutic strategies in TRAIL-based combination treatments of NSCLC and perhaps other human cancers as well.


   Acknowledgments
 
Grant support: Philip Morris USA, Inc., Philip Morris International (N.M. Kneteman), and the Emory Startup Fund (C. Hao).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Doyoun K. Song for her technical support.

Received 10/20/06. Revised 4/ 8/07. Accepted 5/ 8/07.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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