Decoy Receptor 3 Expression in AsPC-1 Human Pancreatic Adenocarcinoma Cells via the Phosphatidylinositol 3-Kinase-, Akt-, and NF-κB-Dependent Pathway¹

Decoy receptor 3 (DcR3)³ is a member of the TNFR superfamily. DcR3 cDNA encodes a 300-aa protein containing the four tandem cysteine-rich repeats characteristic of the TNFR superfamily and lacking a transmembrane sequence (1). DcR3 is therefore regarded as a secreted molecule. Previous studies have identified three ligands that interact with DcR3, namely, Fas ligand (FasL), lymphotoxin-like, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes, and TL1A (1, 2, 3). DcR3 is believed to block the cellular effects caused by the binding of these ligands to their membrane-bound cognate receptors by blocking ligand/receptor binding, as shown for the binding of FasL to Fas and TL1A to death receptor 3 (1, 3, 4). It has recently been reported that, in addition to binding to these ligands, DcR3 may bind to, and cross-link, proteoglycans to induce monocyte adhesion (5).

Evidence is accumulating that DcR3 plays a significant role in immune suppression and tumor progression. DcR3 induces dendritic cell apoptosis (6), modulates the differentiation of dendritic cells and macrophages and impairs macrophage function (7, 8, 9), regulates T cell/B cell activation, prevents T cell/macrophage infiltration in the kidney (10), and inhibits T cell chemotaxis (11). Moreover, several reports link DcR3 with immune disease, e.g., DcR3 increases T cell activation in systemic lupus erythematosus (12), osteoclast formation (9, 13), and adhesion molecule expression on endothelial cells (14), and overexpression of DcR3 is seen in EBV- or human T-lymphotropic virus type 1-associated lymphomas (15). In addition, an association between DcR3 expression and tumor progression is well documented (16, 17). Elevated serum concentrations of DcR3 have been detected in patients with various malignant cancers, e.g., cancers of the esophagus, stomach, glioma, lung, colon, rectum, and pancreas (1, 4, 16, 18, 19). In tumorigenesis, DcR3 not only helps tumor cells to escape immune surveillance by neutralizing FasL- or lymphotoxin-like, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocyte-mediated cell death (1, 2), down-regulating MHC-II expression by tumor-associated macrophages (20), and inducing immune suppression, as described above, but also contributes to the development of a microenvironment suitable for tumor growth, e.g., by inducing angiogenesis (21). DcR3 is therefore a critical factor in tumor progression.

Human pancreatic carcinoma is a highly malignant cancer. This disease is usually diagnosed at a late, incurable stage, and the 5-year survival rate is less than 5% (22). Pancreatic cancer is relatively resistant to cytotoxic therapy (22) and radiation treatment (23). In addition, there is increasing evidence that many cancers, including pancreatic cancer, develop different methods of evading destruction by the immune system, such as resistance to FasL-Fas interaction-mediated apoptotic signals, despite expressing Fas (24). Furthermore, a recent study demonstrated that human pancreatic adenocarcinomas show high expression of DcR3, which blocks the growth inhibition signals mediated by FasL (4). However, the underlying mechanisms involved in modulating DcR3 expression are poorly understood. Kim et al. (25) suggested that LPS treatment of human intestinal epithelial cells induces DcR3 release via activation of ERK1/2/JNK and the transcription factor NF-κB. However, the signaling pathway involved in DcR3 expression in tumor cells is still unclear. In this study, we identified the signal transduction pathway of DcR3 expression in human pancreatic adenocarcinoma cells. Moreover, using small interfering RNA (siRNA) to knockdown DcR3 levels, we evaluated whether reduced DcR3 expression increases the cytotoxic activity of FasL. A clearer understanding of the mechanisms involved in DcR3 expression will help in developing therapeutic strategies for human malignancies.

Rabbit polyclonal Abs against phospho-Akt (Ser⁴⁷³), Akt, phospho-IκB kinase (IKK)α (Ser¹⁸⁰)/β (Ser¹⁸¹), or phospho-p65 (Ser⁵³⁶) and mouse anti-phospho-IκBα (Ser^32/36) mAb were purchased from Cell Signaling Technology. Rabbit polyclonal Abs against insulin-like growth factor (IGF)-1Rβ; mouse mAbs against DcR3, IκBα, GAPDH, or FasL; and protein A/G-PLUS agarose were purchased from Santa Cruz Biotechnology. Mouse mAb against phosphotyrosine (clone 4G10) was obtained from Upstate Biotechnology. Rabbit polyclonal Abs against IKKα and mouse mAb against p65 were purchased from BioVision. HRP-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-mouse IgG, and HRP-conjugated goat anti-rabbit IgG Abs were obtained from Jackson ImmunoResearch Laboratories. The human DcR3 ELISA kit was purchased from R&D Systems. Recombinant human soluble FasL (sFasL) was purchased from PeproTech Asia. IκBαM, a dominant-negative (DN) mutant of IκBα, was provided by B.-C. Chen (Taipei Medical University, Taipei, Taiwan). Myr-Akt (constitutively activated Akt), DN-Akt (a DN mutant of Akt), pGL2-ELAM-κB-luc, the empty expression vector pUSEamp⁺, and the pEGFP-N1 plasmid were provided by C.-M. Teng (National Taiwan University, Taipei, Taiwan). Stealth siRNA for DcR3 (AF104419), nonsilence control RNA, and transfection reagents were purchased from Invitrogen. The dual-luciferase reporter assay kit and pGL4.74[hRluc/TK] vector were obtained from Promega. LY294002 and pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma-Aldrich. Herbimycin A, PD98059, wortmannin, SP600125, AG1478, rapamycin, AG1024, and AG1295 were purchased from Calbiochem. GM6001 was obtained from Millipore. The 17-AAG was obtained from Tocris Cookson. All other chemicals were from Sigma-Aldrich.

The AsPC-1 human pancreatic adenocarcinoma cells, PANC-1 human pancreatic epithelioid carcinoma cells, HT-29 human colon adenocarcinoma cells, and human T cell leukemia Jurkat clone E6-1 cells were obtained from American Type Culture Collection, and cultured in the medium recommended by the vendor (RPMI 1640 medium for AsPC-1 and Jurkat cells, DMEM for PANC-1 cells, and MEM Eagle for HT-29 cells) supplemented with 10% (v/v) FBS (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 μg/ml streptomycin (Biological Industries) at 37°C in a humidified atmosphere of 5% CO₂ in air.

Cell viability was measured by the colorimetric MTT assay. Cells (1 × 10⁴) in 100 μl of medium in 96-well plates were incubated with vehicle or test compound for 48 h. After various treatments, 1 mg/ml MTT was added; the plates were incubated at 37°C for an additional 2 h; the cells were pelleted and lysed in 100 μl of DMSO; and the absorbance at 550 nm was measured on a microplate reader. Each experiment was performed in duplicate and repeated five times.

Cells were incubated for 10 min at 4°C in lysis buffer (20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM β-glycerophosphate, 0.1% Triton X-100, 10% glycerol, 1 mM DTT, 1 μg/ml leupeptin, 5 μg/ml aprotinin, 1 mM PMSF, and 1 mM sodium orthovanadate), then were scraped off, incubated on ice for a further 10 min, and centrifuged at 100 × g for 30 min at 4°C. The whole cell extract (120 μg of proteins) was mixed with an equal volume of SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 1% glycerol, 300 mM 2-ME, and 0.00125% bromphenol blue), the mixture was heated at 95°C for 5 min and electrophoresed on 10% SDS gels, and the proteins were transferred onto polyvinylidene fluoride membranes. Immunoblotting was performed using the relevant rabbit or mouse Ab and the corresponding HRP-conjugated second Ab, followed by detection using ECL reagents (Amersham Biosciences) and exposure to photographic film.

Cell culture supernatants were collected and concentrated 30-fold (v/v) on an Amicon Ultra centrifugal filter device (Millipore), and then 5 mg of concentrated supernatant was immunoprecipitated overnight at 4°C with 1 μg of mouse anti-DcR3 mAb and A/G-agarose beads. The precipitated beads were washed three times with 1 ml of ice-cold cell lysis buffer, and the immune complex was resolved by 10% SDS-PAGE gel electrophoresis, followed by immunoblotting assay using anti-DcR3 Ab.

Cell culture supernatants were collected at various time points, and DcR3 levels were measured using commercial ELISA kits (R&D Systems), according to the vendor’s instructions.

A total of 1 × 10⁶ cells was seeded in 6-well plates in 1 ml of medium without serum 1 day before transfection. Following the manufacturer’s protocol, 5 μl of Lipofectamine 2000 (Invitrogen) in 50 μl of Opti-MEMI reduced serum medium was incubated for 5 min, then 2 μg of plasmid DNA, pEGFP-N1 plasmid, and pGL4.74[hRluc/TK] vector in 50 μl of Opti-MEMI reduced serum medium was added and the mixture was incubated for 20 min at room temperature and added to the cells, which were then incubated for 36 h. Transfection efficiency, determined by fluorescence microscopy, was >60% in all experiments. For the reporter gene assay, 50 μl of reporter lysis buffer (Promega) was added to each well, and the cells were scraped off the dishes, the samples were centrifuged at 16,200 × g for 30 s at 4°C, and the supernatants were collected. Aliquots of cell lysates (5 μl) containing equal amounts of protein (10–20 μg) were placed in the wells of an opaque black 96-well microtitreplate, and 5 μl of luciferase substrate (Promega) was added and the luminescence immediately measured in a microplate luminometer (Packard Instrument). To take into account possible differences in transfection efficiency, the luciferase activity value was normalized using the luminescence from the cotransfected Renilla pGL4.74[hRluc/TK] vector (Promega).

Cells (1 × 10⁶) were plated in 6-cm dishes in 2 ml of medium without serum 1 day before transfection. The cells were transfected with 160 nM DcR3 siRNA duplexes or with DcR3 nonsilence control using Lipofectamine 2000. The siRNA-transfected cells were incubated for 48 h after transfection before analysis. The 21-mer siRNAs were synthesized by Invitrogen. The DcR3 siRNA sequences were as follows: sense sequence, 5′-GCC AGG CUC UUC CUC CCA UdTdT-3′; antisense sequence, 5′-AUG GGA GGA AGA GCC UGG CdTdT-3′. The nonsilence control siRNA sequences were as follows: sense sequence, 5′-GCC CGC UUU CCC UCA GCA UdTdT-3′; antisense sequence, 5′-AUG CUG AGG GAA AGC GGG C-3′.

Cells were harvested, washed twice with FACS washing buffer (1% FBS and 0.1% NaN₃ in PBS), incubated with Abs at 4°C for 30 min, and washed three times with FACS washing buffer; then the fluorescence of the cells was analyzed using a FACScan flow cytometer (BD Biosciences). To detect cell cycle progression, the cells were incubated with or without the indicated agent for 24 h, washed twice with ice-cold PBS, collected by centrifugation, and fixed in 70% (v/v) ethanol for at least 2 h at −20°C. The cells were then incubated with 0.2 ml of DNA extraction buffer (0.2 M Na₂HPO₄ and 0.1 M citric acid buffer (pH 7.8)) for 30 min at room temperature, centrifuged at 3500 × g for 1 min at 25°C, resuspended in 1 ml of propidium iodide staining buffer (0.1% Triton X-100, 100 μg/ml RNase A, and 80 μg/ml propidium iodide in PBS), incubated at 37°C for 30 min in the dark, sorted by flow cytometry (FACScan; BD Biosciences), and analyzed using CellQuest software (BD Biosciences). The cell cycle distribution is shown as the percentage of cells containing G₀/G₁, S, G₂, and M DNA, as judged by propidium iodide staining. The apoptotic population was determined as the percentage of cells with a sub-G₁ (<G₁) DNA content.

Total RNA was isolated from cells using TRIzol reagent (Invitrogen). Single-strand cDNA for a PCR template was synthesized from 10 μg of total RNA using random primers and Moloney murine leukemia virus reverse transcriptase (Promega). The oligonucleotide primers used for the amplification are as follows: human DcR3 (GenBank Accession AF104419) sense (>284–306) 5′-TGC CGC CGA GAC AGC CCC ACG AC-3′ and antisense (723–745) 5′-GAC GGC ACG CTC ACA CTC CTC AG-3′, which produced a product of 461 bp; human IGF-1R (GenBank Accession NM000875) sense (<688–708) 5′-AAA TGT GCC CAA GCA CGT GTG-3′ and antisense (1105–1125) 5′-TGC CCT TGA AGA TGG TGC ATC-3′, which produced a product of 437 bp. GAPDH was used as an internal control. The GAPDH (GenBank Accession NM_002046) primers used were sense (949–972) 5′-TCC TCT GAC TTC AAC AGC GAC ACC-3′ and antisense (1134–1156) 5′-TCT CTC TTC CTC TTG TGC TCT TG-3′, which produced a product of 207 bp. Equal amounts of each reverse-transcription product (1 μg) were PCR amplified using Taq polymerase in 35 cycles consisting of 1 min at 95°C, 1 min at 58°C, and 1 min at 72°C. The amplified cDNA was run on 1% agarose gels and visualized under UV light following SYBR Safe DNA gel stain (Invitrogen). The band intensity was quantified using densitometer. The intensities of the cDNA bands were normalized to GAPDH band intensities.

The isolated RNA subjected to RT-PCR was treated with DNase to avoid amplification of DNA contaminants. The forward and reverse primers were as follows: human DcR3 (GenBank Accession AF104419), CTT CTT CGC GCA CGC TG and ATC ACG CCG GCA CCA G; human IGF-1R (GenBank Accession NM000875), TGG AGT GCT GTA TGC CTC TG and CAC CTC CCA CTC ATC AGG A; and GAPDH (GenBank Accession NM_002046), ATT CCA CCC ATG GCA AAT TC and TGG GAT TTC CAT TGA TGA CAA G. The cycle threshold (C_t) method was used to analyze the results. The C_t value, which is inversely proportional to the initial template copy number, is the calculated cycle number in which the fluorescence signal emitted is significantly above background levels. The mRNA expression level of target genes was normalized to GAPDH using the 2^−ΔΔCt method, in which ΔC_t = target gene C_t – GAPDH C_t, and ΔΔC_t = ΔC_t treatment – ΔC_t control.

As previously described (9), nuclear extracts were prepared and were subjected to the EMSA Gel Shift kit (Panomics), according to the manufacturer’s specifications. Briefly, biotin-labeled NF-κB-specific probes were incubated with 10 μg of nuclear extract at 15°C for 30 min to allow the formation of protein (transcription factor)/DNA complexes. The complexes were run by 6% nondenaturing PAGE in 0.5× TBE at 4°C at 120 V, and then transferred onto Biodyne B nylon membrane. Detection of signals was obtained using an ECL imaging system.

Cells (1 × 10⁵) were inoculated into 24-well plates. After an overnight culture, cells were transfected with 0.8 μg of DN-Akt, IκBαM, empty vector, or 160 nM nonsilence control siRNA, DcR3 siRNA for 48 h. Then, three wells of cells were fixed with 10% TCA to terminate reaction (time zero); other cells were incubated with or without sFasL (50 ng/ml) or PHA-activated Jurkat cells (membrane-bound FasL (mFasL)) for another 24 or 48 h. After incubation, 0.4% SRB (Sigma-Aldrich) in 1% acetic acid was added to each well for 15 min, the plates were washed, and dyes were dissolved by 10 mM Tris buffer. Then, the absorbance was read at a wavelength of 515 nm. Using the following absorbance measurements, such as time zero (T₀), control growth (C), and cell growth in the presence of various treatments (Tx), the percentage of cell growth was calculated as ((Tx − T₀)/(C − T₀)) × 100 for Tx ≥ T₀.

The data are expressed as the mean ± SEM, and were analyzed statistically using one-way ANOVA. When ANOVA showed significant differences between groups, Tukey post hoc test was used to determine the specific pairs of groups showing statistically significant differences. A p value of less than 0.05 was considered statistically significant.

Because DcR3 lacks a transmembrane sequence and is a soluble protein, we used an immunoprecipitation assay to determine the distribution of DcR3 in different human pancreatic cancer cells. As shown as Fig. 1^,A, high levels of endogenous DcR3 protein expression were seen in the human pancreatic adenocarcinoma cell line AsPC-1, but not in the human pancreatic epithelioid carcinoma cell line PANC-1. The human colon cancer cell line HT-29 was used as the negative control and line SW480 as the positive control for DcR3 expression. Using ELISA, expression in AsPC-1 cells peaked after 48-h incubation (6.46 ± 0.32 ng/ml) and remained at this level till at least 60 h (6.24 ± 0.27 ng/ml). Similar result was observed if DcR3 level was normalized with viable cell number (Supplemental Fig. 1).⁴ In addition, no DcR3 was detectable after 60-h incubation in PANC-1 cells (Fig. 1 B).

Extensive studies (26, 27) have demonstrated that growth factors, such as IGF-1, cause tyrosine kinase activation and trigger the PI3K/Akt or MAPK signaling pathways, which play critical roles in tumor growth and development. We next asked whether growth factor-induced tyrosine kinase activation was involved in modulation of DcR3 expression. To address this question, we treated AsPC-1 cells with herbimycin A (a tyrosine kinase inhibitor), PD98059 or SP600125 (MAPK inhibitors), LY294002 or wortmannin (PI3K inhibitors), rapamycin (a mammalian target of rapamycin inhibitor), AG1478 (an epidermal growth factor receptor inhibitor), AG1024 (an IGF-1 inhibitor), AG1295 (a platelet-derived growth factor inhibitor), or PDTC (an NF-κB inhibitor). After treatment for 48 h, only herbimycin A (1 μM), LY294002 (20 μM), wortmannin (20 μM), AG1024 (10 μM), or PDTC (50 μM) significantly inhibited DcR3 expression (Fig. 2). If DcR3 level was normalized with viable cell number, similar inhibitory effects were observed after above-mentioned inhibitor treatment (Supplemental Fig. 2).⁴ None of the treatments had any significant effect on cell viability, assessed using the MTT assay (Fig. 2). These data suggest that growth factor (IGF-1)-mediated tyrosine kinase activation and PI3K/Akt and NF-κB play a role in DcR3 expression in AsPC-1 cells. To make further study, we detected IGF-1R mRNA or protein distribution levels in human pancreatic cancer cells (Supplemental Fig. 3).⁴ High levels of IGF-1R were detected in AsPC-1 cells, but lower in PANC-1 cells. Using an immunoprecipitation assay, we observed different levels of tyrosine-phosphorylated IGF-1R in different human pancreatic cancer cells (Fig. 3^,A), levels being high in AsPC-1 cells and much lower in PANC-1 cells. To examine whether there was a connection between the IGF-1-induced signals (PI3K/Akt and NF-κB activation) and DcR3 expression, we treated AsPC-1 cells with LY294002 (20 μM), wortmannin (20 μM), PDTC (50 μM), or AG1024 (10 μM), and examined levels of phosphorylated and nonphosphorylated Akt, IKKβ, IKKα, IκBα, and p65 using Western blotting. As shown in Fig. 3^,B (left panel), constitutive Akt phosphorylation was seen in AsPC-1 cells. LY294002, wortmannin, or AG1024 treatment not only significantly reduced Akt phosphorylation at Ser⁴⁷³ residue, but also suppressed phosphorylation of IKKβ at Ser¹⁸¹, IKKα at Ser¹⁸⁰, IκBα at Ser^32/36, and p65 at Ser⁵³⁶. However, PDTC treatment suppressed phosphorylation of IκBα and p65, but not of Akt and IKKα/β (Fig. 3^,B). As shown in Fig. 3^,B (right panel), transfection of AsPC-1 cells with 0.8 μg of DN-Akt significantly decreased phosphorylation of Akt, IKKα/β, IκBα, and p65, whereas transfection with a DN mutant of IκBα (IκBαM), which prevents IκBα phosphorylation, only inhibited IκBα and p65 phosphorylation. To directly examine NF-κB activation after blocking Akt and IκBα phosphorylation, AsPC-1 cells were transiently transfected with pGL2-ELAM-κB-luciferase and the turning on of the luciferase gene by NF-κB used as an indicator of NF-κB activation. As shown as Fig. 3^,C, transfection with DN-Akt and IκBαM for 24 h markedly reduced κB-luciferase activity. Similar result was observed in EMSA (Supplemental Fig. 4).⁴ Using RT-PCR (Supplemental Fig. 5A)⁴ and real-time PCR assay (Supplemental Fig. 5B),⁴ DcR3 mRNA levels significantly were down-regulated by transfection with DN-Akt or IκBα plasmids. Furthermore, as shown in Fig. 3 D, transfection of AsPC-1 cells with DN-Akt or IκBαM for 36 h significantly reduced DcR3 expression (left panel), with no effect on cell viability (right panel) or cell growth (Supplemental Fig. 6).⁴ These results clearly demonstrated that endogenous IGF-1 activation of the PI3K/Akt/NF-κB signal pathway is involved in DcR3 expression.

Resistance to apoptosis is believed to be one of the reasons for the failure of cancer treatments. Previous studies have demonstrated that DcR3, acting as a decoy receptor, neutralizes the FasL-mediated apoptotic signal (1, 4). We therefore examined whether FasL-induced apoptosis of pancreatic cancer cells benefited from knockdown of DcR3 expression in AsPC-1 cells. Fig. 4^,A shows that transfection of AsPC-1 cells with DcR3 siRNA significantly reduced DcR3 levels, this effect being first seen at 36 h and maintained for at least 72 h. Fig. 4^,B shows transfection had no effect on cell viability when compared with control group. In addition, FACScan analysis of cell cycle distribution (Fig. 4^,C) showed that DcR3 siRNA transfection (SiR panels) or recombinant human sFasL (50 ng/ml) (sFasL panels) alone had no effect on the number of AsPC-1 cells in sub-G₁ phase, whereas, when cells were transfected with DcR3 siRNA for 24 or 48 h, then were treated with sFasL for 24 h (SiR + sFasL panels), an increase in the number of cells in sub-G₁ phase was seen (35.74 and 44.31%, respectively, at 24 and 48 h compared with controls). Moreover, mFasL is the primary mediator of apoptosis in the immune system (28). A previous study reported that PHA (10 μg/ml) stimulates expression of mFasL in null Jurkat cells (29), and we therefore used this established system to evaluate the effect of DcR3 siRNA on mFasL. Fig. 5^,A shows significant FasL staining was seen following PHA treatment of Jurkat cells. Cell cycle distribution analysis indicated that combined DcR3 siRNA/mFasL treatment resulted in the accumulation of a significant number of cells in sub-G₁ phase (15.81 and 19.16% at 24 and 48 h, respectively) compared with the single treatment groups (4.80 and 4.57%) (Fig. 5 B). Using MTT, SRB assay, or direct cell counting, significant decreasing cell viability or cell growth was observed in DcR3 siRNA transfection-combined sFasL/mFasL groups when compared with each treatment alone group (Supplemental Fig. 7).⁴ These results suggest that siRNA knockdown of DcR3 expression increases the cytotoxic effect of FasL in AsPC-1 cells.

We next examined whether DcR3 was expressed in PANC-1 cells, which did not normally express DcR3, if the cells constitutively expressed active Akt. Transfection of PANC-1 cells with constitutively active Akt (Myr-Akt) resulted in marked phosphorylation of Akt and IκBα in 24 h (Fig. 6^,A), with no effect on cell viability (data not shown). After transfection with constitutively active Akt, DcR3 expression increased from nondetectable levels at 24 h to 0.076 ng/ml at 36 h and 0.445 ng/ml at 48 h (Fig. 6^,B). Again, these data suggest that the PI3K/Akt/NF-κB pathway is involved in the modulation of DcR3 expression. We next examined whether incubation of PANC-1 cells in AsPC-1 cell-conditioned medium (DcR3 rich) could change FasL-induced apoptosis. Fig. 6 C shows that treatment of PANC-1 cells with sFasL (100 ng/ml) resulted in a significant number of cells in sub-G₁ phase (increase from 3.93 to 12.51%), and that this effect was markedly inhibited by replacing the growth medium with AsPC-1 cell-conditioned medium, but not using conditioned medium from AsPC-1 cells transfected with DN-Akt, IκBαM, or DcR3 siRNA (8.67, 11.45, or 11.33%, respectively). Results from detection of cell viability, cell growth, and cell number also support these FACS data (Supplemental Fig. 8).⁴

Taken together, our results demonstrated that IGF-1 activation of the PI3K/Akt/NF-κB signaling pathway is involved in endogenous DcR3 expression in AsPC-1 cells (Fig. 7), and that different DcR3 levels alter FasL-mediated apoptosis in human pancreatic adenocarcinoma cells.

In this study, we investigated the mechanisms of endogenous DcR3 expression and the potential therapeutic application of reducing DcR3 levels combined with FasL treatment in human pancreatic adenocarcinoma. Our data demonstrate, for the first time, that PI3K/Akt-dependent IKKα/β phosphorylation, p65 phosphorylation, and NF-κB activation are involved in DcR3 expression in AsPC-1 cells, and that reducing DcR3 expression by siRNA transfection significantly enhances FasL-induced AsPC-1 cell apoptosis.

Recent studies have revealed that the PI3K/Akt pathway plays an important role in tumor progression (30, 31). This pathway is stimulated by the aberrant activation of upstream signals, such as growth factor receptor tyrosine kinase (32). IGF-1 is one growth factor that activates the PI3K/Akt or MAPK pathway to drive cell survival and growth of different tumor cells (27). In 1994, Sell et al. (33) showed that fibroblasts derived from IGF-1R null mice cannot be transformed by several oncogenes. Our results showed that levels of tyrosine-phosphorylated IGF-1R were different in two different pancreatic cancer cells, being high in AsPC-1 cells and much lower in PANC-1 cells. This implies that different signaling pathways might exist in these cells. In addition, treatment of AsPC-1 cells with AG1024 (a specific IGF-1 inhibitor), herbimycin-A (a tyrosine kinase inhibitor), LY294002 or wortmannin (PI3K inhibitors), or PDTC (an NF-κB inhibitor) significantly reduced DcR3 expression, suggesting that IGF-1 activation of the PI3K/Akt/NF-κB pathway is involved in modulating DcR3 expression in these cells. Indeed, several studies (34, 35) have suggested that constitutive activation of PI3K/Akt and NF-κB is seen in many malignancies, including pancreatic cancer. Moreover, a previous study suggested that constitutive PI3K/Akt and NF-κB activation also confers resistance against gemcitabine-induced cell death (35).

As an antiapoptotic effector, Akt reduces tumor cell sensitivity to apoptosis-inducing stimuli by triggering a multitude of signaling cascades, such as phosphorylation of Bad, thus restoring the antiapoptotic function of Bcl-x_L (36); phosphorylation of caspase-9, inhibiting its catalytic activity (37); phosphorylation of forkhead in rhabdomyosarcoma, preventing its nuclear translocation and the activation of forkhead in rhabdomyosarcoma gene targets (38); phosphorylation of murine double minute-2, counteracting the activity of p53 (39); and phosphorylation of IKK, stimulating the antiapoptotic transcriptional activity of NF-κB (40). Thus, it was not surprising to find that DN-Akt could not completely block NF-κB activity (Fig. 3^,C). Moreover, there is increasing evidence suggesting that NF-κB has antiapoptotic effects that would favor tumor survival (41). NF-κB has been linked to the production of many antiapoptotic proteins, such as cellular inhibitor of apoptosis 2 (a member of the inhibitor of apoptosis protein family), cellular inhibitor of apoptosis 1, and TNFR-associated factors 1 and 2 (42). Besides inducing the expression of these antiapoptotic proteins, NF-κB seems to have other antiapoptosis effects, because Kajino et al. (43) found that IL-1β, a potent NF-κB inducer, blocked TNF-α-induced apoptosis and this effect was not abolished by pretreatment of the cells with the protein synthesis inhibitor cycloheximide or by blocking NF-κB transcription using NF-κB decoy oligonucleotides, showing that the antiapoptotic effect of NF-κB does not need de novo protein synthesis and suggesting that NF-κB has a complex antiapoptic effect. In this study, PI3K inhibitors or DN-Akt treatment significantly attenuated NF-κB activation (Fig. 3, B and C). In addition, treatment with DN-Akt or IκBα (IκBαM) also significantly reduced DcR3 mRNA and protein levels in AsPC-1 cells (Supplemental Fig. 5;⁴ Fig. 3 D). These results suggest that the PI3K/Akt/NF-κB pathway plays an important role in modulating DcR3 levels. However, we have not completely ruled out the possibility that other pathways may also be involved.

Resistance to apoptosis is believed to be one of the hallmarks of cancer cells (44). Recent studies have shown that several cancer cells, including pancreatic adenocarcinomas, have developed mechanisms making them resistant to FasL/Fas-mediated apoptotic signals despite expressing Fas (24, 45). DcR3 is a decoy receptor of FasL, and several studies (46, 47) have demonstrated that there is a significant correlation between DcR3 overexpression and resistance to Fas ligand-mediated apoptosis in cancer cells. Thus, a better understanding of the molecular mechanism of DcR3 expression would help in developing potential therapeutic strategies to increase the apoptosis of FasL-resistant cancer cells by blocking DcR3 expression by cancer cells. Following up this idea, we have developed several natural compounds that reduce DcR3 levels and enhance the apoptotic effect of FasL on FasL-resistant pancreatic cancer cells (data not shown).

In summary, our study demonstrates that PI3K/Akt-mediated IKKα/β phosphorylation, p65 phosphorylation, and NF-κB activation are involved in the modulation of endogenous DcR3 levels in AsPC-1 human pancreatic adenocarcinoma cells, and that reducing DcR3 levels significantly enhances the FasL-mediated apoptotic effect. These results indicate that DcR3 could be a potential therapeutic target in human pancreatic cancer.

The authors have no financial conflict of interest.