β-catenin decreases acquired TRAIL resistance in non-small-cell lung cancer cells by regulating the redistribution of death receptors

  • Authors:
    • Chengcheng You
    • Shimin Zhang
    • Yingming Sun
    • Shiyu Zhang
    • Guiliang Tang
    • Fang Tang
    • Xuefeng Liu
    • Yu Xiao
    • Junhong Zhang
    • Yan Gong
    • Conghua Xie
  • View Affiliations

  • Published online on: August 21, 2018     https://doi.org/10.3892/ijo.2018.4529
  • Pages: 2258-2268
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Abstract

Tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) exhibits antitumor activity in various types of tumor cell and tumor‑bearing animals. However, acquired TRAIL resistance is a common issue that restricts its clinical application. Previous studies have revealed that β‑catenin is associated with TRAIL resistance in melanoma and colorectal tumors. In the present study, an acquired‑resistance non‑small‑cell lung cancer (NSCLC) cell line (H460‑TR) was established from parental TRAIL‑sensitive H460 cells using a gradient ascent model (8‑256 ng/ml TRAIL). Cellular FADD‑like interleukin‑1β converting enzyme inhibitory protein and Mcl‑1 were upregulated and the cell surface distribution of death receptor (DR)4 and DR5 was downregulated in H460‑TR cells compared with the parental H460 cells. The results of reverse transcription‑quantitative polymerase chain reaction and western blot analysis indicated that H460 cells expressed increased levels of β‑catenin and were more sensitive to TRAIL compared with H460‑TR cells. β‑catenin‑knockdown in H460 cells decreased their sensitivity to TRAIL, while upregulation of β‑catenin expression in H460‑TR cells increased their sensitivity to TRAIL, increased the cell surface distribution of DRs and activated caspase‑3/8. Taken together, the results of the present study suggest that β‑catenin impairs acquired TRAIL resistance in NSCLC cells by promoting the redistribution of DR4 and DR5 to the cytomembrane, and inducing TRAIL‑mediated cell apoptosis via caspase‑3/8 activation.

Introduction

Over the past 30 years, lung cancer has had high morbidity and mortality rates worldwide, with 75% of new diagnoses being classified as non-small-cell lung cancer (NSCLC) and advanced tumors at the first visit (1). Chemotherapy and radiotherapy remain the most common treatment methods for advanced cancer (2). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in malignant tumors and has little effect on normal cells (3,4). Recombinant human TRAIL and its receptor agonists are under investigation as promising therapeutic approaches for the treatment of advanced cancer (5). There are 4 different transmembrane receptors in the TRAIL receptor/ligand system, including 2 death receptors (DR4 and DR5), 2 decoy receptors (DcR1 and DcR2), and a soluble receptor, furthermore, osteoprotegerin DRs contain a cytoplasmic death domain transducing the apoptosis signalling pathway (6). Preclinical studies have revealed that recombinant TRAIL and DR agonists inhibit tumor growth in vitro and in vivo without systemic toxicity (79). A percentage of tumor cells were observed to respond to TRAIL therapy, which is called primary TRAIL resistance, while some tumor cells obtained TRAIL resistance following repeated treatments, called acquired TRAIL resistance (10,11). Increasing the existing understanding of the molecular alterations involved in acquired resistance and the cytotoxicity of TRAIL is required to further investigate its therapeutic potential.

A previous study reported that β-catenin and DRs were co-expressed in colonic tumor tissues. DR expression increased during colon carcinoma tumorigenesis, possibly due to upregulation of β-catenin expression (12). However, the mechanisms by which β-catenin regulates TRAIL resistance remain unclear in NSCLC (13). It was, therefore, hypothesized that β-catenin enhanced TRAIL sensitivity by regulating the redistribution of DR4 and DR5.

In the present study, a TRAIL-resistant H460-TR cell line was established to investigate the potential effects of β-catenin on DR redistribution and TRAIL sensitivity. Downregulation of β-catenin expression decreased the redistribution of DR4 and DR5 on the cell surface, and was associated with TRAIL resistance. While β-catenin-knockdown in H460 cells decreased their TRAIL sensitivity, upregulation of β-catenin expression in H460-TR cells rescued TRAIL sensitivity, increased DR distribution on the cytomembrane and activated caspase-3/8. β-catenin may be used as a biomarker to predict TRAIL sensitivity in the future. Patients exhibiting high β-catenin expression may benefit more from TRAIL treatment. Furthermore, the Wnt signaling pathway agonist may be used to promote TRAIL sensitivity during chemotherapy.

Materials and methods

Cells

The human NSCLC cell line, NCI-H460, was provided by the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (both from HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in an atmosphere containing 5% CO2. The TRAIL-resistant H460-TR cell line was established using a gradient ascent model (8, 16, 32, 64, 128 and 256 ng/ml TRAIL) from parental TRAIL-sensitive H460 cells. Cells were continuously exposed to 50 ng/ml TRAIL to maintain their resistant capability.

Reagents and plasmids

TRAIL was purchased from Shanghai Kaibao Pharmaceutical Co., Ltd. (Shanghai, China). Recombinant Wnt-3A was purchased from Peprotech, Inc. (Rocky Hill, NJ, USA). The pCMV-C-flag-β-catenin (pCMV-β-catenin) overexpression plasmid was constructed and identified in our laboratory, as previously described (14). β-catenin-silencing plasmids were purchased from Addgene, Inc. (Cambridge, MA, USA; cat. nos. 19761 pLKO.1.puro shRNA.β-catenin.1248 and 18803 pLKO.1.puro shRNA β-catenin). These will be abbreviated as shRNA1 and shRNA2, respectively.

Cell viability assay

Cell viability was assessed by cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells were seeded in 96-well plates (Corning, Inc., Corning, NY, USA) at a density of 1×106 cells/well. When the cells reached 70–80% confluence, the experimental group was treated with the 0, 10, 25, 50, 100 or 200 ng/ml TRAIL for 24 h. The original medium was discarded and replaced with a basal media mixture containing 10% (v/v) CCK-8 for 1 h. The optical density (OD) was measured at 450 nm using a microplate reader (Rayto Life and Analytical Sciences Co., Ltd., Guangming, China). Cell viability was calculated using the following formula: Cell viability (%) = (OD value of the treated wells - OD value of the blank control wells)/(OD value of the negative control wells - OD value of the blank control wells). All assays contained 5 replicates and were repeated 3 times under the same conditions.

RNA isolation, reverse transcription and RT-qPCR

Total RNA was extracted from cells with TRIzol (Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer’s protocol, and the RNA concentration was detected using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.). Reverse transcription was performed with the SuperScript First-Strand Synthesis system (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocol. The primer sequences were as follows (forward and reverse, respectively): DR4, 5′-AGAGAGAAGTCCCTGCACCA-3′ and 5′-GTCACTCCAGGGCGTACAAT-3′; DR5, 5′-CACCAG GTGTGATTCAGGTG-3′ and 5′-CCCCACTGTGCTTTGTA CCT-3′; DcR1, 5′-ACCAACGCTTCCAACAA-3′ and 5′-AGG GCACCTGCTACACTT-3′; DcR2, 5′-CCTTCTTGCCTGCT ATG-3′ and 5′-GTGGTCACTGTCTCCTCC-3′; FADD, 5′-GCGAGTCTGGAAGAATGTCG-3′ and 5′-GGCTTGTCA GGGTGTTT-3′; Cellular FADD-like interleukin-1β converting enzyme inhibitory protein (c-FLIP), 5′-GGCTCCCCCTGCAT CACATC-3′ and 5′-CGCAGTACACAGGCTCCAGA-3′, and GAPDH, 5′-TGGAAGGACTCATGACCACA-3′ and 5′-TCAGCTCAGGGATGACCTT-3′. The transcriptional level was determined using a SYBR Premix EX Taq II kit (Takara Bio, Inc., Otsu, Japan) and a CFX96 RT-qPCR detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer’s protocol. The thermocycling conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec, then 95°C for 10 sec and melting curve at 65–95°C with interval changes of 0.5°C every 5 sec. The 2−ΔΔCq method was used to analyze the fold-change in gene expression relative to GAPDH (15).

Flow cytometry

Cells were digested with EDTA-free trypsin (Corning, Inc.), harvested and washed twice with PBS. For apoptosis assays, cells were treated with an Annexin V-FITC/PI kit (BestBio Ltd., Shanghai, China), according to the manufacturer’s protocols. A single-cell suspension was established using 400 μl binding buffer and cells were stained with 5 μl Annexin V-FITC for 30 min followed by staining with 7 μl 20 mg/ml propridium iodide (PI) for 5 min at room temperature. To detect cytomembrane DRs, cells were suspended in 50 μl PBS containing 1% goat serum at room temperature for 30 min. Cells were washed with PBS 3 times and incubated with the primary antibodies presented in Table I overnight at 4°C. Subsequently, cells were washed 3 times with PBS and incubated with the secondary antibodies presented in Table II at room temperature for 30 min. Cells were then washed with PBS and suspended in 500 μl PBS. Cells were also processed as described but without primary antibody treatment, as a negative control. The samples were analyzed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). All experiments were repeated 3 times.

Table I

List of primary antibodies used.

Table I

List of primary antibodies used.

AntigenSpeciesMethodDilutionSupplier (cat. no.)
β-catenin, humanRabbit, polyclonalWB1:1,000Abcam (6302)
GAPDH, humanRabbit, polyclonalWB1:5,000Proteintech (10494-1-AP)
BCL-2, humanRabbit, monoclonalWB1:2,000Abcam (34124)
BAX, humanRabbit, monoclonalWB1:1,000Proteintech (50599-2-Ig)
Caspase-3, humanRabbit, polyclonalWB1:1,500Proteintech (19677-1-AP)
Cleaved caspase-3Rabbit, polyclonalWB1:1,000CST (9661)
Caspase-8, humanRabbit, polyclonalWB1:1,500Proteintech (13423-1-AP)
Cleaved caspase-8Rabbit, monoclonalWB1:1,000CST (9496)
FADD, humanRabbit, polyclonalWB1:1,000Abcam (24533)
C-FLIP, humanRabbit, polyclonalWB1:1,000Abcam (ab6144)
Mcl-1, humanRabbit, polyclonalWB1:1,000Abcam (ab32087)
DcR1, humanRabbit, polyclonalWB1:1,000Abcam (ab2087)
DcR2, humanRabbit, polyclonalWB1:1,000Abcam (ab2019)
DR4, humanRabbit, monoclonalWB1:1,000CST (42533)
DR5, humanRabbit, monoclonalWB1:1,000CST (8074)
DR4, humanRabbit, monoclonalIF1:100CST (42533)
DR5, humanRabbit, monoclonalIF1:50CST (8074)
DR4, humanRabbit, monoclonalFC1:50CST (42533)
DR5, humanRabbit, monoclonalFC1:20Bioss (bs-7352R)
Caveolin-1Mouse, polyclonalIF1:100R&D (MAB5736-SP)

[i] IF, immunofluorescence; FC, flow cytometry; WB, western blot; BAX, BCL2 associated X, apoptosis regulator; FADD, Fas-associated death domain; C-FLIP, cellular FADD-like interleukin-1β converting enzyme inhibitory protein; DcR, decoy receptor; DR, death receptor; CST, Cell Signaling Technology, Inc., Danvers, MA, USA; Abcam; Abcam, Cambridge, UK; ProteinTech; ProteinTech Group, Inc., Chicago, IL, USA; R&D, R&D Systems, Inc., Minneapolis, MN, USA.

Table II

Secondary antibodies and DAPI stain.

Table II

Secondary antibodies and DAPI stain.

Secondary detection system antibodyHostMethodDilutionSupplier (cat. no.)
Anti-Mouse-IgG (H+L)-HRPGoatWB1:10,000Sungene (LK2001)
Anti-Rabbit-IgG (H+L)-HRPGoatWB1:10,000Sungene (LK2003)
Hoechst 33342 nucleic acid staining (DAPI)IF1 μg/mlSigma (D8417)
Anti-Mouse-IgG (H+L)-Cy3GoatIF, FC1:100Proteintech (SA00009-1)
Anti-Mouse-IgG (H+L)-FITCGoatIF, FC1:100Proteintech (SA00003-11)
Anti-Rabbit-IgG (H+L)-R-PEGoatIF, FC1:100Proteintech (SA00008-2)

[i] IF, immunofluorescence; FC, flow cytometry; WB, western blot; HRP, horseradish peroxidase.

Immunofluorescence

Cells were fixed with 4% paraformaldehyde (Sangon Biotech Co., Ltd., Shanghai, China) for 30 min and washed 3 times with PBS. Cells were blocked with normal goat serum (MultiSciences Biotech Co., Ltd., Zhejiang, Hangzhou, China) for 30 min at room temperature, and incubated with primary antibodies (Table I) at 4°C overnight. Following incubation with a FITC/Cy3-conjugated secondary antibody (Table II) for 45 min at room temperature, the cells were washed with PBS and the slides were stained with DAPI (1 μg/ml; 100 μl; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at room temperature for 3 min. Images were captured with a confocal laser scanning microscope (Nikon Corporation, Tokyo, Japan).

β-catenin-knockdown in H460 cells using shRNA lentivirus

The sense sequence of shRNA1 was 5′-GTGCTATCTGTCTGCT CTA-3′, and the sense sequence of the negative control (NC) shRNA was 5′-TTCTCCGAACGTGTCACGT-3′. Lentiviral pGMLV was used to construct shRNA1 (cat. no. 19761 pLKO.1.sh.β-catenin.1248). The H460 cells (7×105 cells/well) were infected with shRNA1 or NC lentiviruses (8×105 particles/well), and screened using 3 mg/ml puromycin (Sangon Biotech Co., Ltd.) for 2 weeks. β-catenin levels were subsequently measured using RT-qPCR and western blot analyses.

β-catenin-overexpression in H460-TR cells using plasmid transfection

H460-TR cells were cultured in 6-well plates and transfected with the pCMV-β-catenin plasmid. A total of 5 μl Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and 10 μg plasmid were added to separate 500-μl aliquots of OptiMEM (Gibco; Thermo Fisher Scientific, Inc.), and incubated at room temperature for 5 min. The diluted plasmid was added to the diluted Lipofectamine® 2000 at a 1:1 ratio, and the mixture was incubated at room temperature for 20 min. When the cells reached 70–90% confluence, they were incubated with the plasmid mixture for 6 h. RT-qPCR and western blot analyses were performed to measure the transfection efficiency. For the negative control, a pCMV-C-Tag2C-flag plasmid (pCMV) was transfected following the same protocol.

Western blot analysis

Whole cell lysates were extracted using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with protease inhibitor cocktail (Sangon Biotech Co., Ltd.). Membrane DR4 and DR5 proteins were extracted using ProteoExtract® Transmembrane Protein Extraction kit (Merck KGaA, Darmstadt, Germany), according to the manufacturer’s protocol. The protein content of the supernatant was detected using a bicinchoninic acid assay (Pierce; Thermo Fisher Scientific, Inc.) using bovine serum albumin as a standard. A total of 40 μg protein/lane was separated by 10% SDS-PAGE. The proteins were transferred to polyvinylidene fluoride membranes, which were subsequently probed overnight at 4°C with the primary antibodies listed in Table I. The membranes were washed 3 times with Tris-buffered saline with Tween and incubated with the appropriate secondary antibodies (Table II) at room temperature for 90 min. Membranes were visualized using an enhanced chemiluminescence kit and exposed using a gel imaging analyzer (both from Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analysis

All experiments were repeated 3 times and the results are presented as the mean ± standard deviation. 50% effective concentration (EC50) values were analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA) with the following equation: Y = minimum dose + maximum dose-minimum dose)/1+10LogEC50-X. Differences between 2 groups were analyzed using unpaired Student’s t-tests. P<0.05 was considered to indicate a statistically significant difference.

Results

Establishment and identification of the acquired TRAIL-resistant H460-TR cell line

To explore the molecular mechanism underlying acquired TRAIL-resistance, TRAIL-resistant H460-TR cells were established using H460 cells. No morphological differences were observed between H460-TR and H460 cell lines; both were polygonal, adherent and island-like. When exposed to 80 ng/ml TRAIL for 4 h, the majority of H460 cells presented apoptotic features, while H460-TR cells maintained a typical epithelioid monolayer (Fig. 1A). The toxicity of TRAIL to H460-TR and H460 cells was assessed using CCK-8 assays and was demonstrated to be dose-dependent. The EC50 value of TRAIL in H460-TR cells was 201.4 ng/ml, compared with 65.47 ng/ml in H460 cells (Fig. 1B). Furthermore, the number of Annexin V and TUNEL-positive cells decreased in the H460-TR cell line compared with the parental H460 cell line (Fig. 1C and D). These results indicate the successful establishment of an acquired TRAIL-resistant cell line.

The extrinsic and intrinsic apoptotic signaling pathways were inhibited in acquired TRAIL-resistant NSCLC cells

The effects of TRAIL on the extrinsic and intrinsic apoptotic signaling pathways in the established TRAIL-resistant H460-TR cells were investigated. No significant difference was observed in the expression of FADD or caspase-8 protein between H460 and H460-TR cells; however, the mRNA and protein expression levels of c-FLIP were increased in the TRAIL-resistant cells (Fig. 2A and B). The endogenous anti-apoptotic protein, Mcl-1, was upregulated in H460-TR cells (Fig. 2C). Furthermore, the expression of cleaved caspase-3/8 was significantly decreased in H460-TR cells following 80 ng/ml TRAIL treatment (Fig. 2D). These results indicate that the extrinsic and intrinsic apoptotic signaling pathways are downregulated in cells with acquired TRAIL-resistance compared with cells without.

TRAIL-induced redistribution of DR4 and DR5 to the cytomembrane is inhibited in H460-TR cells

TRAIL-induced apoptosis was reported to be initiated by TRAIL binding to DRs. The mRNA expression of TRAIL receptors was comparable between TRAIL-sensitive and TRAIL-resistant cell lines prior to TRAIL treatment (Fig. 3A). The levels of DR4 and DR5 expression in the cytomembrane were increased in H460 cells following TRAIL treatment, whereas no significant difference was observed in H460-TR cells following treatment compared with pre-treatment levels (Fig. 3B and D). Furthermore, the expression of DcR1 and DcR2 was not affected by TRAIL treatment in H460 or H460-TR cells (Fig. 3E). The lack of TRAIL-induced DR redistribution may be a key factor affecting TRAIL sensitivity in H460-TR cells.

β-catenin is relevant to TRAIL sensitivity

To evaluate whether β-catenin is involved in TRAIL resistance, the expression of β-catenin was analysed using RT-qPCR and western blot analysis. β-catenin was expressed in H460 cells at approximately twice the level of that observed in H460-TR cells (P<0.05; Fig. 4A and B). These results indicate that β-catenin is positively associated with TRAIL sensitivity.

Knockdown of β-catenin reduced drug sensitivity in TRAIL-sensitive H460 cells

shRNA was used to suppress β-catenin transcription in H460 cells and to investigate its effect on TRAIL sensitivity and caspase activation. β-catenin mRNA expression was reduced following infection with shRNA1 to a greater degree than with shRNA2, therefore, shRNA1 was selected for use in further experiments (Fig. 4C). Silencing of β-catenin rendered H460 cells less sensitive to TRAIL, as revealed by the results of a CCK-8 assay (Fig. 4D). Flow cytometry was performed to analyze the apoptosis of H460 cells following treatment with or without 80 ng/ml TRAIL for 8 h. The results further demonstrated that β-catenin-silencing attenuates the cytotoxicity of TRAIL in H460 cells (Fig. 4E and F). The western blotting proteins bands for cleaved-caspase-3/8 were also reduced following β-catenin-silencing (Fig. 4G). These results indicate that β-catenin-silencing decreases TRAIL-sensitivity by inactivating caspase proteins.

Overexpression of β-catenin enhances drug sensitivity in TRAIL-resistant H460-TR cells

To further investigate the association between β-catenin expression and TRAIL sensitivity, TRAIL-resistant H460-TR cells were transfected with pCMV-β-catenin, which was successfully constructed in our previous study (14). Following transfection and puromycin screening, the expression of β-catenin protein was markedly upregulated in H460-TR cells compared with untransfected cells (Fig. 5A). The cells were next treated with TRAIL for 24 h and the results demonstrated that β-catenin overexpression rendered H460-TR cells more sensitive to TRAIL compared with untransfected cells (Fig. 5B). Annexin V-FITC/PI staining also indicated that β-catenin overexpression increased the apoptotic rate of H460-TR cells treated with 80 ng/ml TRAIL for 8 h (Fig. 5C and D) compared with untreated cells. As expected, the protein expression bands of cleaved-caspase-3/8 were larger following β-catenin overexpression (Fig. 5E). These results indicate that β-catenin overexpression increases TRAIL sensitivity via activating caspase.

β-catenin upregulates DR4 and DR5 in NSCLC cells

To determine the effects of β-catenin on DR4 and DR5 in the context of altered TRAIL sensitivity, the expression of these DRs on the cell membrane was assessed following β-catenin-silencing or overexpression. The expression of DR4 and DR5 mRNA and protein was reduced in TRAIL-sensitive H460 cells following β-catenin downregulation (Fig. 6A and B). The expression of DR4 and DR5 was increased in TRAIL-resistant H460-TR cells following β-catenin overexpression (Fig. 6C and D). Flow cytometry confirmed that β-catenin overexpression led to an increase in DR expression in TRAIL-resistant H460-TR cells compared with untransfected cells (Fig. 6E and F). Caveolin-1 is a marker of lipid rafts. The colocalization of Caveolin-1 and DR4/5 was assessed using immunofluorescence and confocal microscopy. The results suggest that β-catenin enhanced the localization and redistribution of DR4 and DR5 to lipid rafts (Fig. 6G). This indicates that β-catenin promotes DR translocation to the cell membrane, allowing them to combine more effectively with TRAIL and activate pro-apoptotic caspase proteins, ultimately inducing apoptosis and reversing TRAIL-resistance.

Discussion

TRAIL is able to selectively target and kill tumor cells without causing damage to normal cells (3,4). DRs are often located on tumor cells and, upon activation by TRAIL, the oligomerization of DRs recruits the linker molecule, Fas-associated death domain (FADD), and pro-caspase-8, which together comprise the death inducing signaling complex (DISC) (6). Activated caspase-8 directly induces apoptosis via activating caspase-3, which cleaves a broad range of apoptosis-associated protein substrates and executes the extrinsic apoptosis pathway (16,17). In addition, activated caspase-8 truncates BH3 interacting domain death agonist along with the pro-apoptotic proteins, BCL-2 associated X, apoptosis regulator and BCL-2 antagonist/killer. However, the clinical application of TRAIL is limited due to the prevalence of drug resistance (16,17). TRAIL-resistance may be intrinsic, occurring at the first exposure to TRAIL, or acquired resistance, developing during treatment (18). At present, the mechanism of acquired TRAIL resistance remains to be elucidated. TRAIL resistance is caused by various factors, including endoplasmic reticulum stress (19), protein synthesis disorders (20), decreased DRs expression (21) and increased anti-apoptotic protein expression (22). Our results revealed that β-catenin expression is positively associated with TRAIL sensitivity via promoting the cytomembrane redistribution of DR4 and DR5.

c-FLIP, which is similar in structure to caspase-8, and competitively binds FADD molecules, thus impeding the cleavage of caspase-8 and subsequent signal transduction in the intrinsic apoptotic signaling pathway (23). It has previously been demonstrated that TRAIL and a DR5 agonist, AD5-10, cleave c-FLIP in H460 cells (24). In human renal carcinoma Caki cells, TRAIL has been reported to downregulate c-FLIP expression and induce apoptosis (25). Mcl-1, an anti-apoptotic protein that belongs to the BCL-2 family, has also been reported to induce TRAIL resistance (26). TRAIL inhibits Mcl-1 expression via activating the pro-apoptotic activity of p38 in the receptor interacting serine/threonine kinase 1-dependent pathway in H460 cells (27). It has also been demonstrated that YM155 sensitizes TRAIL-induced apoptosis via cathepsin S-dependent downregulation of Mcl-1 expression, and nuclear factor-κB-mediated downregulation of c-FLIP expression in Caki cells (28). In the present study, the expression of c-FLIP and Mcl-1 was higher in H460-TR cells compared with H460 cells. These results indicate that the DR-associated apoptotic pathway and the intrinsic apoptotic pathway are responsible for changes in TRAIL-sensitivity.

The binding of TRAIL to its receptors is the first step in TRAIL-induced apoptotic signaling. The cytomembrane expression of DR4 and DR5, rather than the general expression of these DRs, is the main determinant of TRAIL sensitivity (6). SW480 colon cancer cells are characterized as TRAIL-resistant cells that express high levels of DR4, even though cytomembranal DR4 is undetectable (29). The present study revealed that baseline DR4 and DR5 expression was not affected by TRAIL-sensitivity. Nevertheless, the redistribution of DR4 and DR5 to the membrane of TRAIL-sensitive H460 cells was increased following TRAIL treatment, while the cytomembranal expression levels of DR4 and DR5 in TRAIL-resistant H460-TR cells were not significantly altered (Fig. 3B and D). A previous study reported that TRAIL-induced DR transportation into lipid rafts in TRAIL-sensitive cells, while redistribution was not observed in TRAIL-resistant cells (30). As such, the cytomembranal expression levels of DR4 and DR5 after TRAIL treatment is critical for determining TRAIL sensitivity.

Lung cancer comprises a group of molecularly heterogeneous diseases that are characterized by a range of genomic and epigenomic alterations (31). In ongoing experiments by our research group involving a gene expression profiling chip in TRAIL-sensitive H460 cells, the acquired TRAIL-resistant H460-TR cells and the primary resistant A549 cells have demonstrated that multiple targets and genetic alternations may be associated with the resistance process (unpublished). In further experiments, it was demonstrated that β-catenin was highly expressed in TRAIL-sensitive cells compared with resistant cells. Further investigation of the association between β-catenin and TRAIL sensitivity is required.

In the canonical Wnt pathway, β-catenin accumulates in the cytoplasm upon Wnt stimulation and eventually translocates to the nucleus to act as a transcriptional coactivator (32). The Wnt/β-catenin signaling pathway participates in cell adhesion, embryonic development and tumorigenesis (33). β-catenin and DRs are co-expressed in colonic tumor tissues. DR expression gradually increased during colon carcinoma tumorigenesis, possibly due to upregulation of β-catenin expression (12). In TRAIL-resistant melanoma cells, Wnt-3A was revealed to activate Wnt/β-catenin signaling, promote the expression of the apoptotic molecules, BIM and PUMA, and reduce the expression of the anti-apoptotic protein, Mcl-1, thus increasing sensitivity to TRAIL (34). It was also demonstrated that Wnt-3A upregulated β-catenin expression and activated caspase-3/8 in H460-TR cells. β-catenin expression was markedly lower in cells with acquired TRAIL resistance compared with TRAIL-sensitive cells, and thus induced sensitivity to TRAIL.

While the present study demonstrated that β-catenin is a critical determinant of acquired TRAIL resistance in NSCLC cells, the exact molecular mechanisms responsible for TRAIL resistance remain unclear. β-catenin-knockdown or overexpression was used to determine whether β-catenin regulates TRAIL-sensitivity via affecting DR4 and DR5 expression. When β-catenin expression was downregulated in H460 cells, DR4 and DR5 expression levels were reduced and resistance to TRAIL increased. These results were consistent with a previous report that downregulation of β-catenin expression in melanoma cells reduced their sensitivity to TRAIL (34). Accordingly, β-catenin overexpression in H460-TR cells significantly increased DR4 and DR5 expression levels and induced TRAIL-sensitivity. Upregulation of β-catenin using lithium chloride has been reported to sensitize A549 cells to TRAIL (35). A previous study using APC-null colorectal cancer cells revealed that β-catenin upregulated c-MYC expression, which subsequently downregulated c-FLIP expression to promote TRAIL-induced apoptosis (36). It has been reported that Wnt/β-catenin signalling induces apoptosis via a caspase-dependent apoptosis mechanism by downregulating expression of Mcl-1 (37). In the present study, it was revealed that β-catenin expression is positively associated with cytomembranal expression levels of DR4 and DR5, indicating that β-catenin may promote TRAIL-sensitivity by inducing the cytomembranal redistribution of DR4 and DR5.

β-catenin forms a complex with E-cadherin. Interestingly, β-catenin, E-cadherin and DR expression has been detected in the lipid rafts of the cell membrane (38,39). Altering β-catenin and E-cadherin-mediated intercellular adhesion has been reported to induce epithelial-mesenchymal-transition and thus inhibit apoptosis in tumor cells (40). It was therefore speculated that the E-cadherin/β-catenin complex enhanced cytomembranal translocation of DR4 and DR5 in β-catenin-overexpressing H460 cells. As β-catenin expression levels decline in TRAIL-resistant cells, the E-cadherin/β-catenin complex may dissociate and the cytomembranal expression levels of DRs and DISC may be attenuated. As such, low β-catenin expression is associated with acquired TRAIL-resistance in NSCLC cells.

In the present study, an acquired TRAIL-resistant lung cancer cell line, H460-TR, was successfully constructed. It was demonstrated that the expression levels of DRs induced by TRAIL on the cell membrane is a key factor affecting acquired TRAIL resistance. TO the best of our knowledge, the present study it the first to indicate that β-catenin promotes DR-translocation to the cell membrane, allowing them to combine with TRAIL more effectively, to activate the downstream family of caspase pro-apoptotic molecules, to induce apoptosis and reverse TRAIL-resistance. In future studies, an in vivo nude mouse recombinant TRAIL xenograft model should be used to validate our in vitro results. The detailed molecular mechanisms by which β-catenin enhances TRAIL sensitivity remain to be elucidated. β-catenin overexpression or induction using Wnt-3A presents a potential therapeutic strategy to enhance TRAIL sensitivity of NSCLC cells.

Funding

The present study was funded by the Chinese National Natural Science Foundation (grant nos. 81572967, 81372498 and 81773236), Hubei Natural Science Foundation (grant no. 2013CFA006), Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund (grant nos. znpy2016050 and znpy2017049), Wuhan City Huanghe Talents Plan and Chinese National Key Clinical Speciality Construction Program (CX), the Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund (grant no. znpy2017001) and the Fundamental Research Funds for the Central Universities (grant no. 2042018kf0066, YG).

Availability of data and materials

All data generated or analyzed during this study were included in this published article.

Authors’ contributions

CY, SHIMIN Z, YG and CX designed the present study. CY, SHIMIN Z, YS and SHIYU Z acquired the data. CY, SHIMIN Z, GT and FT analyzed the data. XL, YX and JZ interpreted the data. CY and SHIMIN Z drafted the manuscript. CY, SHIMIN Z, YG and CX provided critical revision. All authors approved the version to be published.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Abbreviations:

NSCLC

non-small cell lung cancer

c-FLIP

cellular FADD-like interleukin-1β converting enzyme inhibitory protein

DR

death receptor

DcR

decoy receptor

DISC

death inducing signaling complex

EMT

epithelial-mesenchymal transition

FADD

Fas associated death domain

BAX

BCL-2 associated X protein

BAK

BCL-2-antagonist/killer

Acknowledgments

The authors would like to thank Xiaohua Leng for excellent technical assistance.

References

1 

Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ and He J: Cancer statistics in China, 2015. CA Cancer J Clin. 66:115–132. 2016. View Article : Google Scholar : PubMed/NCBI

2 

Cao C, D’Amico T, Demmy T, Dunning J, Gossot D, Hansen H, He J, Jheon S, Petersen RH, Sihoe A, et al International VATS Interest Group: Surgery versus SABR for resectable non-small-cell lung cancer. Lancet Oncol. 16:e370–e371. 2015. View Article : Google Scholar : PubMed/NCBI

3 

Wiley SR, Schooley K, Smolak PJ, Din WS, Huang CP, Nicholl JK, Sutherland GR, Smith TD, Rauch C, Smith CA, et al: Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 3:673–682. 1995. View Article : Google Scholar : PubMed/NCBI

4 

Hao C, Song JH, Hsi B, Lewis J, Song DK, Petruk KC, Tyrrell DL and Kneteman NM: TRAIL inhibits tumor growth but is nontoxic to human hepatocytes in chimeric mice. Cancer Res. 64:8502–8506. 2004. View Article : Google Scholar : PubMed/NCBI

5 

Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A and Ashkenazi A: Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem. 271:12687–12690. 1996. View Article : Google Scholar : PubMed/NCBI

6 

Ashkenazi A: Directing cancer cells to self-destruct with pro-apoptotic receptor agonists. Nat Rev Drug Discov. 7:1001–1012. 2008. View Article : Google Scholar : PubMed/NCBI

7 

Hotte SJ, Hirte HW, Chen EX, Siu LL, Le LH, Corey A, Iacobucci A, MacLean M, Lo L, Fox NL, et al: A phase 1 study of mapatumumab (fully human monoclonal antibody to TRAIL-R1) in patients with advanced solid malignancies. Clin Cancer Res. 14:3450–3455. 2008. View Article : Google Scholar : PubMed/NCBI

8 

Merchant MS, Geller JI, Baird K, Chou AJ, Galli S, Charles A, Amaoko M, Rhee EH, Price A, Wexler LH, et al: Phase I trial and pharmacokinetic study of lexatumumab in pediatric patients with solid tumors. J Clin Oncol. 30:4141–4147. 2012. View Article : Google Scholar : PubMed/NCBI

9 

Greco FA, Bonomi P, Crawford J, Kelly K, Oh Y, Halpern W, Lo L, Gallant G and Klein J: Phase 2 study of mapatumumab, a fully human agonistic monoclonal antibody which targets and activates the TRAIL receptor-1, in patients with advanced non-small cell lung cancer. Lung Cancer. 61:82–90. 2008. View Article : Google Scholar : PubMed/NCBI

10 

Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA, Blackie C, Chang L, McMurtrey AE, Hebert A, et al: Safty and antitumor activity of recombinant soluble Apo 2 ligand. J Clin Invest. 104:155–162. 1999. View Article : Google Scholar : PubMed/NCBI

11 

Hao C, Beguinot F, Condorelli G, Trencia A, Van Meir EG, Yong VW, Parney IF, Roa WH and Petruk KC: Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apotosis in human malignant glioma cells. Cancer Res. 61:1162–1170. 2001.PubMed/NCBI

12 

Jalving M, Heijink DM, Koornstra JJ, Boersma-van Ek W, Zwart N, Wesseling J, Sluiter WJ, de Vries EG, Kleibeuker JH and de Jong S: Regulation of TRAIL receptor expression by β-catenin in colorectal tumours. Carcinogenesis. 35:1092–1099. 2014. View Article : Google Scholar : PubMed/NCBI

13 

Lu M, Marsters S, Ye X, Luis E, Gonzalez L and Ashkenazi A: E-cadherin couples death receptors to the cytoskeleton to regulate apoptosis. Mol Cell. 54:987–998. 2014. View Article : Google Scholar : PubMed/NCBI

14 

Yang B, Zhang S, Wang Z, Yang C, Ouyang W, Zhou F, Zhou Y and Xie C: Deubiquitinase USP9X deubiquitinates β-catenin and promotes high grade glioma cell growth. Oncotarget. 7:79515–79525. 2016. View Article : Google Scholar : PubMed/NCBI

15 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar

16 

Rudner J, Jendrossek V, Lauber K, Daniel PT, Wesselborg S and Belka C: Type I and type II reactions in TRAIL-induced apoptosis - results from dose-response studies. Oncogene. 24:130–140. 2005. View Article : Google Scholar

17 

Ozören N and El-Deiry WS: Defining characteristics of types I and II apoptotic cells in response to TRAIL. Neoplasia. 4:551–557. 2002. View Article : Google Scholar : PubMed/NCBI

18 

de Miguel D, Lemke J, Anel A, Walczak H and Martinez-Lostao L: Onto better TRAILs for cancer treatment. Cell Death Differ. 23:733–747. 2016. View Article : Google Scholar : PubMed/NCBI

19 

Teng Y, Gao M, Wang J, Kong Q, Hua H, Luo T and Jiang Y: Inhibition of eIF2α dephosphorylation enhances TRAIL-induced apoptosis in hepatoma cells. Cell Death Dis. 5:e10602014. View Article : Google Scholar

20 

Fan S, Li Y, Yue P, Khuri FR and Sun SY: The eIF4E/eIF4G interaction inhibitor 4EGI-1 augments TRAIL-mediated apoptosis through c-FLIP down-regulation and DR5 induction independent of inhibition of cap-dependent protein translation. Neoplasia. 12:346–356. 2010. View Article : Google Scholar : PubMed/NCBI

21 

Haimovici A, Humbert M, Federzoni EA, Shan-Krauer D, Brunner T, Frese S, Kaufmann T, Torbett BE and Tschan MP: PU.1 supports TRAIL-induced cell death by inhibiting NF-κB-mediated cell survival and inducing DR5 expression. Cell Death Differ. 24:866–877. 2017. View Article : Google Scholar : PubMed/NCBI

22 

Mert U and Sanlioglu AD: Intracellular localization of DR5 and related regulatory pathways as a mechanism of resistance to TRAIL in cancer. Cell Mol Life Sci. 74:245–255. 2017. View Article : Google Scholar

23 

Safa AR and Pollok KE: Targeting the anti-apoptotic protein c-FLIP for cancer therapy. Cancers (Basel). 3:1639–1671. 2011. View Article : Google Scholar

24 

Chen F, Guo J, Zhang Y, Zhao Y, Zhou N, Liu S, Liu Y and Zheng D: Knockdown of c-FLIP(L) enhanced AD5-10 anti-death receptor 5 monoclonal antibody-induced apoptosis in human lung cancer cells. Cancer Sci. 100:940–947. 2009. View Article : Google Scholar : PubMed/NCBI

25 

Jeon MY, Min KJ, Woo SM, Seo SU, Kim S, Park JW and Kwon TK: Volasertib enhances sensitivity to TRAIL in renal carcinoma Caki cells through downregulation of c-FLIP expression. Int J Mol Sci. 18:1–12. 2017. View Article : Google Scholar

26 

Murphy ÁC, Weyhenmeyer B, Noonan J, Kilbride SM, Schimansky S, Loh KP, Kögel D, Letai AG, Prehn JH and Murphy BM: Modulation of Mcl-1 sensitizes glioblastoma to TRAIL-induced apoptosis. Apoptosis. 19:629–642. 2014. View Article : Google Scholar :

27 

Azijli K1, Yuvaraj S, van Roosmalen I, Flach K, Giovannetti E, Peters GJ, de Jong S and Kruyt FA: MAPK p38 and JNK have opposing activities on TRAIL-induced apoptosis activation in NSCLC H460 cells that involves RIP1 and caspase-8 and is mediated by Mcl-1. Apoptosis. 18:851–860. 2013. View Article : Google Scholar : PubMed/NCBI

28 

Woo SM, Min KJ, Seo BR and Kwon TK: YM155 sensitizes TRAIL-induced apoptosis through cathepsin S-dependent down-regulation of Mcl-1 and NF-κB-mediated down-regulation of c-FLIP expression in human renal carcinoma Caki cells. Oncotarget. 7:61520–61532. 2016. View Article : Google Scholar : PubMed/NCBI

29 

Jin Z, McDonald ER III, Dicker DT and El-Deiry WS: Deficient tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor transport to the cell surface in human colon cancer cells selected for resistance to TRAIL-induced apoptosis. J Biol Chem. 279:35829–35839. 2004. View Article : Google Scholar : PubMed/NCBI

30 

Ouyang W, Yang C, Zhang S, Liu Y, Yang B, Zhang J, Zhou F, Zhou Y and Xie C: Absence of death receptor translocation into lipid rafts in acquired TRAIL-resistant NSCLC cells. Int J Oncol. 42:699–711. 2013. View Article : Google Scholar

31 

Inamura K: Lung Cancer: Understanding its molecular pathology and the 2015 WHO classification. Front Oncol. 7:1932017. View Article : Google Scholar : PubMed/NCBI

32 

Polakis P: Drugging Wnt signalling in cancer. EMBO J. 31:2737–2746. 2012. View Article : Google Scholar : PubMed/NCBI

33 

Valenta T, Hausmann G and Basler K: The many faces and functions of β-catenin. EMBO J. 31:2714–2736. 2012. View Article : Google Scholar : PubMed/NCBI

34 

Zimmerman ZF, Kulikauskas RM, Bomsztyk K, Moon RT and Chien AJ: Activation of Wnt/β-catenin signaling increases apoptosis in melanoma cells treated with trail. PLoS One. 8:e695932013. View Article : Google Scholar

35 

Lan Y, Liu X, Zhang R, Wang K, Wang Y and Hua ZC: Lithium enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells. Biometals. 26:241–254. 2013. View Article : Google Scholar : PubMed/NCBI

36 

Zhang L, Ren X, Alt E, Bai X, Huang S, Xu Z, Lynch PM, Moyer MP, Wen XF and Wu X: Chemoprevention of colorectal cancer by targeting APC-deficient cells for apoptosis. Nature. 464:1058–1061. 2010. View Article : Google Scholar : PubMed/NCBI

37 

Wu X, Deng G, Hao X, Li Y, Zeng J, Ma C, He Y, Liu X and Wang Y: A caspase-dependent pathway is involved in Wnt/β-catenin signaling promoted apoptosis in Bacillus Calmette-Guerin infected RAW264.7 macrophages. Int J Mol Sci. 15:5045–5062. 2014. View Article : Google Scholar : PubMed/NCBI

38 

Galbiati F, Volonte D, Brown AM, Weinstein DE, Ben-Ze’ev A, Pestell RG and Lisanti MP: Caveolin-1 expression inhibits Wnt/beta-catenin/Lef-1 signaling by recruiting beta-catenin to caveolae membrane domains. J Biol Chem. 275:23368–23377. 2000. View Article : Google Scholar : PubMed/NCBI

39 

Gajate C and Mollinedo F: Cytoskeleton-mediated death receptor and ligand concentration in lipid rafts forms apoptosis-promoting clusters in cancer chemotherapy. J Biol Chem. 280:11641–11647. 2005. View Article : Google Scholar : PubMed/NCBI

40 

Brozovic A: The relationship between platinum drug resistance and epithelial-mesenchymal transition. Arch Toxicol. 91:605–619. 2017. View Article : Google Scholar

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November-2018
Volume 53 Issue 5

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Online ISSN:1791-2423

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Copy and paste a formatted citation
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Spandidos Publications style
You C, Zhang S, Sun Y, Zhang S, Tang G, Tang F, Liu X, Xiao Y, Zhang J, Gong Y, Gong Y, et al: β-catenin decreases acquired TRAIL resistance in non-small-cell lung cancer cells by regulating the redistribution of death receptors. Int J Oncol 53: 2258-2268, 2018
APA
You, C., Zhang, S., Sun, Y., Zhang, S., Tang, G., Tang, F. ... Xie, C. (2018). β-catenin decreases acquired TRAIL resistance in non-small-cell lung cancer cells by regulating the redistribution of death receptors. International Journal of Oncology, 53, 2258-2268. https://doi.org/10.3892/ijo.2018.4529
MLA
You, C., Zhang, S., Sun, Y., Zhang, S., Tang, G., Tang, F., Liu, X., Xiao, Y., Zhang, J., Gong, Y., Xie, C."β-catenin decreases acquired TRAIL resistance in non-small-cell lung cancer cells by regulating the redistribution of death receptors". International Journal of Oncology 53.5 (2018): 2258-2268.
Chicago
You, C., Zhang, S., Sun, Y., Zhang, S., Tang, G., Tang, F., Liu, X., Xiao, Y., Zhang, J., Gong, Y., Xie, C."β-catenin decreases acquired TRAIL resistance in non-small-cell lung cancer cells by regulating the redistribution of death receptors". International Journal of Oncology 53, no. 5 (2018): 2258-2268. https://doi.org/10.3892/ijo.2018.4529