Surface TRAIL decoy receptor-4 expression is correlated with TRAIL resistance in MCF7 breast cancer cells
© Sanlioglu et al; licensee BioMed Central Ltd. 2005
Received: 13 March 2005
Accepted: 25 May 2005
Published: 25 May 2005
Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells. Despite this promising feature, TRAIL resistance observed in cancer cells seriously challenged the use of TRAIL as a death ligand in gene therapy. The current dispute concerns whether or not TRAIL receptor expression pattern is the primary determinant of TRAIL sensitivity in cancer cells. This study investigates TRAIL receptor expression pattern and its connection to TRAIL resistance in breast cancer cells. In addition, a DcR2 siRNA approach and a complementary gene therapy modality involving IKK inhibition (AdIKKβKA) were also tested to verify if these approaches could sensitize MCF7 breast cancer cells to adenovirus delivery of TRAIL (Ad5hTRAIL).
TRAIL sensitivity assays were conducted using Molecular Probe's Live/Dead Cellular Viability/Cytotoxicity Kit following the infection of breast cancer cells with Ad5hTRAIL. The molecular mechanism of TRAIL induced cell death under the setting of IKK inhibition was revealed by Annexin V binding. Novel quantitative Real Time RT-PCR and flow cytometry analysis were performed to disclose TRAIL receptor composition in breast cancer cells.
MCF7 but not MDA-MB-231 breast cancer cells displayed strong resistance to adenovirus delivery of TRAIL. Only the combinatorial use of Ad5hTRAIL and AdIKKβKA infection sensitized MCF7 breast cancer cells to TRAIL induced cell death. Moreover, novel quantitative Real Time RT-PCR assays suggested that while the level of TRAIL Decoy Receptor-4 (TRAIL-R4) expression was the highest in MCF7 cells, it was the lowest TRAIL receptor expressed in MDA-MB-231 cells. In addition, conventional flow cytometry analysis demonstrated that TRAIL resistant MCF7 cells exhibited substantial levels of TRAIL-R4 expression but not TRAIL decoy receptor-3 (TRAIL-R3) on surface. On the contrary, TRAIL sensitive MDA-MB-231 cells displayed very low levels of surface TRAIL-R4 expression. Furthermore, a DcR2 siRNA approach lowered TRAIL-R4 expression on surface and this sensitized MCF7 cells to TRAIL.
The expression of TRAIL-R4 decoy receptor appeared to be well correlated with TRAIL resistance encountered in breast cancer cells. Both adenovirus mediated IKKβKA expression and a DcR2 siRNA approach sensitized MCF7 breast cancer cells to TRAIL.
Cancer still appears to be a challenging disease to treat. According to most recent estimates, more than 10 million new cancer cases were reported in the year 2000 killing around 6 million people . In addition, 10 % of all cancers appear to be the breast cancer. Being the most frequently diagnosed cancer type in women, the breast cancer claims about 370,000 deaths each year around the world . Surgery, radiotherapy and chemotherapy are among the most widely used treatment methods for patients with breast cancer [3–5]. Still, these conventional treatment modalities did not improve the survival rate of patients with locally advanced or metastatic breast cancer. With standard therapy, locally advanced breast cancer has a five year survival rate of 55 % and a ten year survival rate of 35 % . There is a 40 % recurrence rate after ten years following the diagnosis and removal of primary tumor in patients with breast cancer . For all these reasons, novel treatment methods are needed for the treatment of patients with breast cancer.
Induction of programmed cell death known as apoptosis , appears to be a viable alternative to currently employed treatment modalities in the fight against cancer . In order for chemotherapy and radiotherapy treatment options to work as anticancer agents; tumor suppressor gene, p53, is required . Unfortunately, p53 mutations are acquired during the progression of cancer in more than half of the human tumors [11, 12]. Therefore, the resistance to both chemotherapy and radiotherapy is almost unavoidable in tumors lacking p53 . On the other hand, death ligands are capable of inducing apoptosis independently of p53 status of cells . Because of this reason, death ligands are currently considered as anticancer agents . Among the death ligands tested, Tumor Necrosis Factor (TNF) [16–18] and FasL  effectively induced apoptosis in cancer cells. However, due to their systemic toxicity, the application of these agents in cancer gene therapy is very limited. The discovery of a novel death ligand, TRAIL [20, 21], changed this view, since unlike other members of the TNF family, TRAIL selectively killed cancer cells without causing any harm to normal cells . Thus, treating tumor cells with TRAIL ligand appeared as an invaluable way of inducing apoptosis specifically in tumor cells, as normal cells are protected against the death-inducing effects of TRAIL [23, 24]. However, the mechanism of TRAIL resistance in normal cells is not understood  and significant proportions of cancer cells  including those of breast [27, 28] appeared to be TRAIL resistant. Consequently, TRAIL resistance constitutes a barrier if one wishes to use TRAIL as a death ligand in any breast cancer gene therapy approach.
Resistance to TRAIL-induced apoptosis in normal cells was initially considered to be caused by the presence of decoy receptors (TRAIL-R3 and TRAIL-R4), which compete with death receptors (TRAIL-R1 and TRAIL-R2) for binding to TRAIL [29, 30]. So far, no correlation between TRAIL sensitivity and the expression pattern of TRAIL receptors has been demonstrated in cancer cells yet . The presence of intracellular apoptosis inhibitory substances (bcl-xL, c-FLIP, cIAP etc.) was also blamed to be responsible for TRAIL resistance [31–33]. Intriguingly, the engagement of both TRAIL death receptors and TRAIL-R4 decoy receptor also activated NF-kB pathway [24, 34, 35]. Because NF-kB activation is known to hamper the apoptotic pathways in cells by up-regulating the expression of various apoptosis inhibitory molecules such as cFLIP, bcl-xL, c-IAP and the decoy receptor TRAIL-R3 [34, 36, 37], high levels of NF-kB activation might be a strong factor responsible for blocking apoptotic processes in order to establish TRAIL resistance. For this reason, we analyzed both the TRAIL induced as well as endogenous NF-kB activities using Luciferase reporter gene assays in MCF7 breast cancer cells. Because TRAIL-R1, TRAIL-R2 and TRAIL-R4 induced NF-kB activation has been shown to be primarily mediated by TRAF2-NIK-IkappaB kinase alpha/beta signaling cascade , MCF7 breast cancer cells were coinfected with adenovirus vectors encoding a dominant negative mutant to IKKβ(AdIKKβKA)  and hTRAIL (Ad5hTRAIL) in order to test if TRAIL resistance in breast cancer cells is eliminated through the inhibition of IKK, a leading modulator of NF-kB. The molecular mechanism of TRAIL resistance in breast cancer cells (MCF7 and MDA-MB-231) was studied by novel Real Time RT-PCR assays and conventional flow cytometry in order to verify if there is any relationship between TRAIL resistance and the expression pattern of TRAIL receptors. Lastly, a DcR2 siRNA approach was utilized to knock down the expression of relevant TRAIL decoy receptor in order to reveal its connection to TRAIL resistance.
Recombinant adenovirus vector production
Amplification of the vectors Ad5hTRAIL , AdIKKβKA , AdEGFP , AdCMVLacZ  and AdNFkBLuc  was performed as previously described . Amplified vectors were stored at -80°C in 10 mM Tris with 20 % glycerol. AdIKKβKA expresses a dominant negative mutant of IKKβ, which interacts with other IKK subunits to form inactive IKK complexes. The particle titers of adenoviral stocks were in the range of 1013 DNA particles/ml, whereas the typical particle/plaque forming unit ratio was equal to 50.
Infection of breast cancer cells with first generation recombinant adenovirus vectors
Breast cancer cell lines were cultured in RPMI 1640 medium supplemented with 10 % FBS, 2.2 g/l sodium bicarbonate, 1 mM L-glutamine, and 1 % penicillin-streptomycin mixture, at 37°C in a humidified 5 % CO2 atmosphere. Experimental steps of transduction of breast cancer cells with adenoviral vectors can be summarized as follows: Breast cancer cells were infected with an increasing multiplicity of infection (MOI) of AdEGFP (vector expressing enhanced green fluorescent protein (EGFP) reporter gene) vector at 37°C in RPMI 1640 without FBS. Two hours following infection, equal volume of RPMI 1640 supplemented with 20 % FBS was added to increase the serum concentration in the media to 10 %. 48 hours after the infection, the level of transduction was detected by examining of the percentage of GFP (+) cells under a fluorescent microscopy and subsequently by flow cytometry. Propidium iodide exclusion technique was used to determine the cell viability. Overexpression of hTRAIL was provided by Ad5hTRAIL infection. Cells were coinfected with adenovirus vectors encoding IKKβ dominant negative mutant (AdIKKβKA) and Ad5hTRAIL in order to block IKK activity thereby NF-kB activation. NF-kB promoter based Luciferase assay system was utilized to conduct NF-kB transcription activation assays using AdNFkBLuc construct. AdCMVLacZ vector was used as a control.
NF-kB directed transcription activation assays
AdNFkBLuc construct was utilized in order to determine the NF-kB activation status of MCF7 cells. AdNFkBLuc vector  possesses four tandem copies of the NF-kB consensus sequence fused to a TATA-like promoter from the herpes simplex virus-thymidine kinase gene driving the expression of a Luciferase reporter. Transcriptional induction mediated by NF-kB in the presence or absence of TRAIL was measured according to the manufacturer's protocol using the Luciferase assay system with Reporter Lysis Buffer (Promega, Inc.). All measurements of Luciferase activity expressed as relative light units were normalized against the protein concentration.
Cell viability assays
Discrimination of live cells from dead cells was performed using Live/Dead Cellular Viability/Cytotoxicity Kit from Molecular Probes (Eugene, OR). This assay is based on the use of Calsein AM and Ethidium homodimer-1 (EthD-1). Calsein AM is a fluorogenic substrate for intracellular calsein esterase. It is modified to a green fluorescent compound (calsein) by active esterase in live cells with intact membranes, thus serves as a marker for viable cells. Unharmed cell membranes do not allow EthD-1, a red fluorescent nucleic acid stain, to enter inside the cell. However, cells with damaged membrane uptake the dye and stain positive.
Apoptosis detection by Annexin V binding
Annexin V conjugated to fluorochromes such as FITC has successfully been used as probes to detect cells undergoing apoptosis. Annexin V binding assays were carried out according to manufacturer's instructions (Alexis Biochemicals). For this purpose, a FITC conjugated mouse monoclonal antibody to human Annexin V (ALX-804-100F-T100) was employed to detect apoptotic cells via flow cytometry.
The detection of TRAIL receptor expression profile by flow cytometry
Anti-TRAIL receptor flow cytometry set (Cat. ALX-850-273-KI01) was used to detect TRAIL receptor protein expression on cell surface. This kit contains 100μgs of MAb to TRAIL-R1 (clone HS101, Cat. 804-297A), -R2 (clone HS201, Cat.804-298A), -R3 (clone HS301, Cat. 804-344A) and -R4 (clone HS402, Cat. 804-299A). Primary antibodies were used at 5 μg/ml concentration. Biotinylated goat anti-mouse IgG1 (Cat. ALX-211-202) was used as a secondary antibody followed by streptavidin-PE (Cat. ANC-253-050) prior to flow cytometry. Flow analysis was performed according to manufacturer's protocols using BD FACSCALIBUR at the Akdeniz University Hospitals. Purified mouse IgG1 (MOPC 31C, Cat. ANC-278-010) served as an isotype control.
Quantitative Real Time RT-PCR assay for human TRAIL receptors
TRIzol reagent (Life Technologies, Gaithersburg, MD) was used to extract total RNA from breast cancer cells, according to the instructions from the manufacturer. Reverse transcription of 2 μg of total RNA was performed using TaqMan Reverse Transcription Reagents (Applied Biosystems Cat. N8080234). Despite the fact that the sequences for TRAIL-R1 and TRAIL-R2 primers and probes were recently described by our group , we had to design new probe sets for the decoy receptors. Following is the sequence information for TRAIL decoy receptor sets: TRAILR3-5' CCC-TAA-AGT-TCG-TCG-TCG-TCA-T, TRAILR3-3' GGG-CAG-TGG-TGG-CAG-AGT-A, TRAILR3 Probe: 5' 6FAM-TCGCGGTCCTGCTGCCAGTCCTAGC-TAMRA 3'; TRAILR4-5' ACA-GAG-GCG-CAG-CCT-CAA, TRAILR4-3' ACG-GGT-TAC-AGG-CTC-CAG-TAT-ATT, TRAILR4 Probe: 5' 6FAM-AGGAGGAGTGTCCAGCAGGATCTCATAGATC-TAMRA 3'. rRNA was amplified as an internal control in the same reaction. Both the rRNA primers and probes were obtained from PE Applied Biosystems (Cat. 4308329). ΔΔCt method was used as described by Applied Biosystems to calculate the relative quantities of TRAIL receptors. The TaqMan PCR reaction was performed as described by the manufacturer (Applied Biosystems Cat. N8080228).
A DcR2 siRNA approach targeting TRAIL-R4 expression
Posttranscriptional silencing of gene expression became a very useful approach within the last couple of years in research. DcR2 siRNA experiments were performed using DcR2 siRNA (sc-35185), siRNA transfection medium (sc-36868) and siRNA transfection reagent (sc-29528) in MCF7 breast cancer cells as described by the manufacturer (Santa Cruz Biotechnology). Flow cytometry analysis was performed to assess any changes in TRAIL-R4 gene expression. MCF7 cells were infected with Ad5hTRAIL or AdCMVLacZ vectors at increasing doses 35 hours following the transfection. Molecular Probe's Live/Dead Cellular Viability/Cytotoxicity Kit was used to assess the amount of live cells 48 hours following the infection.
MCF7 breast carcinoma cells were efficiently transduced with recombinant adenoviruses
MCF7 breast cancer cells displayed complete resistance to TRAIL
Blocking IKK induced NF-kB activation pathway alone did not cause any reduction in the viability of MCF7 breast carcinoma cells
Because increased NF-kB activity was claimed to be responsible for the resistance to death ligand induced cytotoxicity in some tumors [36, 37], we wanted to test if the inhibition of IKK activity thereby NF-kB would reduce the viability of breast cancer cells. In order to block the intracellular anti-apoptotic NF-kB pathway, MCF7 cells were infected with increasing MOIs of adenoviral vectors encoding a dominant negative mutant of IKKβ(AdIKKβKA), a key molecule involved in the activation of NF-kB. Cell viability was examined 48 hours following the infection under fluorescent microscope (Figure 2). Interestingly, AdIKKβKA vector alone proved inefficient in reducing the viability of MCF7 cells, even at an MOI of 10,000 DNA particles/cell.
Adenovirus delivery of IKKβKA gene expression sensitized MCF7 breast cancer cells to TRAIL-induced apoptosis
Exogenous TRAIL overexpression elevated the basal NF-kB activity in MCF7 cells, whereas IKKβKA expression blocked both TRAIL-induced and basal NF-kB activities
Coinfection of Ad5hTRAIL and AdIKKβKA results in apoptotic cell death in MCF7 breast cancer cells
MCF7 breast cancer cell line displayed significant levels of TRAIL decoy receptor-4 expression
TRAIL sensitive MDA-MB-231 cells displayed very low levels of TRAIL-R4 decoy receptor expression on cell surface
Lowering of TRAIL-R4 gene expression sensitized MCF7 breast cancer cells to TRAIL
Although, conventional treatment modalities could not satisfactorily improve the survival rates of patients with locally advanced and metastatic disease, adenovirus delivery of death ligands represents a feasible choice for the treatment of patients with breast cancer. However, recent observations demonstrating that a considerable portion of human cancers including those of the breast [27, 28] were TRAIL resistant undermined the potential application of TRAIL against cancer. Accordingly, the understanding of the mechanism of TRAIL resistance is the key to resolve primary obstacles in TRAIL mediated gene therapy approach. Based on recent findings from our laboratory and others, we think that NF-kB signaling is one of the most crucial pathways involved in the constitution of TRAIL resistance . Despite the fact that TRAIL-R1, TRAIL-R2 and TRAIL-R4 induced NF-kB activation has been shown to be primarily mediated by TRAF2-NIK-IkappaB kinase alpha/beta signaling cascade , there is some doubt on whether or not NF-kB activation can block TRAIL mediated apoptosis. For example, in one particular study it was reported that NF-kB inhibition by way of IkappaBalpha mutant expression sensitized MCF7 cells to TNF but not TRAIL-induced apoptosis . Considering the fact that there are different ways to activate NF-kB pathway (IkB dependent and independent ways)  we decided to inhibit IKK activity rather than targeting IkappaBalpha itself to look for the possibility of sensitizing MCF7 breast cancer cells to TRAIL.
First of all, in order to find out the efficacy of adenovirus transduction in breast cancer cells, MCF7 cells were infected with increasing MOIs of AdEGFP virus. The transduction profiles analyzed by flow cytometry showed that nearly 100 % of the cells were transduced with AdEGFP at an MOI of 10,000 DNA particles/cell (Figure 1). The efficacy of TRAIL in mediating apoptosis of MCF7 breast cancer cells was assessed using Ad5hTRAIL construct. Interestingly, MCF7 cells displayed complete resistance to TRAIL as no reduction in the level of viable cells was observed even at an MOI of 10,000 DNA particles/cell (Figure 2). IKK inhibiting strategy alone proved inefficient in reducing the viability of MCF7 cells suggesting that an apoptotic stimulus was required in order to induce cell killing (Figure 2). Interestingly, in order to break down TRAIL resistance and to induce cell death, a coinfection of MCF7 cells with Ad5hTRAIL and AdIKKβKA was required (Figures 3 and 4). Luciferase assays confirmed that both the TRAIL induced and endogenous NF-kB activities were drastically reduced by the infection of MCF7 cells with AdIKKβKA virus (Figure 5). Moreover, IKKβKA sensitization of MCF7 breast carcinoma cells resulted in TRAIL induced apoptosis as revealed by Annexin V binding assays (Figure 6). These results suggested that NF-kB activation pathway has a hampering effect on TRAIL-induced cell death in MCF7 cells, and blocking this pathway is essential to sensitize breast cancer cells to TRAIL mediated apoptosis.
So far, no correlation between TRAIL resistance and TRAIL decoy receptor gene expression has been reported. For example, analysis of breast cancer cell lines by just examining the expression levels of TRAIL death receptors (TRAIL-R1 and TRAIL-R2) and TRAIL-R3 decoy receptor using RNase protection assay did not reveal any connection between the expression pattern of TRAIL receptors and TRAIL resistance . But whether or not TRAIL-R4 decoy receptor gene expression in any way contributes to TRAIL resistance in breast cancer cells remains to be tested yet. Quantitative Real Time RT-PCR assays were developed in order to assess the level of TRAIL receptor gene expression in breast carcinoma cells. While all TRAIL receptors were detectable in MCF7 breast carcinoma cell line, the level of TRAIL-R4 decoy receptor gene expression was the highest among the four (Figure 7, Panel A). This intriguing observation is consistent with a previous report suggesting that transient TRAIL-R4 overexpression protected target cells from TRAIL induced cytotoxicity . TRAIL R4 is known to protect cells from apoptosis by acting both as a decoy receptor and an antiapoptotic signal provider. While Real Time PCR assay is useful in assessing the level of gene expression on mRNA levels, obviously this assay does not necessarily reflect TRAIL receptor composition on cell surface. For this reason, conventional flow cytometry analysis was carried out in order to determine the level of TRAIL receptor protein expression on cell surface. Despite the presence of TRAIL death receptors, substantial levels of TRAIL-R4 decoy receptor expression were detectable on the surface of MCF7 breast carcinoma cells (Figure 7, Panel B). On top of that, TRAIL sensitive MDA-MB-231 cell line (Figure 9) displayed very low levels of TRAIL-R4 decoy receptor expression on cell surface (Figure 8, Panel B). Neither of the cell lines expressed detectable levels of TRAIL-R3 decoy receptor on surface. Intriguingly, administration of a DcR2 siRNA approach lowered surface TRAIL-R4 expression and sensitized MCF7 breast cancer cells to TRAIL (Figure 10).
Our results demonstrated that the expression of TRAIL-R4 decoy receptor but not TRAIL-R3 appeared to correlate well with TRAIL resistance phenotype observed in MCF7 breast cancer cells. Further screening of another breast cancer cell line, MDA-MB-231, revealed that low levels of TRAIL-R4 expression on surface were correlated with TRAIL sensitivity. These results strengthen our argument that TRAIL-R4 but not TRAIL-R3 is the decoy receptor which appeared to influence TRAIL sensitivity in breast cancer cells. This is further confirmed by a DcR2 siRNA assay which suggested that down regulation of TRAIL-R4 expression sensitized MCF7 breast cancer cells to TRAIL. In addition, the inhibition of IKK pathway thereby NF-kB sensitized MCF7 cells to TRAIL induced apoptosis despite the expression of TRAIL-R4 decoy receptor on cell surface. Consequently, this complementary gene therapy approach involving IKK inhibition might be necessary to breakdown TRAIL resistance encountered in patients with breast cancer.
- TRAIL= Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand:
EGFP= Enhanced Green Fluorescent Protein, MOI= Multiplicity of Infection, DcR2= Decoy receptor 2.
MDA-MB-231 cell line was kindly provided by Dr. Burhan Savas MD, PhD. This work is supported by grants from Akdeniz University Scientific Research Project Administration Division and the Health Science Institute (to SS).
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