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Mol Oncol. 2015 November; 9(9): 1815–1824.
Published online 2015 June 20. doi:  10.1016/j.molonc.2015.06.003
PMCID: PMC5528717

5‐Fluorouracil preferentially sensitizes mutant KRAS non‐small cell lung carcinoma cells to TRAIL‐induced apoptosis

Abstract

Mutations in the KRAS gene are very common in non–small cell lung cancer (NSCLC), but effective therapies targeting KRAS have yet to be developed. Interest in tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL), a potent inducer of cell death, has increased following the observation that TRAIL can selectively kill a wide variety of human cancer cells without killing normal cells both in vitro and in xenograft models. However, results from clinical trials of TRAIL‐based therapy are disappointingly modest at best and many have demonstrated a lack of therapeutic benefit. Current research has focused on selecting a subpopulation of cancer patients who may benefit from TRAIL‐based therapy and identifying best drugs to work with TRAIL. In the current study, we found that NSCLC cells with a KRAS mutation were highly sensitive to treatment with TRAIL and 5‐fluorouracil (5FU). Compared with other chemotherapeutic agents, 5FU displayed the highest synergy with TRAIL in inducing apoptosis in mutant KRAS NSCLC cells. We also found that, on a mechanistic level, 5FU preferentially repressed survivin expression and induced expression of TRAIL death receptor 5 to sensitize NSCLC cells to TRAIL. The combination of low‐dose 5FU and TRAIL strongly inhibited xenograft tumor growth in mice. Our results suggest that the combination of TRAIL and 5FU may be beneficial for patients with mutant KRAS NSCLC.

Keywords: TRAIL, 5-Fluorouracil, Apoptosis, Lung cancer

Highlights

  • Chemotherapeutic drugs display different levels of synergy with TRAIL in NSCLC cells.
  • 5FU has strongest synergy with TRAIL in inducing apoptosis in mutant KRAS NSCLC cells.
  • The synergy between 5FU and TRAIL is mediated by 5FU‐induced repression of survivin and induction of DR5.

1. Introduction

Lung cancer is the leading cause of cancer‐related death in the United States and worldwide (Siegel et al., 2014). Approximately 85% of cases of lung cancer are classified as non–small cell lung cancer (NSCLC), including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Despite improved treatment strategies, the 5‐year survival rate remains at 5–10% for patients with locally advanced or advanced‐stage NSCLC (Mountain, 2000). Cytotoxic chemotherapy offers modest prolongation of survival for patients with advanced NSCLC. Recent advances in the treatment of lung cancer, including modifications of chemotherapy combinations, the advent of molecular targeted therapies such as bevacizumab and cetuximab, and incorporation of histologic subtype into treatment decisions, have improved the outcomes of patients with advanced NSCLC. However, therapeutic benefits have reached a plateau, with response rates of 20–35% and median survival durations of 8–12 months (Ettinger et al., 2010; Sandler et al., 2006; Scagliotti et al., 2008; Schiller et al., 2002).

It is increasingly clear that NSCLC has molecularly distinct subtypes with distinct patterns of genomic and epigenetic alterations, and different therapeutic approaches are required for each subtype. Mutations in the KRAS gene have been found in 20–30% of cases of NSCLC and occur most frequently in the adenocarcinoma subtype (Aviel‐Ronen et al., 2006). Despite the high prevalence of KRAS mutations in NSCLC, efforts to develop drugs that can target KRAS directly have been unsuccessful. Thus, new therapeutic strategies are needed.

Tumor necrosis factor (TNF)–related apoptosis‐inducing ligand (TRAIL, also called Apo2L) is a membrane‐bound TNF–family ligand (Pitti et al., 1996; Wiley et al., 1995) that interacts with 5 receptors in humans, including the fully functional death receptors 4 and 5 (DR4, DR5), and nonfunctional decoy death receptors 1 and 2 (DcR1, DcR2), and osteoprotegerin (LeBlanc and Ashkenazi, 2003). TRAIL binding to DR4 and DR5 results in receptor aggregation at the membrane and triggers apoptosis through classic death receptor pathway (Schneider and Tschopp, 2000; Sprick et al., 2000). However, interactions of TRAIL with DcR1, DcR2, and osteoprotegerin result in defective death signaling (Falschlehner et al., 2007).

Interest in TRAIL has increased following reports that recombinant soluble TRAIL selectively killed a wide variety of transformed human tumor cell lines in vitro and in xenograft models without harming normal cells. Agonistic anti‐DR4 or anti‐DR5 drugs (e.g., mapatumumab and PRO95780, which bind to TRAIL death receptors and trigger cell death signaling) showed similar activity in preclinical settings. Moreover, various chemotherapeutic agents have shown synergy with TRAIL or TRAIL receptor agonists in killing cancer cells both in vitro and in animal models. However, although TRAIL is well tolerated, results from phase I and II clinical trials of TRAIL signaling–based monotherapy or combination therapy are disappointing (Dimberg et al., 2013). The lackluster response to TRAIL among unselected patients in clinical trials suggests that TRAIL‐based therapy may be effective only in a subpopulation of patients. It is also possible that a specific combination of a chemotherapeutic drug with TRAIL or TRAIL receptor agonists is required to achieve efficient cell death in clinic.

We have previously reported a TRAIL‐based treatment to target mutant KRAS in premalignant lung epithelial cells for chemoprevention of lung cancer (Huang et al., 2011). To develop a TRAIL‐based therapy to target mutant KRAS in malignant lung cancer cells, we examined the effects of TRAIL and various chemotherapeutic agents on mutant KRAS NSCLC cells in vitro and in xenograft models. We found that NSCLC cells with KRAS mutations are more sensitive to TRAIL‐based therapy than NSCLC cells with wild‐type KRAS, and that 5‐fluorouracil (5FU) has stronger synergy with TRAIL than do other chemotherapeutic drugs in inducing cell death in mutant KRAS NSCLC cells in preclinical settings.

2. Materials and methods

2.1. Plasmids, shRNAs, and reagents

The retroviral plasmid expressing a mutant KRAS (KRASV12) and the DR5 expression plasmid used in the current study were described previously (McDonald et al., 2001; Yang et al., 2006). Full‐length survivin cDNA and survivin shRNAs were purchased from Open Biosystems (Lafayette, CO). KRAS siRNA was purchased from Santa Cruz Biotechnology (Dallas, TX). Recombinant soluble human TRAIL (rh‐TRAIL or TRAIL) was prepared according to published results (Wang et al., 2014; Zhang et al., 2010). 5FU, doxorubicin hydrochloride, and paclitaxel were purchased from Sigma–Aldrich (St. Louis, MO) and dissolved in dimethylsulphoxide. Cisplatin [cis‐diammineplatinum(II) dichloride] was obtained from LC Laboratories (Woburn, MA) and dissolved in 0.15M NaCl.

2.2. Antibodies and Western blot analysis

Anti‐c‐FLIP antibody was obtained from ALEXIS Biochemicals (San Diego, CA). Anti‐phospho‐Erk, anti‐Bax, anti‐caspase 8, anti‐Bcl‐XL, and anti‐Bcl‐2 antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti‐β‐actin antibody was purchased from Sigma–Aldrich. Antibodies against KRAS, survivin, Bak, and DR5 were purchased from Santa Cruz Biotechnology. Anti‐DR4 antibody was obtained from Upstate Biotechnology (Lake Placid NY). Anti‐XIAP antibody was purchased from BD Biosciences (San Jose, CA).

Western blot analysis was performed as described previously (Wang et al., 2014; Zhang et al., 2010). Briefly, total protein was extracted from cultured cells using ice‐cold RIPA buffer [50mM Tris HCl, pH 7.4, 150mM NaCl, 1mM EDTA, 1% (v/v) Triton‐X 100, 0.1% (w/v) SDS] supplemented with phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Sigma–Aldrich). Protein concentrations were determined using Bio‐Rad protein assay, according to the manufacturer's instructions (Bio‐Rad, Hercules, CA). Proteins (30 μg) from whole cell lysates were fractionated on 12% SDS‐PAGE gels and transferred to nitrocellulose membranes, and the detection antibodies listed above were used.

2.3. Cell lines, transfection, and drug treatment

Mutant KRAS NSCLC cells (A549, NCI‐H460 (H460), NCI‐H157 (H157), NCI‐H358 (H358), NCI‐H2122 (H2122) and NCI‐H1792 (H1792)), and wild‐type KRAS NSCLC cells (NCI‐H322 (H322), NCI‐H661 (H661)) cells were obtained from American Type Culture Collection, verified yearly by genomic fingerprinting, and maintained in RPMI‐1640 media for no more than 20 passages. Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. For treatment with TRAIL and 5FU, cells growing at the log phase were treated with 5FU at a final concentration of 25 μg/mL for 12 h. Then, TRAIL was added to the media at a final concentration of 100 ng/mL. Cells were harvested after 24 h of treatment with TRAIL. Cell apoptosis was determined using the Annexin V‐FITC Apoptosis Detection Kit (Sigma–Aldrich) or trypan blue dye exclusion (Life Technologies, Grand Island, NY), according to the manufacturer's instructions. The synergistic effects between different agents in in vitro experiments were evaluated using the CompuSyn software (version 3.0.1 [2007]; ComboSyn, Inc.) (Chou, 2007).

2.4. Tumor studies in mice

All animal experiments were reviewed and approved by The University of Texas MD Anderson Cancer Center Animal Care and Use Committee. For xenograft studies, 1 million H460 cells were implanted into the flank of 4‐ to 6‐week‐old male athymic nude mice (National Cancer Institute, Frederick. MD). The animals were randomly divided into 4 groups immediately after inoculation with the cells. Treatments began when the tumor reached approximately 50 mm3. The mice were injected intraperitoneally with 5FU (5 mg/kg), and 12 h later, TRAIL (3 mg/kg) was injected intraperitoneally. Injections were then repeated every 12 h (i.e., alternating 5FU and TRAIL) for a total of 10 injections of each agent. Injections of phosphate‐buffered saline were used as a control. The shortest and longest diameter of the tumors were measured with calipers every day, and tumor volume (mm3) was calculated using the following standard formula: (shortest diameter)2 × longest diameter/2. Mouse weight and body condition were evaluated after each treatment. Twelve hours after the last treatment, the mice were sacrificed and the tumors were collected, processed, and subjected to terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) analysis using the DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI), according to the manufacturer's instructions. The major organs of the mice were fixed in formalin and subjected to histologic examination.

2.5. Statistical analysis

We compared differences between groups using the Student t test. p values <0.05 were considered statistically significant.

3. Results

3.1. 5FU has strongest synergy with TRAIL in inducing apoptosis in NSCLC cells containing mutant KRAS

Although many chemotherapeutic agents have shown synergy with TRAIL in killing various cancer cells, it is not clear which drug has the strongest synergy in NSCLC cells. To address this question, we selected two wild‐type KRAS NSCLC cells (H661 and H322) and two mutant KRAS NSCLC cells (H460 and H157) and analyzed the dose response of 4 chemotherapeutic drugs, 5FU, doxorubicin, paclitaxel, and cisplatin in the presence of 100 ng/mL TRAIL in these cells (Figure 1). A dose‐dependent increase in apoptosis was observed for all drugs in all cell lines (Figure 1). However, cell lines with mutant KRAS were significantly more sensitive to either TRAIL alone, or chemotherapeutic drugs alone, or in combinations than cells with wild type KRAS (Figure 1). Although 5FU is not the strongest drug in inducing apoptosis in NSCLC cells on itself, the combination of 5FU and TRAIL induced greater degree of apoptosis in killing mutant KRAS NSCLC cells (Figure 1). The synergistic effects between chemotherapeutic drugs and TRAIL were analyzed further with ComboSyn software analysis (Table 1). The results showed that 5FU has smallest CI value, thus strongest synergy with TRAIL. More importantly, significant cell apoptosis was observed even with low doses of 5FU in mutant KRAS cells, whereas the wild‐type KRAS cells responded only somewhat to the combination treatment at the highest dose tested (Figure 1). These data suggest that 5FU has the strongest synergy with TRAIL in inducing cell apoptosis in mutant KRAS NSCLC cells compared with other chemotherapeutic drugs tested, and that low doses 5FU is sufficient to sensitize mutant KRAS NSCLC cell to TRAIL‐induced apoptosis.

Figure 1

5‐Fluorouracil (5FU) shows stronger synergy with TRAIL than do other chemotherapeutic drugs in killing mutant KRAS non–small cell lung cancer cells. (A) Cell apoptosis in mutant KRAS (H460 and H157) and wild‐type KRAS cells (H661 ...

Table 1

Combination Index (CI) values of drug combinations in H460 cells.

3.2. Mutant KRAS sensitizes NSCLC cells to treatment with the combination of TRAIL and 5FU

To investigate the role of mutant KRAS in 5FU‐ and TRAIL‐induced apoptosis in NSCLC cells, we first examined cell apoptosis in additional NSCLC cell lines with mutant KRAS treated with either TRAIL, or 5FU, or both (Figure 2A). Similar to H460 and H157 cells, the cell lines with mutant KRAS (A549, H358, H2122, and H1792) were only marginally sensitive to TRAIL or 5FU alone. However, treatment of these cells with the combination of 5FU and TRAIL resulted in marked increases in cell apoptosis (Figure 2A).

Figure 2

Mutant KRAS sensitizes non–small cell lung cancer cells to treatment with the combination of TRAIL and 5‐fluorouracil (5FU). (A) Cell apoptosis induced by TRAIL and 5FU. Non–small cell lung cancer cells with mutant KRAS (A549, ...

To investigate the effect of RAS activation on response to treatment with TRAIL plus 5FU, we introduced the activating mutant KRAS V12 plasmid into wild‐type KRAS H322 and H661 cells (Figure 2B). Constitutive expression of mutant KRAS led to an increase in RAS‐GTPase activity and activation of downstream signaling events, including phosphorylation of Erk, in the H322 and H661 cells (Figure 2B). More importantly, expression of mutant KRAS sensitized H322 and H661 cells to cell apoptosis induced by TRAIL and 5FU (Figure 2C).

We next determined whether mutant KRAS was required in NSCLC cells for the cells to be sensitive to treatment with TRAIL and 5FU. Knockdown of mutant KRAS in H460 and H157 cells reduced the expression of mutant KRAS and inhibited cell apoptosis induced by TRAIL and 5FU in both H460 and H157 cell lines (Figure 2D and E). Taken together, these results suggest that mutant KRAS sensitizes NSCLC cells to cell apoptosis induced by treatment with TRAIL and 5FU.

3.3. 5FU Modulates expression of survivin and DR5

H460 and H157 cells were treated with 5FU and TRAIL and caspase 8 cleavage was analyzed by Western blots (supplemental Fig. S1). 5FU and TRAIL induced strong cleavage of caspase 8, confirming that 5FU and TRAIL induces apoptosis through the activation of caspase 8. As a first step to dissect the underlying mechanisms involved in the effects of treatment with TRAIL and 5FU on NSCLC cells, we analyzed the expression levels of various proteins involved in the apoptotic pathway using Western blot analysis in H460, H157, H322 and H661 cells (Figure 3A). Although protein levels of c‐FLIP, caspase 8, XIAP, Bax, Bak, DR4, Bcl‐XL, and Bcl‐2 showed no obvious change after treatment with 5FU, levels of survivin decreased and levels of DR5 increased significantly after the treatment (Figure 3A and C). More importantly, these changes were observed only in mutant KRAS cells; 5FU had no effect on the protein levels in wild‐type KRAS cells (Figure 3A and C). The 5FU‐mediated induction of DR5 and repression of survivin were detected in other mutant KRAS expressing NSCLC cell lines (supplemental Fig. S2). The induction of DR5 on the cell surface was also observed by FACS analysis after 5FU treatment (supplemental Fig. S3).

Figure 3

5‐Fluorouracil (5FU) inhibits expression of survivin and activates expression of DR5 in mutant KRAS non–small cell lung cancer cells. (A) Western blot analysis showing expression levels of proteins related to the apoptosis pathway in ...

Next, we investigated the effects of treatment with doxorubicin, paclitaxel, or cisplatin on the expression of survivin and DR5. These chemotherapeutic drugs downregulated the expression of survivin, but to a lesser extent than 5FU (Figure 3B and C), and these drugs had no effect on the expression of DR5 (Figure 3B and C). These results suggest that 5FU represses the expression of survivin and induces the expression of DR5 in mutant KRAS NSCLC cells.

3.4. Survivin and DR5 play a functional role in cell apoptosis induced by 5FU and TRAIL

The fact that 5FU downregulates expression of survivin and upregulates expression of DR5 suggests that DR5 and survivin play a role in mediating the synergistic effect of 5FU with TRAIL in mutant KRAS NSCLC cells. To test this hypothesis, we first knocked down survivin in H460 and H157 cells (Figure 4A). Knockdown of survivin significantly sensitized cells to treatment with either TRAIL or the combination of TRAIL and 5FU (Figure 4B). In contrast, overexpression of survivin in these cells significantly attenuated cell apoptosis induced by the combination of TRAIL and 5FU (Figure 4C and D).

Figure 4

Survivin knockdown and overexpression in mutant KRAS non–small cell lung cancer cells affects sensitivity of the cells to TRAIL and 5‐fluorouracil (5FU). (A and B) Knockdown of survivin in mutant KRAS cells. H460 and H157 cells transfected ...

We next investigated the effect of DR5 in mutant KRAS and wild‐type KRAS NSCLC cells (Figure 5). Transfection with DR5 led to a significant increase in DR5 expression in both the mutant KRAS cell lines and the wild‐type KRAS cell lines (Figure 5A and C). In mutant KRAS cells, overexpression of DR5 significantly sensitized the cells to TRAIL and the combination of TRAIL and 5FU without affecting 5FU‐induced cell apoptosis (Figure 5B). In wild‐type KRAS cells, overexpression of DR5 also sensitized the cells somewhat to 5FU, TRAIL, and the combination of TRAIL and 5FU (Figure 5D). In contrast, Knockdown of DR5 in the mutant KRAS cells significantly attenuated cell apoptosis induced by the combination of TRAIL and 5FU (Figure 5E and F). These results suggest that both downregulation of survivin and upregulation of DR5 are critical in the synergistic induction of cell apoptosis by the combination of TRAIL and 5FU in mutant KRAS NSCLC cells.

Figure 5

DR5 overexpression in both mutant KRAS and wild‐type KRAS non–small cell lung cancer cells affects sensitivity to TRAIL and 5‐fluorouracil (5FU). (A and B) Overexpression of DR5 in mutant KRAS cells. H460 and H157 cells transfected ...

3.5. TRAIL and low‐dose 5FU strongly inhibit mutant KRAS NSCLC xenograft growth in mice

Because 5FU showed synergistic effects with TRAIL in mutant KRAS NSCLC cells even at low doses (Figure 2A), we tested the antitumor activity of 5FU at low doses in mice. Mice bearing NCI‐H460 xenograft tumors received 10 consecutive daily treatments with TRAIL (3 mg/kg) and 5FU (5 mg/kg). The longest and shortest diameters of the tumors were measured with calipers daily and tumor volumes were plotted (Figure 6A). Tumor volume measurements showed that the combination treatment strongly inhibited tumor growth and the single‐drug treatments (i.e., TRAIL alone or 5FU alone) only partially reduced tumor growth (Figure 6A).

Figure 6

TRAIL and 5‐fluorouracil (5FU) inhibit the growth of non–small cell lung cancer xenograft tumors in mice. (A) Tumor growth of H460 xenografts over 10 days. Mice bearing H460 xenograft tumors were treated with phosphate‐buffered ...

Consistent with the tumor growth rates observed for the treatment groups, cell apoptosis analysis performed on the tumor samples at the end of the treatment showed that significantly more TUNEL‐positive signals were observed in the combination treatment group than in the single‐drug treatment groups (Figure 6B and C). As expected, very few TUNEL‐positive apoptosis signals were detected in the control group (Figure 6B and C). These results indicate that treatment with TRAIL plus 5FU induces apoptosis in mutant KRAS cells in vivo.

4. Discussion

With advances in understanding of the underlying biology and molecular mechanisms of lung cancer, it has becomes clear that lung cancer is not a single disease entity and can be subdivided into molecular subtypes based on genetic mutations with dedicated targeted and chemotherapeutic strategies. In particular, several advances have been achieved in the management of NSCLC. Understanding the role of activating mutations in EGFR and fusion genes involving ALK in NSCLCs has led to the discovery and successful application of targeted therapies against these alterations. These developments reflect a huge advance in the management of lung cancer, but most patients do not have EGFR mutations or ALK fusions. Identification of more common driver mutations or genetic alterations is needed, along with the development of drugs specifically targeting these genetic aberrations. Activating mutations in the KRAS gene represent one such event. Although the development of a small molecule inhibitor of the constitutively active KRAS protein would be an ideal therapy, research on drugs directly targeting mutant KRAS over the past 25 years has thus far proven to be unsuccessful (Wang et al., 2013). To date, no effective therapy that specifically targets mutant KRAS is available. A number of attempts have been made to target aberrant KRAS signaling using alternative strategies, including the use of synthetic lethality to target mutant KRAS (Luo et al., 2009; Scholl et al., 2009) and inhibition of downstream KRAS effectors (Stephen et al., 2014; Takashima and Faller, 2013). In this report, we showed that mutant KRAS sensitizes NSCLC cells to treatment with the combination of TRAIL and 5FU, offering a potential targeting strategy for mutant KRAS in NSCLC.

The role of RAS signaling pathways, including mutations in KRAS and HRAS, in TRAIL‐induced apoptosis has been the subject of various studies, and the apoptotic effects of TRAIL appear to be dependent on cell type. Oncogenic HRAS has been shown to sensitize normal human cells to TRAIL‐induced apoptosis by enhancing the recruitment of caspase 8 to the death‐inducing signaling complex (Nesterov et al., 2004). Oncogenic RAS has also been shown to increase DR5 expression to promote TRAIL‐induced apoptosis in lung and oral cancer cell lines (Chen et al., 2013; Oh et al., 2012). Moreover, upregulation of HRAS in cancer cells was shown to be associated with decreased DR4 and DR5 expression, leading to TRAIL resistance (Chen et al., 2014). In our study, we observed an increase in TRAIL sensitivity in mutant KRAS NSCLC cells (Figure 1A), but no correlation between KRAS status and DR4 and DR5 expression was observed (Figure 3A), supporting the previous findings that response to TRAIL in mutant KRAS cells depends on the cell type.

It has been reported that ERK/RSK and JNK signaling pathways are involved in RAS‐mediated induction of DR5 through co‐activation cooperative effects among the transcriptional factors CHOP, Elk1, and c‐Jun (Oh et al., 2012). 5FU was shown to have the ability to activate MAPK and JNK pathways (Mohapatra et al., 2011). It is possible that 5FU may enhance the KRAS signaling pathways to induce DR5 expression in mutant KRAS NSCLC cells. The transcription of survivin is regulated by multiple pathways including PI3k/AKT and NF‐κB (Boidot et al., 2014). The ability of 5FU to inhibit these pathways could contribute to its activity in repressing survivin in NSCLC cells (Islam et al., 2007).

Many commonly used standard chemotherapeutic agents, including 5FU, cisplatin, doxorubicin, and paclitaxel, have been shown to synergize with TRAIL (Newsom‐Davis et al., 2009). However, very few studies have examined and compared the synergistic effects of these drugs in specific types of cancer. One of the difficulties in comparing different drugs is that each drug has a different working concentration or specific activity. In this report, we investigated the dose responses of the combinations of TRAIL and the several chemotherapeutic drugs (5FU, cisplatin, doxorubicin, and paclitaxel) in both wild‐type and mutant KRAS NSCLC cells, and we observed that 5FU had the strongest synergy with TRAIL in inducing cell apoptosis in mutant KRAS NSCLC cells even at lower doses (Figure 1).

Given our finding that cisplatin and paclitaxel showed only moderate synergy with TRAIL in mutant KRAS NSCLC cells and no synergy with TRAIL in wild‐type KRAS NSCLC cells, it is unsurprising that previous clinical testing of the addition of TRAIL or its agonist to platinum‐ and paclitaxel‐based therapy in nonselected NSCLC patients showed no added benefit (Soria et al., 2011; von Pawel et al., 2014). Although 5FU is not a standard chemotherapeutic drug for the treatment of NSCLC and it has not yet been tested in clinical trials, our findings support the development of a future clinical trial of the combination of 5FU and TRAIL for the treatment of mutant KRAS NSCLC.

Previous studies have shown that various mechanisms underlie the synergy of chemotherapeutic drugs with TRAIL, such as induction of TRAIL death receptors, upregulation of pro‐apoptotic proteins, downregulation of anti‐apoptotic factors, and enhancement of death‐inducing signaling complex formation (Newsom‐Davis et al., 2009; Pennarun et al., 2010; Yang et al., 2010). A number of reports have shown that 5FU potentiated TRAIL‐induced apoptosis in various cancer cell lines in vitro and in animal models (Galligan et al., 2005; Ganten et al., 2004; Haag et al., 2011; Keane et al., 1999; Lacour et al., 2003; Meurette et al., 2005; Mizutani et al., 2002; Pukac et al., 2005; Shimoyama et al., 2002; Stagni et al., 2010; von Haefen et al., 2004; Zhu et al., 2013). The mechanisms of synergy appear to be specific to the cell line or cancer type. We provided 3 lines of evidence suggesting that survivin and DR5 are downstream mediators of the synergistic effects of 5FU on TRAIL. First, survivin was downregulated and DR5 was upregulated by treatment with 5FU. Second, these modulations were not observed in wild‐type KRAS cells that were not sensitive to treatment with 5FU, and the modulations were observed to a much lesser extent after treatment with other chemotherapeutic drugs. Third, functional analysis demonstrated that both survivin and DR5 play a role in the synergy between 5FU and TRAIL. However, in the only other study of 5FU and TRAIL synergy performed in NSCLC cell lines, Frese et al. did not find that 5FU had stronger synergy with TRAIL than other chemotherapeutic drugs in 6 mutant KRAS NSCLC cell lines, and no wild‐type KRAS NSCLC cell lines were used in that study (Frese et al., 2002). In our study, we used a much lower concentration of 5FU than Frese et al. used, and we performed chemotherapeutic drug pretreatment for 12 h in the 5FU and TRAIL combination treatment experiments, which could be the reason for the discrepancy between the studies.

In summary, we discovered that the combination of TRAIL and 5FU could serve as a novel approach to target mutant KRAS in NSCLC. We demonstrated the efficacy of this approach both in vitro and in vivo. Our data support the development of a future clinical trial to test the combination of TRAIL‐based therapy with 5FU in patients with mutant KRAS NSCLC.

Conflicts of interest

The authors declare no conflict of interests.

Supporting information

The following is the supplementary data related to this article:

Supplementary data

Acknowledgments

We thank Shaoyi Huang, Xiaoyang Ren and Zhengming Xu of UT M.D. Anderson Cancer Center for their technical assistance, and Erica Goodoff for editing the manuscript. This work was supported by CPRIT grant RP110107 (to X. Wu).

Supplementary data 1. 

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.molonc.2015.06.003.

Notes

Wang Haizhen, Yang Tao, Wu Xiangwei, (2015), 5‐Fluorouracil preferentially sensitizes mutant KRAS non‐small cell lung carcinoma cells to TRAIL‐induced apoptosis, Molecular Oncology, 9, doi: 10.1016/j.molonc.2015.06.003.

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