Investigation of the role of tumor necrosis factor-like weak inducer of apoptosis in non-small cell lung cancer
- Authors:
- Published online on: December 11, 2017 https://doi.org/10.3892/or.2017.6141
- Pages: 573-581
Abstract
Introduction
Lung cancer is one of the most lethal malignancies worldwide (1). Targeted therapy drugs that target receptor tyrosine kinases including epidermal growth factor receptor, HER2/neu, echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) improve patient outcomes (2–4). In addition, immunotherapy agents such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed death 1 (PD-1) receptor and its ligands (PD-L1) have been approved for the treatment of lung cancer (5). However, some lung cancer patients exhibit no or limited responses to these treatments. Therefore, identifying unknown tumor-associated molecules and signaling pathways is still a critical strategy by which to develop novel therapeutic strategies.
Recent studies indicate that soluble factors are key factors that affect the behaviors of tumor cells in the tumor microenvironment. Extracellular matrix component-induced signaling pathways regulate the migration and proliferation of tumor cells (6). Various soluble factors including vascular endothelial growth factor A, platelet derived growth factor and osteopntin are involved in the malignant development of lung cancer (7). In addition, tumor-infiltrating macrophage and myeloid-derived suppressor cells are also important components in the tumor microenvironment (8,9). These immune cells that secrete cytokines and chemokines are important factors in malignancy. Chemokine (C-X-C motif) ligand 5 (CXCL5), CXCL8, CXCL12, C-C motif chemokine ligand 5 (CCL5), and receptors C-X-C motif chemokine receptor 4 (CXCR4) and CXCR8 play a role in lung cancer progression (10–12). Tumor necrosis factor-α (TNF-α) is one of the most important inflammatory mediators and induces the apoptosis of tumor cells (13). In contrast, TNF-α also mediates angiogenesis and chronic inflammation through the Jun kinase/nuclear factor-κB (NF-κB) signaling pathway (14). This evidence implies that investigation of soluble factor-associated signaling pathways may identify novel tumor markers or therapeutic targets.
Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK)/tumor necrosis factor superfamily member 12 (TNFSF12) is a member of the TNF superfamily and a ligand of fibroblast growth factor inducible 14 (Fn14)/TNF receptor superfamily member 12A (TNFRSF12A) (15). TWEAK activates the NF-κB signaling pathway through autocrine and paracrine manners (15). Overexpression of Fn14 is correlated with poor treatment outcome in prostate cancer and is a negative prognostic factor in breast and gastric cancer and glioma (16–19). In addition, TWEAK promotes the invasive capacity in prostate and glioma cells via activation of the NF-κB signaling pathway (20,21). Blockage of TWEAK may be a strategy by which to inhibit the metastasis of breast cancer and glioma (22). However, it is interesting to note that TWEAK overexpression in melanoma inhibits its cell growth and invasion even though the NF-κB signaling pathway is activated (21). Decreased TWEAK expression and increased Fn14 expression are observed in cervical cancer cell lines and clinical specimens of squamous cervical carcinoma (23). These findings suggest that the role of the TWEAK/Fn14 signaling pathway is diverse in different types of cancers.
In lung cancer, Src, hepatocyte growth factor receptor (HGFR/MET) and EGFR mutation (exon 19 deletion)-triggered signaling pathways contribute to Fn14 expression (24–26). Src- and MET-driven invasion correlates with FN14 expression (24,26). Although the expression of Fn14 is associated with tumor progression, the role of TWEAK is not fully understood in lung cancer. We investigated these issues by analysis of bioinformatic tools and in vitro human cancer cell lines. In addition, the TWEAK levels in serum samples of lung cancer patients and healthy donors were determined.
Materials and methods
Bioinformatic analysis
TWEAK and Fn14 expression in clinical lung cancer samples and non-tumor samples were determined by accessing the Oncomine Research Edition (http://www.oncomine.org, v4.5; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The expression pattern of TWEAK and Fn14 in tumor and non-tumor regions was obtained from six cohorts including Bhattacharjee's (27), Hou's (28), Landi's (29), Selamat's (30) and Su's cohort (31). The TWEAK expression pattern in 151 lung cancer cell lines was obtained from the Cancer Cell Line Encyclopedia (CCLE) database (expression data was obtained from Oncomine) (32) and the heatmap was made by a web-based tool Morpheus (https://software.broadinstitute.org/morpheus/). TWEAK and Fn14 expression in Calu-3, A549, H1299, CL1-0, CL1-5 and H209 cells was obtained from GEO datasets (accession no. GSE7670 and GSE36133) (31,32).
Assessment of the patient survival rate on KM Plotter database
The survival analysis in lung cancer patients with different expression levels of TWEAK and Fn14 was performed through the KM-Plotter database (33). The prognostic value of each gene was analyzed by splitting patient samples into two groups by the median, after the subtype (adenocarcinoma and squamous cell carcinoma) of lung cancer was chosen. The relapse-free survival rate was analyzed (2016.04.08 update, the lung cancer database included 1,926 samples).
Cell culture
The human lung adenocarcinoma cell lines, CL1-0 and CL1-5, were kindly provided by Dr Pan-Chyr Yang (Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan) (34). Human lung carcinoma cell line A549 and a non-small cell lung cancer cell line H1299 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). CL1-0, CL1-5, A549 and H1299 cells were maintained in RPMI-1640 medium which was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Life Technologies, Grand Island, NY, USA) in an incubator at 37°C in 5% CO2.
Recombinant protein and chemicals
Recombinant human TWEAK was obtained from R&D Systems (Minneapolis, MN, USA). All chemicals and buffer were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Proliferation assay (WST-1 assay)
The effect of TWEAK protein on cell proliferation was evaluated by WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3- benzene disulfonate) (Clontech, Mountain View, CA, USA). Briefly, 5×103 CL1-0, CL1-5, A549 and H1299 cells were respectively seeded in 96-well plates overnight. Before TWEAK treatment, the medium was replaced by RPMI-1640 with 0.5% FBS for 6 h. The proliferation rate was determined at a wavelength of 450 nm on a microplate spectrophotometer (PowerWave X340; BioTek, Winooski, VT, USA).
Transwell migration assay
Transwell cell migration assay was performed through QCM™ 24-well Cell Migration Assay and Invasion System uncoated 8-µm pore size polycarbonate membranes (Millipore, St. Charles, MO, USA) according to the manufacturer's instructions. A549, CL1-0, CL1-5 or H1299 (3×105) cells were seeded into a 24-well insert in 300 µl of serum-free medium, while 500 µl medium with 10% FBS was placed in the lower chamber and then were incubated for 24 h. The Transwell membrane was fixed with 4% formaldehyde solution followed by 1% crystal violet staining. After removal of the cells on the upper surface via a cotton swab, images of the bottom membrane were then captured using an Olympus inverted microscope at ×100 magnification.
Wound healing assay
Lung cancer cells (2×105) were seeded into 24-well plates. When cells reached a complete confluent monolayer, a scratch was made using a 200-µl pipette tip. Cell debris was removed by phosphate-buffered saline washing after scratching. Then, the cells were incubated in RMPI-1640 medium with 10% FBS for 24 h. The images and quantitative results were performed via Leica Applications Suite version 4.5.0™ (LAS v4.5) software (Leica Microsystems, Wetzlar, Switzerland).
Quantification of TWEAK levels in serum
The use of the serum samples in the present study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital. Serum samples from the patients with lung cancer and healthy donors were obtained at Kaohsiung Medical University Hospital (Kaohsiung, Taiwan) after patients provided informed consent. Blood from all donors was drawn in serum separation tubes and then stored in aliquots at −80°C. The serum levels of TWEAK were quantitated using MILLIPLEX MAP Human Cancer/Metastasis Biomarker Magnetic Bead Panel (Millipore) according to the manufacturer's instructions. Data were acquired on Luminex xMAP technology (Millipore). For concentration calculation, a cubic spline-fit method was performed using the Milliplex Analyst Software (Viagene Tech, Carlisle, MA, USA).
Statistical analysis
All bar graphs and statistics were performed using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). Student's t-test and one-way ANOVA were respectively used for analysis of the difference between two groups and more than two groups. P-value <0.05 was considered to indicate a statistically significant difference.
Results
Evaluation of TWEAK and Fn14 expression in human lung cancer samples from online microarray databases
Previous studies have revealed that TWEAK and Fn14 are associated with the progression of several human cancers. In the present study, the expression patterns of TWEAK and Fn14 in human cancers were evaluated through an online database Oncomine (Fig. 1A and B). Compared to TWEAK, high expression of Fn14 was observed in multiple human cancer types except bladder, kidney, leukemia, liver and lung cancer. In Fig. 1A, six independent datasets showed that TWEAK expression in lung cancer samples was significantly lower than that in normal samples. Small cell lung cancer (SCLC; ~10–15%) and non-small cell lung cancer (NSCLC; ~85–90%) are two major types of lung cancer and adenocarcinoma, squamous cell and large cell carcinomas, are main types of NSCLC (35). We further analyzed mRNA expression patterns in 151 human lung cancer samples from the Cancer Cell Line Encyclopedia database (GSE36133) (32). In general, Fn14 expression was found to be relatively low in SCLC compared to NSCLC (Fig. 1C, the list of complete cell lines is shown in Table I). Furthermore, most NSCLC cases expressed relatively low levels of TWEAK and high levels of Fn14. These results suggest that the role of the TWEAK/Fn14 signaling pathways may be different between NSCLC and SCLC.
Adenocarcinoma and squamous cell carcinoma are two major types of NSCLC. In order to further investigate the role of the TWEAK/Fn14 signaling pathways, six independent microarray datasets were collected on the Oncomine database and expression patterns in adenocarcinoma and squamous cell carcinoma were investigated. In adenocarcinoma, TWEAK expression was found to be lower and Fn14 expression was higher compared to the normal among all selected datasets (Fig. 2A). Since the number of squamous cell carcinoma patients was less than the number of adenocarcinoma patients in the clinic, the expression of TWEAK and Fn14 was only available in 1 and 2 datasets, respectively. Relatively low TWEAK expression was observed in squamous cell carcinoma in comparison with the normal cells (Fig. 2B). The correlation of TWEAK and Fn14 expression and the survival rate was evaluated by KM Plotter database. In Fig. 2C-F, high expression of Fn14 was found to be associated with poor outcome in adenocarcinoma. However, the survival rate was not associated with TWEAK in both adenocarcinoma and squamous cell carcinoma. This evidence through bioinformatic analysis suggests that low TWEAK expression may be a characteristic of NSCLC.
Effect of exogenous TWEAK treatment on different types of human NSCLC cell lines
To confirm the role of TWEAK and to mimic the effect of exogenous TWEAK in the tumor microenvironment, four human NSCLC cell lines including adenocarcinoma CL1-0 and CL1-5, lung carcinoma A549, and NSCLC H1299 were treated with recombinant TWEAK. The expression of Fn14 in these cells was firstly evaluated in the CCLE dataset (GSE36133) and another online dataset (GSE7670) (31,32). Compared to the SCLC cell line H209 which was observed to have low TWEAK expression and the NSCLC cell line Calu-3 which is reported to express a high level of Fn14 (22), Fn14 expression in A549 was similar to that in Calu-3 and higher than that in H209 (Fig. 3A). As shown in Fig. 3B, differential Fn14 expression among the four cell lines was observed through analysis of GSE7670. The highest level of TWEAK and Fn14 was observed in A549 cells. Our results showed that the proliferation rate of lung cancer cell lines was not enhanced and inhibited by the treatment of different doses of TWEAK in all cell lines (Fig. 3C-F). In addition, the results of Transwell and wound-healing assays revealed that TWEAK did not affect the migration capacity (Fig. 4A-C). Even though TWEAK expression was different, TWEAK treatment did not promote cell proliferation and migration among the four cell lines.
Detection of serum TWEAK concentration in patients with lung cancer
Bioinformatic and in vitro analysis showed that TWEAK expression in NSCLC and exogenous TWEAK treatment did not promote tumor progression. To further investigate the association between TWEAK and lung cancer, the sera of 64 NSCLC patients which included 47 patients with adenocarcinoma, 10 patients with squamous cell carcinoma, and 7 patients with other types of NSCLC, and 80 normal controls were collected. The characteristics of the patients and normal controls are listed in Table II. Our results showed that TWEAK levels in normal controls were significantly higher than that in NSCLC patients (Fig. 5A). This may imply that TWEAK plays a different role in healthy individuals and NSCLC patients. In addition, NSCLC patients were grouped by their cell type (Fig. 5B), TNM stage (Fig. 5C), and metastasis status (Fig. 5D). There were no significantly different levels in TWEAK among these groups.
Discussion
In multiple types of human cancer, TWEAK/Fn14 signaling pathways promote cell growth, metastasis and invasion in vitro and in vivo. It suggests that TWEAK/Fn14 could serve as a potential therapeutic target. Anti-TWEAK mAb RG7212 exerts an antitumor effect in various types of cancer cell lines, including the NSCLC cell line Calu-3 which expresses a high level of Fn14 (22). In addition, the therapeutic effect of RG7212 is associated with Fn14 expression (22). A study of a phase I clinical trial of RG7212 which is a TWEAK antibody showed that RG7272 treatment is safe (36). In patients with solid tumors (including two NSCLC patients), only high exposure of RG7212 treatment resulted in a decrease in the Fn14 signaling pathway (37). This indicates that targeting TWEAK/Fn14 signaling pathway may be a novel antitumor strategy. Our bioinformatic analysis indicated that Fn14 expression in adenocarcinoma is higher than that in normal tissue. This may imply that RG7212 is beneficial for treating lung adenocarcinoma.
In contrast, bioinformatic analysis showed lower TWEAK expression in both lung adenocarcinoma and squamous cell carcinoma. Therefore, we suppose that TWEAK which is secreted from tumor-infiltrating immune cells may promote tumor progression in physiological condition. Surprisingly, TWEAK treatment did not trigger cell proliferation and migration in four NSCLC cell lines even though endogenous TWEAK and Fn14 expression was found to be different among these cell lines. Furthermore, the serum TWEAK level was significantly low in patient compared to normal tissues. This may imply that endogenous TWEAK expression and TWEAK treatment may not be involved in Fn14 signaling pathway-mediated NSCLC progression.
Unique TWEAK/Fn14 expression and serum TWEAK concentrations were observed in the present study, in ovarian cancer patients and head and neck cancer patients. TWEAK expression was detected in malignant ovarian cancer tissues but the expression of TWEAK/Fn14 did not correlate with the patient subtype, stage, or pathological features (38). In the patients with head and neck cancer, a low serum TWEAK level was found to be associated with a poor recurrence-free survival rate (39). CD163 which is another receptor of TWEAK may be a potential factor which affects TWEAK/Fn14 signaling pathways (40). CD163 is expressed on the surface on monocytes and macrophages and the soluble form of CD163 is constitutively detected in serum (41). Bover et al proposed that CD163 may serve as a scavenger of soluble TWEAK in pathological condition (42). Thus, we hypothesized that the interaction of TWEAK and Fn14 may be a critical factor affecting the TWEAK/Fn14 signaling pathway in the tumor microenvironment in lung cancer, particularly in NSCLC. Investigation of soluble CD163 level and CD163/TWEAK interaction in tumor-infiltrating macrophages may provide a novel regulatory mechanism. In addition, the effect of anti-TWEAK antibody RG7212 on CD163/TWEAK may warrant further investigation in animal tumor models or clinical trials in NSCLC or lung adenocarcinoma.
Compared to normal lung tissues, higher level of Fn14 was observed in the bioinformatic analysis (Fig. 1A). High Fn14 expression was associated with poor survival in lung adenocarcinoma (Fig. 2D). This finding suggests that the Fn14 signaling pathway plays a critical role in tumor progression. Previous studies have demonstrated that Fn14 expression is enhanced by Src, HGFR/MET, and EGFR mutation (exon 19 deletion) (24–26). These factors may also affect CD163 and TWEAK interaction and downstream signaling pathways. Therefore, the role of TWEAK should be investigated in consideration of CD163 and Fn14 expression and the status of other signaling molecules in the clinic.
In NSCLC, our results suggest that TWEAK expression is relatively low and Fn14 expression is high compared to normal lung tissues. In addition, TWEAK expression is not associated with a relapse-free survival rate but high Fn14 expression is associated with poor prognosis in lung adenocarcinoma. We found that TWEAK treatment did not promote cell proliferation and migration. Although the TWEAK-mediated signaling pathways could not be evaluated in serum samples, and the serum sample size was small in the present study, our results revealed that a low serum TWEAK level may be a feature of NSCLC in the clinic (Fig. 6). Further studies on the interaction among TWEAK, soluble CD136, and Fn14 may further clarify the role of TWEAK in NSCLC.
Acknowledgements
The present study was supported by grants from the Ministry of Science and Technology (MOST 106-2314-B-037-016-MY2; MOST 104-2314-B-037-053-MY4; MOST 104-2320-B-037-014-MY3; MOST 103-2320-B-037-006- MY3), the KMU-KMUH Co-Project of Key Research (grant no. KMU-DK 107009 from Kaohsiung Medical University) and the Kaohsiung Medical University Hospital Research Foundation (KMUHS10601; KMUH105-5R14).
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