Abstract
Background/Aim: The aim of this study was to evaluate the role of toll-like receptor 2 (TLR2) in the proliferation of human lung cancer cells and identify the signaling pathway that mediates this effect. Materials and Methods: Adenocarcinoma (A549 and H1650) and adenosquamous (H125) cells were treated with increasing doses of Pam3CSK4, a TLR2 agonist. Cell proliferation and NF-ĸB activation were evaluated. NF-ĸB was inhibited prior to treatment with Pam3CSK4 and proliferation was assessed. Results: TLR2 expression was significantly higher in A549 and H1650 cells compared to H125 cells (p<0.001). TLR2 stimulation induced proliferation in adenocarcinoma cells only and led to a corresponding increase in NF-ĸB activity (p<0.05). Inhibition of NF-ĸB prior to treatment with Pam3CSK4 attenuated this proliferative response. Conclusion: TLR2 activation induced proliferation of lung adenocarcinoma cells through activation of NF-ĸB. Thus, the TLR2 signaling pathway may be a potential therapeutic target in lung adenocarcinoma.
- Lung cancer
- non-small cell lung cancer
- lung adenocarcinoma
- toll-like receptor 2
- nuclear factor-kappa B
- proliferation
In the United States, cancer is the second leading cause of death for all ages and the leading cause of death for adults aged 40 years and older. Lung cancer is the second most common cancer and the most lethal cancer in both men and women, accounting for nearly 25% of all cancer-related deaths (1). Despite advancements in various medical and surgical treatment options, long-term survival remains a significant challenge (2). Small cell and non-small cell lung cancer (NSCLC) are the two primary types of lung cancer in the United States, with 85% of cases classified as NSCLC (3). Adenocarcinoma represents the predominant subtype of NSCLC, accounting for 34% of male and 41% of female lung cancer diagnoses in the United States.
Innate immunity has been implicated in the pathogenesis of multiple different malignancies, with many novel therapeutic targets aimed at modulating specific components of the immune system (4, 5). Although certain aspects of the innate immune system have been studied and successfully targeted in NSCLC, these initial efforts have only made a small impact in the treatment success for patients with NSCLC (6, 7). Toll-like receptors (TLRs) are a family of proteins that play a key role in the innate immune system by recognizing pathogen-associated molecular patterns, or PAMPs. Dysregulation of TLR signaling within the gastrointestinal tract has been associated with cancer development (8-10). Additionally, previous work by our group has demonstrated a potential role of TLRs in the development and progression of esophageal adenocarcinoma (11, 12).
Growing evidence suggests that TLRs may serve as prognostic markers in NSCLC, with the degree of TLR expression correlating with overall survival (13). Pam3CSK4, a highly-specific synthetic agonist of toll-like receptor 2 (TLR2), mimics bacterial lipoproteins commonly present in the cell walls of gram-positive and gram-negative bacteria (14-16). Gowing et al. demonstrated that gram-positive pneumonia can stimulate rapid growth of human lung cancer cells via a TLR2-mediated response (17). In addition, TLR2 has been demonstrated to serve as a specific mediator between lung cancer cells and mesenchymal stem cells present within the tumor microenvironment, facilitating cross-talk that ultimately promotes tumor-supportive phenotypic changes of mesenchymal cells (18).
The specific role and mechanism of TLR2 in promoting tumor growth in NSCLC remains unclear. This study aimed to evaluate TLR2 expression levels in NSCLC subtypes and examine the influence of TLR2 on the growth and proliferation of human lung cancer cells. We hypothesized that TLR2 activation promotes proliferation of lung cancer cells via activation of nuclear factor kappa B (NF-ĸB).
Materials and Methods
Cells and reagents. A549 and H1650 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA), while the H125 cell line was obtained from the University of Colorado Cancer Center Tissue Culture Core (Aurora, CO, USA). All cell lines were maintained in Roswell Park Memorial Institute Medium (RPMI 1640; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS), 0.5% penicillin/streptomycin, and gentamicin/amphotericin (1:500 dilution). Serum-reduced media contained 1% FBS, 0.5% penicillin/streptomycin, and gentamicin/amphotericin (1:500 dilution). Cells were incubated at 37°C in humidified 95% air and 5% carbon dioxide.
Western blot antibodies included phosphorylated NF-ĸB, total NF-ĸB, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Cell Signaling Technology (Beverly, MA, USA), as well as toll-like receptor 4 (TLR4) from Santa Cruz Biotechnology (Dallas, TX, USA) and TLR2 from Boster Biological Technology (Pleasanton, CA, USA). GAPDH was used as the housekeeping protein to standardize protein concentrations for all western blots. Western blot antibodies were diluted in 4% bovine serum albumin (BSA). For immunofluorescence microscopy, we utilized the NF-ĸB p65 (D14E12) XP® (Cell Signaling Technology, Beverly, MA, USA) antibody. This antibody was diluted in phosphate-buffered saline (PBS) containing 1% BSA. The TLR2 agonist Pam3CSK4 (InvivoGen USA, San Diego, CA, USA) was dissolved in 1 ml of molecular grade sterile water for a stock concentration of 1 mg/ml.
Western blotting. This technique was adapted from previously published studies (11, 19, 20). Following cell treatments, cells were washed with PBS twice followed by lysis with 2x Laemmli Sample Buffer (Bio-Rad, Hercules, CA, USA) containing 5% 2-mercaptoethanol. Cell lysates were heated and loaded into a 4-20% linear gradient polyacrylamide gel, and proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose membranes and membranes were then blocked with PBS-Tween 20 containing 5% non-fat milk for 1 h. Membranes were probed for the aforementioned primary antibodies and incubated overnight at 4°C. Rabbit and mouse secondary antibodies were diluted in PBS-Tween 20 with 5% non-fat milk and allowed to incubate for 1 h. Membranes were developed with SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, USA) using Image Lab 5.0 Software (Bio-Rad, Hercules, CA, USA). Protein densitometric analysis was performed with ImageJ Software (National Institutes of Health, Bethesda, MD, USA).
Cell proliferation assay. Cells were plated on 96-well plates at a concentration of 1´105 cells/well. Following cell treatments, cell proliferation was evaluated using the MTS Cell Proliferation Colorimetric Assay Kit according to manufacturer's instructions (BioVision Incorporated, Milpitas, CA, USA). Briefly, 20 μl of MTS reagent was added to each well and allowed to incubate at 37°C for 30 min. Following incubation, optical density was read at 490 nm using a microplate reader.
Enzyme-linked immunosorbent assay (ELISA). NF-ĸB activation in nuclear extracts was measured using the Active Motif TransAM® NF-ĸB Family ELISA kit according to manufacturer's instructions (Carlsbad, CA, USA). In brief, following cell treatments, 5 μg of nuclear extracts from each sample were collected and incubated in a 96-well plate coated with immobilized NF-ĸB consensus oligonucleotides for 1 h. The ELISA plate was washed three times with wash buffer and then incubated with primary antibodies against the NF-ĸB subunit p65 for 1 h. The plate was washed again three times with wash buffer followed by incubation with an HRP-conjugated secondary antibody for 1 h. An HRP substrate was added causing a colorimetric reaction and absorbance was measured at 450 nm.
Immunofluorescence. Cells were plated at 5´104 cells/chamber in Lab-Tek® Chamber Slide™ 8-well glass slides (Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 24 h prior to the start of cell treatments. Imaging was performed as previously described with the following modifications: following treatments, plates were rinsed with PBS two times, permeabilized with 70% methanol/30% acetone for 5 min, washed with PBS, allowed to dry, and fixed with 4% paraformaldehyde for 15 min (19, 21, 22). Slides were then washed with PBS and blocked in 10% rabbit serum for 30 min. Slides were incubated with primary antibody overnight at 4°C using the above-mentioned NF-ĸB p65 antibody (5 μg/ml). After washing with PBS, slides were incubated with Cy3-conjugated secondary antibody for 2 h at room temperature. Bis-benzimide staining of nuclei (4’,6-diamidino-2-phenylindole, DAPI) was imaged on the blue channel, while NF-ĸB staining was imaged on the red (Cy3) channel. Microscopic photography was performed with a Leica CTR5500 digital microscope (Leica Mikroskopie und Systeme GmbH, Wetzlar, Germany).
Measurement of baseline TLR2 expression. Cells were plated onto 12-well plates at a density of 2x105 cells/well and grown in full strength RPMI until 80% confluence was achieved. The cells were then serum-reduced for 24 h followed by cell lysis for western blot analysis. For cells undergoing immunofluorescence, cells were serum-reduced for 24 h followed by development.
Cell proliferation following TLR2 activation. Cells were plated on 96-well plates at a concentration of 1x105 cells/well. Cells were treated with Pam3CSK4 (0 μg/ml, 5 μg/ml, 10 μg/ml) diluted in serum-reduced media for 48 h. The MTS proliferation assay was performed as described above.
Measurement of NF-ĸB activity following TLR2 activation. Cells were stimulated with Pam3CSK4 at a dose of 10 μg/ml diluted in serum-reduced media for increasing time periods (5, 10, 30 min, 1, 2, 4, 8, and 24 h). Controls were incubated in serum-reduced media without Pam3CSK4 for 24 h. Cells were then lysed for western blot or ELISA analysis. Phosphorylated NF-ĸB, total NF-ĸB, and GAPDH protein expression were measured at each time point. Immunofluorescence microscopy for NF-ĸB p65 subunit was performed following treatment with 10 μg/ml Pam3CSK4 for 15, 30, and 60 min. Controls were treated with serum-reduced media without Pam3CSK4 for 1 h.
Cell proliferation following NF-ĸB inhibition and TLR2 activation. Cells were treated with Bay 11-7082 (5 and 25 μM in DMSO), vehicle control (DMSO 0.005%), or negative control (serum-reduced media) followed by treatment with 10 μg/ml Pam3CSK4 for 48 h. The MTS proliferation assay was performed as described above.
Statistical analysis. For all western blot results, the density of the band of interest was divided by the density of GAPDH from the same membrane to create a normalized ratio. The normalized ratio of each treatment group was then divided by the normalized ratio of the control from the same experimental plate and multiplied by 100, giving the control an adjusted percentage value of 100. For western blot results involving NF-ĸB, the phosphorylated and total protein densities were normalized to their respective GAPDH. The normalized phosphorylated protein ratio was then divided by the normalized total protein ratio. This value was divided by the normalized ratio of the control from the same experimental plate and then multiplied by 100, giving the control an adjusted percentage value of 100. For cell proliferation results, the absorbance value of each well was divided by the absorbance value of the control and multiplied by 100, giving the control an adjusted percentage value of 100. By normalizing to the control, the comparison of results between different experiments was allowed.
Treatment in a single cultured group of cells was regarded as n=1. Analysis of variance (ANOVA) with a Fisher's post hoc test was used to compare 3 or more groups. A p-value of <0.05 was considered statistically significant and all tests were two-tailed. The standard error of the mean was used for error bar calculations. All histograms and statistical analysis were generated in Microsoft® Excel® (Redmond, WA, USA) and StatPlus:mac Pro (AnalystSoft Inc., Walnut, CA, USA).
Results
TLR2 is expressed in NSCLC cells. TLR2 expression was readily detectable by western blot analysis in all cell lines (Figure 1). A549 and H1650 cells demonstrated significantly higher levels of baseline TLR2 expression compared to H125 cells (p<0.001). TLR2 expression did not significantly differ between A549 and H1650 cell lines (p>0.05).
TLR2 activation increases proliferation of human lung adenocarcinoma cells. Treatment with Pam3CSK4 induced proliferation of the adenocarcinoma cell lines A549 and H1650 (Figure 2). The maximal growth rate was seen at a dose of 10 μg/ml. Compared to the untreated control, A549 cells showed a 33% increase in proliferation (p=0.004) and H1650 cells showed a 19% increase (p<0.001). TLR2 stimulation of H125 cells resulted in a 16% decrease in growth rate in response to 10 μg/ml Pam3CSK4 (p=0.039).
Activation of TLR2 increases NF-ĸB activity in human lung adenocarcinoma cells. Based on the cell proliferation assay results, we determined 10 μg/ml of Pam3CSK4 to be the optimal dose to treat cell lines. Cells were treated at this dose for varying time intervals up to 24 h and NF-ĸB phosphorylation was assessed (Figure 3). NF-ĸB reached a peak activation in both adenocarcinoma cell lines after 30 min (A549 p=0.0003; H1650 p=0.0013). The adenosquamous cell line (H125) demonstrated a decrease in NF-ĸB activity when treated with Pam3CSK4 at the same dose, with a nadir in activity after 1 h (p=0.0006).
Using the A549 cell line, we also evaluated NF-ĸB activation utilizing an ELISA assay to detect DNA-binding activity of NF-ĸB p65. p65 demonstrated a significant increase in DNA-binding activity after treatment with 10 μg/ml Pam3CSK4 for 30 min (p=0.007; Figure 4A). This result was confirmed by measuring the nuclear translocation of NF-ĸB p65 via immunofluorescence. A549 cells demonstrated an increase in NF-ĸB p65 nuclear concentration between 30 and 60 min following TLR2 activation with Pam3CSK4 (Figure 4B).
NF-ĸB inhibition decreases TLR2-mediated proliferation in NSCLC cells. Given that TLR2 stimulation led to increased proliferation and activation of NF-ĸB, we examined the effect of NF-ĸB inhibition using Bay 11-7082 in the presence of Pam3CSK4. Cells were pretreated with Bay 11-7082 at increasing doses (5 μM or 25 μM) for 1 h and then underwent stimulation with 10 μg/ml Pam3CSK4 for 48 h. The MTS proliferation assay demonstrated a significant decrease in the growth rate of all lung cancer cell lines when treated with the NF-ĸB inhibitor in the presence of the TLR2 agonist. There was no significant difference in proliferation between the untreated control and DMSO vehicle control (p>0.05). When compared to treatment with Pam3CSK4 alone, A549 cells demonstrated a 38% reduction in growth rate when treated with Pam3CSK4 plus 5 μM Bay 11-7082 (p<0.0001) and a 37% reduction in growth rate when treated with Pam3CSK4 plus 25 μM Bay 11-7082 (p<0.0001; Figure 5A). H1650 cells demonstrated a 55% reduction in growth rate and 57% reduction in growth rate when treated with Pam3CSK4 plus 5 μM (p<0.0001) and 25 μM (p<0.0001) Bay 11-7082, respectively (Figure 5B). H125 cells demonstrated a 13% reduction in growth rate with Pam3CSK4 plus 5 μM Bay 11-7082 (p=0.03) and a 14% reduction in growth rate with Pam3CSK4 plus 25 μM Bay 11-7082 (p=0.03; Figure 5C).
Discussion
The aim of this study was to determine the impact of TLR2 stimulation on the growth and proliferation of human NSCLC cells as well as to identify the signaling pathway mediating this effect. Our findings indicate the presence of TLR2 in NSCLC cells, with higher levels of expression in adenocarcinoma cell lines compared to a non-adenocarcinoma cell line. Activation of TLR2 by the specific agonist Pam3CSK4 increases the growth rate of lung adenocarcinoma cells, which is mediated by the TLR2/NF-ĸB pathway. Concurrent stimulation of TLR2 and inhibition of NF-ĸB led to a decrease in the growth rate of adenocarcinoma cells. These results demonstrate the importance of TLR2 and the NF-ĸB pathway in the proliferation of lung adenocarcinoma tumor cells.
Our results are consistent with those of recent studies regarding the impact of TLR2 on lung cancer growth. We have reproduced results reported on TLR2 stimulation and increased proliferation in A549 cells (14). In addition to this, we extended the scope of this finding by demonstrating that NF-ĸB is activated in two different adenocarcinoma cell lines upon TLR2 stimulation, and inhibition of this pathway decreases TLR2-mediated cell proliferation. Recent literature demonstrates that several epithelial-derived carcinomas are dependent upon the expression and activation of TLR2 for further progression of disease (23-25). Here, we examined multiple human lung cancer cell lines and identified varying levels of expression of TLR2. Not surprisingly, cell lines with higher levels of TLR2 expression appeared to be more responsive to growth stimulation by a TLR2 agonist. This differing level of expression and correlating effect on proliferation suggests that TLR2 may represent a potential target for therapy, such that tumors that overexpress TLR2 may be candidates for inhibitor treatment.
Growing evidence supports a link between chronic inflammation and carcinogenesis, with an increasing emphasis on the role of TLRs and NF-ĸB (26-30). A recent study utilizing human tissue samples of intrahepatic cholangiocarcinoma demonstrated that increased TLR2 expression correlated with increased oncologic stage, as well as increased activation of NF-ĸB (25). In this same study, treatment with NF-ĸB inhibitor Bay 11-7082 led to inhibition of tumor cell migration and cytokine production. Likewise, multiple studies have shown that expression and activation of TLR2 may contribute to melanoma metastasis (31, 32). The direct effects of TLR2 stimulation on the lung tissue and subsequent inflammation have been explored, however few studies to date have evaluated the effects of TLR2 stimulation among different NSCLC subtypes (33).
This study has demonstrated for the first time that, in the presence of a TLR2 agonist, the TLR2/NF-ĸB signaling pathway is in part responsible for the increased proliferation of human lung adenocarcinoma cells. We demonstrated that NF-ĸB is not only activated, but also undergoes nuclear translocation and DNA binding when TLR2 is stimulated with Pam3CSK4. Furthermore, inhibition of NF-ĸB resulted in decreased growth of lung adenocarcinoma cells. Some malignancies demonstrate lower levels of TLR2 expression and NF-ĸB activity in comparison to inflamed, benign tissue of the same type (34). This result is consistent with our non-adenocarcinoma H125 cell line, in which TLR2 stimulation caused both NF-ĸB suppression and decreased cell growth rate. This could potentially be explained by increased production of anti-inflammatory cytokines (e.g. IL-6, IL-10, TGF-β), decreased production of pro-inflammatory cytokines (e.g. IL-1, IL-12, IL-18, TNF-a), or the activation of other inhibitory processes that occur in response to TLR2 stimulation in this specific NSCLC subtype.
Our findings suggest that further investigation of the TLR2/NF-ĸB pathway and its impact on proliferation of lung cancer tumor cells is warranted. The downstream effects of TLR2 stimulation should be further evaluated, with a focus on the specific changes in cytokine production that occur across these different NSCLC subtypes. Additionally, further evaluation of the TLR2/NF-ĸB signaling pathway constituents, including MyD88, IRAK-4, IRAK-1, and TRAF6, amongst others, will provide a better understanding of the downstream effects following TLR2 activation. The use of TLR2/NF-ĸB antagonists, such as pyrrogallol or N-benzylideneaniline derivatives, applied to in vitro and in vivo models may provide future potential therapies for investigation in human clinical trials (35, 36).
The results of this study demonstrate that TLR2 stimulation has a direct influence on the growth and proliferation of lung adenocarcinoma cells, which is dependent upon the NF-ĸB signaling pathway. In contrast, non-adenocarcinoma cells exhibit lower baseline expression of TLR2 and decreased growth following TLR2 stimulation, which warrants further investigation. TLR2 and NF-ĸB should continue to be evaluated as important potential therapeutic targets for patients with lung adenocarcinoma.
Footnotes
Authors' Contributions
Anna K. Gergen assisted with data analysis and manuscript writing. Patrick D. Kohtz and Alison L. Halpern assisted with data collection and analysis as well as manuscript writing. Anqi Li, Xianzhong Meng, T. Brett Reece, David A. Fullerton, and Michael J. Weyant assisted with project design and manuscript writing.
This article is freely accessible online.
Conflicts of Interest
The Authors have no conflicts of interest to disclose in relation to this study. The Authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article. This work was funded by the University of Colorado School of Medicine Department of Surgery, Division of Cardiothoracic Surgery. There is no outside funding or grants to report.
- Received June 26, 2020.
- Revision received July 14, 2020.
- Accepted July 15, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved