Introduction
17-β-Estradiol (E2) has been known for decades as the primary reproductive hormone, as it is the major and most potent product synthesized by the ovaries in premenopausal females [
1,
2]. Since estrogen can also be synthesized by the placental syncytiotrophoblast, adipose tissue, skin fibroblasts, the bone, and the brain in both sexes, estrogen is known to play crucial physiological roles in several nonproductive systems, such as the cardiovascular, neuronal and skeletal systems [
3‐
5]. However, a recent Women’s Health Initiative (WHI) study conducted with over 16,000 postmenopausal women receiving estrogens for daily hormone replacement therapy (HRT) revealed an increasing risk of lung cancer mortality in these women compared to women receiving a placebo treatment for 5 years [
6]. Clinical trials indicated that estrogen exposure is a risk factor for lung carcinogenesis. Studies have continued to provide evidence that estrogen receptors (ERs), mainly subtype ERβ, are consistently expressed in lung cancer tissues and adenocarcinoma cell lines [
7‐
9] and that high expression of ERβ predicts a very poor prognosis in lung cancer patients [
7,
10‐
12]. Consistently, our previous studies first detected a significantly higher expression level of ERβ in metastatic lymph nodes of non-small cell lung cancer (NSCLC) patients than in primary tumor tissue. The results further indicated that treatment with estrogen or ERβ activation also significantly promotes lung cancer cell metastasis by upregulating invasiveness-associated matrix metalloprotease 2 (MMP2) both in vitro and in vivo [
13]. Therefore, to markedly depress NSCLC metastasis and to finally detect a new treatment strategy for advanced lung cancer patients, inhibiting the effect induced by estrogen on NSCLC cells is essential.
Antiestrogens demonstrate impressive efficacy in the suppression of tumor progression and metastasis in the context of breast cancer [
2,
3]. Accumulating evidence now supports a potential impactful role for antiestrogen reagents in inhibiting NSCLC development through ERβ. Data from urethane-induced primary NSCLC mouse models indicate that the inhibition of ERβ by fulvestrant can significantly reduce lung cancer formation and pleural metastasis [
14]. In NSCLC cell lines, proliferation-associated IGF-1 and invasion-associated MMP2 expression levels are downregulated in response to antiestrogens in vitro [
13,
15]. A clinical study including 26 women with second primary lung cancer among 6361 diagnosed breast cancer patients provided evidence of a longer cancer-specific survival rate in the patients treated with antiestrogens for breast cancer compared with those treated without antiestrogens [
16]. In current clinical practice, a phase I study showed the safety and potential antitumor activity of the combined use of an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) and fulvestrant in postmenopausal women [
17]. Nonetheless, no significant difference in the progression-free survival rate was detected in another phase II clinical study, which aimed to evaluate whether the addition of fulvestrant enhances the antitumor efficacy of erlotinib (a different EGFR-TKI) [
18]. The results above suggest that it is still uncertain whether NSCLC patients can benefit from fulvestrant treatment. They also reveal that the understanding of antiestrogen theory remains incomplete and that a potential mechanism should be determined to enhance the antimetastatic effect of antiestrogen treatment strategies.
Toll-like receptor 4 (TLR4) is known as an important receptor in innate immunity and the first line of host defense [
19]. The agonist that triggers TLR4 signaling, lipopolysaccharide (LPS), has been proven to be a major component of the outer membrane of Gram-negative bacteria [
20]. Recent studies have provided evidence that TLR4 activation augments NSCLC cell adhesion to murine hepatic sinusoids and the formation of hepatic metastases in vivo, and an upregulation of MMP2 expression in response to LPS induction has been identified [
21]. Other studies highlighted that TLR-4 increased the rate of lung metastasis in inflamed mice through the upregulation of C–C chemokine receptor type 2 (CCR2) expression [
22] and proved that the TLR4/Myd88/NF-κB/MMP2 axis plays a crucial role in cancer metastasis [
23,
24]. CLI-095, also known as TAK-242, is a novel cyclohexene derivative that specifically suppresses TLR4 signaling [
25]. Although novel inhibition of NSCLC cell proliferation induced by knocking down TLR4 expression has been detected in vitro [
26], the antimetastatic effect of specific TLR4 signaling inhibition is still unclear.
For this study, immunohistochemistry was used to detect the expression of ERβ and TLR4 in 180 samples of primary NSCLC tissue and 30 corresponding metastatic lymph node samples, and the relationship between these molecules was analyzed. Then, we used a combination of fulvestrant and CLI-095 to test the hypothesis that inhibiting TLR4 will enhance the antimetastatic effect of fulvestrant by suppressing NSCLC cell migration, invasion and metastasis. Protein expression levels in different groups were detected via Western blot and immunofluorescence analyses. In addition, we also analyzed the duration of formation of the key structure of cell metastasis, the invadopodia, by a 3D spheroid invasion assay and fluorescent gelatin degradation assay. Our results helped us to determine the negative effect of the combination of fulvestrant and CLI-095 on NSCLC metastasis progression and to provide evidence for a new strategy of advanced NSCLC patient treatment.
Materials and methods
Human tissue samples
Tissue samples were obtained from 180 confirmed NSCLC tumors and 30 corresponding metastatic lymph nodes resected between December 2013 and September 2015 and assessed in The Department of Pathology, Affiliated Tongji Hospital of Huazhong University of Science and Technology Tongji Medical College (Wuhan, China). The samples were obtained from 127 males and 53 females (mean age, 50.3 ± 4.7 years; range 21–77 years). Clinicopathological parameters, including age, sex, smoking index, pathological diagnosis and clinical stage, were obtained from the Tongji hospital records. The histological characterization and clinical–pathological staging of the samples was determined according to World Health Organization criteria [
13]. Paraffin-embedded tumor specimens were used to create tissue microarray (Outdo Biotech Co., Ltd., Shanghai, China) blocks with 2-mm diameter cores for immunohistochemical (IHC) staining. Two tissue cores were obtained from each patient. The baseline characteristics of the patients are shown in Table
1. Informed consent was obtained before surgery. All patients involved consented to participate under the Clinical Patient ethical statement. Study approval for the study was obtained from the Research Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IRB ID number 20141101).
Table 1
Correlation between ERβ and TLR4 expression and clinicopathological parameters in 180 cases of primary non-small cell lung carcinoma
Age |
Below median | 78 (43.4) | 28 (15.6) | 0.008 | 0.927 | 86 (47.8) | 20 (11.1) | 0.207 | 0.650 |
Above median | 54 (30.0) | 20 (11.1) | | | 58 (32.2) | 16 (8.9) | | |
Gender |
Male | 91 (50.6) | 36 (20.0) | 0.622 | 0.430 | 102 (56.7) | 25 (13.9) | 0.027 | 0.870 |
Female | 41 (22.8) | 12 (6.7) | | | 42 (23.3) | 11 (6.1) | | |
Smoking index |
< 400 | 84 (46.7) | 35 (19.4) | 1.353 | 0.245 | 90 (50.0) | 29 (16.1) | 0.937 | 0.333 |
≥ 400 | 48 (26.7) | 13 (7.2) | | | 50 (27.8) | 11 (6.1) | | |
Histological type |
Squamous cell | 36 (20.0) | 14 (7.8) | 0.063 | 0.802 | 44 (24.4) | 6 (3.3) | 2.769 | 0.096 |
Adenocarcinoma | 96 (53.3) | 34 (18.9) | | | 100 (55.6) | 30 (16.7) | | |
T stage |
T1–T2 | 112 (62.2) | 36 (20.0) | 2.336 | 0.126 | 119 (66.1) | 29 (16.1) | 0.086 | 0.770 |
T3–T4 | 20 (11.1) | 12 (6.7) | | | 25 (13.9) | 7 (3.9) | | |
N stage |
N0 | 75 (41.7) | 30 (16.7) | 0.468 | 0.494 | 82 (45.6) | 23 (12.8) | 0.571 | 0.450 |
N1–3 | 57 (31.6) | 18 (10.0) | | | 62 (34.4) | 13 (7.2) | | |
Clinical stage |
I–II | 93 (51.7) | 30 (16.7) | 1.029 | 0.310 | 101 (56.1) | 22 (12.2) | 1.085 | 0.298 |
III–IV | 39 (21.7) | 18 (10.0) | | | 43 (23.9) | 14 (7.8) | | |
Details of the 30 metastatic lymph node samples with IIA-IIIB NSCLC and lymph node metastasis were provided in our previous study [
13]. Patients with lymphadenitis or primary malignancies of the lymph node were excluded. Palliative care or surgical biopsy following informed consent was administered to 32 patients with inoperable Stage IIIb-IV primary NSCLC.
Immunohistochemistry
Immunohistochemical staining was carried out using the avidin–biotin peroxidase method [
13,
15]. All sections were deparaffinized in xylene, rehydrated in alcohol, incubated in hydrogen peroxide, blocked in 10% goat serum and then incubated overnight in 0.3% H
2O
2 at room temperature with primary antibodies (rabbit anti-human TLR4 polyclonal antibodies, 1:100, Abcam, Cat: ab13556 and rabbit anti-human ERβ monoclonal antibodies, 1:100, Abcam, Cat: ab3577), Immunoreactivity scores of the cancer tissue samples were determined based on the staining intensity and positive staining area according to the method described in Tang et al. [
14]. A staining index (values 0–16), obtained as the intensity of positive staining [negative (1 scores), weak (2 scores), moderate (3 scores), or strong (4 scores)] and the proportion of immune-staining positive cells of interest [< 25% (1 scores), 25–50% (2 scores), 50–75% (3 scores), ≥ 75% (4 scores)] were calculated. A score of 1–16 was obtained by multiplying the staining intensity and positive cell proportion. A total score > 12 was defined as high expression, a score ≤ 8 was defined as low expression, and a score ≤ 4 was defined as negative expression.
Cell culture and associated reagents
The human NSCLC cell lines A549 and H1793 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The culture condition was described in previous study [
13] and were maintained in RPMI 1640(A549) or DMEM/F-12(H1793) supplemented with 10% fetal bovine serum (FBS). The NSCLC cells were treated with E2 (Sigma-Aldrich, St. Louis, MO, USA), fulvestrant, also known as Faslodex or ICI-182780 (7α-[9-[(4,4,5,5,5-pentafluoropentyl)-sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17β-diol, Cayman Chemical), and CLI-095, also known as TAK242 (ethyl(6R)-6-[N-(2-chloro-4- fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate, InvivoGen, USA) either alone or in combination. The doses of these drugs used in vitro and in vivo were comparable to previously described doses [
13,
15,
27]. Each group of cells was treated for 48 h and harvested for further analysis. Cell culture experiments were performed using reagents formulated in 100% DMSO.
Western blot analyses and Immunofluorescence
Western blot analyses and Immunofluorescence were performed as described by us previously [
13,
27]. Briefly, total protein extract for each cell line was dissolved in lysis buffer and equal amounts of protein (40 μg) were analyzed by immunoblotting. The Immunofluorescence coverslips were observed under a fluorescence microscope (Olympus, Tokyo, Japan).
The primary antibodies used for the Western blot analyses and Immunofluorescence included rabbit anti-human ERβ (1:1,000) from Abcam (Cat: ab3577), rabbit anti-human TLR4 (1:1,000) from Abcam (Cat: ab13556), rabbit anti-human myd88 (1:1,000) from Proteintech (Cat: 23230-1-AP), and mouse anti-human GAPDH (1:10,000) from Cell Signaling Technology (CST; Cat: 51332; USA).
Wound-healing assay
NSCLC cells were seeded in six-well plates for 24 h. After growth to confluence, the surface of the plate was scraped with a 200 μL pipette tip to generate a cell-free zone, and then incubated with RMPI 1640 containing 10% FBS for 24 h. Cells were photographed using a phase-contrast microscope (100×) as previously described [
13].
In vitro Transwell® migration and invasion assays
Transwell migration and invasion assays were performed as described by us previously [
13,
27]. Transwell
® Permeable Supports (24-well supports, inserts were 6.5 mm in diameter) (Corning, NY, USA) were used. In brief, NSCLC cells suspended in serum-free medium were added to the upper chamber at various densities depending on the cell line. Invasion assays were evaluated based on the number of cells invading a Transwell
® membrane coated with Matrigel
® (BD Biosciences, Bedford, MA, USA), and counting was conducted using an Olympus microscope (Olympus, Tokyo, Japan) at 100× magnification. Four fields were randomly selected for analysis. Detailed procedures are described elsewhere.
Migration assays were performed in 24‑well Transwell® chambers containing polycarbonate filters with 8‑μm pores without Matrigel®. The remaining steps were identical to those described for the Transwell® invasion assays.
3D spheroid invasion assay
The Cultrex® 3D Spheroid Cell Invasion Assay Kit (Catalog: 3500-096-K, Trevigen, Gaithersburg, MD) was utilized for this procedure. A total of 1 × 105 cells in 500 mL of medium containing 2.5% Matrigel and 5 ng/mL Spheroid Formation ECM were plated in 24-well plates coated with a collagen/Matrigel mixture. Spheres with protrusions were considered positive for cell invasion. Distances between the invasive cell frontier and spheroid edge were measured at 0, 72 and 144 h using an Olympus IX70 inverted microscope (Olympus, Tokyo, Japan). Each experiment was repeated twice, and each procedure was performed in triplicate.
Fluorescent gelatin degradation assay
A QCM™ Gelatin Invadopodia Assay kit (Green) (Catalog: No. ECM670, Millipore, USA) was utilized for this procedure. Coverslips were cleaned with 20% nitric acid and coated with poly-L-lysine in a 24-well plate. Poly-L-lysine was fixed with 0.5% glutaraldehyde before adding FITC-conjugated gelatin. A thin layer of FITC-conjugated gelatin was applied to the coverslips and crosslinked using glutaraldehyde on ice for 10 min. Crosslinking was continued at room temperature for an additional 30 min. The coverslips were rinsed with PBS, incubated with 5 mg/mL sodium borohydride at room temperature for 3 min, rinsed again with PBS, incubated with 70% EtOH for 10 min, and dried at 37 °C for 15 min in a CO2 incubator. One hour before plating cells, the coverslips were quenched with RPMI-1640 medium containing 10% FBS at 37 °C. Cells were plated on the FITC-conjugated gelatin-coated coverslips and cultured in RPMI-1640 medium for 24 h to quantify the formation of invadopodia. Images were visualized by confocal microscopy (Olympus, Tokyo, Japan).
Statistical analysis
Statistical analysis was performed using SPSS 19.0 statistical software (IBM, New York City, USA). The χ2 test was performed to analyze the correlations among ERβ and TLR4 expression and clinicopathological parameters, and Spearman’s rank correlation was used to analyze the correlations between ERβ and TLR4 expression in primary NSCLC and metastatic lymph node tissue samples. Comparisons between groups were analyzed using an unpaired t test. Data are presented as the mean ± SE. The remaining data are presented as the mean ± SD. p values < 0.05 were considered significant. All statistical analyses were performed using GraphPad Prism (version 7.00, GraphPad, San Diego, CA, USA).
Discussion
Estrogen has recently been shown to be a key promoter in lung cancer development and metastatic progression [
1,
2,
6]. Therefore, estrogen and its receptors, mainly ERβ, have the potential to become prognostic indicators and therapeutic targets in lung cancer [
7,
10,
11]. Similar to hormone replacement therapy clinical trials [
6], our previous work found a marked upregulation of MMP2 expression with increased aggressiveness of NSCLC cells induced by estrogen and the activation of ERβ [
13]. On the other hand, MMP2, which plays roles in degrading the ECM and promoting cell invasion, is also an important component of TLR4-driven cancer metastasis [
24]. Accumulating evidence in vitro and in vivo supports the fact that the activation of TLR4 and the downstream myd88/NF-κB/MMP2 axis via signaling pathways contribute to NSCLC metastasis [
21,
23]. The fact that the mechanism by which TLR4 promotes metastasis is similar to that involving ERβ raises the question of whether a combination therapy targeting both TLR4 and ERβ would show stronger suppression than the use of only a single drug. The answer would help us understand the interaction between ERβ and TLR4 in tumor progression more clearly and provide evidence for a new therapeutic strategy for clinical patients suffering from advanced metastatic NSCLC.
Our results demonstrate the coexpression of ERβ and TLR4 in tissue samples from primary NSCLC tumors and the associated metastatic lymph nodes. To our knowledge, this is the first time that the expression levels of ERβ and TLR4 have been analyzed in lung cancer tissues. However, the fact that ERβ is able to regulate the TLR signaling pathways was previously demonstrated in the physiological immune system [
36]. In dendritic cells and macrophages, multiple studies have shown that ER promotes the production of proinflammatory cytokines in response to TLR ligand stimulation [
37]. A downregulation of TLR4 signaling and decreased secretion of IL-6 and IL-1β have been observed when a ER allele gene is conditionally deleted in myeloid cells [
38]. The results above suggest a molecular basis for the interaction between ERβ and TLR4 in human cells. Consistent with our results, E2 stimulation not only led to upregulated ERβ expression in NSCLC cells but also to increased TLR4 and downstream myd88 expression. Furthermore, the combination of fulvestrant and CLI-095 showed the strongest inhibitory effect on ERβ-mediated suppression, an effect that was stronger than that of fulvestrant alone, which suggests an enhanced inhibitory effect on ERβ signaling by fulvestrant when used in conjunction with a TLR4-specific inhibitor.
We selected fulvestrant for use in these studies, because it is generally regarded as a pure antiestrogen that specifically and selectively inhibits ER signaling in target lung cancer cells [
39,
40]. The antiestrogen inhibitor fulvestrant, as mentioned before, exerts antiproliferative and antimetastatic effects on NSCLC, which have been researched in vitro and in vivo in multiple studies, including our previous research [
13‐
15,
41]. Consistent with the findings of those studies, our results clearly showed that compared with estrogen alone, fulvestrant depressed NSCLC cell migration and invasion and retarded invadopodia formation. Additionally, no significant difference was found between the vehicle and E2 + fulvestrant groups. Therefore, to further enhance the antimetastatic effect of fulvestrant, we combined CLI-095 with fulvestrant in our study. Furthermore, fulvestrant has also been repeatedly added to phase II clinical studies, which mainly aimed to enhance the therapeutic effect of an EGFR-TKI or relieve drug resistance [
17,
18]. However, no significant difference in the progression-free survival rate was detected in these clinical studies, and the clinical effect of monotherapy with an antiestrogen drug remains unknown. In this respect, our results suggested that combined targeting of both ERβ and TLR4 by fulvestrant + CLI-095 may become an efficient way to enhance the clinical effect of antiestrogen therapies. Nevertheless, the indications of fulvestrant will be enlarged by future research, which will help us to understand the mechanism of antiestrogen therapy in NSCLC cells.
In contrast to the effects of targeting fulvestrant, the effects of targeting TLR4 signaling, which is specifically inhibited by CLI-095 (TAK242), have been proven to be significantly effective in inhibiting or blocking cell invasion and metastasis in many malignant tumors, such as ovarian cancer, breast cancer and glioma [
42,
43]. However, research on CLI-095 remains limited to in vitro studies in NSCLC. Currently, the effect of inflammation on tumorigenesis and progression has been widely noted. Research using immunohistochemistry and enzyme-linked immunosorbent assays showed a poor prognosis in TLR4- and programmed cell death ligand 1 (PD-L1)-expressing NSCLC patients [
29]. Increasing numbers of hepatic metastasis nodules of NSCLC cells have been observed in a mouse model treated with LPS [
21]. Furthermore, evidence suggests that knocking down TLR4 expression by siRNA significantly suppresses the constitutive phosphorylation of Akt and PI3K, and this knockdown may contribute to the inhibition of tumor growth [
26]. Although recent studies have provided multiple lines of evidence that the activation of TLR4 and its downstream signaling pathways significantly promotes NSCLC development and metastasis, none of these studies selected CLI-095 to block TLR4 signaling. Our results are the first to prove that CLI-095 treatment can significantly inhibit TLR4 downstream signaling; additionally, our results showed that treatment with CLI-095 suppressed the enhancement of aggressiveness in NSCLC cells induced by estrogen and enhanced the antitumor effect of fulvestrant. The results above may further suggest the possibility that there is an interaction between ERβ and TLR4 at the molecular level.
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