Annexin A1 is a potential biomarker of bone metastasis in small cell lung cancer.

Small cell lung cancer (SCLC) is a subtype of lung cancer with a poor prognosis, with bone metastasis being one of the main causes of treatment failure. Therefore, investigating new biomarkers associated with bone metastasis may result in positive treatment outcomes. The present study detected the expression levels of annexin A1 (ANXA1) in the serum of 82 patients with SCLC using ELISA. ANXA1 expression in patients with SCLC with bone metastasis was significantly higher compared with that in patients without bone metastasis. Receiver operating characteristic analysis revealed that ANXA1 expression was significant in the diagnosis of bone metastasis in SCLC. ANXA1 was inhibited in SBC-5 cells and overexpressed in SBC-3 cells. Results revealed that ANXA1 was able to enhance SCLC cell proliferation, invasion, migration and bone adhesion in vitro. In vivo xenograft bone metastasis assays indicated that ANXA1 had the potential to promote the bone-metastasis ability of SCLC cells in NOD/SCID mice. Furthermore, ANXA1 increased parathyroid hormone-related protein secretion and enhanced Smad2 phosphorylation following TGF-β treatment in SCLC cells. Overall, ANXA1 may be involved in the pathogenesis of bone metastasis in SCLC and may be a potential biomarker for the diagnosis of SCLC. Copyright: © Chen et al.

Introduction

Lung cancer is one of the most frequently diagnosed malignant tumors and the leading cause of cancer-associated death worldwide. For example, in the United States lung cancer caused more deaths than breast, prostate, colorectal and brain cancer combined in 2017, and is projected to account for one-quarter of all cancer-associated deaths in 2020 (1). Histologically, lung cancer can be classified as small cell lung cancer (SCLC) and non-SCLC. SCLC is considered to have the poorest prognosis among all types of lung cancer due to its rapid growth and early distant metastases; clinically, around two-thirds of patients with SCLC present with multiple organ metastases, particularly to the bone (2). Bone metastases are often associated with skeletal-associated events, including pain, hypercalcemia, fracture and nerve compression syndromes, all of which may decrease the quality of life of patients (3,4). The occurrence of bone metastasis directly affects the choice of treatment options (5). Therefore, timely diagnosis of bone metastasis is important to improve the treatment of SCLC. At present, the diagnosis of bone metastasis mainly depends on imaging examination, such as bone scans and computed tomography (CT), which are harmful to patients and not economical; however, due to the rapid progression of SCLC, frequent monitoring of the status of bone metastases is clinically required (6). Therefore, finding biomarkers associated with bone metastasis is important to improve the diagnosis and treatment of patients with SCLC.

Annexin A1 (ANXA1), a member of calcium-dependent phospholipid-binding proteins, serves a role in regulating cell proliferation, apoptosis, phagocytosis and carcinogenesis (7,8). Studies have demonstrated that ANXA1 expression is different in various types of tumor tissue (8,9). For example, ANXA1 expression is upregulated in liver, colorectal and pancreatic cancer, but downregulated in prostate, esophageal and cervical cancer (9). Abnormal ANXA1 expression and the changes of its location may be associated with the differentiation and metastases of malignant tumors (10,11). ANXA1 may be a potential new biomarker for the diagnosis and treatment of tumors; however, the role of ANXA1 in the pathogenesis of SCLC remains unclear. In addition, ANXA1 is involved in regulating bone development and the bone marrow microenvironment (12,13). However, whether this indicates that ANXA1 participates in the bone metastases of tumors requires further confirmation.

The present study analyzed the association between the expression levels of ANXA1 in serum and the clinical characteristics of patients with SCLC, and discussed the diagnostic value of ANXA1 expression in bone metastasis. To further confirm the association between ANXA1 and bone metastasis, ANXA1 was overexpressed in SBC-3 cells and inhibited in SBC-5 cells, and the effects of ANXA1 on the biological behavior of SCLC cells and bone metastasis-associated signaling pathways were investigated.

Materials and methods

Patients and specimens

Serum samples were collected from 82 patients with SCLC who were admitted to Tangdu Hospital of the Air Force Military Medical University (Xi'an, China) between March 2016 and May 2017, and ANXA1 1 expression was detected using a Human Annexin A1 ELISA kit (cat. no. ab222868; Abcam). The present study was based on a cohort of consecutive patients with histopathological diagnosis of SCLC, without inflammatory disease and who had never received chemotherapy or radiotherapy. Detailed information was obtained from the medical records of the enrolled patients in a computerized registry database, including patient age, sex and smoking history. Tumor assessment was performed using CT, radionuclide bone scanning or integrated positron emission tomography-CT. Magnetic resonance imaging or enhanced CT was performed is symptomatic patients. At least one radiologist and one physician confirmed the diagnosis. All tumors were staged according to the pathological tumor/node/metastasis (pTNM) classification (8th edition) of the Union for International Cancer Control (UICC) (14). The study protocol was approved by the ethical review board of Tangdu Hospital. All patients provided written informed consent for use of their medical records and samples for research purposes.

Cell lines and culture

The human SCLC SBC-3 and SBC-5 cell lines were provided by Professor Saburo Sone and Professor Seiji Yano (University of Tokushima School of Medicine, Tokushima, Japan). Both cell lines were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (both Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere of 5% CO2.

Transfection

For ANXA1-knockdown in SBC-5 cells, lentiviral vectors for human ANXA1 short hairpin RNAs (shRNAs) were designed according to the human ANXA1 mRNA sequence (GenBank accession no. NM-000700): shANXA1-1, 5′-AACCATCATTGACATTCTA-3′; shANXA1-2, 5′-CTTGTATGAAGCAGGAGAA-3′; shANXA1-3, 5′-AGCGCAATTTGATGCTGAT-3′; shANXA1-4, 5′-ATTCTATCAGAAGATGTAT-3′; and negative control (non-targeting), 5′-TTCTCCGAACGTGTCACGT-3′. ANXA1 and negative control shRNAs were constructed, packed and purified by Shanghai GeneChem Co., Ltd.. The second generation of self-inactivated lentivirus packaging system was used for virus packaging, which involved three plasmids (Shanghai GeneChem Co., Ltd.): Tool vector plasmid GV248 carrying the target sequence, pHelper 1.0 (carrying gag, pol and rev genes) and pHelper 2.0 (carrying the VSV-G gene). Viral vector generation was obtained by co-transfection of 5×106 293T cells (Shanghai GeneChem Co., Ltd.)/15 ml medium (DMEM with 10% FBS; Gibco; Thermo Fisher Scientific, Inc.) with 20 µg GV248, 15 µg pHelper1.0 and 10 µg pHelper2.0 on 10-cm plates. To overexpress ANXA1 in SBC-3 cells, the cDNA of ANXA1 was transfected into the GV166 virus to build the ANXA1/GV166-LV plasmid, which was then packed into lentiviral vectors. SBC-3 cells and SBC-5 cells were seeded in 6-well plates before transfection. When the cells reached ~80% confluence, SBC-5 cells were either transfected with ANXA1 shRNAs (SBC-5-shANXA1) or the negative control shRNA (SBC-5-NC), and SBC-3 cells were transfected with pcDNA-ANXA1 (SBC-3-ANXA1) or the negative control (empty) vector by co-incubating with Enhance Infection Solution (Shanghai GeneChem Co., Ltd.) for 12 h at 37°C at a multiplicity of infection of 20, after which the medium was replaced by fresh RPMI-1640 with 10% FBS. Three days after transfection, cells were selected using 0.5 µg/ml puromycin and harvested for subsequent experiments.

RT-qPCR

Total RNA was extracted from cells using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT was performed to obtain cDNA using the SuperScript III First-Strand Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. mRNA expression in each cell line was standardized to β-actin. The following primer pairs were synthesized by Shanghai GeneChem Co., Ltd., for qPCR: ANXA1 forward, 5′-TTTTGGCATCAAGAACTAAC-3′ and reverse, 5′-CCTCAGATCGGTCACCCT-3′; parathyroid hormone-related protein (PTHrP) forward, 5′-GGAGACTGGTTCAGCAGTGG-3′ and reverse, 5′-TTGTCATGGAGGAGCTGATG-3′; β-actin forward, 5′-ATCGTGCGTGACATTAAGGACAAG-3′ and reverse, 5′-AGGAAGGAAGGGCTGGAAGAGTG-3′. qPCR was preformed using SYBR Premix Ex Taq™ II (Takara Bio, Inc.). The following thermocycling conditions were used for qPCR: Initial denaturation at 95°C for 3 min; 35 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 1 min; extension at 72°C for 7 min. Each sample was detected in triplicate, and a melting curve was analyzed to confirm amplification specificity. Relative mRNA expression was calculated using the 2−ΔΔCq method (15).

Western blotting

Harvested cells were washed once with PBS and lysed to extract total cellular protein using RIPA buffer (Beyotime Institute of Biotechnology). The proteins were boiled for 5 min and protein concentration was quantified using a Micro-BCA protein assay. Subsequently, 20 µg/lane protein was subjected to 10% SDS-PAGE and transferred to PVDF membranes. The membranes were then blocked in 5% skimmed milk for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Anti-ANXA1 (1:500; cat. no. AMAB90558; Sigma-Aldrich; Merck KGaA), anti-PTHrP (1:200; cat. no. sc-53936; Santa Cruz Biotechnology, Inc.), anti-Smad2 (1:500, cat. no. 5339; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-Smad2 (1:500; cat. no. 18338; Cell Signaling Technology, Inc.) and anti-β-actin (1:1,000; cat. no. A1978; Sigma-Aldrich; Merck KGaA). Subsequently, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (1:5,000; cat. no. ZB-2301; OriGene Technologies, Inc.) or goat anti-mouse IgG (1:5,000; cat. no. ZB-2305; OriGene Technologies, Inc.) secondary antibodies for 1 h at room temperature. Finally, the target proteins were visualized by chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.) and measured using ImageJ software (version 1.52a; National Institutes of Health).

MTT assay

SBC-3 and SBC-5 cells in the logarithmic phase were harvested and seeded into 96-well plates (5×103 cells/well). To assess the relative cell number, 20 µl of 5 mg/ml MTT solution was added to the wells and cells were incubated for 3 h at 37°C. The supernatant was removed and 150 µl DMSO was added to each well to dissolve the blue formazan crystals. The absorbance was determined at a wavelength of 490 nm using a Model 680 microplate reader (Bio-Rad Laboratories, Inc.). To draw the growth curve, the cell number was measured at 24, 48, 72, 96 and 120 h. For accuracy, each assay contained six replicates and was repeated three times.

Colony formation assay

Single-cell suspensions were plated in a 35-mm diameter plate at a density of 1×103 cells/plate and incubated at 37°C with 5% CO2 for 7 days. Subsequently, at room temperature, the colonies were fixed with 4% formaldehyde for 15 min, stained with 0.5% crystal violet for 15 min and counted manually using a light microscope (one colony contained ≥50 cells). Three independent plates were set up for accuracy.

Cell cycle assay

The cells were resuspended at 1×106 cells/ml, fixed and permeabilized with 75% ethanol at 4°C overnight and washed with cool PBS. Subsequently, the cells were stained with staining solution (50 µg/ml propidium iodide and 20 µg/ml RNase in PBS; Sigma-Aldrich; Merck KGaA) at 37°C for 30 min in the dark. The analysis of cellular DNA content was performed using a flow cytometer (FACSCalibur; BD Biosciences) at an excitation wavelength of 488 nm. The distribution of these cells was analyzed using CellQuest v3.3 and ModFit v3.1 softwares (BD Biosciences).

In vitro migration and invasion assay

Both the migratory and invasive abilities of cells were assessed using Transwell inserts (8-µm pore size) in a 24-well plate (Corning, Inc.). The cells were starved for 24 h prior to the migration assay using serum-free RPMI-1640. A total of 3×104 cells in 100 µl RPMI-1640 with 0.1% BSA (Sigma-Aldrich; Merck KGaA) were seeded in triplicates into the upper chamber of the Transwell insert. RPMI-1640 (500 µl) with 10% FBS was added into the lower chamber as a chemoattractant. Following 48 h of incubation at 37°C, cells that did not migrate through the membranes were gently removed using a cotton swab. At room temperature, cells adhering to the lower surface of the membranes were fixed with 4% formaldehyde for 15 min and stained with 0.5% crystal violet for 10 min. Finally, migrated cells were imaged at ×400 magnification using a light microscope and counted manually in five randomly selected fields per well. For the invasion assay, the inserts were coated with 70 µl Matrigel® (1:8 dilution with serum-free RPMI-1640; BD Biosciences) at 37°C for 30 min before the cells were seeded into the upper chamber. Subsequent steps were performed as aforementioned for the migration assay.

In vitro bone adhesion ability assay

A total of 30 female NOD-SCID mice (age, 5 days old; weight, 4.4–5.8 g) were obtained from the Laboratory Animal Research Center of the Air Force Military Medical University (Xi'an, China) and sacrificed by inhaling carbon dioxide (CO2 flow rate, 20% of the container volume per min). As previously described (16), skulls of mice were dissected, swabbed with alcohol wipes and soaked in PBS containing 1×104 U/ml penicillin for 24 h at room temperature. Once the supernatant was removed following centrifugation at 1,000 × g for 15 min at 20°C, collagenase I (5 mg/ml; 400 µl; Sigma-Aldrich; Merck KGaA), collagenase II (5 mg/ml; 400 µl; Sigma-Aldrich; Merck KGaA) and 0.05% trypsin (200 µl) were added to digest the skulls at 37°C for 2 h. The skulls were then washed with PBS twice, and were cut into pieces of 3×3 mm2 for subsequent experimentation. Agar (1%; 0.5 ml/well) was added into a 24-well plate, and the bone pieces were fixed to the solidified agar surface. Subsequently, each well was washed with RPMI-1640 without FBS and incubated in 1 ml RPMI-1640 with 10% FBS in an incubator for 30 min at 37°C. A total of 1×104 cells were planted onto the skulls and incubated for 24 h at 37°C. Subsequently, the bone pieces were taken out, washed with PBS three times, fixed with 95% alcohol for 10 min and stained with 0.5% crystal violet for 15 min, all at room temperature. Finally, cells adhering to the bone surface were visualized under a light microscope (magnification, ×100). Cells adhering to the bone were digested using trypsin and resuspended in PBS to measure the optical density at a wavelength of 600 nm using a spectrophotometer.

In vivo xenograft bone metastasis assay

A total of 10 female NOD/SCID mice (age, 3–5 weeks old; weight, 19–24 g) were obtained from the Laboratory Animal Center of the Air Force Military Medical University and raised under germ-free conditions (temperature, 22°C; ventilation rate, 15/h; light/dark cycle, 12/12 h; food was sterilized with Cobalt-60 irradiation and water was autoclaved; access to food and water was ad libitum). SBC-5-NC/SBC-5-shANXA1 cells were harvested from 80–90% confluent conditions, washed twice and resuspended in PBS. Subsequently, 2×106 cells in 200 µl PBS were implanted into the NOD/SCID mice (4–6 weeks old) via intravenous injection. The mice were divided into two groups according to the expression levels of ANXA1. On the 35th day post-inoculation, mice were anesthetized by intraperitoneal injection of 1% pentobarbital (40 mg/kg). Resultant bone metastases were visualized using X-ray irradiation. At the end of the experiment, the mice were sacrificed by inhaling carbon dioxide (CO2 flow rate, 20% of the container volume per min). All animal experimental procedures, including the aforementioned bone adhesion ability assays, were approved by the Animal Ethics Committee of the Air Force Military Medical University and were in accordance with the ‘Animal Research: Reporting In Vivo Experiments’ guidelines (17).

ELISA and TGF-β treatment

The cells at 80% confluence were harvested, plated into 6-well tissue culture plates (1×105 cells/2 ml/well) and incubated for 24 h at 37°C with 5% CO2. Culture supernatants were collected and the concentrations of PTHrP were determined using the PTHrP ELISA kit (cat. no. CSB-E08649h; Cusabio Technology LLC). For experiments involving TGF-β treatment, 0.5 ng/ml recombinant human TGF-β1 (cat. no. rcyc-htgfb1; InvivoGen) was added to the medium and incubated for 12 h at 37°C with 5% CO2.

Statistical analysis

The data are presented as the mean ± standard deviation. The Kruskal-Wallis test followed by Bonferroni correction for multiple comparisons was used to compare ANXA1 expression in patients with SCLC. Fisher's exact test was used for the association between ANXA1 expression and clinicopathological characteristics. When the variables were normally distributed, unpaired Student's t-test was used to compare two groups and one-way ANOVA was used to compare three groups, followed by Tukey's post hoc test. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic value of ANXA1 for bone metastasis in SCLC. Data analysis was performed using SPSS software (version 25.0; IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

Results

ANXA1 expression is associated with bone metastases in patients with SCLC

Initially, ANXA1 expression was determined in the serum of 82 patients with SCLC (28 females and 54 males; median age, 64 years; age range, 33–83 years). UICC staging evaluation demonstrated stage I disease in 23 patients, stage II disease in 13 patients, stage III disease in 15 patients and stage IV disease in 31 patients. Among the 82 patients with SCLC, distant organ metastasis occurred in 31 cases at initial diagnosis, including 6 cases of brain metastasis, 17 cases of bone metastasis, 13 cases of liver metastasis and 5 cases of adrenal metastasis. ELISA results revealed that ANXA1 expression in the serum ranged between 0.6–6.6 µg/l (Fig. 1A; median, 2.45 µg/l; mean, 2.74 µg/l).

Figure 1.

ANXA1 expression in patients with SCLC and cell lines. (A) ANXA1 expression in the serum of patients with SCLC analyzed via ELISA. (B) Receiver operating characteristic curve of ANXA1 expression in the screening of SCLC bone metastasis. (C) Reverse transcription-quantitative PCR and (D) western blot analysis of ANXA1 expression in SBC-3 and SBC-5 cell lines. *P<0.05. BM, bone metastasis; ANXA1, annexin A1; SCLC, small cell lung cancer; AUC, area under the curve.

Subsequently, the associations between ANXA1 expression and clinicopathological characteristics of the patients were analyzed (Table I). The results revealed that ANXA1 expression was significantly associated with lymphatic invasion (P<0.01), bone metastasis (P=0.03) and TNM stage (P<0.01). No significant association was observed between ANXA1 expression and age (P=0.62), sex (P=0.82), smoking history (P=0.48), tumor invasion (P=0.44), metastasis (P=0.07), liver metastasis (P>0.99) and brain metastasis (P=0.68).

Table I.

Clinicopathological associations of ANXA1 expression in 82 patients with small cell lung cancer.

	ANXA1 expression[a]
Category	N	High	Low	P-value
Age, years
<60	23	13	10	0.62
≥60	59	28	31
Sex
Male	54	26	28	0.82
Female	28	15	13
Smoking history
Yes	73	35	38	0.48
No	9	6	3
Tumor invasion
T1-2	62	29	33	0.44
T3-4	20	12	8
Lymphatic invasion
N0-1	40	13	27	<0.01
N2-3	42	28	14
Metastasis
Yes	31	20	11	0.07
No	51	21	30
Bone metastasis
Yes	17	13	4	0.03
No	65	28	37
Liver metastasis
Yes	13	6	7	>0.99
No	69	35	34
Brain metastasis
Yes	6	2	4	0.68
No	76	39	37
TNM stage
I–II	36	11	25	<0.01
III–IV	46	30	16

Low and high ANXA1 expression was determined according to the median value. ANXA1, annexin A1.

To further clarify the specific association between ANXA1 expression and bone metastasis, patients with SCLC were divided into three groups: ‘No metastasis’ group (n=51); ‘bone metastasis’ group (n=17) and ‘organ metastasis except bone metastasis’ group (n=14). ANXA1 expression in patients with SCLC with bone metastasis was significantly higher compared with the ‘no metastasis’ and ‘organ metastasis except bone metastasis’ groups (3.70±1.44 vs. 2.47±1.56 and 2.56±0.89, respectively; P<0.05), but there was no significant difference between the latter two groups (Fig. 1A). Furthermore, the results of the ROC curve analysis revealed that ANXA1 expression was of diagnostic value for SCLC bone metastasis (area under the curve, 0.74; 95% CI, 0.63–0.83; P<0.05). When the cut-off value was 2.80, the sensitivity was 76.47% and the specificity was 66.15% (Fig. 1B).

SBC-3 and SBC-5 cell lines have similar genetic backgrounds; however, SBC-5 cells are more prone to bone metastasis (18). Total RNA and protein of SBC-3 and SBC-5 cells were extracted to examine ANXA1 mRNA and protein expression using RT-qPCR (Fig. 1C) and western blotting (Fig. 1D), respectively. As expected, the mRNA and protein expression levels of ANXA1 in SBC-5 cells were significantly higher compared with those in SBC-3 cells (Fig. 1C and D).

ANXA1 overexpression enhances the proliferation, migration and invasion of SCLC cell lines

RT-qPCR and western blotting revealed that shRNA-2 was the most efficient shRNA, and this was therefore used for subsequent experiments (Fig. S1A and C). ANXA1 cDNA was transfected to overexpress ANXA1 in SBC-3 cells (Fig. S1B and D). The effects of ANXA1 on cell proliferation were detected using MTT and colony formation assays. In the MTT assay, SBC-3-ANXA1 cells grew at a faster rate compared with SBC-3-vector or parental SBC-3 cells (Fig. 2A). On the other hand, the proliferative rate of SBC-5-shANXA1 cells was lower compared with SBC-5-NC and SBC-5 cells, with a significant difference in the cell proliferation curve observed from day 4 (Fig. 2A). In the planar cloning experiment, colony formation was counted at 1 week after inoculation. ANXA1 overexpression significantly increased cell colony formation ability, while inhibition of ANXA1 expression significantly decreased cell colony formation ability (Fig. 2B).

Figure 2.

Effects of ANXA1 overexpression or knockdown on SCLC cell proliferation, migration and invasion. Effects of ANXA1 on SCLC cell proliferation detected via (A) MTT and (B) colony formation assays. (C) Effects of ANXA1 on the cell cycle detected via flow cytometry. (D) Effects of ANXA1 on invasion and migration of SCLC cells. *P<0.05 vs. parental or control cells. ANXA1, annexin A1; SCLC, small cell lung cancer; OD, optical density; NC, negative control; sh, short hairpin.

Cell cycle analysis was further applied to assess the change in cell proliferation following overexpression or inhibition of ANXA1. As shown in Fig. 2C, the percentage of cells in the G1 phase was higher in SBC-3 and SBC-3-vector cells compared with in SBC-3-ANXA1 cells (61.83±3.06 and 61.32±3.37 vs. 40.47.29±2.44, respectively; P<0.05), while the percentages of cells in S phase were 23.14±2.21, 24.55±2.97 and 38.04±2.88 (P<0.05), respectively. Additionally, SBC-5-shANXA1 cells had a higher proportion of cells in the G1 phase compared with SBC-5 and SBC-5-NC cells (67.59±2.38 vs. 43.09±4.25 and 42.82±2.33, respectively; P<0.05) and a lower proportion in S phase (22.95±2.76 vs. 41.42±2.05 and 41.27±3.71, respectively; P< 0.05) (Fig. 2C).

Transwell assays were performed to determine the effects of ANXA1 on the invasion and migration of SCLC cells. ANXA1-transfected SBC-3 cells exhibited significantly increased migratory and invasive abilities compared with controls (Figs. 2D and S2). Consistently, functional inhibition of ANXA1 significantly impaired the migration and invasion of SBC-5 cells (Figs. 2D and S2).

ANXA1 facilitates the adhesion of SCLC cells to bone in vitro and bone metastases in NOD/SCID mice

The effects of ANXA1 on the bone adhesion ability of SCLC cells were detected via a bone adhesion model in vitro. In the attachment assay, the number of SBC-3-ANXA1 cells that adhered to bone was significantly higher compared with controls (Fig. 3A). On the other hand, functional inhibition of ANXA1 significantly impaired the adhesive ability of SBC-5 cells compared with that of controls (Fig. 3B).

Figure 3.

Association between ANXA1 expression and bone metastasis of SCLC. Representative images and quantification of bone adhesion assay in (A) SBC-3 and (B) SBC-5 cells with overexpressed or inhibited ANXA1 expression in vitro, respectively. Scale bar, 100 µm. (C) Representative images and quantification of xenograft bone metastasis assay in vivo. Scale bar, 1 cm. *P<0.05. ANXA1, annexin A1; SCLC, small cell lung cancer; OD, optical density; NC, negative control; sh, short hairpin; BM, bone metastases.

Furthermore, an in vivo xenograft bone metastasis model in NOD/SCID mice was established. Five weeks after SBC-5-NC and SBC-5-shANXA1 cells were injected into the tail vein, X-ray images displayed significantly decreased lesions and damage on bones in the ANXA1-inhibition group (Fig. 3C). The bone metastasis ability of cells with high or low ANXA1 expression was quantified by the number of bone metastases (3.00±2.51 vs. 0.63±1.06, respectively; P<0.05). Lung and liver metastases were also observed in mice (data not shown). These results indicated that inhibition of ANXA1 significantly decreased bone metastasis in animal models.

Synthesis and secretion of PTHrP in SCLC cells is mediated by ANXA1

To confirm the association between ANXA1 and bone metastasis at the mechanistic level, the effects of ANXA1 on PTHrP expression, which serves an important role in bone metastasis of SCLC (19,20), were investigated. To elucidate whether modulation of ANXA1 affected PTHrP synthesis, western blotting, RT-qPCR and ELISA were applied to quantify PTHrP levels secreted by SCLC cells. SBC-3-ANXA1 cells secreted significantly higher PTHrP levels compared with parental and control SBC-3 cells, while PTHrP synthesis in SBC-5-shANXA1 cells was significantly decreased compared with parental and control SBC-5 cells (Fig. 4A-C).

Figure 4.

ANXA1-mediated PTHrP and p-Smad2 expression in SCLC cells. (A) Western blotting, (B) reverse transcription-quantitative PCR and (C) ELISA of PTHrP expression in SCLC cells. (D) Western blot analysis of Smad2 and p-Smad2 in SCLC cells treated with TGF-β. *P<0.05 vs. TGF-β (−). ANXA1, annexin A1; SCLC, small cell lung cancer; PTHrP, parathyroid hormone-related protein; p, phosphorylated; NC, negative control; sh, short hairpin.

A previous study has demonstrated that TGF-β/Smad signaling participates in regulating PTHrP secretion and serves an important role in bone metastasis (21). Therefore, the expression levels of Smad2 and p-Smad2 after exposure to TGF-β were detected. As shown in Fig. 4D, TGF-β treatment significantly increased Smad2 phosphorylation in the cells. ANXA1 transfection into SBC-3 cells enhanced the function of TGF-β to induce Smad2 phosphorylation, while ANXA1-knockdown in SBC-5 cells impaired Smad2 phosphorylation in response to TGF-β exposure.

Discussion

Bone metastasis hinders the treatment of SCLC, and timely diagnosis of bone metastasis is a prerequisite to improve treatment effects. Therefore, it is of scientific importance and clinical value to identify biomarkers associated with bone metastasis in SCLC.

Compared with other subtypes of lung cancer, SCLC treatment mainly depends on radiotherapy and chemotherapy; hence, access to tumor tissue samples is limited (22). By contrast, blood samples are easy to obtain, and several biomarkers in the blood, such as lactate dehydrogenase, can be effective in reflecting bone metastases (23). Therefore, serum samples of patients with SCLC were used in the present study to study biomarkers for bone metastasis. By detecting the expression levels of ANXA1 in the serum of 82 patients with SCLC, a strong association was identified between ANXA1 expression and bone metastasis. First, compared with patients with SCLC without metastasis, ANXA1 expression was significantly increased in patients with bone metastasis, while no significant increase in ANXA1 expression was detected in patients with other organ metastasis. Second, ROC curve analysis revealed that ANXA1 had diagnostic significance for bone metastasis of SCLC. Additionally, in SBC-3/SBC-5 cell lines with similar genetic background and significantly different bone metastasis ability, ANXA1 expression levels were also associated with bone metastasis abilities. Previous studies have confirmed that ANXA1 is a potential tumor biomarker with abnormal expression in a variety of tumors and is associated with multiple clinical factors, such as metastasis and prognosis (6,11,24). However, the expression and function of ANXA1 are not consistent among different tumors. For example, ANXA1 expression is increased in lung adenocarcinoma, gastric cancer and hepatocellular carcinoma, which is associated with a poor prognosis and decreased disease-free survival and metastasis-free survival (25–27). By contrast, several studies reported that ANXA1 expression is downregulated in head and neck squamous cell carcinoma, nasopharyngeal carcinoma and esophageal carcinoma, and is associated with the differentiation grades (28–30). The aforementioned studies suggest that the use of ANXA1 as a therapeutic or prognostic marker for cancer should be carefully evaluated depending on cancer type, grade and stage.

Next, a series of in vivo and in vitro experiments were conducted to further confirm the role of ANXA1 in bone metastasis. By regulating the expression levels of ANXA1 in SCLC cell lines, the present study confirmed that ANXA1 exerted promoting effects on the proliferation, migration and invasion of SCLC cells. Similar results have been reported in previous studies (9,31). For example, autophagy induced by ANXA1 inhibition promoted nasopharyngeal carcinoma cell invasion and metastasis (32). Bone adhesion experiments in vitro and xenograft bone metastasis assay in vivo further confirmed the role of ANXA1 in promoting bone metastasis in SCLC, consistent with the present results of serological testing on patients.

Bone tissue has a distinct structure and composition; hence, the mechanism involved in bone metastasis is different from that of other organs. PTHrP stimulates osteoclastic bone resorption, resulting in bone destruction and TGF-β release, which is stored in the bone matrix (33,34). TGF-β binds to receptors on the surface of tumor cells and induces the phosphorylation of Smad family members (such as the production of p-Smad2), which then activates downstream signaling pathways and regulates the expression of bone metastasis-associated target genes (21,35). The expression levels of PTHrP and p-Smad2 are closely associated with the degree of bone metastasis (21). To confirm the association between ANXA1 and bone metastasis at the mechanistic levels, the effects of ANXA1 on PTHrP and p-Smad2 expression in SCLC cells were assessed. Using western blotting, RT-qPCR and ELISA, the present study confirmed that ANXA1-overexpressing SCLC cells secreted higher levels of PTHrP, while PTHrP synthesis in ANXA1-knockdown SCLC cells was significantly suppressed. In addition, following exposure to TGF-β, ANXA1-overexpressing SCLC cells exhibited higher levels of p-Smad2 compared with in ANXA1-knockdown SCLC cells.

The present study has some limitations. First, the current study was a single-center study with a limited clinical sample size. Second, due to the limitations of specimen collection, the present study failed to assess the expression levels of ANXA1, PTHrP and pSMAD in tumor tissues, nor the association between ANXA1 and other markers, such as PTHrP in serum. Post-mortem studies of tissue samples may be useful to overcome these problems. Third, the expression level of ANXA1 in patients was not continuously monitored, and methods such as Kaplan-Meier analysis were not used to reflect the influence of ANXA1 on the incidence of bone metastasis. Finally, ANXA1 secretion has not been studied thoroughly. Detection of ANXA1 secretion under different conditions using neutralizing antibodies may help to further clarify the function of ANXA1. In addition, the downstream signaling pathways of ANXA1 in promoting bone metastasis, such as the specific role of PTHrP/Smad2, lack of in-depth study. Overcoming these limitations is important to further confirm the role of ANXA1 in bone metastasis and should be further investigated in future studies.

In conclusion, the present study confirmed the strong association between ANXA1 expression and bone metastasis in SCLC from different perspectives, suggesting that ANXA1 may be a potential marker of bone metastasis and providing new potential for the diagnosis and treatment of SCLC.