Open Access

S100A8 facilitates cholangiocarcinoma metastasis via upregulation of VEGF through TLR4/NF‑κB pathway activation

  • Authors:
    • Shuguang Pan
    • Ying Hu
    • Mengjia Hu
    • Yang Xu
    • Mo Chen
    • Changhong Du
    • Jinchi Cui
    • Ping Zheng
    • Jiejuan Lai
    • Yujun Zhang
    • Jie Bai
    • Peng Jiang
    • Jin Zhu
    • Yu He
    • Junping Wang
  • View Affiliations

  • Published online on: October 30, 2019     https://doi.org/10.3892/ijo.2019.4907
  • Pages: 101-112
  • Copyright: © Pan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

A growing body of evidence indicates that S100 calcium‑binding protein A8 (S100A8) is frequently overexpressed in malignant tumor tissues and regulates tumor progression; however, the role of S100A8 in cholangiocarcinoma (CCA) remains unclear. The present study demonstrated that the protein expression of S100A8 was significantly higher in pathological tissues compared with adjacent normal tissues from patients with CCA. In addition, S100A8 expression was significantly associated with differentiation, lymph node metastasis and poor prognosis in patients following surgical resection of CCA. Furthermore, both in vitro and in vivo experiments revealed that overexpression of S100A6 promoted, while S100A8 knockdown attenuated, the migration and metastasis of CCA cells. Of note, the present results indicated that S100A8 promoted the CCA tumor cell‑induced migration of vascular endothelial cells. Finally, S100A8 was demonstrated to positively regulate the expression of vascular endothelial growth factor (VEGF) in CCA cells, which was mediated by activation of the Toll‑like receptor 4 (TLR4)/NF‑κB pathway. In conclusion, the present study demonstrated that S100A8 had an important role in facilitating CCA cell migration and metastasis via upregulation of VEGF expression by activating the TLR4/NF‑κB pathway. These findings may provide a novel target for CCA treatment.

Introduction

Cholangiocarcinoma (CCA) is currently one of the most common malignant tumors in the biliary tract (1,2). CCA is prone to invasion and metastasis into blood vessels and distant lymph nodes, thereby complicating clinical surgical treatment (1,3). Tumor metastasis is the main cause of cancer-related death (4). Once a tumor metastasizes, the prognosis becomes extremely poor, despite the prominent advances that have been made in cancer treatment over the past few decades (4,5). Tumor invasion and metastasis are complex biological processes involving multiple factors, during which tumor cells detach from the primary site and interact with the extracellular matrix, forming a migration channel for tumor cells (4,6,7). Tumor cells then enter the peripheral blood vessels through the vascular basement membrane, leading to dissemination of the tumor cells in distant tissues, where they finally colonize and form metastatic foci (8,9). However, the underlying molecular mechanisms have not been fully elucidated.

S100 calcium-binding protein A8 (S100A8), also known as myeloid-related protein 8, is a calcium-binding protein recognized as an endogenous ligand of Toll-like receptor 4 (TLR4). S100A8 has been reported to be highly expressed in bone marrow-derived cells, such as neutrophils (10). In addition, S100A8 protein can be secreted in the form of heterodimers by necrotic cells and pathogen-associated molecular pattern-activated immune cells (1012). Of note, S100A8 has been found to positively regulate the progression of malignant tumors and the dissemination of tumor cells (13,14). Consistent with these findings, S100A8 protein is frequently overexpressed in a variety of tumors, such as lung cancer, colon cancer, gastric cancer, pancreatic cancer, and primary liver cancer (4,10,15). However, whether S100A8 is expressed in CCA remains unclear.

Vascular endothelial growth factor (VEGF) is a protein that specifically affects endothelial cells (16,17). The binding of VEGF to its receptor VEGFR, a tyrosine protein kinase, serves a crucial role in promoting endothelial cell proliferation, migration, budding, vascular growth and permeability enhancement (1719). Blood vessels in tumors are known to be critical for malignant tumor growth, invasion and metastasis (2022), and in most cancers, microvessel formation is a prognostic indicator of tumor metastasis, recurrence and survival (20,23). Tumor remodeling and angiogenesis are complex processes that require activation of multiple vascular components: Endothelial cell division, vascular basement membrane degradation, peripheral extracellular matrix degradation and endothelial cell migration (7,24). Previous studies have confirmed that VEGF is secreted by tumor cells and has a major regulatory role in angiogenesis in various types of cancer, by promoting the expansion of endothelial cells (16,17); however, the exact role of VEGF in CCA is poorly understood.

The present study demonstrated that S100A8 protein expression was high in CCA tissues and significantly associated with malignant pathology, differentiation, lymph node metastasis and poor prognosis in patients with CCA. Further investigations revealed that S100A8 activated the TLR4/NF-κB pathway to increase VEGF secretion and to thereby promote CCA invasion and metastasis. Collectively, the findings indicated that S100A8 may be a vital molecule in promoting CCA invasion and metastasis.

Materials and methods

CCA tissue specimens

Pathological specimens were obtained from 134 patients with hilar cholangiocarcinoma who underwent surgical treatment in the Department of Hepatobiliary Surgery at the Southwest Hospital Affiliated with the Third Military Medical University (Chongqing, China) from January 2010 to December 2015. All patients were re-examined using upper abdominal color Doppler ultrasound and computed tomography angiography (CTA) every three months after discharge. Overall survival was calculated from the date of surgery until the date of last contact. All neutrophil counts were derived from routine blood tests. The present study was approved by the Ethics Committee of the Third Military Medical University and followed the guidelines of the Declaration of Helsinki.

Animals

A total of 20 male BALB/c-nu mice (age, 4–6 weeks; weight, 18–22 g) were purchased from the Institute of Laboratory Animal Sciences of the Chinese Academy of Sciences. The mice were randomly divided into four groups and were housed under specific pathogen-free conditions (temperature, 24–26°C; humidity, 40–60%; ventilation, 15 times/h; 12-h light/dark cycle; free access to food and water). All animals received humane care according to the criteria outlined in the 'Guide for the Care and Use of Laboratory Animals' (National Institutes of Health), and the animal experiment protocols were approved by the Institutional Animal Care and Use Committee of the Third Military Medical University. All mice were sacrificed via intraperitoneal injection of excessive sodium pentobarbital.

Cell culture

The human CCA cell lines RBE and HCCC-9810 and the human umbilical vein endothelial cell (HUVEC) line were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) medium containing 10% fetal bovine serum (FBS; Zeta Life, Inc.) and maintained in a humidified incubator containing 5% CO2 at 37°C.

Reagents

The TLR4 inhibitor TAK242 (cat. no. HY-11109) and the NF-κB inhibitor ammonium pyrrolidinedithiocarbamate (PDTC; cat. no. HY-18738) were purchased from MedChemExpress. The TAK-242 (25) and PDTC (26) doses used in the present study were 20 and 40 μmol/l, respectively. The neutralizing anti-human VEGF-A antibody was purchased from Abcam (3 μg/ml; cat. no. ab42609).

Lentivirus and stable cell lines

For S100A8 overexpression, full-length human S100A8 cDNA was amplified via polymerase chain reaction (PCR) and cloned into a lentivirus vector to establish an RBE cell line that stably expressed S100A8. Two clones were tested, and clone A was selected for subsequent experiments. For S100A8 knockdown, lentivirus encoding a specific shRNA against S100A8 was used to establish a stable HCCC-9810 cell line with S100A8 knockdown. Two clones were tested, and clone A was selected for subsequent experiments (target sequence: ACT CTA TCA TCG ACG TCT ACT CGA GTA GAC GTC GAT GAT AGA GT). S100A8 overexpression (HBLV-h-S100A8-3-flag-GFP-PURO) and interference lentiviruses (HBLV-h-S100A8-shRNA1-GFP-PURO) were purchased from Shanghai Genechem Co., Ltd., HCCC-9810 and RBE cells were seeded onto 6-well plates, and lentiviral infection was carried out at 50–70% confluency. For infection, the cells were provided with 1 ml of fresh culture medium, and 30 μl of lentivirus (1×108 transforming units/ml) was added to each well. After 48–72 h, the infection rate was observed using a fluorescence microscope, and S100A8 expression levels were detected by western blotting.

Immunoblotting

Immunoblotting analyses were performed as described previously (27). Antibodies against S100A8 (cat. no. ab180735; Abcam), VEGF (cat. no. ab52917; Abcam), NF-κB p65 (cat. no. 8242; Cell Signaling Technology, Inc.) and phosphorylated (p-) NF-κB p65 Ser536 (cat. no. 3033; Cell Signaling Technology, Inc.) were used at 1:1,000 dilution. Membranes were washed with TBST for 30 min and incubated with an HRP-conjugated anti-rabbit secondary antibody (1:4,000; cat. no. ab6721; Abcam) for 2 h at room temperature.

Immunohistochemistry (IHC)

The expression of S100A8 in paraffin-embedded clinical samples was investigated using IHC, according to a previously described protocol (28). Antibodies against S100A8 (1:200; cat. no. ab180735; Abcam) and VEGF (1:200; cat. no. ab52917; Abcam) were used. IHC staining of S100A8 and VEGF revealed both membrane and cytoplasmic localization. Hematoxylin and eosin (H&E) staining of liver and spleen tumor tissue sections from mice was performed, as previously described (29). The immunostained tissue slides were scored according to stain intensity (0, no staining; 1, light staining; 2, moderate staining; and 3, intense staining) multiplied by a distribution score (1, staining of 0–33%; 2, staining of 33–66%; and 3, >66% staining). The final score was grouped as low expression [negative, (0) or low (12) scores], medium expression (moderate score, 3–4) or high expression (high score, 6–9) for further non-parametric tests. Each immunostained slide was scored by two pathologists in a double-blind manner using a light microscope.

Tumor cell migration assay

Tumor cell migration assays were performed using Transwell inserts (Corning, Inc.) with 8 μm pores. Tumor cells (2×105) in 0.2 ml of serum-free medium were seeded in the upper chamber of the insert, and 0.8 ml of medium containing 10% FBS was added to the lower chamber. Cells were cultured for 12 h in a humid environment at 37°C with 5% CO2. Cells remaining in the upper part of the transwell were removed with a cotton swab. Migrated cells were then stained with 0.5% crystal violet, and the number of cells per field was determined using a light microscope (Olympus Corporation; magnification, ×200).

Tumor cell/HUVEC co-culture migration assay

HUVEC migration assays were performed using Transwell inserts (Corning, Inc.) with 8 μm pores. HUVECs (1×105) in 0.2 ml of serum-free medium were seeded in the upper chamber, and 0.8 ml of tumor cell culture supernatant was added to the lower chamber. For the tumor cell supernatant, tumor cells (5×105) were cultured for 24 h in 1.0 ml of medium containing 10% FBS in a humid environment at 37°C and with 5% CO2. HUVECs were allowed to migrate for 5 h. Then, cells remaining in the upper part of the Transwell were removed with a cotton swab. Migrated cells on the lower part of the Transwell were stained with 0.5% crystal violet, and the number of cells per field was determined using a light microscope (Olympus Corporation; magnification, ×200). In some experiments (Fig. S2), tumor cells (2×105/ml) in 0.2 ml of serum-free medium were added to the upper chamber and HUVECs were placed into the lower chamber, and migration of lentivirus-transduced tumor cells or control cells was observed.

HUVEC tube formation assay

For HUVEC tube formation assays, 0.3 ml of Matrigel (BD Biosciences) was pipetted into a 24-well plate and then allowed to solidify for 1 h at 37°C. HUVECs (1×105) in 0.2 ml of conditioned medium were seeded into each well and incubated for 10 h. The average number of HUVEC tubule structures per field was determined using a light microscope (Olympus Corporation; magnification, ×100).

ELISA

VEGF levels in tumor cells and tissues were detected using a human VEGF ELISA kit (cat. no. DY293B; R&D Systems, Inc.), according to the manufacturer's instructions. In addition, the S100A8 levels in the CCA tissue samples were determined using an ELISA kit (cat. no. DY4570-05; R&D Systems, Inc.), according to the manufacturer's instructions.

In vivo experiments

After anesthetizing with 1% sodium pentobarbital (60 mg/kg; Sigma-Aldrich; Merck KGaA) by intraperitoneal injection, a median abdominal incision of 1 cm was made under the xiphoid of nude mice. For experimental liver metastasis, RBE and HCCC-9810 cells were trypsinized and resuspended in PBS, and 5×105 cells or the indicated number of cells (in 0.1 ml of PBS) were injected into the spleen of the mice. After 45 days, a 7.0 T small animal MRI (Bruker Biospec) was used to scan the mice and to observe metastasis. Mice were sacrificed at 45 days after injection (preliminary experiments were performed to determine the optimal time after injection to evaluate metastasis; data not shown), livers were isolated and fixed in 10% neutral-buffered formalin for 24 h at room temperature, and then dehydrated and embedded in paraffin. Metastatic foci on the surface of the livers were counted under a dissecting microscope, and the nodule size was measured.

Distant initial dissemination of tumor cells can be observed with the first few hours after the tumor cells enter the blood circulation, which is a key step to the metastatic cascade (30). For experimental initial liver colonization, 5×105 RBE and HCCC-9810 cells were preloaded with Vybrant DiO Cell-labelling Solution (cat. no. V22886; Invitrogen; Thermo Fisher Scientific, Inc.) for 15 min at 37°C following the manufacturer's instructions. Cells were then injected into the spleen of the mice, as aforementioned. After 6 h, livers were isolated, embedded in OCT compound (Tissue-Tek; Sakura Finetek USA, Inc.) and frozen in liquid nitrogen. Liver sections (8 μm thickness) were analyzed using an Olympus fluorescence microscope (VS120; Olympus Corporation), and the number of RBE and HCCC-9810 cells (green signal) was determined.

To assess tumor volumes, the longest (length) and shortest (width) diameters of the metastatic tumor foci in the liver were measured, and the volumes were calculated using the following formula: Length × width2 ×0.52. The maximum volume of liver tumors was 91 mm3, and the maximum diameter was 7 mm.

Statistical analysis

Data were analyzed with Prism 6.0 (GraphPad Software, Inc.) or SPSS 19.0 software (IBM Corp.). For categorical data, chi-square analysis or Fisher's exact test was used. Kaplan-Meier and Cox proportional hazards regression analysis were applied to assess overall survival. Experiments were repeated at least three times independently. Comparisons among multiple groups were made using ANOVA followed by least significant difference post hoc test. Normally distributed data are presented as the mean ± standard error and were analyzed using Student's t-test. Non-normally distributed data were analyzed using Mann-Whitney U test. Spearman rank correlation coefficient was used to analyze the association between the protein expression levels of S100A8 and VEGF. P<0.05 was considered to indicate a statistically significant difference.

Results

S100A8 is highly expressed in CCA, and its expression is closely associated with CCA severity

To investigate the role of S100A8 in CCA, pathological CCA tumor tissue and adjacent normal tissue specimens were collected from 41 patients with CCA. S100A8 expression was found to be higher in CCA tissues compared with adjacent normal tissues, as detected by western blot analysis (Fig. 1A). In addition, increased S100A8 expression was observed in CCA primary tumor tissues compared with the adjacent normal tissues by immunohistochemical staining (Fig. 1B and Fig. S1A). To further evaluate the association between S100A8 protein expression and CCA malignancy, the S100A8 protein levels were detected in 134 pathological specimens from patients with CCA via ELISA. S100A8 expression levels were significantly associated with tumor differentiation status and lymph node metastasis, but not with age or sex (Table I). In accordance with this finding, S100A8 expression levels were also significantly associated with poor prognosis in patients with CCA following surgical resection, as evaluated by multivariate Cox proportional hazards regression analysis (Table II). In addition, Kaplan-Meier survival analysis revealed that high S100A8 expression was correlated with a significant reduction in overall survival of patients with CCA (Fig. 1C). Because S100A8 is known to be highly expressed by neutrophils, the relationship between S100A8 expression and neutrophil numbers was investigated in the 134 patients with CCA in the present study, but no significant correlation of S100A8 expression with the number of neutrophils in the peripheral blood was observed (Fig. S1B). The present results indicated that S100A8 expression was significantly associated with the severity of CCA.

Table I

Association between S100A8 expression levels and clini-copathological features in 134 patients with cholangiocarcinoma.

Table I

Association between S100A8 expression levels and clini-copathological features in 134 patients with cholangiocarcinoma.

Clinicopathological featureS100A8 expression
P-value
Low 68 (%)High 66 (%)
Sex0.468
 Male43 (63)46 (70)
 Female25 (37)20 (30)
Age (years)0.729
 <5530 (44)32 (48)
 ≥5538 (56)34 (52)
Differentiation0.037
 Poor19 (28)24 (36)
 Moderate43 (63)42 (64)
 High6 (9)0 (0)
Lymph node metastasis0.01
 Yes30 (44)44 (67)
 No38 (56)22 (33)

[i] S100A8, S100 calcium-binding protein A8.

Table II

Multivariate Cox proportional hazards regression analysis for S100A8 expression levels and overall survival in patients with cholangiocarcinoma.

Table II

Multivariate Cox proportional hazards regression analysis for S100A8 expression levels and overall survival in patients with cholangiocarcinoma.

FactorHazard ratio (95% CI)P-value
Expression of S100A8 (low vs. high)1.654 (1.130–2.421)0.01
Sex0.850 (0.568–1.272)0.429
Age1.404 (0.960–2.055)0.08
Differentiation0.610 (0.420–0.884)0.009
Lymph node metastasis0.312 (0.206–0.473)<0.001

[i] S100A8, S100 calcium-binding protein A8; CI, confidence interval.

S100A8 facilitates CCA cell invasion and migration in vitro

Next, the present study hypothesized that S100A8 may be involved in promoting CCA metastasis. To investigate this, the RBE CCA cell line, which has low intrinsic S100A8 protein expression, was transduced with lentivirus to overexpress S100A8. The transduction efficiency was confirmed by immunoblotting; the results revealed that S100A8 protein expression was significantly upregulated in the RBE-overexpressing cells compared with the negative control cells (Fig. 2A; clone A was selected for further experiments). In vitro Transwell assays revealed that S100A8-overexpressing RBE cells displayed a significant increase in migration ability compared with control cells (Fig. 2C). Transwell invasion assays (with Matrigel-coated inserts) gave similar results for cell invasion (data not shown).

To determine whether silencing of S100A8 expression could attenuate the migration of CCA cells, another CCA cell line, HCCC-9810, which has high intrinsic S100A8 expression, was used. Knockdown of S100A8 in HCCC-9810 cells was established via a shRNA-encoding lentivirus, and successful transduction was confirmed by western blotting (Fig. 2B; clone A was selected for further experiments). In vitro Transwell assays revealed that S100A8-silenced HCCC-9810 cells had significantly reduced migration ability compared with control cells (Fig. 2D). These results indicated that S100A8 may participate in promoting CCA migration.

S100A8 enhances the tumor-induced migration of vascular endothelial cells in vitro

Because angiogenesis is a key process contributing in malignant tumor growth, invasion and metastasis, the role of S100A8 in promoting angiogenesis was investigated next. The ability of RBE cells overexpressing S100A8 to promote migration of HUVECs was analyzed using a Transwell assay. Conditioned media from S100A8-overexpressing and control RBE cells was placed into the lower chamber of the Transwell system, and HUVECs were placed into the upper chamber. The number of migratory HUVECs was found to be significantly increased towards the conditioned media of the S100A8-overexpressing RBE cells compared with the control group (Fig. 3A). However, no migration of lentivirus-transduced RBE tumor cells or control cells was observed when HUVECs were placed into the lower chamber and lentivirus-transduced RBE cells and control cells were placed into the upper chamber (Fig. S2). In addition, these observations were further confirmed using the second CCA cell line, HCCC-9810; S100A8 knockdown reduced the ability of HCCC-9810 to induce HUVEC migration (Fig. 3B). These findings indicated that S100A8 had a pro-angiogenesis function in CCA.

S100A8 promotes CCA dissemination and metastasis in vivo

To investigate whether S100A8 increases CCA metastasis in vivo, a liver experimental metastasis model was established in mice. Control RBE and S100A8-overexpressing RBE cells were injected into the spleens of wild-type mice, and then, liver metastasis was monitored. Both S100A8-overexpressing RBE cells and control RBE cells formed metastatic foci in the liver, but S100A8-overexpressing RBE cells resulted in significantly increased numbers of metastatic foci (Fig. 4A, C and D). By contrast, S100A8 knockdown reduced the metastatic ability of HCCC-9810 CCA cells in vivo (Fig. 4B). To investigate whether S100A8 protein overexpression or knockdown affects metastatic growth, the volume of the liver metastatic foci was measured, and no significant difference was found between the S100A8-overexpressing, the S100A8-silenced and the control CCA cells (data not shown).

Tumor metastasis is primarily dependent on hematogenous dissemination in the blood circulation (30). To further investigate whether S100A8-overexpressing RBE cells increase metastatic dissemination in the liver, equal quantities of fluorescently-labelled RBE and S100A8-overexpressing RBE cells were injected into the spleen of mice. Compared with control cells, S100A8-overexpressing RBE cells resulted in a significantly increased liver metastatic tumor burden (Fig. 4E). In addition, fluorescence microscopy analysis revealed that the tumor cells were primarily distributed around the vasculature (Fig. 4E), indicating that these tumor cells may have entered surrounding tissues through the blood vessels to form metastases. These data suggested that S100A8 may enhance the dissemination and metastasis ability of CCA cells in vivo.

S100A8 upregulates VEGF expression in CCA cells

Given that VEGF is a key factor in tumor invasion and metastasis (7,22), the present study next evaluated the VEGF expression levels in the patient tissues. The results demonstrated that VEGF was more highly expressed in CCA tissues compared with adjacent normal tissues from patients with CCA (Fig. 5A and B, and Fig. S3). These results were similar to the S100A8 expression results. Consistently, VEGF expression was also significantly associated with tumor differentiation status, lymph node metastasis and poor prognosis in patients with CCA following surgical resection (Tables III and IV). In addition, Kaplan-Meier survival analysis revealed that high VEGF expression was associated with a significant decrease in the overall survival of patients with CCA (Fig. 5C).

Figure 5

S100A8 upregulates VEGF expression in CCA cells. (A) VEGF protein expression levels were determined by western blotting in 41 tumor tissues and adjacent normal tissues from patients with CCA. Representative blots from 4 paired samples are shown. (B) Representative images from immunohistochemical staining of VEGF in CCA tumor (high expression) and adjacent normal tissues (low expression). (C) Kaplan-Meier plot for overall survival of 134 CCA patients with low or high VEGF expression (based the mean expression level). (D) Representative images and quantification of vascular tube-like formation in HUVECs treated with supernatants from RBE cells with or without S100A8 overexpression (n=3). (E) RBE cells with or without S100A8 overexpression were cultured in 1.0 ml of medium containing 10% FBS for 24 h. The total VEGF levels were then determined by ELISA. (F) HCCC-9810 cells with or without S100A8 knockdown were cultured in 1.0 ml of medium containing 10% FBS for 24 h. The total VEGF levels were then determined by ELISA. (G) Correlation between S100A8 and VEGF protein levels in 134 patients with CCA, as determined by ELISA. (H) Migration of RBE cells (with or without S100A8 overexpression) following treatment with a neutralizing VEGF antibody. **P<0.01 and ***P<0.001, with comparisons indicated by brackets. S100A8, S100 calcium-binding protein A8; VEGF, vascular endothelial growth factor; CCA, cholangiocarcinoma; HUVEC, human umbilical vein endothelial cell; Ca, carcinoma; Aj, adjacent normal; OE, overexpression; KD, knockdown; NC, negative control.

Table III

Association between VEGF expression levels and clinicopathological features in 134 patients with cholangiocarcinoma.

Table III

Association between VEGF expression levels and clinicopathological features in 134 patients with cholangiocarcinoma.

Clinicopathological featureVEGF expression
P-value
Low 68 (%)High 66 (%)
Sex0.361
 Male41 (62)48 (71)
 Female25 (38)20 (29)
Age (years)0.392
 <5528 (42)34 (50)
 ≥5538 (58)34 (50)
Differentiation0.008
 Poor16 (24)27 (40)
 Moderate44 (67)41 (60)
 High6 (9)0 (0)
Lymph node metastasis0.015
 Yes29 (44)45 (66)
 No37 (56)23 (34)

[i] VEGF, vascular endothelial growth factor.

Table IV

Multivariate Cox proportional hazards regression analysis for VEGF expression levels and overall survival in patients with cholangiocarcinoma.

Table IV

Multivariate Cox proportional hazards regression analysis for VEGF expression levels and overall survival in patients with cholangiocarcinoma.

FactorHazard ratio (95% CI)P-value
Expression of VEGF (low vs. high)1.781 (1.213–2.614)0.003
Sex0.842 (0.563–1.259)0.401
Age1.498 (1.020–2.201)0.039
Differentiation0.619 (0.424–0.903)0.013
Lymph node metastasis0.307 (0.202–0.466)<0.001

[i] VEGF, vascular endothelial growth factor; CI, confidence interval.

Treatment of HUVECs with supernatants from S100A8-overexpressing RBE cells distinctly promoted the formation of vascular tube-like structures (Fig. 5D). In addition, a previous study has reported that S100A8 can promote the secretion of vasoactive factors, including VEGF (7). These findings suggested that VEGF may be responsible for the role of S100A8 in facilitating CCA dissemination and metastasis. Notably, overexpression or knockdown of S100A8 significantly increased or reduced the secretion of VEGF from CCA cells, respectively (Fig. 5E and F). In addition, S100A8 expression levels were significantly correlated with VEGF expression levels in the tissues of the 134 patients with CCA (Fig. 5G). Multivariate Cox proportional hazards regression analyses revealed that combined high S100A8 and high VEGF expression levels were significantly associated with poor prognosis in patients with CCA following surgical resection (Table V). Finally, inhibition of VEGF via a neutralizing antibody abrogated the S100A8-enhanced migration of RBE cells (Fig. 5H). Therefore, these results suggested that S100A8 may play a crucial role in CCA progression through upregulation of VEGF.

Table V

Multivariate Cox proportional hazards regression analysis for combined S100A8/VEGF expression levels and overall survival in patients with cholangiocarcinoma.

Table V

Multivariate Cox proportional hazards regression analysis for combined S100A8/VEGF expression levels and overall survival in patients with cholangiocarcinoma.

FactorHazard ratio (95% CI)P-value
Expression of high1.819 (1.210–2.734)0.004
S100A8/high VEGF
Sex0.896 (0.599–1.340)0.593
Age1.380 (0.944–2.018)0.096
Differentiation0.652 (0.447–0.950)0.026
Lymph node metastasis0.316 (0.208–0.480)<0.001

[i] S100A8, S100 calcium-binding protein A8; VEGF, vascular endothe-lial growth factor; CI, confidence interval.

S100A8 increases VEGF expression by activating the TLR4/NF-κB pathway

Finally, the present study sought to determine the underlying mechanism by which S100A8 upregulates VEGF expression. TLR4 has been reported to be a receptor of S100A8, and NF-κB, which is downstream of TLR4, is responsible for VEGF production (31,32). Therefore, it was speculated that S100A8 might promote VEGF expression through the TLR4/NF-κB pathway. As anticipated, overexpression of S100A8 in RBE cells activated the NF-κB pathway (Fig. 6A). Notably, TLR4 and NF-κB inhibition completely abrogated the S100A8 overexpression-induced upregulation of VEGF expression in RBE cells (Fig. 6E), and significantly inhibited RBE cell migration (Fig. 6C). In addition, results from these assays in HCCC-9810 cells revealed that the NF-κB pathway was crucial to the migration of the CCA cells and the secretion of VEGF (Fig. 6B, D and F). Taken together, the present findings demonstrated that the TLR4/NF-κB pathway mediated the role of S100A8 in promoting VEGF expression in CCA cells.

Figure 6

S100A8 increases VEGF expression by activating the TLR4/NF-κB pathway. (A) p-p65, total p65 and VEGF protein expression levels were determined by western blotting in RBE parental, NC and S100A8-overexpressing cells, with or without treatment with the TAK242 and PDTC inhibitors. (B) p-p65, total p65 and VEGF protein expression levels were determined by western blotting in HCCC-9810 cells with or without treatment with the TAK242 and PDTC inhibitors. (C) RBE parental, NC and S100A8-overexpressing cells, with or without treatment with the TAK242 and PDTC inhibitors, were allowed to migrate in Transwell chambers for 12 h, then the numbers of migrated cells were determined by microcopy (magnification, ×200). (D) HCCC-9810 cells with or without treatment with the TAK242 and PDTC inhibitors were allowed to migrate in Transwell chambers for 12 h, then the numbers of migrated cells were determined by microcopy (magnification, ×200). (E) RBE parental, NC and S100A8-overexpressing cells, with or without treatment with the TAK242 and PDTC inhibitors, were seeded separately into 24-well dishes in 1 ml of medium containing 10% FBS and cultured for 24 h. The total VEGF levels were then determined by ELISA. (F) HCCC-9810 cells with or without treatment with the TAK242 and PDTC inhibitors were seeded separately into 24-well dishes in 1 ml of medium containing 10% FBS and cultured for 24 h. The total VEGF levels were then determined by ELISA. *P<0.05, **P<0.01 and ***P<0.001, with comparisons indicated by brackets. S100A8, S100 calcium-binding protein A8; VEGF, vascular endothelial growth factor; TLR, Toll-like receptor; p-, phosphorylated; NC, negative control; OE, overexpression; KD, knockdown.

Discussion

Patients with CCA have a poor prognosis and high mortality due to the increased invasiveness and early metastasis of CCA (3335). Although many studies have focused on CCA, the molecular mechanisms underlying CCA invasion and metastasis have not been fully elucidated. The present study demonstrated for the first time that S100A8 promoted CCA migration and metastasis via the TLR4/NF-κB/VEGF signaling axis.

Previous studies have shown that S100A8 protein is localized in the cytoplasm of multiple cell types and that it has multiple biological functions, including regulating cell cycle progression, affecting cell differentiation and inducing cell apoptosis (3638). S100A8 can also function as a cytokine that is primarily secreted by neutrophils to activate monocytes and macrophages (39,40). Notably, other studies have observed increased expression of S100A8 protein in multiple tumor types (37,41), however, the expression and role of S100A8 protein in malignant bile duct tumors have not been previously elucidated. The present study reported that S100A8 was more highly expressed in CCA tissues compared with adjacent normal tissues, suggesting that S100A8 may have a unique role in CCA. Additionally, S100A8 expression was significantly associated with CCA severity and poor clinical prognosis. Furthermore, in vitro and in vivo experiments revealed that S100A8 had a crucial role in CCA tumor migration and metastasis.

New blood vessels in tumor tissues are required for tumor growth and metastasis (4244), and VEGF is well-established as a factor required to promote angiogenesis, which markedly enhances tumor invasion and metastasis (23). In the present study, through analysis of pathological specimens, it was demonstrated that VEGF expression levels were increased in CCA tumor tissues compared with adjacent normal tissues, and that they were significantly associated with the poor prognosis of CCA, which was similar to the S100A8 findings. Furthermore, in vitro overexpression or knockdown of S1000A8 in CCA cells resulted in increased or decreased, respectively, expression of VEGF. According to a previous study, the migration of endothelial cells induced by tumor cells greatly increases the microvessel density in tumors, thus serving a pivotal role in tumor invasion and metastasis (7,45). In line with this notion, the present study confirmed that S100A8-overexpressing CCA cells increased the migration of endothelial cells, therefore potentially leading to an enhancement in tumor dissemination and metastasis ability. However, other studies have reported that a low S100A8 concentration promotes endothelial cell migration and expansion and enhances vascular permeability and a high S100A8 concentrations leads to necrosis of endothelial cells (4648). Thus, the biological functions of S100A8 may be different depending on the cell or tumor type.

Multiple studies have reported that the NF-κB pathway has an important role in the progression multiple types of cancer, by promoting cell proliferation and inhibiting cell apoptosis (49,50). In addition, the NF-κB pathway is responsible for generation of many of the cytokines associated with tumor angiogenesis, such as VEGF (51,52). The current study provided experimental evidence that S100A8 promoted CCA cell migration, at least in part, through the TLR4/NF-κB/VEGF signaling pathway. However, the possibility that there could be other mechanisms that also mediate the role of S100A8 in CCA migration and metastasis cannot be excluded, and further research will be required to fully elucidate this.

In conclusion, the present study highlighted the importance of S100A8 in upregulation of VEGF expression through activation of the TLR4/NF-κB pathway, thereby providing a favorable microenvironment for tumor invasion and metastasis. Therefore, S100A8 may serve as a potential target for tumor metastasis. Additionally, the present findings suggested that S100A8 may be a candidate prognostic marker for CCA.

Supplementary Data

Funding

This work was supported by grants from the National Natural Science Fund of China (nos. 81725019, 81502755 and 81500087) and the Scientific Research Project of The Chinese People's Liberation Army (grant no. AWS16J014).

Availability of data and materials

The datasets used and analyzed in the current study are available from the corresponding author upon reasonable request.

Authors' contributions

JW and YH designed and supervised all experiments. MC and PZ prepared the materials and contributed to data acquisition. YH, YX, CD, JL, YZ, JB and JZ assisted with the animal and molecular biology experiments. JC and PJ analyzed the data. SP and MH drafted the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Experiments were approved by the Ethics Committee of the Southwest Hospital of the Third Military Medical University of China. Written, informed consent was obtained from all participants.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

We thank the Institute of Hepatobiliary Surgery of Southwest Hospital for providing human cholangiocarcinoma tissue. We also thank the Statistical Teaching and Research Department of the Third Military Medical University for technical assistance.

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January 2020
Volume 56 Issue 1

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APA
Pan, S., Hu, Y., Hu, M., Xu, Y., Chen, M., Du, C. ... Wang, J. (2020). S100A8 facilitates cholangiocarcinoma metastasis via upregulation of VEGF through TLR4/NF‑κB pathway activation. International Journal of Oncology, 56, 101-112. https://doi.org/10.3892/ijo.2019.4907
MLA
Pan, S., Hu, Y., Hu, M., Xu, Y., Chen, M., Du, C., Cui, J., Zheng, P., Lai, J., Zhang, Y., Bai, J., Jiang, P., Zhu, J., He, Y., Wang, J."S100A8 facilitates cholangiocarcinoma metastasis via upregulation of VEGF through TLR4/NF‑κB pathway activation". International Journal of Oncology 56.1 (2020): 101-112.
Chicago
Pan, S., Hu, Y., Hu, M., Xu, Y., Chen, M., Du, C., Cui, J., Zheng, P., Lai, J., Zhang, Y., Bai, J., Jiang, P., Zhu, J., He, Y., Wang, J."S100A8 facilitates cholangiocarcinoma metastasis via upregulation of VEGF through TLR4/NF‑κB pathway activation". International Journal of Oncology 56, no. 1 (2020): 101-112. https://doi.org/10.3892/ijo.2019.4907