Background
Gallbladder cancer (GBC) is rare but represents the most common cancer of the biliary tract, accounting for 80–95 % of biliary tract malignancies [
1,
2]. GBC is a highly aggressive disease with very poor prognosis (5-year survival rate < 5 % [
3,
4]), due to its tendency to metastasize to the lymph nodes in early stages. More than 50 % of all patients with GBC exhibit lymph node metastases (LNM) [
5]. Therefore, understanding the mechanism underlying lymphatic metastasis in GBC is helpful to improve patient treatment and prognosis. However, the specific mechanisms underlying lymphatic metastasis in GBC are largely unknown.
In 1863, Virchow first observed that inflammatory cells can be found in tumors [
6]. Since then, many studies have examined the relationship between inflammation and cancer. It has been generally accepted that chronic inflammation promotes cancer [
7], including some cancers of the liver [
8], intestine [
9,
10] and lung [
11]. Cytokines secreted by inflammatory cells, including TNF-α, IL-1, and IL-6, play important roles in cancer-related inflammation [
7,
12‐
15]. Tumor necrosis factor alpha (TNF-α), a key pro-inflammatory cytokine that was first identified as a mediator of tumor cell death, is now also known to promote the tumor progression, proliferation, epidermal-mesenchymal transition (EMT), angiogenesis, invasion and metastasis [
16‐
19]. Lymphatic metastasis is one of the major forms of tumor metastasis. However, the relationship between TNF-α and lymphatic metastasis requires further research.
Recently, we confirmed that TNF-α can promote lymphangiogenesis and lymph node metastasis of GBC through upregulation of vascular endothelial growth factor C (VEGF-C) downstream of NF-κB [
20]. Furthermore, we determined that vascular endothelial growth factor D (VEGF-D), another key lymphangiogenic factor similar to VEGF-C, is associated with lymphangiogenesis and lymph node metastasis of GBC [
21]. Thus, we aimed to further explore whether VEGF-D is involved in TNF-α-induced lymphatic metastasis of GBC and the underlying mechanisms.
In this study, we first analyzed the relationship between TNF-α levels and VEGF-D expression in clinical specimens and demonstrated that TNF-α can upregulate VEGF-D expression in the NOZ and GBC-SD cell lines. Previous studies have demonstrated that TNF-α promotes the expression of target genes mainly through NF-κB and (or) AP-1 signaling pathways [
22]. We further sought to determine whether TNF-α upregulates VEGF-D expression and enhances its promoter activity through these two pathways. Furthermore, we determined that TNF-α can promote tube formation of human dermal lymphatic endothelial cells (HDLECs), lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D
in vitro and
in vivo.
Methods
Patient samples and cell culture
20 GBC tissues and the matching bile used in present study were obtained from the patients admitted to Fujian Medical University Union Hospital in China. The informed consents of agreement to use the samples for further study were signed pre-operation. The samples were collected according to the protocol approved by the Ethics Committee of the Medical Faculty of Fujian Medical University, according to the Declaration of Helsinki. The details of the patients including the age and sex of the patient, clinical stage, grade of the tumor and lymph node metastasis (LNM) had been described in [
20]. The human GBC cell lines: NOZ (obtained from Health Science Research Resources Bank in Japan), GBC-SD (purchased from Shanghai Institutes for BiologicalSciences in China) and SGC-996 (provided by the Tumor Cytology Research Unit, Medical College, Tongji University, China) were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco, USA) supplemented with 10 % fetal bovine serum (Gibco). Human dermal lymphatic endothelial cells (HDLECs, purchased from Sciencell, San Diego, California, USA) were incubated in endothelial cell medium (Sciencell). All of the cells were incubated at 37 °C under 95 % air and 5 % CO2.
Immunohistochemistry
The VEGF-D expression and lymphatic vessels of GBC specimens were detected by immunohistochemistry as previously described [
21]. The primary antibodies were VEGF-D (ab155288, Abcam) at a 1:80 dilution and LYVE-1 (AF2125, R&D Systems) at a 1:150 dilution. The method used to measure the VEGF-D expression has been described previously [
23]. The density of LYVE-1-positive vessels (lymphatic vessels density, LVD) was assessed according to the method described by Qiang Du [
24].
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from GBC cells with TRIzol reagent (Invitrogen). RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo). PCR reactions were performed with Fast Start Universal SYBR Green Master Mix (Roche), and fluorescence was measured using the 7500 quantitative real-time thermocycler (Applied Biosystems). GAPDH served as an internal control. All procedures followed the manufacturer’s instructions.
Enzyme-linked immunosorbent assay (ELISA)
VEGF-D levels in cell culture media were measured by double antibody sandwich enzyme-linked immunosorbent assay using Quantikine ELISA Kits from R&D Systems following the manufacturer’s instructions. VEGF-D Standards for drawing standard curve were prepared before the antibody reaction. 100 μL of Assay Diluent RD1X was added to each well, and then 50 μL of Standard, sample or control were added to each well and incubated for 2 h at room temperature. Wash each well with wash buffer (400 μL) for four times. Add 200 μL of VEGF-D Conjugate to each well and incubate for 2 h at room temperature. Wash each well again and add 200 μL of Substrate Solution to each well. Add 50 μL of Stop Solution to each well after incubation for 30 min (protect from light). The wells were read at 450 nm with a Model 550 Microplate Reader (Bio-Rad, Hercules, CA, USA). Each reaction was run in triplicate.
A series of 5′-deletion DNA fragments of the VEGF-D gene promoter were amplified by PCR with primers containing an XhoI or BglII (Thermo) restriction site, which were connected to the pGL4.10-Basic vector (Promega) carrying a firefly luciferase report gene. These recombinant VEGF-D promoter luciferase reporter plasmids were named PGL4-2148 (−2148 to +117, relative to the transcription start site “ATG”), PGL4-1621 (−1621 to +117), PGL4-988 (−988 to +117), PGL4-717 (−717 to +117), PGL4-444 (−444 to +117), PGL4-325 (−325 to +117), PGL4-154 (−154 to +117), and PGL4-57 (−57 to +117). Forty-eight hours after transfection with promoter vector, cells were lysed and the intracellular luciferase activity of the lysates was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. The relative luciferase units were obtained by comparison with the luciferase activity of the pRL-TK plasmid (plasmid carrying a renilla luciferase report gene as an internal reference).
Identification of putative transcription factor binding sites
Site-directed mutagenesis
The site-directed mutagenesis was performed by overlap extension PCR as previously described [
20,
25]. The primers targeting the two mutation sites of the AP-1 binding sites were as follows: AP-1mut1 (−401 to -393 nt), (forward), 5′-CATCTGCTGCCAATGCTACACAGAAAGCAATC-3′ (reverse); AP-1mut2 (−345 to -337 nt), 5′-CTTAAGCAATCCCACCGAGATACAAAGGTC-3′ (forward), 5′-GACCTTTGTATCTCGGTGGGATTGCTTAAG-3′ (reverse).
Nuclear extraction and electrophoretic mobility shift assay (EMSA)
Nuclear proteins were extracted from NOZ cells using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, JiangSu, China), and electrophoretic mobility shift assay (EMSA) was performed with the LightShift Chemiluminescent EMSA kit (Thermo Scientific, Inc.) according to the manufacturers’ recommendations. Two biotin-labeled oligonucleotide probes (5′biotin-CTTTCTGTGTGTCATTGGCAG-3′, which contained −401 to −393 nt, and 5′biotin-ATCCCACTGAGATACAAAGGT-3′, which contained −345 to −337 nt) were used to confirm the DNA binding of AP-1. For competition analysis, we used 100-fold excess of unlabeled competitive probes, including cold probes and mutational cold probes (5′-CTTTCTGTGTAGCATTGGCAG-3′, and 5′-ATCCCACCGAGATACAAAGGT-3′, mutation sites underlined).
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay was performed according to the manufacturer’s instructions using the EZ-Magna ChIP kit (Merck Millipore, Darmstadt, Germany). An antibody against AP-1 (c-Jun, phosphor S63, Abcam), a negative control normal rabbit IgG, and a positive control anti-acetyl histone H3 antibody were used for immunoprecipitation. The primers for PCR were as follows: 5′-TTGCATGTATGGATGGATGTTTT-3′ (forward) and 5′-AAGAAGGGACCTCAGATGCTCAT-3′ (reverse); and 5′-GAGCATCTGAGGTCCCTTCTTAA-3′ (forward) and 5′-AAGAAGGGACCTCAGATGCTCAT-3′ (reverse).
AP-1(c-Jun) siRNA oligonucleotide treatment of cells
The AP-1 (c-Jun) siRNA interference sequence has been described previously [
26] (named siAP-1, sense: 5′-GAUGGAAACGACCUUCUAUdTdT-3′, anti-sense: 5′-AUAGAAGGUCGUUUCCAUCdTdT-3′), and the non-targeting control (named siNC) were synthesized chemically by GenePharma Co., Ltd. (Suzhou, China). The transient transfection was performed according to the manufacturer’s instructions.
Western blotting
Western blot analysis was performed as described previously [
27]. Cells were washed twice with ice cold PBS and then incubated on ice with 100 μL of RIPA buffer with 100 mM PMSF (phenylmethylsulfonyl fluoride) for 15 min. Plates were scraped and lysates were centrifuged at 13,000 rpm for 5 min at 4 °C. The protein concentrations of cell lysates were measured in duplicate using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). The appropriate amount of 5× loading buffer was mixed with the protein lysates and boiled for 5 min at 100 °C. Equal amounts of total protein were resolved by 10 % SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis and transferred to PVDF (polyvinylidene fluoride) membranes. The PVDF membranes were then blocked with 5 % nonfat milk in Tris Buffered Saline with Tween (TBST; 10 mM Tris–HCl, 150 mM NaCl, and 0.05 % Tween) for 2.5 h. The appropriate diluted primary antibodies, including anti-VEGF-D, anti-AP-1 (c-Jun, phospho-S63), anti-phosphorylated AP-1 (p-AP-1) antibodies (1:1000, Abcam) and the β-actin antibody (1:1000, Santa Cruz), were then incubated with the membranes overnight at 4 °C. The appropriate secondary antibody conjugated with horseradish peroxidase diluted in TBST was added for 1 h at room temperature. Immunoreactivity was detected using a chemiluminescence western blot immunodetection kit (Invitrogen) according to the manufacturer’s instructions and recorded on Hyperfine-ECL detection film. The amounts of each protein were semiquantified as ratios to β-actin indicated on each gel.
Construction of a stable NOZ cell line with lentiviral VEGF-D shRNA
We previously identified an siRNA sequence (5′-GCUAUGGGAUAGCAACAAAUG-3′) that effectively knocked down VEGF-D gene expression in NOZ cells [
21]. To establish a stably expressing cell line, we used lentiviral vector expressing a VEGF-D shRNA construct (named LV-siVEGF-D) and a control vector containing a non-targeting sequence (named LV-siNC). Both vectors were constructed by Genepharma Co., Ltd. (Suzhou, China) and were used to infect NOZ and GBC-SD cells; puromycin was used to screen for stably infected cells.
To assess the role of the TNF-α-VEGF-D axis in the tube formation of HDLECs, NOZ or GBC-SD cells stably transfected with LV-siVEGF-D were co-cultured with HDLECs previously labeled by DiI (a cell membrane dye emitting red fluorescence; Beyotime Institute of Biotechnology, ShangHai, China) in a three dimensional coculture system following the method described by Yiping Zeng [
28]. Briefly, 7.5 × 10
3/well of GBC cells and 7.5 × 10
3/well of HDLECs were seeded to the same well of microwell-plate (ibidi) which was previously painted with matrigel. Tube formation of HDLECs was observed by inverted fluorescence microscopy (Nikon, Japan), and images were digitally captured at 1 h, 3 h, 5 h, 8 h and 24 h after cell seeding. The total number of tube-like structures formed in each well were measured with Axiovision Rel 4.1 software (Carl Zeiss AG, Jena, Germany).
Establishment of the orthotopic xenograft model
Thirty male athymic BALB⁄c nude mice 4–6 weeks-old were obtained from Slaccas Laboratory Animal Co. (Shanghai, China) and raised in the specefic pathogen free (SPF) laboratory animal room. All experiments in this part were carried out in accordance with institutional guidelines and were approved by the Ethics Committee of the Medical Faculty of the Fujian Medical University. The orthotopic xenograft models were established following the method by Qiang Du [
20,
24]. Two weeks later, exogenous TNF-α (2 μg/kg) was injected into the peritoneal cavity every 3 days for 3 weeks. Five weeks after injection of cells, the mice were euthanized by exposure to CO
2, and primary tumors were dissected and excised.
Statistics
Results are presented as the mean ± SD from at least three independent experiments. Data were analyzed by Student’s t-test. A two-sided P-value <0.05 was considered statistically significant.
Discussion
As previously mentioned, the relationship between inflammation and cancer was first appreciated by Vichow in 1863. It is currently estimated that approximately 25 % of the malignancies worldwide are induced by chronic inflammation [
29,
30]. The characteristics of this chronic inflammation is the infiltration of a large number of inflammatory cells that secrete various cytokines [
31]. TNF-α, mainly secreted by macrophage, is a key player in cancer-related inflammation. Chronic inflammation induced by gallstones, infection, or other factors is one of the leading causes of GBC according to epidemiological investigations, [
32,
33] and TNF-α has been detected in the inflammatory environment of the gallbladder [
34,
35]. Consistent with these reports, our laboratory recently observed that the level of TNF-α in the bile of GBC patients was higher than that of patients with cholecystic polypus (without obvious inflammation) and demonstrated the ability of TNF-α to promote lymphangiogenesis in GBC [
20].
Lymphangiogenesis is thought to be an important step in cancer metastasis [
36]. Our previous study have confirmed that TNF-α can promote lymphangiogenesis of GBC through upregulation of VEGF-C. Meanwhile, we found that the effect of TNF-α-induced lymphangiogenesis in GBC was only partially inhibited with knock-down of VEGF-C expression. This interesting phenomenon promoted us to speculate that there should be other molecular mechanisms involved in the TNF-α-induced lymphangiogenesis in GBC. Similar to VEGF-C, VEGF-D is another key lymphangiogenic factor which is associated with lymphangiogenesis and lymph node metastasis of GBC [
21]. Therefore, we hypothesized that VEGF-D may be involved in the TNF-α-induced lymphatic metastasis of GBC.
In the present study, we found that the level of TNF-α in the bile of GBC patients was correlated with the expression of VEGF-D in the tissue. Subsequently, we confirmed in vitro that TNF-α significantly increased the mRNA and protein expression of VEGF-D in NOZ and GBC-SD cell lines within the dose range of 10–50 ng⁄mL in a dose- and time-dependent manner. We further to reveal that TNF-α can upregulate the protein expression and promoter activity of VEGF-D through the ERK1/2 - AP-1 pathway. Moreover, we determined that TNF-α can promote tube formation of HDLECs, lymphangiogenesis and lymph node metastasis of GBC by upregulation of VEGF-D in vitro and in vivo. In the tube formation assay, HDLECs were previously labeled by DiI before co-culture with GBC cells and observed by the inverted fluorescence microscope after co-culture. This method can effectively exclude the interference of GBC cells when observation. In addition, the orthotopic xenograft model of GBC in nude mice is more able to reflect the growth pattern of GBC in human body.
Many studies have focused on the relationship between VEGF-D and lymphatic metastasis [
21,
37‐
40]. However, few investigations have concentrated on the regulation of VEGF-D promoter activity. To date, only two studies have suggested that orphan receptor hepatocyte nuclear factor 4α (HNF-4α), chicken ovalbumin upstream promoter transcription factors 1 and 2 (COUP-TF1 and COUP-TF2) and AP-1 bind to the VEGF-D promoter [
41,
42]. A large number of studies have demonstrated that the downstream effector molecules associated with tumor progression are NF-κB or AP-1 [
22,
43]. To determine whether TNF-α regulates VEGF-D promoter activity through these two transcription factors, we used the TFbind and Promoter Scan programs to search for potential binding sites of NF-κB or AP-1 in the three fragments of VEGF-D promoter with higher activities (−988 to -717 nt,-444 to -325 nt,and −154 to -57 nt), and found that the −444 to -325 nt region contains two putative AP-1 binding sites, whereas NF-κB sites were not found. Subsequently, we confirmed that both the AP-1 sites could bind to the VEGF-D promoter and that TNF-α could enhance the combination by site-directed mutagenesis, EMSA, and ChIP analysis. Further, we used siRNA to knock down AP-1, and the protein level of VEGF-D and the activity of the PGL4-444 plasmid were consequently decreased in the both groups with or without TNF-α treatment.
It is demonstrated that the multiple effects of TNF-α in cancers are due to the different downstream signaling pathways activated by the combination of TNF-α and its receptor (mainly through NF-κB and (or) AP-1 pathway). There are two AP-1 binding sites (no NF-κB site) in the core region of VEGF-D promoter, which revealed that TNF-α-induced upregulation of VEGF-D is mainly through the AP-1 pathway. Two signaling pathways associated with AP-1 have been clarified in previous studies: the TNF-α - TNFR1 - signaling complex - MAP3K (ASK1) - JNK or p38 MAPK - AP-1 pathway and the TNF-α - TNFR1 - Ras - Raf - MEK1 - ERK1/2 - AP-1 pathway [
44]. To further determine which pathway is involved in the TNF-α - VEGF-D axis, we employed three reagents, SP600125, SB203580 and PD98059, to selectively inhibit JNK, p38 MAPK and ERK1/2, respectively. The protein expression of AP-1, p-AP-1, and VEGF-D and the activity of the PGL4-444 construct were significantly inhibited in the PD98059 treatment group, which indicated that TNF-α upregulated VEGF-D promoter activity and protein expression primarily through the ERK1/2/AP-1 signaling pathway.
The active Ras proteins combine with the guanosine triphosphate (GTP) and then activates the downstream signaling pathways including the MAPK pathway [
45]. The alteration of Ras protein conformation caused by Ras gene mutation makes it lose the GTPase activity and leads to the continuous activation of the downstream signaling which accordingly promotes cell proliferation and invasion. K-ras gene is a member of the Ras family and K-ras mutation has been reported in various malignancies including GBC and NOZ cell line [
45‐
48]. As mentioned above, the Ras protein is an effector between TNFR and ERK1/2. Thus we can speculate that K-ras mutation could enhance the activity of the “TNF-α/ERK1/2/AP-1/VEGF-D” pathway in NOZ cells which might accordingly enable the nude mice bearing human GBC in the present study more prone to appear lymphatic metastasis.
In this study, we first discovered the relationship between the TNF-α - VEGF-D axis and the lymphangiogenesis and lymphatic metastasis of GBC. Subsequently, we demonstrated that the regulatory mechanism between TNF-α and VEGF-D is dependent on the ERK1/2/AP-1 signaling pathway. Furthermore, we determined the core activity region of the VEGF-D promoter and identified two AP-1 binding sites in these regions. The regulatory mechanisms of inflammation-induced tumor metastasis are very complicated, but our work helps elucidate some of these mechanisms.
Together with our previous study, these results reveal that TNF-α can promote lymphangigenesis and lymph node metastasis of GBC at least by two signaling pathways: the NF-κB/VEGF-C pathway and the ERK1/2/AP-1/VEGF-D pathway. But, which pathway is dominated or both are equally important, needs further study.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CYL, SFF, HHJ and JL contributed to concept design, discussed results. HHJ also performed immunohistochemistry, relative luciferase reporter assay, mutant constructs, EMSA, and ChIP assay; participated in cell culture and animal experiment; and wrote the manuscript. LYF participated in cell culture and tube formation assay; performed PCR and ELISA. HCL carried out protein isolation and Western blotting, and also participated in animal experiment. ZGW participated in the sequence alignment. DQ and TNH performed the statistical analysis. WXQ gave assistance with several technical performances. All authors read and approved the final manuscript.