Background
Intrahepatic cholangiocarcinoma (IHCC) is a malignancy whose pathogenesis involves abnormal biliary epithelial differentiation [
1]. It is the most frequent primary malignant liver tumor next to hepatocellular carcinoma and is highly fatal because of its early invasion, widespread metastasis, and the lack of an effective therapy [
2],[
3]. Therefore, it is urgent to uncover the molecular mechanisms of IHCC and identify potential therapeutic targets to improve the treatment. Chemokine receptors form a large family of proteins that mediate chemotaxis of cells towards a gradient of chemokines. Many studies have shown that chemokines and their receptors are implicated in the development of different types of cancers [
4]-[
6]. One of the best studied chemokine receptors is CXCR4. CXCR4 is a G protein-coupled chemokine receptor, encoded on chromosome 2 [
7]. During embryonic development, CXCR4 is expressed on progenitor cells, allowing the migration from their birthplace to their final destination where they will differentiate into organs and tissues. In the late 1990s, CXCR4 expressed on CD4+ T cells was found to serve as a co-entry receptor for human immunodeficiency virus HIV-1 [
8]. The following-up studies also found that CXCR4 can mediate the metastasis of a variety of cancers [
4],[
6],[
9],[
10]. CXCR4 selectively binds the CXC chemokine ligand-12 (CXCL12, or SDF-1), which has been found to be important in the tumorigenesis, proliferation, metastasis, and angiogenesis in cancers [
11],[
12]. CXCR4 has been reported to be upregulated in more than 20 cancers, including ovarian [
13], prostate [
14], esophageal [
15], melanoma [
16], neuroblastoma [
17], and renal cell carcinoma [
18], and plays an important role in the communication of cancer cells with their microenvironment [
19],[
20]. Moreover, CXCR4-positive cancer cells can migrate toward distant organs in response to CXCL12 gradient. By inhibition of CXCR4, the growth and invasion of cancer cells can be impaired [
21]-[
23]. In 2014, T. Yu et al. [
24] found that suppressing expression of CXCR4 by MicroRNA-9 could inhibit the proliferation of oral squamous cell carcinoma cells both
in vitro and
in vivo through the Wnt/β-catenin signaling pathway, and activation of CXCR4 expression led to the constitutive activation of β-catenin, implying the important role of Wnt/β-catenin in CXCR4 signaling, which was consistent with the previous reports in colorectal cancer [
25], ovarian cancer [
26], pancreatic cancer [
23], and bone marrow stromal cells [
27].
In cholangiocarcinoma, Ohira et al. [
28] demonstrated that CXCR4 was mainly expressed in IHCC cells and CXCL12 in stromal fibroblasts, and the interaction of CXCL12 released from fibroblasts and CXCR4 expressed on IHCC cells may be actively involved in IHCC migration, suggesting CXCR4 could be a therapeutic target to prevent IHCC invasion. This possibility was confirmed by Gentilini et al. [
29] using AMD3100, a non-peptide antagonist of the CXCR4, and Tan et al. [
30] using siRNA targeting at CXCR4. In 2012, CXCL12/CXCR4 was further reported to mediate angiotensin II-enhanced epithelial-to-mesenchymal transition (EMT) in IHCC [
31]. More recently, Leelawat K. et al. [
4] found that CD24 could induce CXCR4 expression in cholangiocarcimoma cells, which may assist invasion of the cancer cells. When treated by AMD3100, the motility and invasiveness of CD24 (+) cells were decreased, implying the importance of CXCR4 in cholangiocarcinoma cell invasion. However, the precise function of CXCR4 and the signal transduction pathways following CXCR4 activation in IHCC remain elusive. The aim of this study was to define the role of CXCR4 in IHCC and elucidate the underlying mechanism.
Methods
Cell culture
Human intrahepatic cholangiocarcinoma cell lines, HuCCT1 (ATCC, Manassas, VA, USA), HCCC-9810 ( Keygen Biotech, China), RBE ( Keygen Biotech, China), and Huh28 (Keygen Biotech, China) were cultured at 37°C in RPMI 1640 medium (Hyclone) supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin in humidified atmosphere containing 5% CO2.
Immunohistochemistry
Samples including 122 primary IHCCs, 75 matched metastatic lymph nodes, and 122 adjacent non-cancerous liver tissues containing normal intrahepatic bile ducts (at least 5 cm distant from the tumor edge) were obtained from the Department of Pathology, Shandong Provincial Hospital. Immunohistochemical staining for CXCR4 was performed using the SABC kit (Boster, Wuhan, China) according to the manufacturer’s instruction. Primary antibody for CXCR4 (1:50, polyclonal, Abcam, MA, USA) was used for overnight incubation at 4°C. For the evaluation of CXCR4 IHC staining, a semi-quantitative scoring criterion was used, in which both the staining intensity and positive areas were recorded. A staining index (values 0–12), obtained as the intensity of CXCR4-positive staining (weak, 1; moderate, 2; strong, 3) and the proportion of immune-positive cells of interest (0%, 0; <10%, 1; 10–50%, 2; 51–80%, 3; >80%, 4), were calculated. The cases were grouped into low (score 0–6) and high (scores 8–12) CXCR4 expression. The study was approved by the Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong University, as stipulated by the Declaration of Helsinki, with written informed consent for the use of the specimens from all enrolled patients.
Construction and transfection of CXCR4 shRNAs
This study utilized three CXCR4 shRNA targeting different regions of the CXCR4 [GenBank: NM_003467]. The shCXCR4-1 targeted CXCR4 mRNA at nucleotides 1093-1111 with sense: 5′- AGCGAGGTGGAC ATTCATC-3′, and antisense: 5′- GATGAATGTCCACCTCGCT -3′; The shCXCR4-2 targeted CXCR4 mRNA at nucleotides 741-759 with sense: 5′- CTGTCCTGCTATTGCATTA -3′, and antisense: 5′- TGACAGGACGACGATAACGTAAT -3′; The shCXCR4-3 was designed to be homologous to nucleotides 206-224 of the human CXCR4 with sense: 5′-TGAGAAGCATGACGGACAA-3′, antisense: 5′-TTGTCCGTCATGCTTCTCA-3′ [
23]. A negative control, targeting at no region in human genome, was designed with sense: 5′-TTCTCCGAACGTGTCACGT-3′, antisense: 5′-ACGTGACACGTTCGGAGAA-3′. These shRNA oligos were cloned to lentiviral vector pLKO.1 following the instruction provided by Addgene (Boston, MA, USA). All constructs were verified by sequencing. Stable transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Quantitative real-time RT-PCR
RNA was isolated from cells and reverse-transcribed. Real-time RT-PCR Primers specific for target genes were as follows: CXCR4, forward 5′-GATCAGCATCGACTCCTTCA-3′ and reverse 5′-GGCTCCAAGGAAAGCATAGA-3′; β-catenin, forward 5′-AAAATGGCAGTGCGTTTAG-3′ and reverse 5′-TTTGAAGGCAGTCTGTCGTA-3′; c-myc, forward 5′-AATGAAAAGGCCCCCAAGGTAGTTATCC-3′ and reverse 5′-GTCGTTTCCGCAACAAGTCCTCTTC-3′; CD44, forward 5′-AGAAGGTGTGGGCAGAAGAA-3′ and reverse 5′-AAATGCACCATTTCCTGAGA-3′; Vimentin, forward 5′-TGTCCAAATCGATGTGGATGTTTC-3′ and reverse 5′-TTGTACCATTCTTCTGCCTCCTG-3′; Slug, forward 5′-TGTTGCAGTGAGGGCAAGAA-3′ and reverse 5′-GACCCTGGTTGCTTCAAGGA-3′. GAPDH (forward: 5′-AACGGGAAGCTTGTCATCAATGGAAA-3′, reverse: 5′-GCATCAGCAGAGGGGGCAGAG-3′) served as an internal control. Experiments were repeated three times in duplicates. Relative gene expression was calculated using the 2-ΔΔct method.
Cell proliferation and cell cycle assays
Cell proliferation was measured using a Cell Counting Kit-8 (CCK-8) (Dojindo Molecular Technologies, Kumamoto, Japan). Control cells or cells stably transfected with sh-CXCR4 or negative control were seeded into 96-well plates at 2000 cells per well and incubated overnight with or without CXCL12 (R&D, MN, USA) at 100 ng/ml. Viability of cells were measured using a Cell Counting Kit-8. Briefly, 10 μl of CCK-8 solution was added to each well after 1, 2, 3, 4 and 5 days for proliferation measurement, respectively. In viable cells, WST-8 was metabolized producing a chromogen that was detected at 450 nm using a Spectra Max M2 spectrophotometer (Molecular Devices, Sunnyvale, California, USA).
For cell cycle analysis, transfected cells were cultured for 24 h, collected, fixed into 70% ethanol at -20°C for 24 h, stained with 50 μg/ml propidium iodide (Kaiji, Nanjing, China) and analyzed with a FACS Calibur (Epics XL-4; Beckman Coulter, Brea, California, USA).
A quantity of 500 cells transfected with either shCXCR4 or negative control were cultured in 6-well plates with or without CXCL12 for 2 weeks in regular culture medium. Colonies with more than 50 cells per colony were counted, fixed with methanol for 15 min, and stained with hematoxylin and eosin (H&E). All the experiments were performed in triplicate wells and repeated at least three times.
β-Catenin/Tcf transcription reporter assay
Briefly, 1 × 105 cells were seeded each well in a 24-well plate before transfection with the construct of TOPflash or FOPflash reporter plasmid (Millipore, Billerica, MA, USA). TOPflash comprised three copies of the Tcf/Lef sites upstream of a thymidine kinase (TK) promoter and the Firefly luciferase gene. FOPflash comprised three mutated copies of Tcf/Lef sites and was used as a control for measuring nonspecific activation of the reporter. All transfections were performed using 0.8 μg of TOPflash or FOPflash plasmid and 2 μl of Lipofectamine 2000. To normalize the transfection efficiency in reporter assays, the cells were co-transfected with 0.02 μg of an internal control reporter plasmid, containing Renilla reniformis luciferase driven by the TK promoter. Twenty four hours after transfection, the luciferase assay was performed with the Dual Luciferase Assay System kit (Promega Corp., Madison, WI, USA). Relative luciferase activity was reported as the fold induction after normalization for transfection efficiency.
Wound healing and matrigel invasion assays
Cells transfected with negative control or shCXCR4 were seeded in 6-well plates and cultured. Upon reaching appropriate confluence, cells were serum starved for 24 h, and then the cell layer was scratched with a sterile plastic tip, immediately washed twice with PBS, and cultured in serum free 1640 medium with or without CXCL12 at 37°C in a humidified incubator with 5% CO2. At 24 h, the plates were photographed under a microscope.
For invasion assay, cells were re-suspended in serum-free medium and seeded in the top chambers of Matrigel-coated (invasion) chambers (24-well insert, 8 μm pore, Corning Costar Corp., Cambridge, MA, USA) at the concentration of 2 × 105 per 200 μl medium. The lower chambers were filled with 0.5 ml of normal culture medium with or without CXCL12 (100 ng/ml). After 24 h, the cells on the upper surface of the membrane were removed using cotton tips, and cells that migrated to the lower surface were fixed in 4% paraformaldehyde for 15 min at room temperature, stained with hematoxylin and eosin (H&E), and counted under the light microscopy.
Western blotting
Whole cell extracts were prepared in lysis buffer as described previously [
32]. The cell lysates were separated by electrophoresis in 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, blocked in 5% nonfat milk, and incubated with primary antibodies against CXCR4 (1:1000 dilution, Abcam), phospho-CXCR4 (1:1000 dilution, Abcam), β-catenin (1:1000 dilution, Abcam), Vimentin (1:1000 dilution, Abcam), MMP-9 (1:1000 dilution, Abcam), and β-actin (1:5000 dilution; Abcam) at 4°C overnight. After incubation with corresponding peroxidase-conjugated secondary antibodies (1:5000 dilution, Abcam), protein bands were detected using an enhanced chemiluminescence reagent (Sigma, Ronkonkoma, New York, USA).
Tumorigenicity assay in nude mice
Five to six-week-old male nude mice used in the studies were purchased from the Institute of Zoology, Chinese Academy of Sciences (Beijing, China). After 4 days of acclimatization, a total of 2 × 106 IHCC cells stably transfected with either sh-CXCR4 or negative control were injected subcutaneously into either side of the groin of each mouse (left: negative control cell, right: sh-CXCR4 for group A, and inversely for group B). Each group contained 3 mice. The mice were killed on the 28th day after injection. The mice were manipulated according to the guidelines approved by the Shandong University Institutional Animal Care and Use Committee (IACUC).
Statistical analysis
The data are presented as percentages of control ± SEM or means ± SEM from multiple experiments. The statistical significance between groups was determined using the Student’s t-test. Overall survival was counted from the first day of surgery to the date of death or the last follow-up visit and the estimated value was calculated by the Kaplan-Meier method and compared between groups via the log-rank test. SPSS version 12 (SPSS Inc. Chicago, IL, USA) software was used for all data analyses.
Discussion
Compared with other malignancies, IHCC is generally characterized by strong proliferation, invasion, and early metastasis. Many factors such as adhesion molecules, proteases, cytokines, and chemokine are involved in these processes. CXCR4 and CXCL12 play an essential role in tumor growth, metastasis, and cancer cell-microenvironment interaction. CXCR4 has been known to be overexpressed in more than 20 human tumor types [
13]-[
18], and CXCR4 antagonists inhibit tumor growth in multiple experimental orthotopic [
33],[
34], subcutaneous human xenografts [
35],[
36], and transgenic mouse models [
37]. Preclinical cancer models have revealed that directed metastasis of cancer cells is mediated by CXCR4 activation and migration of cancer cells is towards CXCL12 expressing organs [
14],[
35],[
38] while targeting CXCR4 impairs the spread of cancer cells and development of metastasis [
34],[
37],[
38]. Moreover, high levels of CXCL12 expressed by cancer cells and tumor-associated stromal cells directly stimulate the proliferation and invasiveness of breast cancer cells in the autocrine and paracrine manners [
19]. High CXCL12 levels in the tumor attract CXCR4-positive inflammatory, vascular and stromal cells into the tumor mass, where they will eventually support the tumor growth by secreting growth factors, cytokines, chemokines, and pro-angiogenic factors [
19],[
39]. In addition, CXCR4 positive cancer cells can be recruited to CXCL12-rich mesenchymal stroma niches. This recruitment mimics the homing of normal stem cells to the bone marrow [
39],[
40], and cancer cells homed to bone marrow reside in a microenvironment that protects them in a CXCR4-dependent manner from chemotherapy [
41]. In this study, we demonstrated that the overall survival rate of IHCC patients with high CXCR4 expression is significantly lower than those with low CXCR4 expression. Elevated CXCR4 expression is related to vascular invasion, lymph node metastasis, and the TNM stages. This is similar to previous reports that CXCR4 may be a useful marker for cancer progression [
6],[
42],[
43]. We also found that CXCR4 shRNA not only significantly reduced the expression of CXCR4, but also notably decreased phosphorylation of CXCR4 at serine 339. Considering the findings that the phosphorylation of CXCR4 at serine 339 may be a way to activate CXCR4 on the cells [
44], our data further confirmed that CXCR4 shRNA could effectively inhibit CXCR4 function in IHCC cancers.
Tumorigenesis is the result of cell cycle disorganization, leading to uncontrolled cell proliferation and cancer progression. In this study, we have demonstrated that the blockade of CXCR4 can decrease IHCC cancer cell growth and cell cycle by prolonging the G0–G1 cycle and reducing the G2 and S phases, and inhibit tumorigenesis both
in vitro and
in vivo. The Wnt/β-catenin pathway plays a major role in intrahepatic cholangiocarcinoma cell growth, metastasis, and cancer susceptibility [
4],[
28]-[
31]. Dysregulation of β-catenin and other Wnt components leads to activation of Wnt target genes, including c-myc, cyclin D1, and MMP-9 [
45]-[
47], and the enhancement of tumor formation [
48]. Our data showed the TOPflash luciferase activity was sharply decreased by the inhibition of CXCR4 whereas FOPflash luciferase activity was nearly unchanged in the β-catenin/Tcf assay. Moreover, the expression of Wnt target genes, including β-catenin, c-myc, and MMP-9, was markedly decreased, suggesting that the TCF-binding activity could be effectively inhibited by CXCR4 knockdown, which may suppress theWnt/β-catenin signaling and Wnt target genes expression.
Next, we analyzed the expression of invasion-related genes Vimentin and Slug. These two genes are typical mesenchymal markers associated with the EMT process, which may influence carcinoma metastasis [
49]-[
54]. Our data showed that Vimentin and Slug were downregulated in CXCR4 knockdown cells, together with the decreased ability of invasion and migration as shown in transwell and wound healing assays. This is consistent with the previous report that CXCR4 could influence EMT formation and cancer invasion [
31],[
55]-[
60]. However, an intriguing phenomenon was also observed in the clinical trial of plerixafor (a CXCR4 inhibitor) as a combined treatment with intensive chemotherapy in heavily pre-treated relapse AML patients [
61]. In the phase II study of 46 patients, a two-fold mobilization in leukemic blasts into the peripheral circulation was found, which was in modest correlation with CXCR4 expression. Furthermore, in a recent phase I study of another CXCR4 inhibitor LY2510924 for advanced cancer [
62], the circulating tumor cell (CTC) counts were included as one of the study endpoints in addition to safety, pharmacokinetics, efficacy, and pharmacodynamics. In some (7/42) patients the CTC numbers were increased after the treatment with the CXCR4 inhibitor. Although the significance of these studies are inconclusive due to small sample sizes, these intriguing observations should prompt to investigate the mobilizing effects of CXCR4 inhibition in tumors in more details.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SZ and CQ conceived and designed the experiments. SZ and JW performed the experiments. SZ and CQ analyzed the data. SZ and JW performed the statistical analysis. SZ and CQ wrote the manuscript. All authors read and approved the final manuscript.