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SDF-1/CXCR4 induces cell invasion through CD147 in squamous cell carcinoma of the hypopharynx

Hypopharyngeal squamous cell carcinoma (SCC) has a poor prognosis due to local invasion and metastasis. The chemokine receptor CXC chemokine receptor type 4 (CXCR4) and its ligand, stromal cell-derived factor 1 (SDF-1), play roles in tumor progression through unclear mechanisms. For the present study, we used a hypopharyngeal SCC cell line, FaDu, expressing CXCR4. We found that SDF-1 promotes migration and invasion of the FaDu cells. In addition, AMD3100, a specific antagonist of CXCR4, inhibited the binding of SDF-1 to CXCR4, resulting in a significant decrease in the FaDu cell migration induced by SDF-1. Stimulation of CXCR4 with SDF-1 induced an increase in the expression of CD147, a cell membrane protein; and this CD147 upregulation was abrogated by AMD3100. CD147 function-blocking antibodies also abolished the SDF-1-induced FaDu invasiveness. Our results suggested that SDF-1/CXCR4 mediate hypopharyngeal SCC cell migration and that CD147 is involved in the SDF-1/CXCR4-related tumor progression.


Introduction
Hypopharyngeal cancer (mostly squamous cell carcinoma (SCC)) is most common in the head and neck. Treating patients is difficult and they experience poor life quality due to problems eating, swallowing, vocalizing, and breathing; moreover, the prognosis remains particularly ominous, even among the head and neck cancers (1). These cancers also present high local invasiveness with neck lymph node and distant metastases (2), and approaches are needed to improve adjuvant therapy for hypopharyngeal cancer.
Chemokines are a superfamily of small cytokine-like proteins that can bind to the transmembrane domain and activate chemokine receptors, a family of seven transmembrane G protein-coupled receptors (3). The chemokine family currently comprises over 40 members that are subdivided into groups based on their first two N-terminal cysteine residue motifs and binding to G protein-coupled receptors segmented based on chemokine subgroups (CXCR, CCR, CX3R1, XCR1) (4). Chemokines have various roles in vivo, including promoting mitosis and regulating apoptosis, survival, and angiogenesis (5,6). These functions are beneficial for tumor progression, and chemokines are now recognized to be expressed in many tumor types (7).
In particular, the interaction between the CXC chemokine receptor type 4 (CXCR4) and its ligand stromal derived factor 1 (SDF-1), also known as CXCL12, has been shown to affect cell survival, proliferation, and migration in malignant tumors (8,9). It was recently discovered that the expression of CXCR4 is involved in the metastasis of breast and prostate cancer cells cooperating with SDF-1 (10,11). Thus, SDF-1/CXCR4 plays a role in the tumorigenesis of many cancers (12,13).
CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a member of the immunoglobulin superfamily that is highly expressed in cancer cells. It causes a variety of malignant tumors, including HNSCC (23,24). Studies have attempted to elucidate the mechanisms underlying CD147-induced tumorigenesis in various types of cancer (3,25), and the number of studies on the contribution of CD147 to the progression of HNSCC is increasing. We have previously reported that CD147 increases cell invasiveness, proliferation, and drug resistance via interaction with its ligand, cyclophilin A, in HNSCC cells [including in a hypopharyngeal cell line; (26)]. Moreover, CD147 expression is associated with cervical lymph node metastases in patients with tongue SCC (27). However, the role of CD147 in HNSCC tumorigenesis and its underlying mechanisms are not fully understood. In particular, although the relationship between CD147 and cytokines has been reported in various diseases (28,29), many uncertainties remain in patients with cancer. The present study was aimed to investigate the mechanisms of tumor progression by SDF-1/CXCR4 in patients with hypopharyngeal cancer, thus assessing their association with CD147.

Materials and methods
Cell lines and cell culture. We purchased HSC-3, a human tongue SCC cell line, from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). FaDu, the cells from a human hypopharyngeal SCC cell line, were kindly gifted by the Department of Cell Biology and Morphology, Akita University Graduate School of Medicine (Akita, Japan); we used both cell lines for in vitro studies. All cells were maintained in the Dulbecco's modified Eagle's medium (DMEM; Merck KGaA) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO 2 at 37˚C.
Western blotting. We detected protein expression using western blot analysis with actin as an internal control. We lysed cell lines in detergent containing 1% NP-40, 150 mmol/l NaCl, 1 mmol/l EDTA, 0.1 mmol/l phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin and determined the protein levels using the Bio-Rad Protein Assay method (Bio-Rad Laboratories). We separated 40 µg of the total protein on 8% SDS-PAGE gels and transferred them to nitrocellulose membranes using a semidry transfer machine (Bio-Rad Laboratories). Next, we blocked membranes with 5% skimmed milk/TBS with Tween-20 solution for 1 h at room temperature, and incubated with primary antibodies in 5% skimmed milk in TBS-T overnight at 4˚C. After washing with TBS-T three times, we incubated the membranes for 1 h with horseradish-peroxidase-conjugated secondary antibody (Bio-Rad Laboratories) 1:3,000 diluted in 5% skimmed milk in TBS-T. We rinsed the filters with TBS-T three times and developed the blot using Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by autoradiography. The band intensities were analyzed using the ImageJ software (U. S. National Institutes of Health). We used the following primary antibodies: Rabbit anti-CXCR4 (1:1,000; Bioss), rabbit anti-CD147 (1:1,000; Santa Cruz Biotechnology), and mouse anti-β-actin (1:5,000; Merck Millipore).
Matrigel invasion and cell migration assays. We evaluated cell invasion and migration using in vitro using Matrigel-coated semipermeable modified Boyden inserts with a pore size of 8 µm (BectonDickinson/Biocoat). We plated cells in duplicate at a density of 5x10 3 cells/well for the invasion assay or at 3x10 4 cells/well for the migration assay. Plating was carried out on serum-free DMEM with SDF-1 (0.1 µg/ml; Pepro Tech), AMD3100 (10 ng/ml; Abcam), anti-CD147 function-blocking antibody (10 µg/ml, UM-8D6, cat. no. 10R-CD147aHU; Research Diagnostics), for which the blocking activity has been published (30,31), or a combination of SDF-1 and AMD3100 for the migration assay or anti-CD147 function-blocking antibody for the invasion assay in the inserts. We plated the cells in 96-well plates to serve as loading controls. Both the insert and the holding well were filled with the same medium composition, but without serum. The insert contained no serum, whereas the lower well contained 10% FBS that served as a chemoattractant. After a 24-h treatment at 37˚C in a 5% CO 2 incubator, we gently wiped away the cells in the insert using a cotton swab. Cells on the reverse side of the insert were fixed and stained with Diff-Quik ® (Sysmex) according to the manufacturer's instructions. Cells plated in 24-well plates were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, and we normalized the cell numbers across the groups. We also adjusted the number of invading or migrating cells accordingly.
Proliferation assay. FaDu cells were plated in triplicate at a density of 3x10 4 cells/well and allowed to seed overnight in a 12-well plate. Cells were then treated with SDF-1 (0.1 µg/ml), anti-CD147 function-blocking antibody (10 µg/ml), or a combination of SDF-1 and anti-CD147 function-blocking antibody in DMEM with 10% FBS. At selected time-points, we trypsinized the cells and stained them with trypan blue, and viable cells were counted using a hemocytometer.
Statistical analysis. Statistical analyses were performed using Statcel 3 (OMS Publishing). One-way ANOVA with post-hoc Tukey test was used to assess the statistically significant differences in proliferation, invasion, and migration studies. Data are presented as the mean ± SD from experiments that were repeated at least three times. P<0.05 was considered to indicate a statistically significant difference.

Results
Hypopharyngeal SCC cell expresses CXCR4. To investigate the function of CXCR4 in hypopharyngeal SCC, we measured the expression of CXCR4 in FaDu cells (established from hypopharyngeal SCC) by western blotting. At the same time, we also analyzed the expression of CXCR4 in HSC-3 (a cell line established from SCC of the tongue) as a control (32). Our results showed that FaDu cells express CXCR4 protein; however, the expression level was weak compared to that in the tongue SCC cell line HSC-3 (Fig. 1).

SDF-1/CXCR4 increases cell migration in hypopharyngeal SCC cells.
As tumor cell invasion and migration are important during the tumor progression cascade, controlling cell mobility is viewed as a target for clinical tumor suppression (33,34). To understand the mechanisms underlying hypopharyngeal SCC cell invasion, we seeded FaDu cells into Boyden chambers and stimulated them with SDF-1.
The invasiveness of FaDu cells increased when the cells were co-cultured with SDF-1 (P<0.05) is shown in Fig. 2A and B. To determine whether this increased migration was mediated by an SDF-1-CXCR4 interaction, we added AMD3100, a CXCR4 antagonist (35). The AMD3100 treatment reversed the increased migration of FaDu cells (P<0.05), suggesting that SDF-1 induces hypopharyngeal SCC cell FaDu cell migration increased in response to SDF-1 stimulation, whereas the addition of AMD3100 reversed this increase. Experiments were repeated three times, and fold migratory relative to control was expressed as the mean ± standard deviation. * P<0.05. (C) Proliferation assay of hypopharyngeal SCC cells treated with SDF-1 (0.1 µg/ml) and/or AMD3100 (10 ng/ml). Cells were plated in 12-well plates in DMEM with 10% FBS, and after 24 h, the growth media was replaced with media containing each agent. Cells were harvested and counted by vital dye exclusion. Cell counts on days 1, 2, 3 and 4 from three independent experiments are presented as the mean ± standard deviation. * P<0.05. SCC, squamous cell carcinoma; CXCR4, CXC chemokine receptor type 4; SCC, squamous cell carcinoma; SDF-1, stromal cell-derived factor 1. migration by stimulating CXCR4 ( Fig. 2A and B). In addition, we evaluated the ability of SDF-1 to induce FaDu cell proliferation because during tumor progression, an increase in cell number is as important as cell mobility.
On day 4 the cells treated with SDF-1 increased significantly in number as compared to the untreated cells (P<0.05) (Fig. 2C). To determine whether this increase in proliferation ability was mediated by an SDF-1-CXCR4 interaction, we added AMD3100. AMD3100 treatment reversed the increase in proliferation of FaDu cells (P<0.05), suggesting that SDF-1 induces hypopharyngeal SCC cell proliferation by stimulating CXCR4 (Fig. 2C).
Our results suggest that the SDF-1/CXCR4 axis plays a key role in hypopharyngeal SCC cell mobility and proliferation, and may contribute to tumor progression.

SDF-1/CXCR4 induces expression of CD147 in hypopharyn-
geal SCC cell. CD147 expression is associated with tumor progression and poor prognosis in various types of solid tumors (including HNSCC). We previously found that CD147 mediates invasion, proliferation, and drug resistance in FaDu cells (26). Thus, for this study we hypothesized that CD147 is associated with CXCR4-induced tumorigenesis. We treated FaDu cells with SDF-1, and confirmed by western blotting that CD147 protein expression was up-regulated in these cells. In addition, AMD3100 suppressed the CD147 upregulation induced by SDF-1. Our results suggest that SDF-1/CXCR4 regulates CD147 expression in hypopharyngeal SCC (Fig. 3). Figs. 2A-B and 3 indicate that SDF-1/CXCR4 induce cell mobility and CD147 expression in the hypopharyngeal SCC cell line FaDu. But, whether CD147 was associated with the SDF-1/CXCR4-induced increment of mobility of the cells was unclear. Thus, we planned an invasion assay with FaDu cells using a function-blocking antibody of CD147 in the presence or absence of SDF-1. As shown in Fig. 4, SDF-1 induced invasiveness, and this effect was significantly diminished by addition of the CD147 function-blocking antibody. This suggests that SDF-1/CXCR4 induces invasion in hypopharyngeal SCC and that CD147 may play a role in SDF-1/CXCR4 induced tumorigenicity.

Discussion
The importance of SDF-1/CXCR4 in cancer progression has been reported, especially in the metastasis and proliferation of cancer cells. The association between solid cancers and SDF-1/CXCR4 interaction was first reported as a chemokine receptor that is highly expressed in primary and metastatic breast cancer lesions (10). In subsequent studies, signaling via SDF-1/CXCR4 in various solid tumors has been established as a chemokine receptor pathway that plays an important role in cancer progression and malignancy. SDF-1/CXCR4 has roles in oncogenesis, proliferation, metastasis, and angiogenesis in many cancers types, including lung cancer, melanoma, esophageal cancer, ovarian cancer, glioblastoma, and basal cell carcinoma cells (8,9,12,13,36,37).
Analyses using molecular biology techniques have revealed that communication between CXCR4 and SDF-1 activates multiple signaling pathways to enhance tumor cell invasion and distant metastasis (38). In addition, CXCR4 cooperates with other transcription factors, such as NF-κB and Nanog, to help maintain the stemness of cancer stem cells as well as to induce metastatic behavior (39,40). Therefore, SDF-1/CXCR4 is considered a new drug target for cancer treatment. Research on sSDF-1/CXCR4 molecules is also undergoing in patients with head and neck cancer. CXCR4 promotes migration and invasion of oral cancer cells (18) and SDF-1/CXCR4 is involved in the metastasis of laryngeal and hypopharyngeal cancers (14). For this process, MMP production via the ERK signaling pathway is essential, and this mechanism of SDF-1/CXCR4 molecules in patients with head and neck cancer has been elucidated (14). Studies have reported that CXCR4 expression in primary tumors is involved not only in the local recurrence of head and neck cancer (22) but also in cervical lymph node metastasis and distant metastasis of hypopharyngeal cancer (14). Moreover, SDF-1/CXCR4 expression has been shown to be a negative prognostic factor after postoperative radiotherapy for head and neck cancer (21,41).
Therefore, clarifying the mechanisms of SDF-1/CXCR4 is important to overcome the therapeutic resistance of HNSCC.
Through our experiments, we confirmed CXCR4 expression in FaDu cells. A study has confirmed that FaDu cells express CXCR4 but not its ligand SDF-1 (42). Thus, we used exogenous SDF-1 for experiments on the interaction between SDF-1 and CXCR4.
In this study, we found that CXCR4 was involved in FaDu cell invasion and proliferation.
This was similar to the result of a study that used oral cancer cells (16). On the other hand, it was reported that CXCR4 was not involved in cell proliferation in HEp-2, an SCC of the larynx cell line (43). Therefore, these reports indicate that CXCR4 has different effects depending on the sub-location even within the same type of head and neck cancer and that detailed examination is necessary depending on the tumor site.
CD147 is associated with various physiological actions in cells, mainly via the production and activation of MMP (44). These are also beneficial for tumor progression. The correlation between the expression of CD147 in tumors and poor prognosis has been widely observed in individuals with solid tumors including HNSCC (45). It has been reported that CD147 is involved in MMP production, cell invasion, cell proliferation, and EMT in head and neck cancer cells (26,46). Moreover, we have reported that CD147 expression in tongue cancer patients is positively correlated with lymph node metastasis (23). Thus, the involvement of CD147 in cancer progression has been gradually established. Furthermore, the role of CD147 as a therapeutic target for cancer has been studied, and its antitumor effects are expected for head and neck cancer (47,48).
It is known that a targeting strategy is very important and epidermal growth factor receptor (EGFR) antibody is already in clinical use for head and neck cancer (49). In addition, recent studies have reported that the antitumor effect is further enhanced by simultaneously targeting multiple tumorigenic factors (34,50). We also have reported that head and neck cancer progression can be suppressed synergistically by combining the inhibition of EGFR and CD147 (51).
These results suggest that because of the complex pathways of cancer progression, inhibition of a single tumor-related factor is not sufficient for tumor control. For a more effective antitumor effect, it is necessary to simultaneously inhibit a plurality of factors related to the factor of interest.
In this study, we showed that CD147 expression was enhanced after the stimulation of CXCR4 by SDF-1. This enhanced expression of CD147 was abrogated by AMD3100, an inhibitor of CXCR4. In addition, the enhancement of cell infiltration induced by SDF-1/CXCR4 was suppressed by inhibiting CD147. These results indicate that CD147 acts as a mediator of the SDF-1/CXCR4 pathway and that it is involved in the invasion induced by SDF-1/CXCR4 in hypopharyngeal cancer cells.
These results suggest a possibility that not only would SDF-1/CXCR4 be a therapeutic target, but also that targeting CD147 simultaneously with SDF-1/CXCR4 may be a new and effective strategy for treating hypopharyngeal cancer.
On the other hand, the inhibition of infiltration induced by the SDF-1/CXCR4 was only partial after CD147 inhibition. This suggests that one of the SDF-1/CXCR4-induced tumor progression pathways is via CD147. Elucidation of these pathways is required for more powerful control of head and neck cancer progression.