Pro-Inflammatory Cytokine IL-1β Up-Regulates CXC Chemokine Receptor 4 via Notch and ERK Signaling Pathways in Tongue Squamous Cell Carcinoma

Chronic inflammation contributes to tumor development through the induction of oncogenic mutations, genomic instability, early tumor promotion, and enhanced angiogenesis. Here, we report that IL-1 receptor 1 (IL-1R1) was expressed in 40 of 41 human tongue squamous cell carcinomas (TSCC). IL-1β up-regulated the expression of CXCR4, a CXC chemokine receptor that mediates cancer growth and metastasis, at both mRNA and protein levels in Tca8113 TSCC cells. IL-1β treatment of Tca8113 cells promoted migration in response to CXCR4 ligand stromal-derived factor α (SDF-1α). The inhibition of IL-1R1 by its antagonist IL-1Ra or RNA interference significantly reversed the up-regulation of CXCR4 induced by IL-1β. IL-1R1 activation also up-regulated the expression of IL-1β itself, suggesting a positive feedback regulation of CXCR4 expression. Furthermore, IL-1β induced the activation of Notch, which was originally considered a stem cell regulator. Pharmacological inhibition of Notch signaling reversed the up-regulation of CXCR4 induced by IL-1β, suggesting that Notch signaling may be involved in the growth and metastasis of cancers via up-regulation of CXCR4. In addition, IL-1β induced the activation of extracellular signal regulated kinase (ERK) and ERK inhibition decreased the up-regulation of CXCR4 induced by IL-1β, suggesting the involvement of ERK signaling in cancer metastasis. Taken together these data suggest that IL-1β and IL-1R1 promote cancer growth and metastasis by up-regulating CXCR4 expression and that CXCR4 may be a link between inflammation and cancer.


Immunohistochemical analysis
Human tissue specimens were approved for use without informed consent by the Ethics Committee of Changsha Central Hospital. Primary tongue squamous cell carcinoma tissue from routine diagnostic biopsy specimens were obtained from the Department of Pathology, the Second Xiangya Hospital of Central South University, and the Department of Pathology, Changsha Central Hospital. Tissues were fixed with 4% formaldehyde, embedded in paraffin, and sectioned. Endogenous peroxidase activity was blocked by hydrogen peroxide (0.3%). Slides were stained with IL-1R1 antibodies (1:50), followed by incubation with a biotinylated anti-mouse secondary antibody (1:1000) and horseradish peroxidase-conjugated streptavidin. Color was developed with diaminobenzidine, and sections were counterstained with hematoxylin.

Quantitative real time RT-PCR (qRT-PCR)
qRT-PCR was performed as described by Sun et al. [31]. Briefly, total RNA from the cells was isolated and reverse transcribed as described above. The cDNA was amplified using TaqMan Universal PCR master mix (Applied Biosystems, Foster City, CA, USA) and an ABI Prism 7500 sequence detection system (Applied Biosystems). The amplification of the target genes was normalized using the amplification levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control. The efficiency of the PCR was tested by amplification of the target from serially diluted cDNA generated by reverse transcription of a stock set of human RNA. The data analysis and calculations were performed using the 2 −ΔΔCT comparative method, as described by the manufacturer. Gene expression is shown as the fold induction of a gene in IL-1β-treated samples relative to samples cultured with control medium.

Flow cytometry analysis
For CXCR4 protein detection, Tca8113 cells were cultured for 24 h in 6-well plates, harvested and washed with fluorescence-activated cell sorting (FACS) buffer (5 mM EDTA, 0.1% NaN3, and 1% FCS, in Dulbecco's phosphate-buffered saline (PBS)). After incubation with a monoclonal antibody against human CXCR4 for 40 min on ice, the cells were stained with a FITClabeled secondary antibody and examined for protein expression by flow cytometry (BD Biosciences).

Transwell migration assays
In vitro cell migration assays were performed using Transwell chambers with polyethylene terephthalate membrane (24-well inserts, 8.0 μm, Corning). Briefly, 2.5 × 10 4 cells, treated with medium or IL-1β for 24 h, were seeded onto the upper chambers. Medium containing 10% FBS with 20 ng/ml SDF-1α was placed in the lower chamber to serve as a chemoattractant. Twentyfour hours later, the cells on the upper surface of the filter were removed by gently wiping with a cotton swab. The cells that migrated to the lower surface of the filter were fixed with 95% alcohol for 5 min and incubated with a mixture of 50 μg/ml PI (Sigma-Aldrich) and 25 mg/ml RNase A (Sigma-Aldrich) at 37°C for 30 min. Migrated cells were visualized by fluorescence microscopy (Leica, Wetzlar, Germany). Cells in three fields per filter were counted under the microscope.

shRNA plasmid transfection
Cells were cultured in six-well plates and transfected with 1 μg of a plasmid containing hairpin RNA targeting human interleukin-1 receptor 1 (IL-1R1) or with 1 μg of a control plasmid containing non-specific hairpin RNA with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Expression of IL-1R1 in the transfected cells was examined by western blot 48 h after transfection. For stable transfection, G418-resistant cells were selected after incubation with 800 μg/ml G418 for 3 weeks.

Statistical analysis
All experiments were performed at least three times, and representative results are shown. The results are expressed as the mean ± S.D. Differences between groups were examined for statistical significance using Student's t test, and P values equal to or less than 0.05 were considered statistically significant (n = 3 for each qRT-PCR test).

Results
Tongue squamous cell carcinomas express IL-1 receptor 1 We first detected IL-1R1 protein expression in biopsy specimens of human tongue squamous cell carcinomas. Sections from 40 of 41 tongue carcinoma tissues stained positively for IL-1R1 protein by immunohistochemistry (Fig 1A and 1B). Eight of 9 poorly differentiated tongue carcinomas (grade III), all 11 moderately differentiated tongue carcinomas (grade II), and all 21 well differentiated tongue carcinomas (grade I), were IL-1R1 positive ( Fig 1A). When we observed the staining patterns of IL-1R1, we found that tongue squamous cell carcinomas were positive in both the cytoplasm and nucleus ( Fig 1B). In poorly differentiated carcinomas, perinuclear staining was observed ( Fig 1B). Little membrane staining was observed ( Fig 1B). In addition, some positive nuclear staining was also observed in stromal cells. These results indicated that IL-1R1 is widely expressed in tongue squamous cell carcinoma.

IL-1β up-regulates CXCR4 expression
IL-1β has been reported to up-regulate CXCR4 expression in astroglioma cells [27]. We measured the effect of IL-1β on CXCR4 expression in human tongue squamous cell carcinoma Tca8113 cells. IL-1β upregulated the levels of CXCR4 transcripts in a dose-dependent manner (Fig 2A). However, IL-1β did not up-regulate CXCR4 mRNA levels in human laryngeal epidemoid carcinoma Hep2 cells ( Fig 2B). IL-1β did not regulate CCR6 or CCR7, two other CC chemokine receptors related to cancer metastasis [32,33], in either Tca8113 or Hep2 cells (Fig 2A  and 2B). Quantitative real-time RT-PCR showed that the up-regulation of CXCR4 transcript induced by IL-1β was significant in Tca8113 cells ( Fig 2C). IL-1β-induced up-regulation of CXCR4 was also time-dependent (Fig 2D and 2E). Two peaks were found, with one peak at 1 h and another peak at 24 h (Fig 2D and 2E). FACS results showed that IL-1β up-regulated CXCR4 protein levels in a dose-dependent manner ( Fig 2F). Transwell assay results showed that IL-1β dose-dependently promoted cell migration in response to the CXCR4 ligand SDF-1α ( Fig 2G and 2H)

IL-1RI is responsible for the induction of CXCR4
To determine why IL-1β induced the up-regulation of CXCR4 in Tca8113 cells but not in Hep2 cells, we measured the expression of IL-R1 and 2 in these cell lines. RT-PCR results showed that Tca8113 cells expressed IL-1R1 but not IL-1R2 ( Fig 3A). However, Hep2 cells expressed neither IL-1R1 nor 2 ( Fig 3B). In addition, western blot results showed that treatment of Tca8113 with IL-1β up-regulated the protein levels of IL-1R1 ( Fig 3C). These results suggest that the expression of IL-1R1 was responsible for the induction of CXCR4 induced by IL-1β. As IL-1Ra is a strong antagonist of IL-1R1, we tested the effect of IL-1Ra on the induction of CXCR4 induced by IL-1β. IL-1Ra pre-treatment of Tca8113 cells significantly inhibited the up-regulation of CXCR4 induced by IL-1β at both the mRNA and protein levels (Fig 3D,  3E and 3F). In addition, when IL-1R1 was down-regulated by RNA interference (Fig 3G), the IL-1β-induced up-regulation of CXCR4 was prevented (Fig 3H and 3I). Positive feedback regulation of IL-1β and CXCR4 expression An IL-1β-mediated positive-feedback loop has been reported to play a protective role against Clostridium difficile infection [34]. In this study, we found that IL-1β treatment of Tca8113 induced a significant up-regulation of IL-1β in a dose-and time-dependent manner at the transcript level (Fig 4A, 4B, 4C and 4D). As a control, IL-1β did not up-regulate the expression of TNF-α ( Fig 4A). Up-regulation of IL-1β transcript was also dependent on IL-1R1, as IL-1Ra pre-treatment also inhibited the up-regulation of IL-1β (Fig 4E and 4F). This IL-1β positive feedback loop induced a sustained up-regulation of both IL-1β and CXCR4. IL-1β and CXCR4 up-regulation persisted for 4 days after a single treatment of Tca8113 cells with IL-1β (Fig 4G,  4H and 4I). The Notch signaling pathway is involved in the up-regulation of CXCR4 induced by IL-1β Notch signaling has been reported to modulate CXCR4 expression [35]. To determine whether the observed IL-1β-induced CXCR4 up-regulation was related to the activation of Notch signaling, we first measured the activation of Notch1 induced by IL-1β in Tca8113 cells. Western blot results showed that treatment with IL-1β induced the cleavage of Notch1 to form the activated fragment NICD (Notch intracellular domain) in a dose-and time-dependent manner (Fig 5A and 5B). RT-PCR showed that IL-1β treatment up-regulated the mRNA levels of the Notch-targeting gene Hes1 mildly but significantly (Fig 5C). These results suggest that IL-1β activates Notch signaling [36]. Moreover, Notch inhibition by the inhibitor L685458 decreased the up-regulation of CXCR4 induced by 1 h of IL-1β treatment (Fig 5D and 5E). Notch inhibition also decreased the induction of IL-1β transcript induced by IL-1β (Fig 5F and 5G). To determine the effect of Notch inhibition on the long-term expression of CXCR4 mRNA, Tca8113 cells were pre-treated with L685458 for 30 min and then treated with IL-1β for 24 h. RT-PCR showed that Notch inhibition reversed the IL-1β-induced up-regulation of CXCR4 mRNA (Fig 5H). Western blot results also showed that Notch inhibition reversed IL-1βinduced CXCR4 protein up-regulation (Fig 5I). These results demonstrate that the Notch pathway is involved in the induction of CXCR4 induced by IL-1β.

IL-1β-induced ERK activation is involved in the up-regulation of CXCR4 expression
In response to IL-1β binding to IL1R1, a complex sequence of combinatorial phosphorylation and ubiquitination events results in activation of the nuclear factor NF-κB and MAPK signaling pathways, which induce the expression of IL-1β target genes [37]. We measured the activation of MAPKs, including ERK, JNK and p38, induced by the treatment of Tca8113 cells with IL-1β. Western blot results showed that IL-1β significantly induced phosphorylation of ERK and slightly induced the activation of JNK and p38 in a time-dependent manner (Fig 6A). IL-1β had little effect on the activation of NF-κB or the degradation of IκB-α (Fig 6B). We therefore focused on ERK signaling and measured the effect of ERK inhibition on IL-1β-induced CXCR4 up-regulation. RT-PCR results showed that the inhibition of ERK by the specific inhibitor U0126 significantly reversed the up-regulation of both CXCR4 (Fig 6C and 6D) and IL-1β (Fig 6E and 6F) induced by IL-1β treatment for 1 h. To determine the effect of ERK inhibition on the long-term expression of CXCR4 mRNA, Tca8113 cells were pre-treated with U0126 for 30 min and then treated with IL-1β for 24 h. RT-PCR showed that ERK inhibition also reversed the IL-1β-induced up-regulation of CXCR4 mRNA (Fig 6G). Western blot results also showed that ERK inhibition reversed IL-1β-induced CXCR4 protein up-regulation (Fig 6H). These results demonstrate that the ERK signaling pathway is also involved in the up-regulation of CXCR4 induced by IL-1β.

Discussion
Oral tongue squamous cell carcinoma is one of the most prevalent tumors of the head and neck region. Despite advances in treatment, the survival of patients has not significantly improved over the past several decades. Human papillomavirus infection has been reported to be related to the pathogenesis of tongue carcinoma [38][39][40]. Neutrophils, CD8+ and CD4 + lymphocytes infiltrate tongue carcinomas, and infiltrating immune cells produce pro-inflammatory cytokines [41,42]. The pathogen recognition receptor toll-like receptor 5 has been reported to play an important role in the pathophysiology of tongue carcinoma and might represent a link between bacterial infection and cancer [43]. This suggests that tongue squamous cell carcinoma is inflammation-related cancer.
IL-1β is up-regulated in solid cancers, including breast, colon, lung, head and neck cancers, and melanomas. Patients with IL-1β-producing tumors have generally bad prognoses [44]. IL-1α and IL-1β transcript levels have been evaluated in melanoma, non-small cell carcinoma, colon, and squamous cell cancer cell lines via real-time qRT-PCR, and several of these cell lines exhibit significantly increased levels of IL-1α or IL-1β [18]. IL-1 exhibits autocrine behavior by stimulating the tumor cell to invade and proliferate or exerts a paracrine effect on stromal cells in the microenvironment [44]. In this study, we did not detect the expression of IL-1β in resting Tca8113 cells, but treatment with IL-1β induced an up-regulation of IL-1β in a dose-and time-dependent manner, suggesting that IL-1β exhibits autocrine behavior in Tca8113 cells. Metastasis is the major factor that limits survival in cancer patients. The chemokine receptor CXCR4 promotes cancer metastasis in breast cancer and melanoma [45]. CXCR4 is also expressed in many types of cancer, and plays important roles in cancer metastasis [22]. CXCR4 is also related to cancer growth and proliferation [22]. HIF-1α is a major factor that promotes CXCR4 expression [25], and other factors may also regulate CXCR4 expression. In our study, we found that the activation of IL-1R1 significantly up-regulated CXCR4 levels. We have also found that another pro-inflammatory cytokine, tumor necrosis factor (TNF) α, up-regulated CXCR4 expression at both transcript and protein levels in nasopharyngeal carcinoma and colorectal cancer (data not shown). These studies suggest that pro-inflammatory cytokines may function to promote cancer growth, proliferation and metastasis via up-regulation of CXCR4.
Target-based therapies are widely considered to be the future of cancer treatment. The evidence that ERK signaling promotes cell proliferation, cell survival and metastasis, along with the high frequency at which this pathway is aberrantly activated in cancer, has led many labs to focus on developing ERK inhibitors for cancer therapy [46]. Tumor fibroblast-derived epiregulin, a 46-amino acid protein that belongs to the epidermal growth factor family, promotes the growth of colitis-associated neoplasms through ERK [47]. Periostin, an extracellular matrix protein that functions in tooth and bone remodeling, is produced by cancer-associated fibroblasts and supports growth of gastric cancer through the activation of ERK [48]. The blockade of ERK signaling inhibits tumor growth in various types of cancer, including gastric cancer [49], renal cell carcinoma [50], and rhabdomyosarcoma [51]. Conversely, ERK activation facilitates cancer invasion and metastasis [52,53], and the inhibition of ERK signaling inhibits metastasis [50]. In our study, we found that ERK signaling is involved in the up-regulation of CXCR4 induced by IL-1β. The inhibition of ERK signaling prevented the up-regulation of CXCR4 induced by IL-1β. These results suggest that ERK may be a target for the prevention and control of tongue squamous cell carcinoma.
The Notch pathway is an evolutionarily conserved signaling system that regulates proliferation, differentiation, cell fate determination, and self-renewal of stem and progenitor cells in both embryonic and adult organs [54]. Notch signaling is a double-edged sword in cancer, as it can be either oncogenic or tumor suppressive, depending on the cellular context [55]. On the one hand, it is well established that Notch acts as an onco-protein in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL), an aggressive tumor that occurs mainly in children and adolescents [55]. Notch signaling is also related to growth, invasion, and metastasis in other solid tumors, such as melanoma [56], glioma [57], breast cancer [58], and lung adenocarcinoma [59]. On the other hand, Notch signaling can function as a tumor suppressor. In conditional Notch1 knockout mice, an increased incidence in skin cancers has been reported, and the incidence of papilloma formation in Notch1 null skin was markedly increased by exposure to chemical carcinogens plus phorbol ester [60]. The expression of a specific inhibitor of the Notch transcription complex, dominant negative MAML1 (DN-MAML), in murine skin led to the development of cutaneous squamous cell carcinoma [61]. Several studies have reported conflicting results concerning Notch regulation of CXCR4 expression. Notch signaling has been reported to positively control CXCR4/SDF-1 expression and functions in myeloma cell lines, and forced CXCR4 activation partially rescues tumor cells from the effects of Notch inhibition [62]. By up-regulating CXCR4, Notch signaling is involved in lipopolysaccharideinduced maturation of bone marrow-derived dendritic cells [63]. In our study, we found that Notch is involved in IL-1β-induced CXCR4 up-regulation. Inhibition of Notch signaling decreased CXCR4 expression. However, the inhibition of Notch signaling by a γ-secretase inhibitor or knockout of the down-stream transcription factor RBP-J has been reported to significantly increase cell surface, total protein, and mRNA levels of CXCR4 in mesenchymal stem cells [64]. In endothelial cells, the expression of the Notch ligand Dll4 inhibited attachment and migration in response to stromal-derived growth factor 1 (SDF1) due to the downregulation of CXCR4 [65]. Thus, the regulation of CXCR4 in response to Notch signaling seems to be cell type dependent.