CCL18 promotes the metastasis of squamous cell carcinoma of the head and neck through MTDH‐NF‐κB signalling pathway

Abstract Metastasis is one of the primary causes for high mortality in patients with squamous cell carcinoma of the head and neck (SCCHN). Our previous study showed that chemokine (C‐C motif) ligand 18 (CCL18), derived from tumour‐associated macrophages (TAMs), regulates SCCHN metastasis by promoting epithelial‐mesenchymal transition (EMT) and preserving stemness. However, the underlying mechanism needs to be further investigation. Interestingly, metadherin (MTDH) expression was induced when SCCHN cells were stimulated with recombinant CCL18 protein in this study. Suppressing MTDH expression reversed CCL18‐induced migration, invasion and EMT in SCCHN cells. Furthermore, the NF‐κB signalling pathway was involved in the MTDH knock‐down cells with CCL18 stimulation. We performed ELISA to evaluate the CCL18 levels in the serums of 132 treatment‐naive SCCHN patients, 25 patients with precancerous lesion and 32 healthy donors. Our results demonstrated that serum CCL18 levels were significantly higher in SCCHN patients than patients with precancerous lesion and healthy individuals. CCL18 levels were found to be significantly correlated with tumour classification, clinical stage, lymph node metastasis and histological grade in SCCHN patients. Thus, our findings suggest that CCL18 may serve as a potential biomarker for diagnosis of SCCHN and promote SCCHN invasion, migration and EMT by MTDH‐NF‐κB signalling pathway.


| BACKG ROU N D
Squamous cell carcinoma of the head and neck (SCCHN) is the sixth most aggressive malignancy worldwide. 1 Although effective multidisciplinary treatments, including surgery, radiotherapy and chemotherapy, have been applied in clinical practice for decades, the prognosis of SCCHN patients remains poor. 2 Although metastasis is the primary cause of death in SCCHN patients, there is no explanation for its causal role. [3][4][5] Elucidation of the underlying mechanism might help generate an effective surveillance of tumour progression and improve the survival outcome of SCCHN patients.
The tumour microenvironment, which consists of a variety of nonmalignant stromal cells, such as tumour-associated macrophages (TAMs), tumour-associated dendritic cells and tumour-infiltrating T cells, is an indispensable participant in tumour progression and metastasis. 6,7 TAMs, which produce chemokines, are the key players in creating the tumour microenvironment, and they are the most notable migratory hematopoietic cell type. [8][9][10][11] Chemokine (C-C motif) ligand 18 (CCL18), also known as pulmonary and activationregulated chemokine (PARC), dendritic cell-derived CC chemokine-1 (DC-CK1), alternative macrophage activation-associated CC chemokine-1 and macrophage inflammatory protein-4, has been reported to attract naive T cells or the mantle-zone B cells, and it primarily targets lymphocytes and immature dendritic cells. 12 CCL18, a member of the CC chemokine family, is mainly secreted by M2-type TAMs. 7,8 Excessive secretion of CCL18 has been observed in multiple human malignancies, including breast carcinoma, [9][10][11]13 hepatocellular carcinoma, 14 ovarian cancer, 15,16 nasopharyngeal carcinoma, 17 lung cancer, [18][19][20] colon cancer 21,22 and pancreatic ductal adenocarcinoma. 23,24 Expression of CCL18 is associated with tumour initiation and progression, and therefore, could be used to predict the prognosis of patients with breast cancer. 9 Previously, we demonstrated that CCL18 derived from M2 TAMs promoted the migration and invasion of SCCHN cells through the induction of epithelial-mesenchymal transition (EMT) and cancer stemness. 25 However, the molecular mechanism by which CCL18 induced metastasis and EMT in SCCHN cells remains unknown.
Metadherin (MTDH), also known as astrocyte-elevated gene-1 and LYsine-RIch CEACAM1 co-isolated protein, was initially cloned as HIV-1 and tumour necrosis factor α-inducible gene in primary human foetal astrocytes. [26][27][28][29] MTDH is a downstream target of Ha-ras and c-myc and helps in mediating their biological effects, suggesting a potential role of MTDH in tumour initiation and progression. 30,31 Overexpression of MTDH in normal immortal cloned rat embryo fibroblasts was found to induce anchorageindependent growth in soft agar and enhance invasion in nude mice. 32 Several studies have demonstrated that elevated MTDH expression is closely related to poor prognosis of various types of cancers, including breast cancer, 33 oesophageal squamous cell carcinoma, 34 gastric cancer, 35 hepatocellular carcinoma 36 and non-small cell lung cancer. 37 Thus, MTDH is a potential prognostic biomarker for human malignancies. Our previous studies have also confirmed that overexpression of MTDH is highly correlated with lymph node metastasis and shows negative association with poor prognosis in SCCHN patients. 38,39 MTDH has been demonstrated to be a valuable biomarker for SCCHN progression, and it regulates the metastasis of SCCHN via EMT in vitro. 38,40 Activation of NF-κB signalling is associated with a variety of cellular responses, such as inflammation, cell survival, differentiation and proliferation. NF-κB signalling pathway has also been demonstrated to play an important role in malignant cell mobility. [41][42][43][44] Accumulated evidence suggests that MTDH mediates the biological effect of the NF-κB signalling pathway through several mechanisms. 45 However, it is unknown whether the NF-κB signalling pathway is involved in the regulation of metastasis by CCL18 in SCCHN cells.

| Enzyme-linked immunosorbent assay
CCL18 levels in the serum of SCCHN patients, precancerous lesions of SCCHN patients and healthy volunteers were determined quantitatively using a human PARC (CCL18) ELISA kit (Raybiotech, Atlanta, GA) according to the manufacturer's protocol. Each experiment was performed in triplicate.

| Quantitative real-time PCR
Total RNA was extracted from samples using TRIzol reagent (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. After cDNA synthesis (All-in-One First-Strand cDNA Synthesis kit, GeneCopoeia Inc, Santa Cruz, CA), quantitative real-time PCR (qRT-PCR) was carried out using All-in-One qPCR Mix (GeneCopoeia Inc, USA) on ABI 7500HT System (Applied Biosystems, Foster City, CA) using primers described in Table 1. PCR conditions were as follows, 95°C for 10 minutes followed by 40 cycles of 95°C for 10 seconds, 60°C for 20 seconds and 72°C for 27 seconds. The specificity of each qRT-PCR reaction was verified by melting curve analysis. β-Actin was used as an internal control. Duplicate reactions were run for each sample and relative change in gene expression was calculated using the 2 −ΔΔCT method. All the samples for each experiment were run in duplicate.

| Western blot analysis
Whole cell protein extracts were collected and Western blot analysis was performed as described previously. 40 Each experiment was repeated in triplicates.

| Immunofluorescence
Tu686, 6-10B and FaDu cells were seeded in six-well plates and in-

| Wound healing and Transwell invasion assay
For wound healing assay, Tu686, 6-10B and FaDu cells were seeded in six-well plates and allowed to reach 90% confluency.

| Correlation of serum CCL18 levels with clinicopathological parameters in SCCHN patients
Since serum CCL18 levels were elevated in SCCHN patients, we analysed the relationship between serum CCL18 levels and the major clinicopathological parameters in SCCHN patients ( Table 2).
As shown in Table 2, serum CCL18 levels were found to be significantly associated with tumour classification (T1 + T2 vs T3 + T4;

SCCHN patients
To evaluate the performance of serum CCL18 in diagnosis of SCCHN  Figure 1B). AUC for evaluating the diagnostic accuracy of precancerous lesions from healthy controls was 0.629 (specificity: 0.94, sensitivity: 0.28, cut-off value: 36897.26 pg/mL, Figure 1C).
In conclusion, a cut-off value of 29927.73 pg/mL for serum CCL18 (SCCHN vs. healthy controls) was applied for further analysis and clinical diagnosis.
Furthermore, EMT is closely related to cancer metastasis, which can typically be characterized by different molecular markers. 38,39 In this study, treatment with rhCCL18 down-regulated the expres-  Figure 3B and C).

| MTDH is essential for CCL18-induced migration, invasion and EMT in SCCHN cells
In our previous study, we found that MTDH plays a very important role in the invasion, metastasis and EMT of SCCHN cells. 38,39 To  Figure 6A) and protein levels ( Figure 6B). These results suggest that the biological effect of CCL18 in promoting EMT in SCCHN cells is dependent on the expression of MTDH.

| NF-κB activation is involved in CCL18/MTDHmediated metastasis and EMT of SCCHN cells
To further investigate the mechanism by which CCL18/MTDH mediates migration, invasion and EMT in SCCHN cells, we measured the phosphorylation status of p65 and IκB-α using Western blotting. As shown in Figure 7B, treatment with rhCCL18 significantly increased the expression of p65 and also increased the phosphorylation of p65  Previous studies have reported that production of CCL18 by TAMs induced NF-κB activation in hepatocellular carcinoma metastasis. 14 This is in agreement with our results showing that NF-κB nuclear translocation and the classic signalling pathway was activated in CCL18-stimulated SCCHN cells. However, the mechanism by which expression of NF-κB is regulated is still not known. The F I G U R E 7 NF-κB activation is essential for CCL18/MTDH-mediated cell migration, invasion and EMT in SCCHN cells. A, Representative immunofluorescence images show that CCL18 induces nuclear translocation of p65. B, Expression of NF-κB-associated protein subsequent to rhCCL18 stimulation. C and D, rhCCL18 enhanced the EMT property featured by E-cadherin and Vimentin expression (**P < 0.01). This effect of CCL18 is reduced by repressing NF-κB activation in Tu686 cells. (C) mRNA levels and (D) protein levels. CCL18, chemokine (C-C motif) ligand 18; EMT, epithelial-mesenchymal transition; MTDH, metadherin; rhCCL18, recombinant human CCL18; SCCHN, squamous cell carcinoma of the head and neck present study demonstrated that NF-κB signalling was mediated by MTDH in CCL18-stimulated SCCHN cells. MTDH regulates multiple signalling pathways, including NF-κB signalling. MTDH has been shown to facilitate the translocation of NF-κB into the nucleus and interacts with the p65 subunit of NF-κB which acts as a coactivator for NF-κB to modulate gene expression of targeted genes. 45,55 Moreover, IκB kinase β-mediated MTDH phosphorylation is critical for IκB-α degradation as well as NF-kB-dependent gene expression and cell proliferation, which correlates with survival in cancer patients. 56 Thus, to summarize, our study demonstrated that serum levels of CCL18 may serve as potential biomarker for diagnosis of SCCHN, and CCL18 plays an important role during invasion, metastasis and EMT of SCCHN. Moreover, MTDH is at the core of CCL18-induced biological effects. Activation of NF-κB is involved in CCL18/ MTDH-induced EMT, which intrinsically enhances the ability of cell migration and invasion, suggesting that MTDH may be used as a therapeutic target for the inhibition of SCCHN metastasis.