CD147 promotes progression of head and neck squamous cell carcinoma via NF‐kappa B signaling

Abstract CD147/basigin (BSG) is highly upregulated in many types of cancer, our previous study has found that CD147/BSG is highly expressed in head and neck squamous cell carcinoma (HNSCC) stem cells, but its role in HNSCC and the underlying mechanism is still unknown. In this study, we investigated the role of CD147 in the progression of HNSCC. Real‐time PCR, western blot and immunohistochemistry were used to detect the expression of CD147 in total 189 HNSCC tissues in compared with normal tissues. In addition, we used proliferation, colony formation, cell cycle and apoptosis, migration and invasion as well as wound‐healing assay to determine the biological roles of CD147 in HNSCC. Then, a xenograft model was performed to evaluate tumor‐promoting and metastasis‐promoting role of CD147 in HNSCC. The results showed that upregulated CD147 expression was associated with aggressive clinicopathologic features in HNSCC. In addition, CD147 promoted proliferation, migration and reduced the apoptosis phenotype of HNSCC cells in vitro as well as tumor initiation and progression in vivo. Furthermore, we demonstrated that CD147 promoted HNSCC progression through nuclear factor kappa B signaling. Therefore, we concluded that CD147 promoted tumor progression in HNSCC and might be a potential prognostic and treatment biomarker for HNSCC.

widely expressed on the surface of cancer cells. 6 Reports showed that CD147 was over-expressed in many types of cancer, such as human malignant melanoma, hepatocellular carcinoma, lung cancer, gastric cancer, breast cancer and so on. [7][8][9][10][11][12] CD147 is also reported to involve in tumor progression, angiogenic function and immune response. 13,14 Furthermore, many studies demonstrated that CD147 could stimulate the activation of nuclear factor kappa B (NF-kappa B) pathway. [15][16][17] The NF-kappa B pathway is reported to elevate cancer cell proliferation, metastasis and reduce cancer cell apoptosis. 11,18 And the family of NF-kappa B transcription factors consists of RelA/p65, c-Rel, RelB, NF-κB1 (p50), and NF-κB2(p52) and are kept silenced in the cytoplasm. 11 When there are stimuli, the NF-kappa B complex degrades and p65 enters the nucleus to regulate their target genes. 19,20 However, direct evidence for the crosslink between CD147 and the NF-kappa B pathway in HNSCC has been lacking.
In this study, we firstly investigated the expression patterns of

| Immunohistochemistry
Tissues slides were deparaffinized, rehydrated, and then were heated with citric acid buffer for antigenic retrieval. After cooled at room temperature, the sections were submerged into 0.3% hydrogen peroxide for 15 minutes to block endogenous peroxidase activity. After washed in phosphate-buffered saline (PBS) for 5 minutes, sections were blocked with 5% bovine serum albumin at room temperature for 1 hour. The tissues were incubated with indicated primary antibodies in a humidified chamber at 4°C overnight. After several washes, horseradish peroxidase (HRP)-labeled goat antimouse or goat anti-rabbit secondary antibody (Gene Tech, China) was incubated for 50 minutes at room temperature. Hematoxylin and dehydration were used to counterstain the nuclear and slides were dehydrated, and covered with coverslips. Primary antibodies were used at the following dilutions: rabbit CD147 antibody (1:200; CST, USA), rabbit Ki67 antibody (1:200; Abcam, UK).
A tissue microarray containing 101 HNSCC specimens and 10 normal tissues at Shanghai Ninth People's Hospital were used in this study. The immunohistochemistry (IHC) stain score is the sum of positive cell score and staining intensity score for each sample. The positive cell score were classified into five categories: 0%-25%, scored 1; >25%-50%, scored 2; >50%-75%, scored 3; >75%, scored 4. The staining intensity score was defined as 0, 1, 2, and 3. Finally, the above two scores were multiplied for an overall score (ranging from 1 to 12). A total score of 1-6 was considered low expression; 7-12 was considered high expression.

| Lentivirus transfection
We silenced three sequences of the CD147 gene, namely, CD147-Homo-550 (siRNA), CD147-Homo-647 (siRNA) and CD147-Homo-702 (siRNA). We checked the efficiency of the three interference sequences and selected the interference of CD147-Homo-550 (siRNA), then packaged it as lentivirus to use in this study

| Cell proliferation assay
Cells transfected with shCD147 or shNC were plated at a density of 1 × 10 3 cells per well in 96-well plates. CCK-8 kit (Dojindo, Japan) was used to determine the OD value at 450 nm after 2 hours following the kit instructions. All experiments were performed in triplicate, and the mean proliferation rate was reported.

| Colony formation assay
Cells transfected with shCD147 or shNC were plated at a density of 1 × 10 3 cells per well in 6-well plates. After 2 weeks, cells were washed with PBS, fixed in 4% paraformaldehyde for 15 minutes, stained with 0.5% crystal violet for 1 hour and were counted at least three times.

| Animal experiments
To evaluate the role of CD147 in tumor progression, 1 ×

| Statistical analyses
The data were compiled using with SPSS 19.0 statistical software.
Values are presented as means ± SEM. The results of the real-time PCR, cell proliferation assays, cell cycle assays, colony formation assays, cell scratching assays, cell migration and invasion assays and cell apoptosis assays were compared using Student's t test. Kaplan-Meier survival analyses were used to analyse the relationship between CD147 level and the clinicopathologic features. The staining intensity score at 6 was considered to be median score: 1-6 was  Figure 1A). Correspondingly, western blot showed that the protein levels of CD147 were also significantly upregulated in the HNSCC tissues compared to the non-tumor F I G U R E 6 CD147 activated NF-kappa B signaling in HNSCC. (A) After being starved with serum-free DMEM overnight, HN4 shCD147 and negative control cells were treated with 5 ng/mL TNFα for 0, 5, 15, 30, 60, 120 min. Lysates were prepared, and CD147, IKKα, p-IKKα, IκBα, p-IκBα, p65, p-p65 were analysed by western blotting. Tublin was used as a loading control. (B) After being starved with serum-free DMEM overnight, HN30 shCD147 and negative control cells were treated with 5 ng/mL TNFα for 0, 5, 15, 30, 60, 120 min. Lysates were prepared, and CD147, IKKα, p-IKKα, IκBα, p-IκBα, p65, p-p65 were analysed by western blotting. Tublin was used as a loading control. (C) After being starved with serum-free DMEM overnight, HN6 shNC and shPTK7 cells were treated with 5 ng/mL TNFα or serum-free DMEM for 5 min, and HN6 was treated with PDTC for 5 min, then the location of p65 was detected by laser scanning microscopy. (D) After being starved with serum-free DMEM overnight, HN30 shNC and shPTK7 cells were treated with 5 ng/mL TNFα or serum-free DMEM for 5 min, and HN30 was treated with PDTC for 5 min, then the location of p65 was detected by laser scanning microscopy adjacent tissues ( Figure 1B). The relationship between variable CD147 expression levels and clinicopathologic features was detected by IHC with a tissue microarray containing 101 HNSCC specimens and 10 normal tissues at the Shanghai Ninth People's Hospital. The results showed relative negative, weak, moderate and strong CD147 staining images from HNSCC patients compared with normal tissues ( Figure 1C). As shown in Table 1, CD147 expression levels were significantly associated with gender, nodal status, differentiation and prognosis of HNSCC, but was not significantly associated with age, smoking, drinking and tumor size.
Kaplan-Meier was used to analysed the OS probability and DFS probability of HNSCC patients and the results revealed that patients with high CD147 expression had a significantly low OS probability and low DFS probability (both P < 0.0001) ( Figure 1D and E). The COX regression analyses revealed that CD147 expression was significantly correlated with poor OS in HNSCC patients and was an independent predictor of prognosis for patients with HNSCC ( Table 2).

| CD147 promoted the proliferation and reduced the apoptosis phenotype of HNSCC cells
To explore the role of CD147 in proliferation of HNSCC, we first analysed protein expression level of two normal mucosa cells and seven HNSCC cells. The results showed that relatively moderate to high CD147 expression levels were observed in HNSCC tumor cells, whereas low levels were observed in normal mucosa cells ( Figure S2A). We then knocked down CD147 in HN4 and HN30 cells with GFP-tagged lentivirus. Western blot showed that endogenous CD147 expression was efficiently suppressed compared with negative control cells (Figure 2A and Figure S2B). CD147 knockdown significantly reduced proliferation rate of HN4 (P < 0.01) and HN30 cells (P < 0.001) ( Figure 2B and C). In addition, colony formation capacities of HN4 (P < 0.001) and HN30 (P < 0.01) cells were significantly reduced compared with negative control cells ( Figure 2D). Cell cycle arrest and apoptotic cells in HN4 and HN30 transfected with shNC and shCD147 were all consistent with these findings ( Figure 2E and F). Taken together, we demonstrated that CD147 promoted proliferation and reduced apoptosis of HNSCC cells.

| CD147 promoted the migration and invasion phenotype of HNSCC cells
Based on the role of CD147 in the proliferation of HNSCC cells, we further detected the effect of CD147 on tumor migration and invasion. Scratch wound healing assays showed that CD147 knockdown significantly reduce the migration of HN4 and HN30 cells (both P < 0.01) ( Figure 3A). In addition, migration and invasion assays were also associated with the results (Figure 3B and C). Collectively, these data showed that CD147 promoted the migration and invasion phenotype of HNSCC cells.

| Knockdown of CD147 in HNSCC cells reduced tumor growth in vivo
To examine the effect of CD147 on tumorigenicity in vivo, we established a xenograft model in BALB/C nude mice. A total of 1 × 10 6 HN6 negative control cells and shCD147 cells in 100 μL fresh DMEM medium were subcutaneously injected into the left or right flank of the BALB/C nude mice. Tumor growth was recorded for overall 20 days and mice were killed ( Figure 4C). The results showed that the tumor volume and weight of HN6 shCD147 cells were significantly inhibited than HN6 negative control cells (P < 0.05) ( Figure 4A, B and D). There were no morphological difference between HN6 negative control group and HN6-shCD147 group in xenografted tumors ( Figure 4E). Immunohistochemistry assays showed that CD147 knockdown reduced the expression of Ki67 in xenograft tumors (P < 0.0001) ( Figure 4F). Consistent with the data in vitro, these studies demonstrated that knockdown of CD147 in HNSCC cells reduced tumor growth in vivo.

| CD147 expression was higher in lung metastasis tissues in vivo
To investigate the role of CD147 in lung metastasis, a total of 2 × 10 6 Rca-T cells were injected into the lateral tail vein of nude mice and mice were killed at 0, 1 day, 4 days, 1 week and 2 weeks respectively. Then the lungs were isolated and pictures were taken ( Figure 5A). H&E staining revealed that the metastatic colony numbers in lungs at 1 week and 2 weeks were significantly higher compared to control groups ( Figure 5B). Lung weight were also significantly elevated at 1 week (P < 0.01) and 2 weeks (P < 0.001), but not significantly elevated at 1 day and 4 days compared with 0 day ( Figure 5C). We further explored the CD147 expression level at different points, and the results demonstrated that CD147 expression levels were higher in lungs of 1 week (P < 0.001) and 2 weeks (P < 0.0001) compared with 0 day ( Figure 5D). Therefore, we proposed that CD147 may take part in the metastatic clones formation in vivo.

| CD147 activated NF-kappa B signaling in HNSCC
As studies have shown, CD147 enhanced tumor growth through NFkappa B signalling. 21,22 To observe the time-dependent activation of NF-kappa B signaling, negative control cells and shCD147 cells were treated with serum-free DMEM overnight, subsequently stimulated with TNFα and cells were harvested at 0, 5, 15, 30, 60 and 120 min.
Interestingly, the protein expression of p-IKKα, p-IκBα and p-p65 in shNC cells significantly increased from 5 min after stimulated with TNFα, but the protein expression in shCD147 cells had no obvious upregulation in both HN4 and HN30 cells ( Figure 6A and B). Consistent with these results, the immunofluorescence assay also demonstrated that when treated with TNFα, p65 of HN4 and HN30 negative control cells entered into the nucleus, but p65 of HN4 and YU ET AL.
| 963 HN30 shCD147 cells was still in the cytoplasm. The HN4 and HN30 cells that were treated with PDTC were used as negative control ( Figure 6C and D). Taken 11,30,35,36 There are reports shown that CD147 promoted the expression of MMP-9, which might be a mechanism that CD147 promoted tumor progression and invasion in HNSCC via regulating NF-kappa B signaling. 30 In addition, inhibition of CD147 by using the CD147 monoclonal antibody (5A12) was reported to reduce the proinflammatory cytokine production and thus suppressed the activation of NF-kappa B pathway. 29

CONFLI CT OF INTEREST
The authors declare no conflict of interest.

AUTHOR CONTRI BUTION
MY and WC performed conception and design; BY and YZ contributed to experiments performing and wrote the manuscript; BY and YJ performed data analysis; KW collected the tissue samples information of patients and generated the data; LW analysed the staining and results of pathological section; MY and WC provided financial support and final approval of manuscript.