FcGBP was upregulated by HPV infection and correlated to longer survival time of HNSCC patients

FcGBP was normally found in intestinal and colonic epithelia, gallbladder, cystic duct, bronchus, submandibular gland, cervix uteri and in fluids secreted by these cells in humans, and was down-regulated during colon carcinogenesis. We found FcGBP gene expression was decreased in HNSCC tissues compared to surgical safety border tissues while TGF-β expression level increased in HNSCC tissues, and higher FcGBP expression level was correlated to longer OS time of HNSCC patients. FcGBP expression level was higher in HPV-positive HNSCC tissues compared to HPV-negative HNSCC tissues, while TGF-β expression level was lower in HPV-positive HNSCC tissues. Gene expression level of FcGBP and TGF-β was negatively correlated in HNSCC tissues. FcGBP expression level increased after HPV E6 overexpression in HPV-negative HNSCC cells, and TGF-β could inhibit the up-regulation of FcGBP after HPV E6 or FcGBP overexpression in HPV-negative HNSCC cells. The migration capability was inhibited after FcGBP overexpression, and TGF-β could counteract the inhibition of migration caused by FcGBP overexpression. FcGBP gene expression level was correlated to the expression levels of EMT markers. In conclusion, FCGBP expression was upregulated by HPV infection while inhibited by TGF-β, and was correlated to the prognosis of HNSCC patients.


INTRODUCTION
Head and neck squamous cell carcinoma (HNSCC) accounts for about 6% of all human cancers, with about 650,000 new cases reported and 350,000 HNSCCrelated deaths per year worldwide [1]. Tobacco, alcohol consumption and human papillomavirus (HPV) infection are the main etiological factors [2][3][4]. The biological behavior, prognosis and genetic landscape are significantly different between HPV-positive and HPV-negative HNSCC patients, and many studies have found that HPV-positive HNSCC patients have better prognosis, with increased survival time and less chance of recurrence [5][6][7][8].

Research Paper
important role in anti-inflammation and cell protection in epithelium cells [15]. The mutation of FcGBP has been previously identified in CRC [16]. Furthermore, FcGBP was significantly enriched in the function of cell adhesion. Metastasis is facilitated by the cell-cell interactions between the endothelium and tumor cells in targeted organs. Cell adhesion occurring in vasculature of specific organs is an essential step in cancer metastasis [17].
In this paper, we found FcGBP gene expression was downregulated in HNSCC tissues and correlated with overall survival time in HNSCC patients. Furthermore, we found FcGBP gene expression level was higher in HPVpositive than HPV-negative HNSCC tissues while TGF-β gene expression level was lower in HPV-positive HNSCC tissues, and FcGBP gene expression was inhibited by TGF-β. This network played an important role in HPV related tumorigenesis and HNSCC progression.

FcGBP gene expression was down regulated in HNSCC and negative correlated to TGF-β gene expression
First, we investigate FcGBP mRNA expression level in GEO dataset GSE59102, and we found that FcGBP was down regulated in HNSCC tissues compared to surgical safety border tissues (p=0.0015) ( Figure 1A). Then we detect FcGBP protein expression level in 62 paired HNSCC and surgical safety border tissues, and we found that FcGBP protein expression level was lower in HNSCC compared to surgical safety border tissues (p<0.0001) ( Figure 1C), and similar results generated in IHC experiment (p<0.0001) ( Figure 1B). After that, we investigate TGF-β mRNA and protein expression levels in HNSCC and surgical safety border tissues, and we found that TGF-β mRNA and protein expression levels were both increased in HNSCC compared to surgical safety border (p<0.0001) ( Figure 1A, 1B, 1C). Also, we found mRNA and protein expression level of FcGBP and TGF-β was significantly negative correlated in HNSCC and surgical safety border tissues (GSE59102: r=-0.3835, p=0.0122; WB: r=-0.6767, p=0.0011) ( Figure 1A, 1C).

FcGBP gene expression level was higher in HPVpositive HNSCC and correlated to longer OS time in HNSCC patients
After we determined the expression level of FcGBP and TGF-β in HNSCC and surgical safety border tissues, we pay attention to verify the gene expression difference of FcGBP and TGF-β in HPV-positive and HPV-negative HNSCC tissues. We found FcGBP mRNA expression level was higher in HPV-positive HNSCC tissues compared to HPV-negative HNSCC tissues in GSE40774 (p<0.0001), while TGF-β mRNA expression level was lower in HPV-positive HNSCC tissues compared to HPV-negative HNSCC tissues in GSE40774 (p<0.0001) (Figure 2A). Next we detect FcGBP and TGF-β protein expression level in HPV-positive and HPV-negative HNSCC tissues. Similar to GEO dataset analysis, FcGBP protein expression level was higher in HPV-positive HNSCC tissues (p<0.001), and TGF-β protein expression level was lower in HPV-positive HNSCC tissues (p<0.001) ( Figure 2B). Also, FcGBP and TGF-β mRNA expression level was negative correlated in HPV-positive and HPV-negative HNSCC tissues (GSE40774: r=-0.3291, p=0.0001) (Figure 2A).
We further investigated the role of HPV status and FcGBP expression level in the prognosis of patients with HNSCC. The mRNA expression data from GSE65858 showed that FCGBP mRNA expression level and HPV status were highly correlated with OS and RFS time in patients with HNSCC. HPV-negative HNSCC patients had shorter OS and RFS time than HPV-positive HNSCC patients (OS: HR=0.3750, p=0.0157; RFS: HR=0.4434, p=0.0080) ( Figure 2C). Meanwhile, HNSCC patients with higher expression level of FcGBP mRNA had longer OS time than HNSCC patients with lower FcGBP mRNA expression level (OS: HR=0.5081, p=0.0054) ( Figure 2C).

FcGBP overexpression and exogenous TGF-β treatment could modulate proliferation, migration capability in HNSCC cells
Since we identify that FcGBP gene expression decreased in HNSCC and correlated with poor prognosis in patients with HNSCC, we ectopic overexpress FcGBP in HNSCC FaDu and Cal-27 cells (Figure 3). We found that proliferation rate of FaDu and Cal-27 cells both significantly decreased after FcGBP overexpression, and the inhibition rate of proliferation of FaDu and Cal-27 was 49.6% and 53.2% at 72h. Meanwhile, proliferation rate of FaDu and Cal-27 cells both decreased after 5ng/ml TGF-β treatment, and the inhibition rate of proliferation of FaDu and Cal-27 was 30.1% and 31.2% at 72h ( Figure 4A, 4B).
We found that FcGBP gene expression level decreased in FaDu and Cal-27 cells after 5ng/ml TGF-β treatment for 48h. Next, we investigated FcGBP gene expression level in FaDu/FcGBP and Cal-27/FcGBP cells after 5ng/ml TGF-β treatment for 48h, and we found the same results ( Figure 5A, 5B).
Also, we found that FcGBP gene expression level was negatively correlated to Vimentin and N-Cadherin (CDH2) gene expression level while positively correlated with CDH1 gene expression level in GSE17708 and GSE20247. Meanwhile, data from GSE17708 and GSE20247 showed that CDH2 and Vimentin expression level increased while CDH1 expression level decreased in consequence of EMT activation after exogenous TGF-β treatment ( Figure 6B, 6C, 6D, 6F, 6G, 6H).

DISCUSSION
Head and neck squamous cell carcinoma (HNSCC) accounts for ~6% of all human cancers, with ~650,000 new cases reported and 350,000 HNSCC-related deaths per year worldwide. Although the treatment has been technically and medically improved during the last decades, the 5-year overall survival (OS) rate is still stagnating at about 50% [18], local recurrence and metastasis were the main cause of death in HNSCC patients. Meanwhile, the treatment effect of many other types of tumors has been improved, the patient's survival time is prolonged, and the quality of living is improved [19][20][21].
HPV infection is an important etiology of HNSCC, but HPV-positive and HPV-negative HNSCC have different biological behavior and clinical outcomes due to HPV infection. HPV-positive HNSCC patients have a significantly better prognosis than HPV-negative patients, with the 3-and 5-year survival at 84% and 62% for HPV-positive patients compared to 57% and 26% for  HPV-negative patients, respectively [7]. In present study, we referenced the results of GSE65858, and found that HPV-positive correlated with longer OS and RFS time compared to HPV-negative HNSCC patients.
FcGBP was first identified as an Fc portion of the IgG molecule binding site in intestinal and colonic epithelia, gallbladder, cystic duct, bronchus, submandibular gland, cervix uteri and in fluids secreted by these cells in humans. It might play a role in cell protection and anti-inflammation in tissues [15]. FcGBP gene expression is significantly decreased in colon cancer carcinogenesis [22]. Griffith et al. showed that FcGBP was differentially expressed in normal thyroid tissue, thyroid adenomas and thyroid carcinomas [23]. We found FcGBP gene expression was down regulated in HNSCC tissues, and correlated to OS time of HNSCC patients. The proliferation and migration capability was inhibited after FcGBP over-expression in HNSCC FaDu and Cal-27 cells.  FcGBP is composed of many repeated domains, such as thirteen Von Willebrand factor D domains, and twelve cysteine rich (Cys-rich) and twelve trypsin inhibitorlike domains. The Cys-rich domains are essential in the formation of many disulfide bridges with another molecules, plausibly resulting in a net-like scaffold within the mucosal barrier [12]. In the intestine, FcGBP protein exists in a large complex containing Muc2 and Trefoil peptide [24,25]. These results suggest that it may be important in the maintenance of homeostasis and integrity of epithelium.
Meanwhile, we found that FcGBP was lower in HPV-negative HNSCC tissues compared to HPV-positive HNSCC tissues. We assume that FcGBP gene expression could be modulated by HPV infection. FcGBP gene located in 19q 13.2, and genome research found that gain of 19q chromosome occurs in about 23.1% HPV-positive HNSCC patients [26], and this might explain why FcGBP gene expression level was higher in HPV-positive HNSCC patients.
In present investigation, we found that both mRNA and protein expression level of FcGBP was negative correlated with TGF-β in HNSCC and surgical safety border tissues. Currently TGF-β is understood to act as a tumor-suppressor early in tumorigenesis, and then in later phases it enhances the malignant phenotype. Thus, the expression levels of TGF-β are usually reduced during tumorigenesis, and the expression of TGF-β is increased during malignant progression [27,28]. TGF-β activities bifurcate to exert anti-proliferative and pro-EMT actions in HPV-transformed cells [29]. In present investigation, we found that TGF-β expression level was higher in HNSCC tissues than in normal tissues along with another reports, also we found that TGF-β gene expression was higher in HPV-negative HNSCC patients compared to HPV-positive HNSCC patients. Małgorzata et al. found that TGF-β level was lower in saliva of patients with HPV infection [30]. We found TGF-β gene expression was down regulated after HPV E6 transfection in HPVnegative HNSCC FaDu and Cal-27 cells. These results indicated that TGF-β gene expression was inhibited by HPV infection in HNSCC patients. We found TGF-β could inhibit FcGBP gene expression in vitro, and the results of GSE17708 and GSE20247 confirmed these findings.
EMT is a key incident in HNSCC development, and TGF-β is a primary factor triggering EMT in HNSCC [31]. In present investigation, we found that EMT could be activated by exogenous TGF-β treatment, and EMT could be inhibited by FcGBP over-expression in HNSCC cells. Exogenous TGF-β could counteract the EMT inhibition caused by FcGBP overexpression.
In conclusion, FcGBP gene expression was decreased in HNSCC, and lower FcGBP expression was correlated with shorter OS time of HNSCC patients. HPVpositive was a good prognostic factor in HNSCC patients, and TGF-β expression was inhibited by HPV infection. FcGBP gene expression was modulated by HPV infection.
TGF-β mediated EMT activation played an important role in this progress (Figure 7).

HNSCC associated database obtained from GEO database
In this study, 5 HNSCC associated datasets, GSE17708, GSE20247, GSE40774, GSE59102 and GSE65858, were downloaded from GEO database, respectively. GABRP mRNA expression data was extracted and analyzed. Detailed information could be found in Table 1.

HNSCC tissue samples
62 patients were selected to enter the study ( Table  2). The diagnosis and HPV status were confirmed based on clinical, and histopathological or cytological examination. This study was approved by the ethics committee of The Sixth Affiliated Hospital, Wenzhou medical University, and all specimens were collected from patients provided written informed consent in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice Guidelines.

Immunohistochemical staining and evaluation of results
IHC staining was performed as described previously [32]. The primary mouse anti-FcGBP antibody (1:100) was used. For IHC staining evaluation, immunoreactivity was assessed by two blinded independent observers using light microscopy (Olympus BX-41 light microscope). FCGBP protein intensity and frequency were transformed into a Composit Expression Score (CES) utilizing the formula CES=Intensity× Frequency. The range of CES was from 0 to 12. The CES was scored as negative (0), weak positive (1~4), positive (5~8), strong positive (9~12). www.impactjournals.com/oncotarget

Cell proliferation test
We use Cell Counting Kit-8 (CCK-8) to investigate the proliferation of HNSCC cell lines. Cells at logarithmic phase were seeded in 96 well plates with 1×10 3 cells per well for normal culture. Six double wells were set in every group, and a blank control was also used. After 24 h, 48 h and 72 h culture, 10μl of CCK-8 regent was added in each well, and incubated at 37°C for 1 hour, and absorbance value was measured at 450 nm.

Cell migration and invasion test
Cell migration and invasion assays were performed using Transwell migration chambers according to the manufacturer's protocol. In brief, 1x10 4 cells were suspended in 200 μl serum-free RPMI-1640 medium. The cells were seeded in the upper chamber; the lower chamber contained RPMI-1640 medium with 10% FBS as the chemoattractant. After incubation for 48 h at 37˚C in a humidified atmosphere of 5% CO2, any cells that had not penetrated the membrane were removed using cotton swabs; the cells that had successfully migrated to the bottom surfaces of the membranes were fixed with 4% polyoxymethylene and stained with 0.1% crystal violet for 20 min. They were counted under a microscope.

Western blotting
Total cell protein extracts were prepared following the manufacturer's instructions. The lysates were resolved using 10% Bis-tris GEL, transferred to PVDF membranes and immunoblotted with primary antibodies against HPV E6, FcGBP, TGF-β, E-Cadherin, Vimentin and GAPDH. Following incubation with secondary antibodies, the protein bands were detected using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Rockford, IL, USA).

The statistical analysis
All experiments were performed three times. All statistical analyses were performed using the SPSS 16.0 software. Quantitative data was expressed as means ± SD. Student's t-test was used to compare the differences of means between each two groups. One-way ANOVA was used to analyze the differences between multiple sets of data Pearson Correlation Coefficient analyzed correlation between two groups. The Kaplan-Meier method, Hazard ratios (HR), 95% confidence intervals (CI) and log-rank test were used to evaluate the correlation between FcGBP expression and patient survival. The criterion for statistical significance was established at P<0.05.