Comprehensive Analysis of the Expression and Prognosis for GBPs in Head and neck squamous cell carcinoma

Guanylate binding proteins (GBPs) belongs to the interferons (IFNs) induced guanylate-binding protein family (Guanosine triphosphatases, GTPases) consisting of seven homologous members, termed GBP1 to GBP7. We used multidimensional survey ways to explore GBPs expression, regulation, mutations, immune infiltration and functional networks in head and neck squamous cell carcinoma (HNSCC) patient data based on various open databases. The study provides staggered evidence for the significance of GBPs in HNSCC and its potential role as a novel biomarker. Our results showed that over expressions of 7 GBPs members and multivariate analysis suggested that N-stage, high expressions of GBP1 and low expression of GBP6/7 were linked to shorter OS in HNSCC patients. In addition, B cells of immune infiltrates stimulant the prognosis and might have a medical prognostic significance linked to GBPs in HNSCC. We assume that GBPs play a synergistic role in the viral related HNSCC. Our results show that data mining efficiently reveals information about GBPs expression in HNSCC and more importance lays a foundation for further research on the role of GBPs in cancers.

Until now, seven different GBPs and six in mouth have been identified and human GBPs show a high level of homology. GTPases are involved in process of signal transduction, cell proliferation, differentiation and intracellular protein transportation 13,14 . Inside host cells, GBPs quickly assemble into big antimicrobial defense complexes that fight various bacterial pathogens 15 . Studies found the immune roles of human GBPs in regulating not only pyroptosis, but also apoptosis 16 . Most previous studies the function of GBPs in bacterial pathogens, but more and more studies explore the role of GBPs in tumor mechanism. Study found that GBP1 acts specifically as a tumor suppressor in colorectal carcinoma and the reduction of GBP1 expression might imply cancer evasion from the IFN-γ-dominated Th1 immune response 17 . Nevertheless, the mechanism by which GBPs are depressed or activated still remained unclear in the development and progression of HNSCC. Until now, a comprehensive bioinformatics analysis has not yet to analysis the potential role of GBPs in HNSCC. we explored the mutations and expressions of various GBPs based on thousands of variations in copy numbers or gene expressions in patients whom with HNSCC in detail to provide new insights into the potential functions, expression patterns, molecular mechanisms and distinct prognostic underlying GBPs regulation.

Over-expression of different GBPs family members in HNSCC.
In order to explore the potential therapeutic value and distinct prognostic of different GBPs members in HNSCC patients, the expression GBPs were explored by ONCOMINE database, TCGA database and HPA database. We first contrast the transcriptional levels of GBPs in cancers and normal samples and significantly higher mRNA expressions of GBP1/4/5 were found in HNSCC tissues in multiple datasets (Fig. 1). In Peng Head-Neck statistics 18 , GBP1 over-expression was found in oral cavity squamous cell carcinoma tissues compared with normal tissues with a fold change of 7.854 (P = 4.15E-24), while Talbot 19 observed 2.962-fold increase in GBP1 mRNA expression in tongue samples (P = 3.10E-12), Ginos 20 found 5.397-fold increase in GBP1 mRNA expression in HNSCC tissues (P = 2.70E-13), Estilo 21 found 4.286-fold increase in GBP1 mRNA expression in tongue tissues (P = 3.51E-9) and Ye 22 found 4.450-fold increase in GBP1 mRNA expression in tongue tissues (P = 5.39E-5, Table 1). Significant up-regulation of GBP4 was also found in HNSCC tissues compared to normal tissues. In Peng Head-Neck statistics 18 , GBP4 over-expression was found in oral cavity squamous cell carcinoma tissues compared with normal tissues with a fold change of 3.256 (P = 2.06E-12). Similarly, In Peng Head-Neck statistics 18 , GBP5 over-expression was found in oral cavity squamous cell carcinoma tissues compared with normal tissues with a fold change of 14.065 (P = 3.61E-26), while Ye 22 observed 3.376-fold increase in GBP5 mRNA expression in tongue samples (P = 1.53E-4). We then tried to analysis the protein expression patterns of GBPs in HNSCC by the HPA database. As the result shown GBP1/4/5 proteins were not expressed in normal head and neck tissues, whereas high and medium expressions of them were observed in HNSCC tissues. In addition, low protein expressions of GBP3/7 were expressed in normal tissues, while medium and high protein expressions of them were observed in tumor tissues. Moreover, medium protein expression of GBP2 was observed in normal tissues and high expression in HNSCC tissues (Fig. 2). However, no expression and low protein expression of GBP6 was observed in normal and tumor tissues. In total, our results suggested that transcriptional and proteinic expressions of GBPs were over-expressed in patients with HNSCC.

Association of mRNA expression of different GBPs family members with clinicopathological
parameters of HnScc patients. Using the GEPIA dataset, we first contrast the mRNA expression difference of GBPs between cancer and normal tissues. The results demonstrated that the expression of GBP1 and GBP5 were higher in HNSCC tissues, while the expression level of GBP6 was lower in cancer tissues (Fig. 3). Moreover, we also explored the expression of GBPs with cancer stage for HNSCC and we found GBP1/5/6 groups significantly varied, but GBP2/3/4/7 groups did not significantly differ (Fig. 4).
predicted functions and pathways of GBps in HnScc. We next explored the GBPs alterations and networks via the cBioPortal online tool and we further analyzed 50 neighbor genes which were significantly related to GBPs mutations. In the 528 sequenced HNSCC patients, genetic alteration was found in 115 patients and the mutation rate was 22%, GBP5 ranked the highest genes with genetic alterations with the mutation rates were 7% and we also shown the network for GBPs and the 50 most frequently altered neighbor genes (Fig. 6). The top 10 type and frequency of GBPs neighbor gene alterations in HNSCC including ADAR, IRF6, IRF9, IRF2, CD44, STAT1, FCGR1A, NFATC2, ICAM1 and STAT2 (Table 2). In addition, functions and pathways of GBPs and their 50 frequently altered neighbor genes were analyzed by GO and KEGG in DAVID online database (Supplementary Table 3). GO analysis results showed that changes in biological processes (BP) were significantly   Table 4).

Discussion
Although certain GBPs members have been shown to play a key role in cancer, the different roles of GBP family members in HNSCC remain to be elucidated. So, we first explored the mutations and expressions of various GBPs based on thousands of variations in copy numbers or gene expressions in patients whom with HNSCC. Our study showed that high expressions of mRNA in all the GBPs family members, and mRNA expression of GBPs was significantly related to patients' individual cancer stages in HNSCC patients. Multivariate analysis suggested that N-stage (HR = 1.33, 95% CI: 1.03-1.72, P < 0.05), high expressions of GBP1 and low expression of GBP6/7 were linked to shorter OS of cancer patients. www.nature.com/scientificreports www.nature.com/scientificreports/ The occurrence of HPV-related HNSCC has steadily rised over the past two decades and now indicate a majority of HNSCC cases 23 . Epstein-Barr virus (EBV) infection is found in 90% to 100% of nasopharyngeal carcinoma (NPC) cases in endemic regions 24 . GBPs belongs to the IFNs induced guanylate-binding protein family, while IFNs are the main antiviral factors released during the process of infecting host cells by viruses or other pathogens, and have cell surface receptors that bind to responsive cells, regulating host antiviral status, anti-malignant cell proliferation and immune response 15 . Functions and pathways of GBPs and their 50 frequently altered neighbor genes were analyzed by GO and KEGG demonstrated that changes in BP of DEGs were significantly enriched in regulation of immune response and virus and cytokine production, MF were mainly enriched in protein/ region DNA/nucleic acid binding and CC were mainly enriched in intracellular membrane-bounded organelle and MHC class I protein complex et al. KEGG were mainly enriched in antigen processing and presentation and viral infection. So, we presume that GBPs play a synergistic role in the HPV-HNSCC or other viral related cancers.
GBP1 expression is excited not only by type I and type II interferons (IFN), including IFN-γ, and also by interleukin-1α, IL-1β, and tumor necrosis factor-α [25][26][27][28] . In vivo, GBP1 is related to the existence of inflammation and has been adhere to in inflammatory bowel diseases (IBD), autoimmune diseases and cancers. GBP1 regulates the suppression of endothelial cells invasion and proliferation in reaction to inflammatory cytokines and exhibit antiviral activity 27 . Yu et al. reported that GBP1 positively regulating the invasion and migration of oral cavity squamous cell carcinoma cells 29 . A recent study showed that GBP1 may promote lung adenocarcinoma invasiveness by stimulate cell motility and via manage of GBP1 expression may development of new therapeutic ways for tumor 30 . GBP1 also found as a fetal T lymphocyte-induced protein that cause breast cancer cells to cross the blood-brain barrier 31 . Our results showed that higher mRNA expressions of GBP1 were found in HNSCC tissues, and that mRNA expression of GBP1 was significantly related with patients' cancer stages and abundance of immune infiltrates. Moreover, previous studies focus on the role of T immune cell in cancers, while our cumulative survive showed that B cells significantly stimulant the prognosis and might have a medical prognostic significance linked to GBPs in HNSCC. GBP2 is strongly induced by IFN-γ. GBP2 is obviously higher in esophageal squamous cell carcinomas than in adjacent normal epithelium cells and is consistently increased in a p53-dependent manner and also followed by an rise in protein levels of IRF-1 32 . Zhang et al. 33 first found that GBP2 inhibits cell metastasis and mitochondrial fission in breast cancer cells not only in vitro but in vivo and another study found an interesting result that GBP2 related to a recently generally T cell signature, suggesting tumor infiltration with T cells in breast cancer 34 . Other study found GPB2/5 shown wide antiviral activity by suppressing the activation of the virus-dependency factor furin 35 . GBP2 was similarly relocalized in cells in which autophagy was impaired 36 . Our study results shown that higher mRNA expression of GBP2 were significantly related to longer OS and RFS of HNSCC patients. So, we guess GBP2 may play a critical role in cancer autophagy and needs more study to convince.
Xu et al. 37 first found that GBP3 lead to the proliferation of glioma cells via SQSTM1-ERK1/2 pathway. GBP3 also has been reported to contributed to anti-influenza activity via suppressing viral transcription   I~Stage IV) for HNSCC and we found GBP1/5/6 groups significantly varied, but GBP2/3/4/7 groups did not significantly differ. The method for differential gene expression analysis is one-way ANOVA, using pathological stage as variable for calculating differential expression. The larger F, the better the study fit and P-Value < 0.05 as statistically significant. (GEPIA Database). www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ and replication 38 . GBP1 and GBP3 exhibit highest homology between the seven members (88% amino acid sequence) 12 so GBP3 may be a novel proto-oncogene. Similarly, our results showed that higher mRNA expressions of GBP3 and related to shorter OS in HNSCC tissues. Study reported that GBP4 is a negative regulator of virus-induced IFN-I production resulting in impaired TRAF6-mediated IRF7 ubiquitination and it is deemed as a novel protein targeting IRF7 and suppressing its function 39 . Therefore, GBP4 may affect ubiquitination-mediated pathways, thereby inhibiting viral infection. IRF regulation of tumors is mainly through regulation of the cell cycle, apoptosis or tumor suppressor gene p53, but also regulation of anti-tumor immunity, or regulation of interferon signaling Pathway (eg IRF9) 40,41 . Our results found the 50 most frequently altered GBPs neighbor gene alterations in HNSCC including interferon regulatory factors 6 (IRF6), IRF9 and IRF2, indicated that IRFs and GBPs coordinate the development of tumors and requires more study. GBP5 have splice variants (GBP5a, GBP5b, GBP5ta) before 1992 42 . Krapp et al. 43 reported that GBP5 has a broad spectrum of antiretroviral capabilities. Wehner et al. 44 reported that only the a/b splice variant is rise in healthy cells, but the truncated splice variant has been discovered in each melanoma and lots of lymphoma cell lines detected. Study also found GBP5 act as an only rheostat for NLRP3 inflammasome activation and thus alert the immune system to the exist of infection or tissue damage 45 . Our results showed that higher mRNA expressions of GBP5 in HNSCC tissues and may related with patients' tumor immune microenvironment. Until now, about the function of GBP6/7 is rare. HuGBP-6 lacks a GAS or NF-κB site and therefore, would not be expected to be induced by IFNs 46 . In GEPIA database analysis we found high GBP6 expression in normal tissue and related with patients' cancer stages, while in TCGA database analysis we found high expression in cancer tissue, in addition, in HPA databased we found no expression and low protein expression of GBP6 was observed in normal and tumor tissues. Both GBP6/7 significantly related to longer OS of HNSCC patients. GBP7 can triggered an IFIT5-IRF1/3-RSAD5 pathway in the DF-1 cells which potentially limited the viral replication cycle in the initial infection stage 47 . Thus, more researches should conduct to provide new ideas for the development of anti-tumor drugs and the treatment of human diseases.
There were some limitations in our study, one is that all the data analyzed in our study was retrieved from the online databases and cannot provides precise clinical data, larger sample sizes are needed to validate our findings, another is that we did not evaluate the potential diagnostic and therapeutic roles of GBPs and tumor markers can vary widely due to the histological type of HNSCC and multiple anatomical sites, so future related researches are required. Finally, due to insufficient data, we were unable to compare differences in function of GBPs between HPV-positive and HPV-negative HNSCC patients and we will do a lot of work in the future. In conclusion, our results showed that over expressions of 7 GBPs members and multivariate analysis suggested that N-stage, high expressions of GBP1 and low expression of GBP6/7 were linked to shorter OS in HNSCC patients. In addition, B-cell infiltration of immune cells affects prognosis and may be related to GBPs in HNSCC. We hypothesized that the GBPs plays a synergistic role in virus-associated HNSCC. This research uses online tools that rely on the most popular bioinformatics theories to perform target gene analysis of tumor data information from open databases, and authorizes large-scale HNSCC genomics research and follow-up functional exploration.

Materials and Methods ethics statement.
As the work is a bioinformatics analysis article, so the ethical approval was not necessary and all the data were retrieved from the free online databases. oncoMine database. The DNA copy number and mRNA expression of GBPs in HNSCC were investigated inside the Oncomine 4.5 database. Oncomine (www.oncomine.org) contains 715 gene expression data sets and 86,733 samples, is also the biggest oncogene chip database and incorporated data mining platform worldwide 48 . This analysis drew on a series of HNSCC studies and GBPs expression was involved in evaluated in HNSCC tissue in respect to its expression in normal tissue, and P < 0.05 as the cutoff criterion considered statistically significant.
Human protein atlas. The Human Protein Atlas (https://www.proteinatlas.org/about/licence) is a website that involve immunohistochemistry-based expression data for distribution and expression of 20 tumor tissues, 47 cell lines, 48 human normal tissues and 12 blood cells, the results are represented by at least 576 immunohistochemical staining maps, which have been read and indexed by professionals. These tissues were from 144 different tcGA database. TCGA uses large-scale genome sequencing to map out the genomic variation maps of all human cancers, and conduct systematic analysis to understand the mechanism of cancer cell occurrence and development, and on this basis, to obtain new diagnostic and therapeutic methods 52 . In our analysis, clinicopathological parameters of 512 HNSCC patients and mRNA expression of GBPs of 529 HNSCC patients were downloaded from the TCGA. The listwise deletion technique was utilized to deal with missing data, which excluded the entire sample from the investigation if any single value was absent. Cox analysis was utilized to evaluate the impact of GBPs expression on survival alongside other clinical attributes (such as age, gender, stage, distant metastasis).
cBioportal. The cBioPortal (http://cbioportal.org) 53 is an open-access asset gives visualization, analysis and download of substantial scale cancer genomics data sets. We utilized c-BioPortal to analyze GBPs alterations in the TCGA HNSCC samples and shows an overview of genetic alterations per test in GBPs. A tab biological interaction network of the GBPs and their co-expression genes was analyzed and neighboring genes with alteration frequencies were included. Functions of GBPs mutations and 50 genes significantly related to GBPs mutations were performed by GO and KEGG in the Database for Annotation, Visualization, and Integrated Discovery (DAVID) online tool. P-Value<0.05 as statistically significant. tiMeR analysis. TIMER (https://cistrome.shinyapps.io/timer/) 54 is a comprehensive asset for systematical investigation of immune infiltrates over various malignancy types. The abundances of six immune infiltrates (CD8+ T cells, B cells, CD4+ T cells, Macrophages, Neutrphils and Dendritic cells) are assessed by our statistical method, which is approved using pathological estimations. Using Gene module to explore correlation between GBPs expression and abundance of immune infiltrates in HNSCC; Survival module to draw Kaplan-Meier plots for immune infiltrates and GBPs to picture the survival differences. P-Value < 0.05 as statistically significant.