Fc fragment of IgG binding protein is correlated with immune infiltration levels in hepatocellular carcinoma

The Fc fragment of IgG binding protein (FCGBP) has been confirmed to play an important role in various cancers. However, the specific role of FCGBP in hepatocellular carcinoma (HCC) remains undefined. Thus, in this study, the enrichment analyses (Gene Ontology [GO], Kyoto Encyclopedia of Genes and Genomes [KEGG], and Gene Set Enrichment Analysis [GSEA]) of FCGBP in HCC and extensive bioinformatic analyses using data of clinicopathologic characteristics, genetic expression and alterations, and immune cell infiltration were perfomed. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to verify the expression of FCGBP in both HCC tissues and cell lines. The subsequent results confirmed that FCGBP overexpression positively correlated with poor prognosis in patients with HCC. Additionally, FCGBP expression could effectively distinguish tumor tissues from normal tissues, which was verified by qRT-PCR. The result was further confirmed by using HCC cell lines. The time-dependent survival receiver operator characteristic curve exhibited the strong ability of FCGBP to predict survival in patients with HCC. Additionally, we revealed the strong relationship between FCGBP expression and a number of classic regulatory targets and classical oncogenic signaling pathways of tumors. Finally, FCGBP was involved in the regulation of immune infiltration in HCC. Therefore, FCGBP has potential value in the diagnosis, treatment, and prognosis of HCC and may be a potential biomarker or therapeutic target.


Introduction
Primary liver cancer is one of the most common malignancies worldwide. Hepatocellular carcinoma (HCC) accounts for 75%-85% of the histologic forms of primary liver cancer, ranking third in cancer-related deaths [1]. Thus, an overwhelming burden has been imposed on public health and finance globally. Impressive progress has been made in HCC diagnosis and treatment [2], but most patients miss the optimal treatment opportunity at the time of diagnosis due to the characteristic of the occult onset of HCC [3]. Consequently, the clinical benefit and treatment options for patients are extremely limited. Generally, tumorigenesis is closely related to multiple factors, such as genetic alteration, tumor signal transduction pathways, tumor immune microenvironment, etc [4][5][6][7]. The roles of the immune system in anti-cancer strategies are vital [8]. The tumor microenvironment significantly influences the immunotherapy response and prognosis of patients [9]. Immunotherapies targeting tumor microenvironment are revolutionary in anti-cancer therapy [10]. Previous studies have shown that immunotherapy has achieved promising anti-tumor effects in many cancers, including HCC [8,[11][12][13][14][15]. Therefore, identifying the biomarkers closely related to the occurrence, development, and poor prognosis of HCC is crucial for early HCC diagnosis and proper treatment.
The Fc fragment of IgG binding protein (FCGBP) is expressed in many types of mucin-secreting cells and its secreted fluids, such as cells from the bronchus, colon, and cervix uteri [16]. FCGBP has been reported to be involved in maintaining the mucosal structure as a gel-like component of the mucosa [17]. Meanwhile, numerous studies revealed that FCGBP plays an important role in tumorigenesis and progression. FCGBP expression is different in various tumor types [18][19][20]. Cilibrasi et al. [21] revealed that FCGBP is highly expressed in glioblastoma. Wang et al. [22] confirmed that high FCGBP expression was associated with poor survival in ovarian cancer. In contrast, FCGBP was downregulated in gallbladder cancer [23]. Moreover, FCGBP expression levels were reported to positively correlate with the survival rate in colorectal cancer, including primary lesions and liver metastases [24]. However, the role of FCGBP in the prognosis and biological function of HCC is poorly understood.
This study analyzed the prognostic and diagnostic value of FCGBP in HCC. The correlation between FCGBP expression and the clinicopathological characteristics of patients was demonstrated. The potential pathogenic signaling pathways involving FCGBP were examined in HCC development. The correlation between FCGBP expression and immune infiltration was evaluated. Our finding provides insights into the mechanism by which FCGBP contributes to HCC.

Materials and methods
Analysis of prognosis and diagnosis R software package "survminer" was used to construct the FCGBP expression levels on pan-cancer prognosis. Moreover, the receiver operator characteristic (ROC) curves, including diagnostic ROC curve and time-dependent survival ROC curve, nomogram model, and calibration plot, were constructed using R packages "pROC," "ggplot2," "timeROC," "rms," and "survival."

Expression analysis and genetic alteration analysis
The FCGBP gene expression data collected from the TCGA database included 50 patients with HCC with their paired adjacent normal liver tissues and 50 normal liver tissues with 374 HCC tissues. Meanwhile, the TIMER, GEO (GSE14520), Clinical Proteomic Tumor Analysis Consortium (http://ualcan. path.uab.edu/ analysis-prot.html), and Human Protein Atlas databases (https://www.proteinatlas.org/) were used to examine the differential FCGBP expression in pan-cancer and HCC. Additionally, the correlation between FCGBP expression levels and clinicopathological characteristics, including T stage, pathologic stage, and vascular invasion status, was computed using the R software package "ggplot2." Moreover, three datasets of HCC (INSERM, Nat Genet 2015; AMC, Hepatology 2014; TCGA, Firehose Legacy) collected from the cBioportal website (www.cbioportal.org/) were used to analyze the FCGBP genomic profiles. Meanwhile, the Catalogue of Somatic Mutations in Cancer (COSMIC) database (https:// cancer.sanger.ac.uk/cosmic) was used to investigate the mutation types of FCGBP in HCC.

Clinical tissue samples and cell lines
In 2020, 25 pairs of HCC and paratumor tissue samples were collected from Tianjin Medical University Cancer Institute and Hospital (Tianjin, China). These patients were pathologically diagnosed with HCC (without other malignancies) and had not received any special treatment preoperatively. The cell lines, LO2, HepG2, Huh7, and Hep3B, were purchased from ATCC (ATCC, Manassas, USA).

Difference and enrichment analysis
Data collected from the TCGA database for identifying the related differentially expressed genes according to the high-level and low-level groups of FCGBP were analyzed using the R software package "ggplot2." Differences of |logFold-Change| of >1 and adjusted P value <0.05 were considered statistically significant. Then, we conducted GO term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis with R software packages "clusterProfiler," "GOplot," "ggplot2," and "org.Hs.eg.db" on the collected differentially expressed genes to learn more about FCGBP information. Additionally, we further explored the potential biological signaling pathways by Gene Set Enrichment Analysis (GSEA), which collected the gene set of c2 (c2.cp.v7.2.symbols.gmt) from the Molecular Signatures Database (MSigDB). The adjusted P value < 0.05 and q-value (false discovery rate) < 0.25 indicated significant enrichment.

Immune infiltration analysis
The TCGA database was utilized for analyzing the correlation of FCGBP expression levels with 24 types of immune cell infiltration in HCC using the R software package "GSVA." Additionally, we evaluated the relationship between FCGBP expression and immune checkpoints using the TIMER and TCGA databases.

Ethical statement
All specimens were anonymized in accordance with ethical and legal standards. This study was approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital (NO. E20210074) and all patients signed the written informed consents.

Statistical analysis
This study used the R software V3.6.3 for data processing and statistical analysis. We used the Wilcoxon test for differential expression analysis. KM method, Cox regression, and log-rank test were used to evaluate the prognosis. Spearman's correlation coefficients were calculated for correlation analysis. A
The diagnostic ROC curve and time-dependent survival ROC curve were constructed to evaluate the accuracy of FCGBP expression levels in HCC diagnosis and prognosis prediction. The ROC analysis revealed that the different FCGBP expression levels could effectively distinguish the tumor tissues from the normal tissues (area under the curve [AUC]: 0.647) ( Figure 1G). Moreover, the time-dependent survival ROC curve exhibited the strong discriminant ability of FCGBP to predict 1-year (AUC: 0.669), 2-year (AUC: 0.573), and 3-year (AUC: 0.571) OS for patients with HCC ( Figure 1H), suggesting FCGBP as a potential diagnostic biomarker. We further constructed a nomogram model to predict the survival probabilities at 1, 2, and 3 years for patients with HCC, C-index: 0.794 (0.763-0.824) by integrating clinicopathologic factors, including FCGBP expression levels, T stage, M stage, TP53, adjacent hepatic tissue, inflammation, histologic grade, prothrombin time, and status of vascular invasion ( Figure 1I). Additionally, a calibration plot was used to test the accuracy of the prediction model. Our data revealed that the predicted probabilities of our model fitted well with the actual probabilities ( Figure 1J). Thus, we conclude that the prediction based on our nomogram model is reliable.

Expression levels of FCGBP in pan-cancer and HCC
We investigated FCGBP expression levels in various malignancies according to the paired samples and unpaired samples from the TCGA database to better understand the FCGBP functionality. The results revealed different FCGBP expression in various cancer types ( Figure S5a and S5b). Interestingly, FCGBP was upregulated in various tumors (e.g., cholangiocarcinoma, glioblastoma multiforme, and HCC), but downregulated in some tumors, such as head and neck squamous cell carcinoma, kidney chromophobe carcinoma, kidney renal clear cell carcinoma, etc. Meanwhile, we analyzed the FCGBP expression levels in different cancer types using the data from the TIMER database, which were similar to the above data ( Figure S5c). Moreover, we evaluated the FCGBP expression levels in HCC tissues. Our results demonstrated that FCGBP was highly expressed in HCC tissues at the mRNA and protein levels (Figures 2A, 2B, 2E-2G, and S2b). Meanwhile, qRT-PCR was used to validate this in 25 pairs of HCC and paratumor tissue samples ( Figure 2C and Table S1) and HCC and normal cell lines ( Figure 2D), suggesting that FCGBP overexpression may play an important role in HCC development.
Analysis of FCGBP expression, clinicopathological parameters, and genetic alterations in HCC The characteristics of 374 patients with HCC with complete RNAseq and clinical data collected from the TCGA database are shown in Table S2. Interestingly, we found that the FCGBP expression levels were significantly different in the different subgroups of T stage, pathologic stage, and status of vascular invasion (P < 0.05) ( Figure 3A-3C). FCGBP expression levels were higher in patients with HCC with T2/T3/T4, stage II/III/IV, and vascular invasion than those in patients with HCC with T1 (P = 0.0025), stage I (P = 0.0021), and non-vascular invasion (P = 0.03). Additionally, we conducted a logistic regression analysis ( Table 1) and found that FCGBP expression was closely associated with the T stage (odds ratio [OR] = 0.628 for T1 vs T2/T3/T4, P = 0.026), pathologic stage (OR = 0.589 for stage I vs stage II/III/IV, P = 0.014), and vascular invasion status (OR = 0.623 for no vs yes, P = 0.046), suggesting that FCGBP expression levels might be used to predict the HCC stage.
Next, we evaluated the FCGBP genetic alterations in HCC using three databases (TCGA, Firehose Legac; AMC Hepatology 2014; and INSERM, Nat genet 2015). Three types of genetic alterations were found (missense mutation, truncating mutation, and amplification), with a 4% rate of FCGBP genetic alteration (33/851 patients), and the missense mutation was the most prevalent type of genetic alteration ( Figure S6a). The mutation frequency of FCGBP was 3% (26/851 patients) ( Figure S6b). The rate of FCGBP genetic alteration ranked from 3.29% (8/243 patients) to 4.51% (17/377 patients) and the mutation frequency ranged from 2.6% (6/231 patients) to 3.29% (8/243 patients) ( Figure S6b). We performed an analysis using the COSMIC database to gain further insight into the FCGBP mutations. The missense substitution remained the most prevalent type of FCGBP mutations (63.47%, 384/605 samples) ( Figure S6c). C>T was the most prevalent type of substitution mutation (40.07%, 216/539 samples) ( Figure S6d). Additionally, we assessed the effect of the FCGBP genetic alterations on patients' survival using KM plots and log-rank tests and found little effect of this alteration on OS and disease-free survival ( Figure S6e and S6f).
Differential expression analysis associated with FCGBP and enrichment analysis A total of 2219 differentially expressed genes were identified by single gene differential expression analysis of highand low-expression FCGBP samples using the TCGA database ( Figure S7). Next, we performed the GO and KEGG enrichment analysis, including GO:0010574, GO:0046425, GO:0048863, GO:1904894, GO:0005911, GO:0070371, GO:0017147, hsa04151, hsa05202, and hsa04015 ( Figure 4A and Table S3), suggesting that FCGBP expression is strongly related to some classic regulatory targets of tumors and classical oncogenic signaling pathways, such as extracellular regulated protein kinases (ERK), signal transducer and activator of transcription (STAT), phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway, etc. GSEA based on the single gene differential expression analysis was conducted to further explore the differentially activated signaling pathways associated with FCGBP expression levels. The finding revealed that the signaling pathways related to immune checkpoints, PI3K/AKT, Janus kinase (JAK)/STAT, mitogen-activated protein kinase (MAPK), and cancer were  significantly enriched in the group of high FCGBP expression ( Figure 4B and 4C), suggesting the oncogenic role of FCGBP by regulating multiple targets and activating multiple signaling pathways in HCC.

Correlation between FCGBP expression levels and immune infiltration in HCC
Generally, the occurrence and development of malignant tumors and immunity are closely interlinked [25]. Therefore, we analyzed the relationship between FCGBP expression levels and 24 types of immune cells in HCC ( Figure 5A). Figures 5B, 5C, and 6A show that the FCGBP expression was positively associated with some immune cells, such as macrophages (r = 0.394, FCGBP and immune checkpoints expression levels was analyzed using TCGA and TIMER databases ( Figure 6B and 6C). We found that the FCGBP expression was positively correlated with that of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PDCD1) (P < 0.001), suggesting that FCGBP might play an important role in HCC occurrence and development by regulating immune infiltration.

Discussion
HCC remains a global public health challenge due to the low rates of early diagnosis and high mortality and incidence rates. The low rates of early diagnosis are one of the major causes of death in patients with HCC [26]. Importantly, its mortality and incidence rates are increasing [27]. By 2025, more than 1 million people are expected to suffer from liver cancer each year, which will put a huge economic burden on society, especially in East Asia and Africa [27,28]. Thus, improving early detection rates and exploring new effective treatments for patients with HCC is urgently needed. In recent years, we can study more fundamental molecular mechanisms of HCC with the rapid advancement of precision medicine and sequencing technologies. Many studies have confirmed that FCGBP plays an important role in various malignancies, including ovarian cancer [22,29], glioblastoma [21], colorectal cancer [20,24,30,31], gallbladder cancer [23], head and neck squamous cell carcinoma [32,33], prostate cancer [34], papillary thyroid carcinoma [35], and osteosarcoma [36]. However, to date, the role of FCGBP in HCC has not been reported. Herein, we conducted a comprehensive analysis of FCGBP in HCC using multiple public databases, clinical tissue samples, and cell lines. This study revealed that the higher the FCGBP expression levels, the worse the prognosis of patients with HCC. Meanwhile, ROC curve analysis and the nomogram based on FCGBP expression revealed that FCGBP has certain values in diagnosing and determining the prognosis of patients with HCC. Additionally, aberrant FCGBP expression was found in most human cancers, including liver cancer. We confirmed significantly upregulated FCGBP expressions in HCC samples at mRNA and protein levels compared with normal samples. FCGBP expression was positively associated with clinicopathological characteristics, including T stage, pathologic stage, and vascular invasion status. Therefore, FCGBP may be a potential prognostic and diagnostic biomarker for patients with HCC.
Next, we studied FCGBP mutations in patients with HCC. Our finding revealed that the mutation frequency of FCGBP was 3%, with the predominant type being a missense mutation. However, no significant correlation was found between these mutations and prognosis, which may be due to the small sample size. Hence, we will further expand the sample size to explore its clinical significance.
We performed GO, KEGG, and GSEA enrichment analyses to further elucidate the underlying molecular mechanisms of FCGBP in HCC. GO analysis indicated that FCGBP was involved in the tumor initiation and progression through various pathways, including ERK1 and ERK2 cascade, JAK-STAT cascade regulation, vascular endothelial growth factor production regulation, etc. These ways played a crucial role in HCC occurrence and development [37][38][39]. KEGG analysis revealed that FCGBP was related to multiple oncogenic signaling pathways, especially PI3K/AKT signaling pathway. Several studies have confirmed the cancer-promoting effects of these signaling pathways in HCC [40][41][42][43]. Additionally, we performed GSEA to further explore the oncogenic signaling pathways of FCGBP. We found that the multiple typical carcinogenic pathways and immune escape-related pathways were significantly enriched, such as PI3K/AKT, MAPK, JAK/STAT, and CTLA-4 and PDCD1 signaling pathways, in which FCGBP expression was increased. The main results of these enrichment analyses were in accordance. Thus, we conclude that FCGBP plays an important role in HCC occurrence and development by regulating multiple tumorigenic targets and activating multiple carcinogenic signaling pathways.
Studies revealed that immune cells played an important role in regulating the malignant behaviors of tumor cells [44][45][46]. Therefore, we performed an analysis for immune infiltration, which indicated that FCGBP expression levels were closely related to multiple immune cells in HCC, which was consistent with the results of the previous report [47]. Additionally, immune checkpoint inhibitors provided a new direction for treating malignancies [48]. Our study revealed that FCGBP expression was closely associated with CTLA-4 and PDCD1. It suggests that FCGBP is a potentially novel therapeutic target in HCC.
Our study comprehensively evaluates the potential value of FCGBP and explores its molecular mechanisms in HCC occurrence and progression using the cross-validation method across multiple databases and qRT-PCR; however, the above findings need to be further verified in the wet laboratory.

Conclusion
Taken together, our study preliminarily identified the role and mechanisms of FCGBP in HCC occurrence and development.
Our finding provides insights into the role of a potential HCC biomarker in terms of diagnosis, prognosis, and treatment.