The tumor-suppressive function of the unique H domain of FCGBP and its potential as a therapeutic target in colorectal cancer

Abstract

Background

Colorectal cancer (CRC) is a major global health issue, characterized by high incidence and mortality rates. This study aims to further elucidate the function of H domain of Fc fragment of IgG binding protein (HFCGBP), as a candidate tumor suppressor gene, in the progression of CRC and its clinical implications.

Methods

A comprehensive methodological approach was employed, encompassing the collection of clinical samples, proliferation and migration assays, immunohistochemistry, and in-depth data analysis.

Results

The downregulation of FCGBP expression was significant negatively correlated with tumor stage and grade in CRC tissues. Importantly, the overexpression of HFCGBP in CRC cells resulted in a notable inhibition of cell proliferation and migration, highlighting its potential as a therapeutic target. Combined with bioinformatic analysis of RNA-seq, the key signaling pathways modulated by HFCGBP, such as neuroactive ligand-receptor interaction and platelet activation, were identified, thereby offering valuable insights into its regulatory mechanisms. Furthermore, the analysis of TCGA and Gene Expression Omnibus (GEO) databases further confirmed the reduced expression of FCGBP in various tumor types, with a particular focus on CRC, reinforcing its potential as a prognostic biomarker. It underscores the crucial role of HFCGBP in CRC and its promising potential as a therapeutic target.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-026-15718-8.

Background

Cancer is a major global health challenge. In recent years, although the methods of early diagnosis and treatment have emerged one after another, the morbidity and mortality are still rising, particularly among younger populations. Such a rising trend places a significant burden on healthcare systems and families [1, 2]. Colorectal cancer (CRC) is characterized by a complex etiology influenced by various factors, including genetic predisposition, environmental exposures, and lifestyle choices [3]. Current treatment strategies for CRC include surgical intervention, chemotherapy, radiotherapy, and immunotherapy [4, 5]. Nevertheless, these methodologies are frequently limited by late-stage diagnosis and variable treatment responses. Although several molecular biomarkers, such as carcinoembryonic antigen and carbohydrate antigen19-9, have been implemented clinically, their specificity and sensitivity remain inadequate for early diagnosis [6, 7]. This highlights the urgent necessity for the discovery and investigation of novel biomarkers and therapeutic targets that could improve early diagnosis and management, ultimately enhancing patient prognosis and survival rates.

Fc fragment of IgG binding protein (FCGBP), a highly glycosylated mucin-like glycoprotein initially identified in intestinal goblet cells, plays a pivotal role in maintaining mucosal barrier integrity and modulating the tumor immune microenvironment [8–10]. The full-length cDNA of FCGBP is 17,000 bp, of which with a calculated molecular weight of 500 kDa [9]. The special structure of FCGBP reveals the presence of H, R, and T domains. The determination of crystal structure of R and T domains clarifies their molecular mechanism as the core scaffold protein of the mucosal barrier, providing a promising target for treating mucosal diseases [11, 12]. The H domain is an IgG Fc-binding region located at the N-terminus of the FCGBP protein and composed of 450 amino acids. It is deeply involved in the formation of bioactive mature proteins. It is likely that it processes the FCGBP peptide segments, transforming them into a form with Fc-binding activity [9]. Additionally, it can assist in precisely targeting the relevant proteins to the Golgi apparatus for subsequent processing and modification. The presence of H domain is closely related to the Fc-binding activity. Thus, if the H domain is deleted, this Fc-binding activity may be lost accordingly. However, up to now, the research on the role of HFCGBP in tumors is blank. Studies have reported the presence of FCGBP in various malignancies, including lung cancer, gallbladder cancer, invasive breast cancer, diffuse large B-cell lymphoma, glioma, pancreatic cancer, ovarian cancer, gastric cancer, and head and neck squamous cell carcinoma [9, 13–15]. Elevated levels of FCGBP have been observed in the serum of patients with conditions such as ulcerative colitis and Crohn’s disease, indicating its potential as a biomarker for autoimmune diseases [16]. Furthermore, FCGBP expression is notably increased in the bloodstream of patients with mycoplasma pneumonia, suggesting its involvement in systemic inflammatory responses [16]. In the context of CRC, FCGBP appears to exhibit tumor-suppressive properties, as evidenced by studies showing a progressive decline in its expression from intestinal inflammation to colorectal adenomas and ultimately to CRC [9, 15]. This trend is further exacerbated in metastatic lesions, emphasizing the necessity for a deeper understanding of its molecular mechanisms and clinical significance in CRC [15, 17]. Despite the growing body of evidence regarding FCGBP’s important role in tumor biology, its specific molecular mechanisms and clinical implications in CRC remain inadequately characterized, highlighting a significant gap in the current research landscape [17, 18]. To address the role of HFCGBP in CRC, we constructed an overexpression vector of the H domain to elucidate its specific functions in CRC. These findings underscore the need for further investigation to elucidate its precise functions within the tumor microenvironment and its potential as a therapeutic target.

In this study, we aim to elucidate the molecular mechanisms of HFCGBP in CRC by integrating clinical sample analysis, cellular experiments, and bioinformatics approaches. That is, by evaluating the expression patterns of HFCGBP in clinical samples and its impact on cell proliferation, migration, invasion, and cell cycle, we seek to provide a comprehensive understanding of its functional roles in CRC. Additionally, we will explore the potential of HFCGBP as both a prognostic biomarker and a therapeutic target, contributing to the advancement of personalized treatment strategies for CRC patients.

Methods

Clinical sample collection

Paraffin-embedded tissues and corresponding para-cancerous tissues of 32 cases diagnosed as CRC were obtained from Pathology Department of Tangdu Hospital (Xi’an, China) between January and October 2024. All specimens were fixed in 10% neutral formalin solutions within 30 min after operation and finally embedded in paraffin. Clinical information for the corresponding patients was gathered. The research strategies concerning human tissues were all approved by the IEC of Institution for National Drug Clinical Trials, Tangdu Hospital Fourth Military Medical University (No. K-HG-202505-14) and written informed consent was provided by the patients.

Cell culture and transfection

The cell lines SW480 and HCT116 were donated by the Cancer Prevention and Treatment Center of Tangdu Hospital. The cells were cultured in high-glucose DMEM (BBI, K517fc0251, China) containing 10% fetal bovine serum (Cell-Box, AUS-01s-02, Australia) and 1% penicillin-streptomycin mixture, and placed in an incubator at 37 °C and 5% CO₂ humidity. The 1284 bp ORF of FCGBP was cloned into pCDH-CMV-MCS-COpGFP-T2A-Puro vector to construct the HFCGBP overexpression vector (HFCGBP-OV). Then a total of 8 µg recombinant HFCGBP overexpression plasmid were transfected into CRC cell lines in logarithmic phase with 60–70% cell confluency by 10 µL of Lipofactmine 2000 (Invitrogen, 11668019, China). The cells were collected for subsequent function analysis 48 h after transfection.

CCK8 cell proliferation assay

Two methods were employed to detect cell proliferation: CCK8 assay and colony formation assay. For CCK8 assay, about 2000–3000 cells were seeded in a 96-well plate, with at least 5 replicates for each treatment. At 24, 48, 72, and 96 h after seeding, 10 µl of CCK8 solution was added to each well and incubated at 37 °C for 1 h. Cell viability was determined by measuring the absorbance at 450 nm using a microplate reader. For colony formation assay, 600–1000 cells were seeded in a 6-well plate and cultured for approximately 2 weeks until visible cell colonies formed. The cells were fixed with 4% paraformaldehyde and stained with crystal violet at 37 °C for 30 min. The stained colonies were quantified using Image J software.

Flow cytometry

Cell Cycle detection was performed using the Cell Cycle Analysis Kit (Beyotime, C1052, China). Cells in the logarithmic growth phase were collected, gently digested with trypsin (without EDTA), and centrifuged at 1500 rpm for two 5-minute cycles according to the manufacturer’s instructions. The cells were then resuspended in precooled PBS, admixture of propidium iodide (PI, 40 g/mL) and RNase (100 g/mL) was configured and stained at 37 °C for 30 min. Flow cytometry were performed using a 200-purpose filter. Finally, the results were analyzed using FlowJo software.

Migration and invasion assay

After 48 h of overexpression transfection, cells in logarithmic growth phase were collected for transwell and scratch experiments. For the transwell assay, 1 × 10⁵ serum -free cells were inoculated into the upper chamber of a transwell system (12-well plate), while the lower chamber was a medium containing only 5% serum. After incubation for 48 h, the cells were fixed with methanol, stained with 0.05% crystal violet, and the number of cells passing through the membrane was counted using Image J software. 1 × 10⁶ cells without serum were inoculated into a 6-well plate. After the cells adhere firmly to the wall, the same gun head was used to draw a line along the ruler in the 6-well plate using the same force. Finally, the detached cells were washed with sterile PBS, and the healing process was monitored and photographed every 24 h.

Immunohistochemistry (IHC)

IHC staining of FCGBP was performed after 3 μm-thick paraffin sections were dewaxed in xylene, hydrated with gradient alcohols, and treated with microwave for antigen retrieval for 15 min (Citric acid, pH 6.0). Specifically, 50 µl FCGBP antibody was firstly dropped on the tissue sections for 1 h at room temperature (Abcam, Ab121202, rabbit, 1:300). Then, the MaxVisionTM 2/HRP reagent (EliVision Super DAB, Fuzhou Maixin Biotechnology, China) was then added on the slice and incubated at room temperature for 18 min. Enhanced DAB (EliVision Super DAB, Fuzhou Maixin Biotechnology, China) was added to the slice for color development. The immunohistochemical results of each patient’s cancer tissues and adjacent normal tissues were scored based on the intensity of cell staining and the proportion of positive cells, respectively. (1) Cell staining intensity: 1 point, light yellow, weakly positive; 2 points, brownish yellow, moderately positive; 3 points, brown strong positive. (2) The proportion of positive cells in all cells: the number of positive cells < 5% for 0 point, 5% -25% for 1 point, 26% -50% for 2 points, 51% -75% for 3 points, and > 75% for 4 points. The final score was calculated by multiplying the above two scores including cell staining intensity and the proportion of positive cells in all cells, with ≥ 6 being high expression and < 6 being low expression. Finally, the average total score of cancer tissue and para-cancer normal tissue was recorded as the final score of the patient. The IHC results were scored independently by three experienced pathologists.

Data collection

To ascertain the expression level of FCGBP, we employed “colorectal cancer (CRC)” as the search keywords in the TCGA, Gene Expression Profiling Interactive Analysis (GEPIA) database, and the Gene Expression Omnibus (GEO) database. After excluding samples lacking complete prognostic information, the final sample size from TCGA was 644. We selected datasets GSE21510, GSE32323, and GSE25071 from the GEO database for further analysis, as detailed in Table 1. GSE21510 dataset included 123 CRC samples and 25 para-cancer control samples, GSE32323 dataset included 17 pairs of cancerous and non-cancerous tissues, and GSE25071 dataset consisted of 46 CRC tissues and 4 normal colonic mucosa tissues. Using the cBioPortal gene mutation database (https://www.cbioportal.org/) to calculate the mutation rate of FCGBP in different tumors and clinical features of CRC.

Table 1.

GEO datasets used in the study

Accession ID	Platform	CRC tissues (N)	Normal tissues(N)	Years	Contributor(s)
GSE21510	GPL570	123	25	2011	Tsukamoto S et al.
GSE32323	GPL570	17	17	2012	Ahmed K et al.
GSE25071	GPL2986	46	4	2011	Agesen TH et al.

Clinicopathological features and survival analyses

Clinical information of all patients, including gender, age, pathologic T/N/M stage, primary therapy outcome, nerve invasion, histological type, and lymph node involvement were obtained from TCGA. We pre-specified the median (50th percentile) of log2^{(TPM +1)} transformed FCGBP expression as the dichotomization threshold within the TCGA cohort, yielding “high” and “low” groups. Ties were handled by splitting at the median. Kaplan-Meier (K-M) /COX survival analysis and Cox regression models were used to compare OS (Overall Survival), DSS (Disease Specific Survival) and PFI (Progress Free Interval) between the high and low FCGBP groups.

Correlation and enrichment analyses

In order to better understand the subtle changes in SW480 cells after FCGBP overexpression and the specific mechanisms causing functional changes in tumor cell proliferation, migration and invasion, the whole transcriptome sequencing was performed (Wuhan matware company). Differential expression analysis was performed with a | log²(fold change) |≥ 1, and p value < 0.05 as criterion. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed in https://cloud.metware.cn/, to observe the biological process and signaling pathways affected by FCGBP. Additionally, differentially expressed genes (DEGs) were selected for gene cluster analysis based on data from TCGA, GEO, and our transcriptome sequencing. Using the Calculate and draw custom Venn diagrams (http://bioinformatics.psb.ugent.be/webtools/Venn/) to take the intersection of all DEGs.

Statistical analyses

Data download and processing was performed through the network platform Xiantao (http://www.xiantao.love). K-M analysis was used to assess the prognostic value of FCGBP in CRC patients. Pearson correlation analysis was employed to examine the co-expression of genes with FCGBP in CRC. In correlation analysis, p < 0.05 and R > 0.20 were considered as significant positive correlation. Statistical significance was defined as p < 0.05.

Results

Observation of abnormal FCGBP expression in CRC

To detect the expression of FCGBP in CRC, we first analyzed its expression in pan-cancer using the GEPIA online database. Compared with normal tissues, the expression of FCGBP was significantly lower in several tumors, including BRCA, COAD, HNSC, KICH, KIRP, PCPG, PRAD, READ, STAD, and THCA, particularly in CRC (COAD+READ) (Fig. 1A). To further validate these findings, the expression profiling analysis was performed using GEO datasets GSE21510, GSE32323, and GSE25071. Consistent with our initial findings, FCGBP mRNA level was significantly reduced in tumor specimens compared with the normal control group (Fig. 1C-E). Similar results were observed in paired tissue samples from the TCGA database (Fig. 1B). At the same time, our immunohistochemical results further confirmed the results, namely the protein expression level of FCGBP was significantly reduced in CRC tissues compared with normal intestinal epithelium (Fig. 1F-G). In summary, these results indicated that FCGBP was significantly downregulated in CRC tissues and might as a tumor suppressor.

Fig. 1.

Different expression of FCGBP between normal tissues and tumor tissues. A The expression of FCGBP in pan-cancer (showing only significant differences). B FCGBP expression in paired CRC tissues from the TCGA database. C FCGBP expression levels in GSE21510. D FCGBP expression levels in GES25071. E FCGBP expression levels in GSE32323. F Representative immunohistochemical images of FCGBP expression in CRC and paired para-carcinoma tissues. G FCGBP expression was significantly lower in the CRC tissues than in the paired para-carcinoma tissues. n = 32, * p < 0.05, ** p < 0.01, *** p < 0.001

Correlation between abnormal expression of FCGBP and clinical pathological features

The correlation analysis between FCGBP expression and clinical pathological characteristics showed that the differential expression of FCGBP significantly was correlated with tumor clinical staging, pathological grading, and tumor histological classification. However, no significant correlation was observed with tumor size, patient gender, age, and the presence or absence of lymph node metastasis (Table 2). These findings suggested that FCGBP might play a crucial role in tumor progression.

Table 2.

Correlation between abnormal expression of FCGBP and clinical pathological features in TCGA

Characteristics	Low expression of FCGBP	High expression of FCGBP	P value
n	322	322
Pathologic N stage, n (%)			0.012
N0	168 (26.2%)	200 (31.2%)
N1	77 (12%)	76 (11.9%)
N2	73 (11.4%)	46 (7.2%)
Pathologic M stage, n (%)			0.003
M0	229 (40.6%)	246 (43.6%)
M1	58 (10.3%)	31 (5.5%)
Pathologic stage, n (%)			0.017
Stage I	52 (8.3%)	59 (9.5%)
Stage II	108 (17.3%)	130 (20.9%)
Stage III	95 (15.2%)	89 (14.3%)
Stage IV	58 (9.3%)	32 (5.1%)
Primary therapy outcome, n (%)			0.367
PD	19 (6.1%)	14 (4.5%)
SD	1 (0.3%)	4 (1.3%)
PR	9 (2.9%)	7 (2.2%)
CR	123 (39.4%)	135 (43.3%)
Gender, n (%)			0.813
Female	149 (23.1%)	152 (23.6%)
Male	173 (26.9%)	170 (26.4%)
Age, n (%)			0.203
<= 65	146 (22.7%)	130 (20.2%)
> 65	176 (27.3%)	192 (29.8%)
Histological type, n (%)			< 0.001
Adenocarcinoma	303 (47.9%)	247 (39%)
Mucinous adenocarcinoma	16 (2.5%)	67 (10.6%)
Perineural invasion, n (%)			0.924
No	95 (40.4%)	80 (34%)
Yes	33 (14%)	27 (11.5%)
Lymphatic invasion, n (%)			0.636
No	168 (28.9%)	182 (31.3%)
Yes	116 (19.9%)	116 (19.9%)

Overexpression of HFCGBP inhibited proliferation of CRC cell

Infinite proliferation is one of the characteristics of tumor development. To investigate the effect of HFCGBP on CRC cells, we transfected HFCGBP overexpression plasmid into SW480 and HCT116 CRC cell lines and measured the expression of HFCGBP mRNA. Compared with the control group, transfection with lipofectamine 2000 resulted in a 400-fold increase in HFCGBP mRNA expression in SW480 cells (Fig. 2A), and a 150-fold increase in HCT116 cells (Fig. 2B), confirming successful transient transfection. After 48 h of transfection, CCK-8 assay showed that overexpression of HFCGBP significantly inhibited cell proliferation in SW480 cells and HCT116 cells (Fig. 2C-D). It was suggested that FCGBP was a tumor suppressor gene. This conclusion was further supported by the colony assay. As shown in Fig. 2E-F, HFCGBP overexpression resulted in a significant decrease in cell proliferation (p < 0.01).

Fig. 2.

Overexpression of HFCGBP inhibited the proliferation of CRC cells. A RT-PCR analysis verified the effective overexpression of HFCGBP mRNA in SW480 cells. B The effective overexpression rate of HFCGBP mRNA in HCT116 cells. C-D CCK-8 assay was used to measure the proliferation of SW480 and HCT116 cells. E-F The clonogenic ability of SW480 and HCT116 cells was inhibited by overexpression of FCGBP. * p < 0.05, ** p < 0.01, *** p < 0.001

Overexpression of HFCGBP inhibited migration and invasion of CRC cell

Tumor metastasis is a key characteristic of tumor progression. This study explored how overexpression of HFCGBP affected the migration and invasion of tumor cells using wound healing and transwell assays. HFCGBP overexpression significantly inhibited the wound healing ability of SW480 and HCT116 cells (Fig. 3A-B). Furthermore, transwell with or without matrix gel demonstrated that HFCGBP overexpression significantly inhibited the migration and invasion ability of tumor cells. Compared with HFCGBP-NC group, the number of migratory and invasive cells in HFCGBP-OV group significantly decreased (Fig. 3C-F).

Fig. 3.

Overexpression of HFCGBP inhibited migration and invasion of SW480 and HCT116. A-B SW480 and HCT116 cells were treated with FCGBP-OV plasmid and inoculated into 6-well culture dishes. After the cells adhere firmly to the wall, the same gun head was used to draw a line in the 6-well plate form a wound with the same force and take photos of the injured cells at a designated time. The yellow line represents the boundary of the wound area. The relative wound healing rates between 72h and 0 h are calculated by Image J software. SW480 and HCT116 cells were treated with FCGBP-OV plasmid and inoculated into 12-well culture dishes. C and E Transwell chamber without matrix gel was used to detect tumor cell migration ability. D and F Transwell chamber with matrix gel was used to detect tumor cell invision ability

Enrichment analysis of DEGs

To further elucidate the specific mechanisms by which HFCGBP induces alterations in proliferation, migration, invasion, and cell cycles, we performed RNA transcriptome sequencing to identify downstream genes and signaling pathways regulated by HFCGBP overexpression. A total of 184 DEGs were identified (| log2 (Fold Change) |≥1, and p < 0.05), among which 78 were upregulated and 106 were downregulated (Fig. 4B). In the cluster heatmap (Fig. 4A, red and blue represented the increase and decrease of expression, respectively), we found that the overexpression of HFCGBP significantly changed the gene expression pattern. GO and KEGG analysis were performed on these 184 DEGs. In terms of function enrichment, DEGs were mainly enriched in the biological processes closely related to the occurrence, development and metabolism of human diseases, such as the transmembrane transport of calcium ions into the cytoplasm, the activity of metal ion transmembrane transporters, the activity of cation channels and calcium ion homeostasis (Fig. 4C). The enriched signaling pathways were primarily enriched in neuroactive ligand-receptor interaction, platelet activation, glycine, serine and threonine metabolism, inflammatory mediator regulation of TRP channels and renin secretion (Fig. 4D). We performed a vene cross-over of all the DEGs involved and found that FCGBP and PRSS2 were shared differential genes (Fig. 4E), which suggested that FCGBP and PRSS2 may play an important role in the development of CRC. Moreover, further analysis revealed a significant positive correlation between FCGBP and PRSS2 in CRC (R = 0.259 and p < 0.001) (Fig. 4F), namely PRSS2 also showed a similar trend of change as the change of FCGBP expression. Thus, both FCGBP and PRSS2 had high diagnostic value in CRC (Fig. 4G). Certainly, further experimental data are required to confirm the specific functions of FCGBP and PRSS2 in CRC cell lines. Although K-M survival curve results suggested that FCGBP expression correlated with patients’ treatment response and survival (Fig. 4H-I), this association did not remain significant after adjusting for age, TNM stage, and histological type in multivariate Cox regression (HR = 0.959, 95% CI: 0.633–1.453; P = 0.844; Supplementary Table 1).

Fig. 4.

Transcriptomics Analysis for HFCGBP overexpression transfection of SW480 cells. A After 48 h of transfection with the HFCGBP overexpression vector, the cells in the logarithmic growth phase were collected for differential expression analysis. B Volcanic map of all differentially expressed genes. C GO enrichment analysis of FCGBP-OV treatment of SW480 cells. D KEGG enrichment analysis of HFCGBP-OV treatment of SW480 cells. E The transcriptome data, TCGA and GEO data were intercrossed to identify common differential genes. F The correlation between FCGBP and PRSS2. G Analysis of the diagnostic value of FCGBP and PRSS2. H Survival analysis for FCGBP in CRC. I survival analysis for PRSS2 in CRC

Overexpression of HFCGBP decreased the S phase distribution of CRC cells

Overexpression of HFCGBP significantly affected the cell cycle distribution of CRC cells SW480 and HCT116, especially in reducing the proportion of S phase cells. In SW480 cell line, HFCGBP overexpression promoted the transformation from the S phase to the G2 phase (Fig. 5A-C). In contrast, in the HCT116 cell line, HFCGBP overexpression slowed down the transition from G1 phase to S phase (Fig. 5D-F). These results suggested that HFCGBP may inhibit tumor cell proliferation by modulating cell cycle regulation mechanism.

Fig. 5.

Overexpression of HFCGBP decreased the S phase distribution of CRC cells. A-C Effects of HFCGBP overexpression on cell cycle distribution in SW480. D-F Effects of HFCGBP overexpression on cell cycle distribution in HCT116. The representative results of cell cycle were shown in left panel and corresponding quantification of cell cycle distribution was depicted in right panel. Data were shown as the mean ± SD. * p < 0.05, ** p < 0.01

The mutation of FCGBP in tumor

We found that the mutation rate of FCGBP exceeded 1% in 23 out of 30 tumors by analyzing the ‌cBioPortal gene mutation database (https://www.cbioportal.org/). The FCGBP gene had the highest mutation rate in melanoma, up to 24%. In CRC, the total mutation rate of FCGBP was 9.93%, of which 9.26% was mutation and 0.67% was amplification (Fig. 6A). The major mutation types of FCGBP included mutation, amplification and deep deletion. In CRC, a total of 69 sites exhibited missense mutations, 4 sites had truncation mutation, and 1 site had an in-frame mutation (Fig. 6B). Using the TCGA database, CRC samples were classified and compared according to the mutation and non-mutation status of FCGBP. The results demonstrated that FCGBP mutations were associated with significant differences in tumor type, number of mutations, microsatellite instability (MSI) and tumor mutation burden (TMB) status in CRC (p < 0.001) (Fig. 6C-D). However, no significant differences were observed in disease-free survival between the non-mutated and mutated FCGBP groups in terms of gender, age of onset, weight, and prognosis evaluation (Fig. 6E). These above results indicated that the mutations of FCGBP gene may play a significant role in tumor development and progression, especially in melanoma and CRC, where the mutation frequency was high. This highlights the potential clinical significance and therapeutic implications of FCGBP gene mutations in these cancers.

Fig. 6.

Relationship between FCGBP gene mutation and non-mutation groups in clinical features of CRC. A FCGBP gene mutations in various cancers. B Display of different FCGBP mutation sites in CRC. C Difference between FCGBP gene mutation and non-mutation groups in Colon Adenocarcinoma (COAD) and Rectal Adenocarcinoma (READ). D Significant difference between FCGBP gene mutation and non-mutation groups in number of mutations, MSI and TMB status in CRC (p < 0.001). E There were no significant differences in sex, diagnosis age, patient weight, and prognosis evaluation between FCGBP gene mutation group and non-mutation group (p > 0.01)

Correlation analysis between abnormal expression of FCGBP and immune infiltration

Utilizing the CIBERSORT algorithm from the R package GSVA [1.46.0] [19], we explored the immune infiltration patterns associated with high and low expression of FCGBP across different immune cells, based on the 24 immune cell markers provided by Bindea [20] (Fig. 7A). The results revealed significantly the association between FCGBP expression and the infiltration levels of various immune cells, including dendritic cells (DC), cytotoxic cells, activated DC (aDC), immature DC (iDC), mast cells, neutrophils, NK CD56bright cells, T cells, TFH, and Th2 cells, with differences observed between tumor samples with high and low FCGBP expression (all p < 0.05) (Fig. 7C). Further validation of these findings showed FCGBP expression was significantly linked to the infiltration of B cell naïve, plasma cells, T cells CD4 memory resting, T cells CD4 memory activated, T cells regulatory regulatory T cells (Tregs) and Macrophages M0 (all p < 0.05) (Fig. 7D). When we focused on macrophages alone, we found that FCGBP expression was negatively correlated with the infiltration of M0 and M1 macrophages, indicating that lower FCGBP expression was associated with higher macrophage infiltration (Fig. 7E).

Fig. 7.

The patterns of immune infiltration associated with high and low expression of FCGBP were calculated by different algorithms. A Overlay histograms by CIBERSORT algorithm. B Bar chart of ImmuneScore by ESTIMATE algorithm. C ssGSEA algorithm. D CIBERSORT algorithm. E Correlation between high and low expression of FCGBP and macrophage infiltration

The correlation between FCGBP expression levels and immune scores was determined using the ESTIMATE algorithm (Fig. 7B). The results indicated that higher FCGBP expression was associated with higher immune scores in tumors, and high immune score was correlated with better prognosis for CRC patients, including longer disease-free survival (DFS) and OS, and potentially a greater likelihood of benefiting from immunotherapy. All in all, the variations in FCGBP expression were closely associated with the composition and status of immune cells in the tumor microenvironment. These changes may influence tumor growth, invasion, metastasis, and response to treatment, highlighting the potential importance of FCGBP in modulating immune responses within tumor microenvironment.

Discussion

Clinical relevance of HFCGBP as a potential biomarker in CRC

CRC is the third most prevalent malignant tumor globally and the second leading cause of cancer-related mortality [21, 22]. Key factors such as poor lifestyle, harsh living environment, genetic mutations, genetic susceptibility, and other factors significantly contribute to the development of colon cancer [23]. The lack of early symptoms or irregular physical examination leads to late onset of CRC in nearly half of patients, adversely affecting their survival rates [24]. Studies have shown that about 90% of CRC originate from adenomas, that is, it develops progressing from normal mucosa to polyps, then to adenomatous polyps, dysplasia, and ultimately to carcinoma [25]. This progression typically spans a period of 10 to 15 years and makes CRC one of the most suitable cancers for early screening and prevention. Thus, it is a crucial strategy to identify novel biomarkers to assist early diagnosis and therapeutic intervention for reducing the incidence and mortality associated with CRC [26]. Recent studies highlight the role of molecular markers, including FCGBP, in tumor suppression and immune regulation [27]. Moreover, reduced HFCGBP expression has been linked to more aggressive tumor phenotypes and poorer patient prognoses, highlighting its potential as a prognostic biomarker in CRC [28–30].

Functional and structural insights into the tumor-suppressive tole of HFCGBP in CRC

This study comprehensively investigated the functional role of HFCGBP in CRC by clinical sample analysis, in vitro experimental validation, and bioinformatics methodologies. Our aim was to elucidate the biological role of HFCGBP in CRC and its relationship with tumor progression. The results showed a significant reduction in FCGBP expression in CRC tissues, which correlated with advanced tumor grades and stages, suggesting that FCGBP played a crucial role in tumor progression. Moreover, the increased expression levels of HFCGBP inhibited tumor proliferation, migration, and invasion in CRC cell lines HCT116, which further supported that FCGBP acted as a tumor growth inhibitor. The result was highly consistent with that described by Jiefu Wang et al. [31]. As a multi-domain protein, FCGBP has a remarkable functional modularity feature. Determination of the crystal structure of R and T domains has clarified their molecular mechanism as the core scaffold protein of the mucosal barrier, providing a promising target for treating mucosal diseases [11, 12, 32]. The presence of H domain was closely correlated with the Fc-binding activity [9]. To address the domain specificity, structural analyses from AlphaFold predictions and recent cryo-EM studies reveal that FCGBP consists of an N-terminal H domain (approximately 450 amino acids, containing the unique IgG Fc-binding region with low cysteine content), a central R domain (12 tandem repeats of cysteine-rich von Willebrand D-C8-TIL-TILR motifs responsible for glycosylation and mucus scaffolding), and a C-terminal T domain (170 amino acids, a von Willebrand D domain aiding in protein stability and dimerization via disulfide bonds) [11,33]. This modular structure implies functional divergence: the H domain’s Fc-binding likely enables specific immune modulation, such as transporting IgG-antigen complexes and acting as a viral trap which may contribute to tumor-suppressive effects in CRC by enhancing immune surveillance in the tumor microenvironment, distinct from the R and T domains’ primary roles in maintaining mucosal barrier integrity through interactions with mucins like MUC2 and trefoil factors [9, 32, 34]. While full-length FCGBP has been implicated in tumor suppression across cancers (e.g., stabilizing p53 by binding MDM2 in CRC), our focus on the H domain represents a novel exploration in CRC, though direct experimental comparisons (e.g., overexpressing full-length vs. H domain) are planned for future work to confirm uniqueness, as noted in the limitations [17, 31]. In contrast, our study also involved experiments on the proliferation, migration, invasion, and cell cycle of HFCGBP in SW480 cells, and all these results showed a similar trend of influence to those in HCT116 cells. Our quantitative assays demonstrated that overexpression of the H-domain led to robust expression of HFCGBP, with a 400-fold increase in FCGBP mRNA levels in SW480 cells and a 150-fold elevation in HCT116 cells. To our knowledge, this study represents the first report revealing the tumor-suppressive role of the HFCGBP in CRC cells. These findings further suggest a functional divergence between the H-domain and R or T domain in mediating cancer-related pathways.

Transcriptomic pathway analysis revealed multidimensional tumor-suppressive effects of HFCGBP

RNA transcriptome sequencing analysis demonstrated that HFCGBP overexpression influenced the key signaling pathways associated with cell migration, proliferation, and the cell cycle, as well as those related to neuroactive ligand-receptor interactions and platelet activation [28]. This suggested that FCGBP may exert its tumor-suppressive effects through multiple molecular pathways, impacting both tumor cell behavior and the tumor microenvironment. These findings aligned with recent reports linking platelet activation to CRC metastasis via tumor microenvironment remodeling and neuroactive ligand–receptor signaling to tumor cell behavior in glioma [35–38]. However, as a bioinformatics-driven study, our results were associative and required validation [39]. Future work will prioritize replication in independent cohorts, causal inference to identify HFCGBP’s upstream regulators (e.g., via multi-omics integration and Mendelian randomization), and survival-oriented analyses to further delineate its indirect effects through immune or platelet-related mediators, as exemplified in recent CRC studies [39]. Targeted perturbation experiments will further clarify HFCGBP’s mechanistic role in CRC progression. The disruption of these pathways may elucidate the aggressiveness observed in CRC cases with diminished FCGBP expression. Furthermore, our findings were consistent with the existing literature on the role of FCGBP in other malignancies, such as gliomas and oral squamous cell carcinoma, indicating that FCGBP may have a broader significance across various cancer types and ensuring further investigation as a clinical outcome biomarker [10, 40, 41]. By elucidating the molecular mechanisms and signaling pathways involved, our study enhances the understanding of FCGBP’s impact on cellular behaviors, such as migration and invasion, which are crucial to cancer progression. The observed reduction in migratory and invasive capabilities following FCGBP overexpression underscores its potential to inhibit metastasis, a critical factor in the poor prognosis of CRC patients [42]. The potential application of FCGBP as a biomarker for CRC prognosis and treatment response is promising and deserves further exploration in clinical settings [17, 28].

Prevalence and uncertain prognostic impact of FCGBP mutations in CRC

Gene mutations have complex effects on tumor cells, potentially influencing signaling pathways that regulate the cell cycle and apoptosis, enhancing tumor cell invasiveness, and affecting immune cell infiltration and angiogenesis in the tumor microenvironment [43–45]. Mutations may also contribute to resistance to chemotherapy, radiotherapy, or targeted therapy [17, 46–48]. Notably, FCGBP mutations have been identified as significant contributors to the progression of CRC, lung cancer induced by smoking, and other malignancies. In CRC, the overall mutation rate of FCGBP is about 9.93% (9.26% missense, 0.67% amplifications), indicating its potential key role in CRC progression. However, despite the high mutation rate and its potential impact on tumor biology, FCGBP mutations did not affect disease-free survival in CRC patients when age, sex, and weight were considered. This differs from other oncogenes or tumor suppressor genes such as KRAS and TP53 mutations, which are well established in CRC [49]. Thus, while FCGBP mutations are prevalent in CRC and other tumors, the impact of mutation frequency changes on tumor behavior and patient prognosis warrants further investigation.

Pathway-level insights into the role of HFCGBP in CRC progression

Both we and Wang et al. conducted functional clustering and pathway analysis of the molecules affected by FCGBP [28]. The GO results reveal that FCGBP participates in the transmembrane transport of calcium ions into the cytoplasm, the role of metal ion transmembrane transporters, the functioning of cation channels, and calcium ion homeostasis. Furthermore, KEGG pathway analysis indicates that FCGBP is implicated in neuroactive ligand-receptor interactions, platelet activation, glycine, serine, and threonine metabolism, inflammatory mediator regulation of TRP channels, and Renin secretion. At the same time, FCGBP acts as an early response gene after microbial infection, its expression is strongly induced by various cytokines, which can regulate the adhesion of pathogenic microorganisms to mucosal surfaces and play an important role in the innate immune defense of mucosal epithelium such as the intestine and lungs [50, 51]. In addition, FCGBP is also involved in the regulation of inflammatory responses, with significantly increased expression in the serum of patients with inflammatory bowel diseases such as ulcerative colitis and Crohn’s disease [16, 46]. It also plays an important role in airway diseases such as pneumonia and may affect disease progression by promoting mucus secretion and regulating immune cell responses [16]. These analyses imply the involvement of FCGBP in a wide array of immune- and inflammation-related biological processes.

Association between FCGBP expression and immune cell infiltration in the tumor microenvironment

Immune cells within the tumor microenvironment are essential constituents of tumor tissue, and mounting evidence underscores the clinicopathologic importance of immune cell infiltration in forecasting the survival and treatment responsiveness of cancer patients [52, 53]. Given that the FCGBP expression patterns vary across different tumors, its role in carcinogenesis may differ. Using the ssGSEA algorithm from the R package GSVA, we identified significant variations in immune cell infiltration levels between CRC samples with high and low FCGBP expression, particularly in aDC, cytotoxic T cells, and Th2 cells. This observation aligns with previous studies that immune cell infiltration serves not only as a marker of immune response but also as a determinant of tumor behavior [54]. For instance, Jiang et al. report that increased immune cell infiltration is associated with a higher incidence of invasive lesions in pre-invasive tumors, highlighting the role of immune cells in tumor progression [55]. These studies support the hypothesis that FCGBP expression may serve as a biomarker for immune cell infiltration, providing insights into the tumor microenvironment and its implications for cancer progression. Moreover, Wang et al. demonstrate that FCGBP expression is associated with immune infiltration scores in ovarian cancer, where elevated FCGBP expression correlates with increased M2 macrophage infiltration [28]. Utilizing the CIBERSORT algorithm, we found that FCGBP expression was significantly associated with the infiltration of various immune cells, including naive B cells, plasma cells, resting CD4+ memory T cells, activated CD4+ memory T cells, Tregs, and M0 macrophages (p < 0.05). Notably, in our investigation of macrophages, we identified that FCGBP expression was inversely correlated with M0 and M1 macrophages infiltration, suggesting a potential link between reduced FCGBP levels and increased macrophage presence. Previous studies have reported similar associations between FCGBP and macrophage markers such as CD163, Mrc1, and TGFb1, as well as its positive correlation with M2 and negative correlation with M1 polarization in ovarian cancer [28]. However, these results should be interpreted cautiously. Bulk deconvolution algorithms such as CIBERSORT are limited in accurately resolving macrophage subtypes or rare populations, particularly when transcriptional profiles overlap [56]. Recent single-cell studies further reveal the high plasticity and heterogeneity of tumor-associated macrophages [57]. Thus, Our findings further support the complex interactions between immune cell infiltration and tumor biology, highlighting that FCGBP could serve as a biomarker for depicing the immune microenvironment in tumors. However, validation using single-cell or spatial transcriptomics approaches is still required.

Limitations

Although substantial experimental and analytical efforts were made, several limitations were worth noting. These limitations become apparent when comparing our work to recent studies emphasizing multigene prognostic modeling, causal inference, and advanced analytical frameworks. First, compared with studies such as Xie et al., which developed multigene prognostic models using LASSO regression and multivariable Cox analysis, our study focused on a single gene, aiming to explore the biological and immunological relevance of HFCGBP [58]. While this approach provided valuable insights, it did not offer a multigene or multi-omic prognostic model, which might limit its immediate predictive applicability across independent cohorts. Second, our transcriptomic and immune infiltration analyses identified several pathways and microenvironmental features associated with HFCGBP. However, these results remained associative, and causal relationships or indirect effects through molecular or cellular mediators were not explored. As noted in Zhang et al., a more advanced causal inference or path-based analysis would be beneficial for further understanding the mechanisms underlying HFCGBP’s role. Finally, immune cell infiltration was inferred from bulk transcriptomic data using deconvolution algorithms, which had limitations in distinguishing closely related immune subsets, such as macrophage phenotypes [59]. Liu et al. emphasized the importance of using uniform cutoffs across datasets to improve the reproducibility and generalizability of immune infiltration estimates [60]. Despite these limitations, our study provided important contributions to the understanding of HFCGBP in colorectal cancer, particularly in identifying its biological and immunological roles. Future research incorporating causal analysis, external validation cohorts, and higher-resolution technologies, such as single-cell or spatial transcriptomics, will help refine our findings and strengthen their clinical and mechanistic relevance.

Conclusion

The study has conducted a systematic and comprehensive investigation into the functional role of FCGBP, addressing a significant gap in the existing literature, which often lacks functional analyses of FCGBP H domain in CRC. We have demonstrated a marked decrease in FCGBP expression as CRC progresses, with protein levels trending towards undetectability. Notably, HFCGBP overexpression inhibited tumor proliferation, migration, and invasion, inducing cell cycle arrest in the S phase. These results suggest that FCGBP holds potential as a prognostic biomarker, providing valuable insights for early diagnosis and personalized treatment strategies. Further exploration of FCGBP’s clinical applicability could lead to innovative approaches in CRC management, ultimately enhancing patient prognosis. However, our study has some limitations, such as the relatively small sample size, which may limit the generalizability of the results and the potential diagnostic and prognostic potential of FCGBP requires validation in larger cohorts. Furthermore, the absence of long-term follow-up data on clinical samples limits our ability to fully assess the clinical implications of our findings. While structural data support domain divergence, experimental validation of H-specific versus full-length FCGBP effects is needed, as our current findings rely on inferential evidence from the H domain’s unique IgG Fc-binding properties. High mutation rates warrant domain-focused mutagenesis studies to elucidate how alterations in the H, R, or T domains impact tumor-suppressive functions. Broader implications for FCGBP’s pan-cancer roles require multi-omics integration to explore its interactions across diverse tumor types. In subsequent research, we will continue to expand the sample size, complete the study on the functions of the FCGBP R and T domains in cancer, and conduct direct experimental comparisons (e.g., overexpressing full-length vs. H domain) to confirm the H domain’s unique role.

Abbreviations

CRC

Colorectal cancer

FCGBP

Fc fragment of IgG binding protein

HFCGBP

H domain of Fc fragment of IgG binding protein

GEO

Gene Expression Omnibus

GEPIA

Gene Expression Profiling Interactive Analysis

GO

Gene Ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

MSI

Microsatellite instability

TMB

Tumor mutation burden

DFS

Disease-free survival

OS

Overall survival

DSS

Disease Specific Survival

PFI

Progress Free Interval

DC

Dendritic cells

Tregs

Regulatory T cells

Authors’ contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by QL, HYJ, YYW, LLZ, NW, CL, JT, and QNW. The Figure 1 was prepared by YYW and NW. The Figs. 2, 3 and 4 were prepared by QL, HYJ, and JT. The Fig. 5 was prepared by QL and CL. The Figs. 6 and 7 were prepared by QL, HYJ, and QNW. The first draft of the manuscript was written by QL. The language of the full text was polished by LG and YL. WZ, NN, LG and YL re-wrote the paper. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

The present study was supported by the Key Research and Development Program of Shaanxi (No. 2024SF-GJHX-33, 2024JC-YBQN-0842 and 2025SF-YBXM-327). The Talent Launch Program and of the “Phoenix Attraction Initiative” at the Second Affiliated Hospital of the Air Force Medical University (2023YFJH005).

Data availability

The datasets GSE21510, GSE32323, and GSE25071 can be found here: (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21510) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE32323) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25071). RNA sequencing data are in (https://cloud.metware.cn/) , the account and password are available from the corresponding author on reasonable request. All data generated or analyzed in this study are fully available within the manuscript.

Declarations

Ethics approval and consent to participate

The research strategies concerning human tissues were all approved by the IEC of Institution for National Drug Clinical Trials, Tangdu Hospital Fourth Military Medical University (No. K-HG-202505-14) and were in accordance with the Declaration of Helsinki and the US Public Health Service Policy on Human Care and written informed consent was provided by the patients.

Competing interests

The authors declare no competing interests.

Consent for publication

Not applicable.