IGG FC BINDING PROTEIN (FCGBP) IS DOWN-REGULATED IN METASTATIC LESIONS AND PREDICTS SURVIVAL IN METASTATIC COLORECTAL CANCER PATIENTS

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Relevance:

Colorectal cancer

The research objective:

In this study, we are aimed to explore the significance of FCGBP in liver metastatic CRC (LMCRC) patients.

Methods:

Patients and Tissue Samples

In this study 135 paired specimens including normal mucosa, primary tumor and liver metastasis tissue were all collected from CRC patients who were diagnosed with no other metastasis according to CT scan. All the patients were taken surgical operation from January 2006 to February 2007 and followed up to December 2012. The diagnosis was all confirmed by Pathology Department of Cancer Institute and Hospital and all cases can provide completely clinical information, including age, gender, the location of tumor, histologic classification, TNM stage, follow-up information and so on. All the patients were followed-up regularly every 3 months until to the 5th year after the resection.

 

FCGBP Expression Analyses in GEO and TCGA Databases

To evaluate FCGBP expression between PC and LMCRC, we summarized the expression profiling microarray data from Gene Expression Omnibus (GEO) database with the accession number GSE41258 and GSE68468 (Affymetrix Human Genome U133A Array). The standardized FCGBP expression was divided into Normal (normal tissue), Primary and Metastasis in the datasets above. For data from GEO, the different expression genes were analyzed by limma package in Supplementary Table 1 and data from TCGA, the different expression genes were analyzed by UCSC XENA in Supplementary Table 2. The cohort was divided into four groups, Normal (normal mucosa), Primary, Metastasis and Recurrence.

 

Tissue Microarray and Immunohistochemistry

Tissue microarrays (TMAs) were adopted after HE staining verification. 1 mm punched samples were measured taken from the tumor center. Different specimen collected from the same patient were placed on the same TMA.

  Immunohistochemical staining was performed on the TMAs slides with FCGBP (#HPA003564; 1:500; Sigma-Aldrich, United States) rabbit polyclonal antibody. The immunohistochemistry score (SI) was calculated by the staining intensity (0, negative; 1, weak; 2, moderate; 3, strong) multiplied by the positive rate of stained cells (0%-5%, 0; 6%-25%, 1; 26%–50%, 2; 51%–75%, 3; >75%,4). In this study, SI=4-12 was defined as positive staining, while SI=0-3 was defined as negative staining. Positive rate=positive samples/ (positive samples + negative samples). Positive and negative controls for FCGBP immunohistochemistry were shown in Figure 2 (F) and (G). Main point of our study was elucidated as a flowchart in Figure 1.

 

Cell Transfection, RNA Extraction and qRT-PCR Analyses

Transfections were carried out using lipofectamine 2000 reagent (Invitrogen) according to manufactures instructions. Si-RNAs GenePharma (China) were used to knockdown gene expression.

The total RNA was extracted from cells using TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcribed complementary DNA was synthesized with random primers or microRNAs specific stem-loop primers. Subsequently, the cDNA was subjected to real-time PCR on a 7500 real-time PCR system (AB Applied Biosystems, Mannheim, Germany). GAPDH was used as internal controls. Primers and si-FCGBP sequences were listed in Supplementary Table3.

 

Cell Migration Assay

The cell migration assay was performed using the Transwell Chamber from BD Biosciences (San Jose, CA, USA). Briefly, cells (1×105) were seeded in the upper chamber in serum-free media. The lower chamber was filled with corresponding media with 10% FBS as a chemoattractant. Cells were incubated for 24h at 37 °C, fixed in formaldehyde and stained with crystal violet for 10 min. Cells that migrated to the bottom surface of the filter were counted as invaded cells.

 

Statistical Analysis

The statistical analyses were performed using Paired t-test or one-way ANOVA. Survival curves were calculated by Kaplan–Meier method and the log-rank test was used to compare the overall survival (OS) and disease-free survival (DFS). Univariate and multivariate analysis for prognosis were using Cox proportional hazards regression model. The calculations were carried out using SPSS Statistics 23.0 or GraphPad Prism 8.0. P value less than 0.05 was considered statistically significant.

The results and discussions:

The expression data of FCGBP was obtained from Gene Expression Omnibus (GEO) and TCGA database, FCGBP RNA expression was evaluated between primary lesions (PC) and liver metastatic lesion (LM). FCGBP RNA was down-regulated in PC and LM, and especially lower in LM (p<0.001). Next, the expression of FCGBP protein was evaluated by immunohistochemistry array in 135 paired primary tumor tissue and metastatic tissue. We also found FCGBP protein was down-regulated in primary lesion and metastatic lesion, especially in metastatic lesion. According to immunohistochemistry score (SI), each cohort was divided into FCGBP-positive (SI=4-12) and FCGBP-negative (SI=0-3) group. In both groups, the level of CEA (PC group, 3.880 vs 77.049, p<0.001; LM group, 3.890 vs 14.239, p=0.008) and CA19-9 (PC group, 8.610 vs 111.700, p<0.001; LM group, 7.660 vs 19.380, p=0.037) was lower than those in FCGBP-negative group. FCGBP-positive in LM cohort was an independent risk factor both in OS (HR 1.573, 95% Cl [1.017-2.433], p=0.042) and DFS (HR 1.869, 95% Cl [1.256-2.781], p=0.002).

Discussion:

In this study, immunohistochemical staining was performed in LMCRC TMAs with FCGBP antibody. We compared the IHC score of FCGBP between primary tumor tissues and liver metastasis tissues and found that FCGBP expression was decreased with disease development. We have verified the phenomenon based on TMAs in the GEO and TCGA datasets to confirm that FCGBP mRNA expression was decreased with the progression of the disease. As for clinical information, we found that CEA and CA19-9 level was lower in FCGBP-positive group. The percentage of cN0 in FCGBP-positive group was higher and the percentage of cN2 was lower than that in FCGBP-negative group. In LM cohort, we found that FCGBP-positive was an independent risk factor both in OS and DFS. FCGBP might be a prognostic factor for LMCRC patients.

   At present, metastasis remains an important factor contributing towards the majority of cancer-associated mortalities, as metastatic cancer is often resistant to conventional therapies. Cell adhesion occurring in vasculature of specific organs is an essential step in cancer metastasis, FCGBP was significantly enriched in the function of cell adhesion. In the intestine, FCGBP protein exists in a large complex containing Muc2 and Trefoil peptide, suggest that it may be important in the maintenance of homeostasis and integrity of epithelium. However, it is not currently available to detect reliable and sensitive methods for early metastasis CRC. Metastasis is facilitated by the cell-cell interactions between the endothelium and tumor cells in distant tissues. Previous studies have found that down-regulated FCGBP was associated with lots of malignant diseases and it was known as a prognostic marker in a variety of cancers. Ma R et al had performed whole exome-sequencing analysis of 63 CRC cases, and found that with the deficiency of FCGBP, CRC developed and showed worse survival rates. At stage I or II CRC, it has reported that FCGBP was positively associated with the prognosis of CRC. Onstenk W et al measured CTCs of liver metastasis CRC patients and their primary tumor tissues and found that FCGBP was down-regulated[19]. Meanwhile in other kinds of cancer, FCGBP also has positive effects on prognosis. In HPV-infected patients FCGBP expression was upregulated and negatively correlated with TGF-β in head and neck squamous cell carcinoma, it was meaningful to the prognosis of patients. The current study indicated that FCGBP expressions could be further evaluated as biomarkers for predicting survival of patients with gallbladder cancer and FCGBP could be promising targets in the control of gallbladder cancer progression. Xiong L et al found that immunohistochemical staining of FCGBP was decreased and evaluated it as a biomarker for predicting survival of patients with gallbladder cancer. They found that NT5E was the most decreased gene, while FCGBP is the most decreased gene in the TGF-βinduced gallbladder cancer cells, which suggest these two genes may play essential roles in EMT. It would be a potential target in the control of gallbladder cancer progression. Using the univariate Cox model and Kaplan-Meier survival curve, a correlation between FCGBP expression and overall survival in ovarian adenocarcinoma was observed.

As we known, metastasis is the main death reason of CRC patients especially liver metastasis CRC that has much poorer prognosis. Previous works found that FCGBP can be evaluated as biomarkers in CRC all stages broadly. But according our present study, we found that FCGBP-positive in LMCRC has longer survival. Multivariate Cox analysis illustrated that FCGBP-positive was an independent prognostic factor for OS and DFS in liver metastasis cohort but not in primary tumor cohort. We analyzed that FCGBP can predict prognosis more adequately in metastasis tumor. We first reported that FCGBP can predict prognostic in LMCRC and we thought it could be a potential biomarker for LMCRC patients. The pattern of FCGBP expression is gradually down-regulated with the cancer development and we speculate that FCGBP may be a tumor suppressor gene. The function of FCGBP and mechanism that FCGBP is down-regulated with cancer development deserve further investigation.

Conclusion:

This study has found the relationship between FCGBP and clinical information of LMCRC patients and FCGBP expression was decreased with disease development. The expression of FCGBP in liver metastasis is associated with the OS and PFS. Our works illustrate that FCGBP can be a promising prognostic factor for LMCRC.

About the authors

Ziming Yuan

Department of Colorectal Surgery, the Second Affiliated Hospital of Harbin Medical University

Author for correspondence.
Email: yuanziming123456@163.com
ORCID iD: 0000-0002-8658-8268

Doctorate    Oncology

China

References