Background
DNA damage caused by endogenous or exogenous genetic toxicants can contribute to genomic instability and directly lead to a variety of cancers. Cells have evolved a series of DNA repair pathways to avoid the deleterious result [
1]. Nucleotide excision repair (NER) can identify many types of underlying damage and cut damaged DNA strands at precise distances on both sides of the lesion, as well as base-damaged oligonucleotide fragments [
2]. NER pathway has four main steps which are damage identification, damage partitioning and unwinding, damage incision and new strand synthesis [
3,
4]. XPF(xeroderma pigmentosum complementation group F), locating on chromosome 16p13.12, has 11 exons with a span of 28.2 kb [
4]. The heterodimer of XPF-ERCC1 is involved with the 5′ incision step of the NER pathway. The catalytic area located in XPF can determine the activity of NER [
5]. It is an essential human gene in the NER pathway responsible for the removal of UV-C photoproducts and large volume adducts from DNA [
6,
7]. Cells or animals that lack XPF cannot perform the NER pathway [
4].
Given its important function in the NER pathway, XPF may be involved in diseases associated with imbalance between DNA damage and repair. A number of researches have focused on its role in different cancers. XPF has been reported in the literature that its expression in renal cell carcinoma is significantly higher compared with bladder cancer and testicular cancer, and is related to the clinical features and chemotherapy sensitivity [
6]. XPF expression is increased in gastric cancer (GC) tissue, and the prognosis of patients with high expression of XPF is poor [
7]. XPF is also highly expressed in oral cancer tissue, while its high expression indicates a low survival rate [
8]. Colorectal cancer (CRC) is a malignant tumor which is the third cause of cancer death in China [
9]. There have been some previous studies focusing on the relationship between XPF polymorphism and the risk of CRC. The results showed that there was an association between XPF polymorphisms and the risk of CRC [
10‐
12]. So far, although there have been small sample studies investigating the relationship between XPF expression and the risk of CRC [
13], the pathological process from colorectal benign diseases to precancerous lesions to cancer has not been studied, and large sample size studies on the relationship between XPF expression and CRC are needed. In the current study, we first studied the expression tendency of XPF in the progression from anal benign disease to adenoma to CRC. Further, we analyzed the association of XPF expression with clinicopathological parameters and survival of CRC patients, thus to investigate the effect of XPF on development, progression, and prognosis of CRC. By performing bioinformatics analyses, we studied the function and regulation network of ERCC4 in CRC.
Methods
Patients and tissue specimens
The design of this study was approved by the Human Ethics Committee of China Medical University. Each subject participated in the study provided the written informed consent. The patients undergoing surgery were from the First Hospital of China Medical University between November 2012 and June 2016. We enrolled a total of 824 cases of colorectal tissue for risk study, including 276 cases of CRC and 284 adjacent non-tumor tissue (248 cases of CRC had survival time, 230 pairs had cancer tissue and its matched adjacent tissue), 202 cases of adenoma and 62 cases of anal disease; and 248 cases of CRC tissue with survival time were used for prognosis study.
We collected the tissue of CRC, which were derived from the histological results, and the collection was according to the World Health Organization standards. The TNM staging of CRC was evaluated based on the International Union Against Cancer (UICC)/United Joint Cancer Committee (AJCC) (7th edition in 2010) [
6]. There were 3 cases of CRC patients that needed to be excluded: (1) patients with XP disease; (2) patients who received chemotherapy or radiation therapy before surgery; (3) patients with hereditary nonpolyposis colorectal cancer (HNPCC).
Follow-up study was conducted until April 2018. We performed prognostic analysis of 248 patients enrolled (the follow-up time was 12 to 63 months, the average survival time was 48.15 months, and there was no death). We excluded 14 patients who lacked visits in the OS analysis. Patients with the habit of smoking at least one cigarette a day for at least 1 year were considered to have a history of smoking. In the meantime, the study defined the drinking history as an average daily intake of at least 50 grams of alcohol for at least 1 year. Clinical characteristics of cancer patients included gender, age, whether smoking or drinking, tumor location, TNM stage, invasive extent, lymph infiltrative, distant metastasis, tumor deposit, perineural invasion, vessel carcinoma embolus, growth pattern, differentiation degree, maximum diameter and family history.
Immunohistochemistry
The tissue was fixed in formalin and embedded in paraffin, then cut into 4 μm thick sections, and the sections were mounted on glass slides [
14]. Antigen retrieval was performed after routine dewaxing. The tissue sections were washed with phosphate buffered saline (PBS, pH 7.4). Then the sections were blocked with 10% normal goat serum for 10 min. The expression of XPF protein was detected with mouse anti-XPF monoclonal antibody (ab-85,140, 1: 200 dilution; Abcam, Cambridge, UK), and the primary antibody was used to incubate at room temperature for 1 hour. We spined off the primary antibody on the slice, and then used a biotinylated secondary antibody (goat anti-rabbit antibody, Fujian Maixin) to incubate the tissue for 10 min. The tissue was rinsed with PBS for 10 min. After that, we used streptavidin Biotin-biotin peroxide at a temperature of 24–27 °C for incubating the tissue for 10 min, and stained with DAB (DAB-0031, Maixin City, Fujian Province, China) on a glass slide. When the tissue stain become brown (about 30 s), we rinsed the DAB with PBS. Finally, the slides were dehydrated, the tissue was fixed with resin and the coverslips were covered to observe the staining.
Evaluation of immunohistochemistry
Two experienced pathologists scored XPF’s expression in different tissue independently, and this process followed the double-blind principle. The pathologists scored the staining intensity and staining area of XPF respectively. If there are differences in the scores of the pathologists, two pathologists will discuss and summarize the final scores. Semi-quantitative scoring criteria were used to assess the expression of XPF. Scoring standard: (1) staining intensity was classified into four levels, including 0 (no staining), 1 (light brown), 2 (brown staining), and 3 (heavy brown staining); (2) percentage of stained cells was divided into: 0(0–5); 1(6–25); 2(26–50); 3(51–75); 4(76–100%). We got the final IS (immunoreactivity score) by multiplying staining intensity and percentage of stained scores. Finally, the IS score was classified as: negative (−), score = 0; weak positivity (+), score = 1–4; medium positivity (++), score = 5–8; and strong positivity (+++), score = 9–12.
Oncomine analysis
Oncomine, a cancer microarray database and web-based data mining platform, aiming to analyze genome-wide expression for cancer types and provide transcriptome data of cancer tissue [
15,
16]. We compared the mRNA expression of XPF in normal colon and rectum tissue vs. colon adenocarcinoma by Oncomine. We choosed 1.5 fold change,
P value = 0.05 and top 10% gene rank as threshold.
The function and regulation network of XPF by GO and KEGG analysis
STRING is a database designed to collect, score and integrate all public sources of information on protein–protein interactions [
17]. Gene ontology (GO) analysis is a major bioinformatics tool that unifies the characterization of genes and gene products through the three components of biological processes, cell composition and molecular function [
18]. Kyoto Encyclopedia of Genes and Genomes (KEGG) is a set of databases whose main purpose is to study genetic pathways, and contains information about biological pathways, genomes, chemicals and diseases [
19]. The Database for Annotation, Visualization and Integrated Discovery (DAVID; v.6.8;
https://david.ncifcrf.gov/home.jsp; accessed on September 16, 2020) was applied to perform the enrichment analyses of GO and KEGG [
20]. DAVID is an online portal that provides comprehensive annotation analysis of large gene lists. GO analysis comprises groups of molecular function, cellular function and biological process [
21]. We used STRING to explore the genes closely correlated with XPF. XPF and its interacting genes were enriched and analyzed by David for GO and KEGG pathways, respectively. The ggplot2 package in the R platform (Version 3.6.3) was used to show the obtained results. Gene Set Enrichment Analysis (GSEA) is an analysis method for whole-genome expression profiling chip data, which compares genes with predefined gene sets [
22]. By analyzing the gene expression profile data, we can understand the expression status of XPF in a specific functional gene set, and whether there is some statistical significance in this expression status. We searched the expression of XPF in normal and cancerous colorectal tissue in the Oncomine database [
15].
Statistical analysis
All the statistical analyses were conducted using SPSS 20.0 software (IL, Chicago). The difference of XPF expression between CRC and adjacent non-tumor tissue was compared by non-parametric tests. We performed Mann–Whitney U test of nonparametric test to evaluate the relationship between XPF expression and clinicopathological parameters of CRC. Survival analysis was performed by Kaplan–Meier method. When we compared the differences between subgroups, and the log-rank test was used. The Cox proportional hazard model was used to evaluate the effect of XPF expression on CRC prognosis. P < 0.05 was considered as statistically significant.
Discussion
As a key gene of NER system, XPF plays an indispensable role in keeping the integrity and stability of genome, thus influencing the occurrence of cancer [
13]. Although there have been some studies on the correlation between XPF expression and CRC [
10‐
13,
23], this is the first research on XPF expression that covers dynamic CRC development. In addition, this study explored the relationship between XPF expression and clinical traits of CRC. Our results showed that XPF expression was upregulated in CRC tissue compared with adjacent non-tumor tissue, adenoma and anal benign disease. Overexpression of XPF was related to poor prognosis of CRC patients with T1-2 invasive extent. XPF expression was associated with Ubiquitin like protein specific protease activity WNT signaling pathway and so on.
Firstly, we detected the expression of XPF in cancerous and non-cancerous tissue in multiple dimensions and different levels. It is found that the XPF protein expression was significantly higher in CRC tissue than that in adjacent colorectal tissue. The mRNA level of XPF expression came out with similar results: XPF was highly expressed in colonadenocarcinoma than in colon and rectal normal tissue. Subgroup analysis revealed significant difference in male, female, age ≤ 60, age > 60, smoking, no smoking, colon cancer, rectum cancer, drinking, no drinking, lymph node metastasis and other clinicopathological factors. The consistent results of stratified analysis showed that the expression of XPF in CRC was higher than that in adjacent non-tumor tissue regardless of other factors. Previous studies of XPF expression in other tumors have yielded similar results: Li.P et al. [
7] found a significant increase of the XPF expression in GC tissue compared with adjacent tissue. Meanwhile, XPF protein played a vital role in the occurrence and progress of GC [
7]. Moreover, our results indicated that XPF expression showed an obvious trend of increasing with the development from anal disease, adenoma to CRC. Most sporadic CRC develops from intestinal adenoma. Adenomas represented by conventional, tubular, or tubulovilious adenomas are considered as precancerous lesions of CRC [
24]. We conjecture that DNA damage accumulates more along with the dynamic process from normal intestinal tissue to adenoma to CRC. When CRC occurs, cells need the NER system for damage repair, and a large amount of XPF is required to be highly expressed in CRC. As a result, XPF can be a potential biomarker for CRC risk.
Further analysis combined with clinicopathological features of patients brought to light that increased expression of XPF was closely related to clinical features, including rectal cancer and cloddy/nested pattern. XPF expression was related to the invasion of hepatic capsules and microvascular tumor embolus in human hepatocellular carcinoma [
25]. The expression of XPF was significantly related to some clinical features such as family history and Laurén classification in GC [
7]. XPF abundance was associated with positive ER status in breast cancer, through clinicopathological parameter analysis [
26]. Therefore, XPF may have a certain significance for predicting the biologic activities and the progression of CRC. Our study results demonstrated that XPF expression correlates tightly with growth patterns or positions in cancer. As we know the growth patterns of carcinoma may have an influence on the proliferation and invasion of tumor cells. Besides, we suppose that different growth patterns or tumor site may cause distinct degrees of DNA damage, thereby inducing the translation of XPF with diverse activities, resulting in the differences in XPF expression. Further study is warranted to investigate the relationship between XPF expression and clinopathological features in CRC.
In this study, we also explored the association between XPF expression and prognosis of CRC patients. The results showed that the XPF expression was not significantly associated with the survival time in overall analysis. As for patients in stage T1-T2, those with low XPF expression can survive longer than those with high expression. Previously, low XPF expression was also found to predict better prognosis in other types of cancer. Li et al. reported that XPF-positive patients had shorter survival time than XPF-negative patients in GC [
7]. Mesquita et al. found that low ERCC1/XPF expression was related to better progression-free survival in 331 ovarian cancer patients [
27]. Vaezi et al. revealed that Low XPF expression correlated with longer survival time in patients of squamous cell carcinoma of the head and neck [
8]. It has been reported that XPF-ERCC1 endonuclease is required in the repair pathways such as NER, DSB, ICL responsible for the repair of helix-distorting DNA lesions as well as interstrand crosslinks aroused by radiation and platinum compounds [
28,
29]. We may extrapolate that low XPF expression are more sensitive to DNA damage agents such as cisplatin. With the increase of damage in cancer tissue, damage repair activities increase, so the high expression of XPF in colorectal cancer tissue also indicates a worse prognosis. The correlation between low expression of XPF and longer survival time may be applicable to CRC, but its molecular mechanism still needs further research to be clarified.
The expression pattern of XPF in CRC and its potential prognostic role inspired our understanding of XPF in development and progression of CRC. Therefore, we further performed PPI and functional enrichment analysis to reveal the interacting network and the biological function of XPF. By STRING database, we queried the genes interacted with XPF, which were ERCC1, XPA, ERCC5, MSH2, XPC, ERCC3, ERCC2 and so on. Firstly, functional analysis of this PPI network showed that positive regulation of DNA secondary structure binding and damaged DNA binding were the most significant. Besides, the network was also correlated with UV protection and DNA repair complex. XPF is an essential human gene in the NER pathway responsible for the removal of UV-C photoproducts and large volume adducts from DNA [
4]. KEGG analysis also showed that XPF-related PPI network was mainly enriched in nucleotide excision repair pathway. Studies have mentioned that XPF was in charge of the 5′ incision process in the NER pathway [
27]. GSEA analysis showed that with the expression of XPF increased, some pathways can be activated, such as ubiquitin like protein specific protease activity, WNT signaling pathway and calcium modulating pathway. There are reports in the literature showing that high expression of ubiquitin-specific protease 6 N-terminal-like protein can regulate proliferation activity of CRC cell via Wnt/β-catenin pathway [
22]. Therefore, we suspected that increased XPF expression may be related to CRC risk and progression by activating the above mentioned pathways.
Conclusions
In conclusion, we investigated the expression of XPF in adjacent non-tumor tissue, benign disease, adenoma and CRC by immunohistochemistry. We found that the expression of XPF was gradually increased with the progress of CRC. Besides, XPF protein expression was associated with tumor location and growth patterns of CRC. XPF may be a promising biomarker for CRC risk, and also showed potential as a prognosis predictor in T1-T2 stage patients with CRC.
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