Upregulation of ECT2 is associated with transcriptional program of cancer stem cells and predicts poor clinical outcome in gastric cancer
- Authors:
- Published online on: July 27, 2020 https://doi.org/10.3892/ol.2020.11915
- Article Number: 54
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Copyright: © Gong et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Cancer stem cells (CSCs), also known as tumor-initiating cells, are characterized by stem-like properties, including self-renewal and ability to generate daughter cells. Cancer initiation, dissemination and recurrence are closely associated with CSCs (1). CSCs were first detected in acute myeloid leukemia and have since been identified in several solid tumors, including gastric cancer (1). Furthermore, certain populations of gastric CSCs abilities to self-renewal and undergo multipotent differentiation have been detected in gastric cancer (2). Villin+ and Lgr5+ gastric stem cells have been detected in the antrum, while Troy+ chief cells have been found in the corpus (3). Additionally, Sox2+ gastric stem cells are present in both the antrum and the corpus (3).
Gastric cancer is the seventh most common cancer and the third leading cause of cancer-associated mortality worldwide (4). In 2018, 1,033,701 new gastric cancer cases and 782,685 mortalities were reported worldwide (4). Gastric cancer has been extensively investigated in the biomedical field due to its high morbidity and mortality rates (4). It is speculated that gastric carcinogenesis may be associated with Helicobacter pylori infection, inherited susceptibilities, and environmental and dietary factors (5,6). In recent years, the prevailing hypothesis that the occurrence and progression of gastric cancer is associated with CSCs has been partially proven (7).
Epithelial cell transforming 2 (ECT2) is a proto-oncogene gene encoding a guanine nucleotide exchange factor for the Rho GTPases (8). When expressed in NIH/3T3 fibroblasts, ECT2 promotes their malignant transformation (9). Increased ECT2 expression has been detected in several types of human tumor, including glioma and liver, pancreatic and lung cancer (10–13). ECT2 upregulation significantly enhances the activity of RhoGPase, prevents cell apoptosis and induces cancer cell metastasis (10). Conversely, ECT2 downregulation suppresses activation of the ERK signaling pathway and impairs the migration of cancer cells (10). However, whether and how ECT2 contributes to gastric cancer malignancy remains elusive.
The present study aimed to investigate the association between ECT2 expression and the clinicopathological characteristics of patients with gastric cancer. The expression levels of ECT2 were investigated using immunohistochemical analysis, combined with Gene Expression Omnibus database and gene set enrichment analysis, and it was revealed that gastric tumors with elevated ECT2 levels exhibited transcriptional traits of CSCs. In addition, high ECT2 expression predicted poor clinical outcome, suggesting its use as a novel prognostic indicator for gastric carcinoma. Further investigation into the role of ECT2 may provide alternative therapeutic targets for the treatment of gastric cancer.
Materials and methods
Clinical tissue samples
A total of 130 primary gastric cancer tissues and 108 paired adjacent normal tissues (some paired adjacent normal tissues were not harvested during the operation due to patients clinical conditions) were collected from patients who underwent surgery at the Hospital of Chengdu University of TCM (Chengdu, China) between March 2012 and December 2015, and retrospectively analyzed. Paraffin-embedded tissue samples were stored at room temperature. None of the patients had received anticancer treatment prior to diagnosis and no additional malignancies were present. Pathological staging was based on the Union for International Cancer Control/American Joint Committee on Cancer Tumor-Node-Metastasis (TNM) Classification (8th edition of 2016) (14). The present study was approved by the Institutional Review Board of the Teaching Hospital of Chengdu University of TCM (Chengdu, China) (approval no. 2018KL-023) and written informed consent was provided by all patients prior to the study start.
Immunohistochemistry (IHC)
The tissue samples were fixed in 4% paraformaldehyde >24 h at room temperature, then dehydrated in graded ethanol series (30, 50, 70, 95 and 100%), and embedded in paraffin. For IHC analysis, paraffin-embedded samples were cut into 3-µm-thick sections, dewaxed with xylene at room temperature and rehydrated in a descending ethanol series (100, 95, 85 and 75%). For antigen retrieval, sections were heated at 97°C for 20 min. Following a brief proteolytic digestion with 0.1% trypsin at 37°C for 10 min and peroxidase blocking with 3% hydrogen peroxide solution at room temperature for 15 min, the sections were incubated with primary antibodies against: ECT2 (1:400; cat. no. 07-1364; Sigma-Aldrich, Merck KGaA), BUB1 (1:200; cat. no. DF6698; Affinity Biosciences) and E2F transcription factor 7 (E2F7; 1:200; cat. no. DF2444; Affinity Biosciences) overnight at 4°C. Following the primary antibody incubation, the sections were incubated with a HRP/Fab secondary antibody at room temperature for 20 min (freshly prepared solution from the kit; cat. no. PV-6000-D; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.). Tissue sections were stained with diaminobenzidine substrate for 5 min and counterstained with hematoxylin for 20 sec at room temperature. Each slide was analyed using light microscopy (H-7650; Hitachi, Ltd.). The magnification used was ×200.
A total of two independent investigators, without prior knowledge of the clinicopathological data, evaluated the ECT2 staining in a semiquantitative manner. The final immunoreactivity scores (IRS) were determined according to the sum total of the percentage of positive cells (0 points, 0–5% positive cells; 1 point, 6–25%; 2 points, 26–50%; 3 points, 51–75% and 4 points, 76–100%), and staining intensity scores (0 points, no staining; 1 point, weak staining; 2 points, moderate staining and 3 points, strong staining). A final IRS >4 indicated strong positivity, while scores <4 indicated weak positivity.
ECT2 analysis in the Gene Expression Omnibus (GEO) database
ECT2 expression was assessed in several independent gastric cancer clinical datasets (15–18) available from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The GSE13861 dataset (15) included a collection of 65 primary gastric adenocarcinoma and 19 surrounding normal tissues. The GSE29272 (16) dataset included a cohort of 134 gastric adenocarcinoma and paired surrounding normal tissues. The GSE51575 (17) dataset consisted of a cohort of 27 advanced gastric carcinoma and paired surrounding normal tissues. The GSE65801 (18) dataset consisted of a cohort of 32 gastric cancer tissues and paired surrounding noncancerous tissues. The normalization procedures employed for gene expression intensity data are stated in the individual datasets and related publications.
Gene set enrichment analysis (GSEA)
In order to determine how biological processes and signaling pathways are differentially regulated in gastric cancer with low or high ECT2 expression, transcriptomic data of gastric cancer were retrieved from The Cancer Genome Atlas (TCGA) database and analyzed using GSEA. TCGA gastric cancer cohort, consisting of 407 samples and transcriptional profiles, was downloaded from TCGA Data Portal (https://tcga-data.nci.nih.gov/docs/publications/tcga). GSEA was performed using GSEA software (v2.2.2; www.broadinstitute.org/gsea). The median ECT2 expression level (cut-off value=11.13) was used to dichotomize samples into low and high expression groups. A total of 1,000 permutations were used to calculate the P-values. All other parameters were set based on their default values.
Survival analysis
The prognostic value of ECT2 in gastric cancer was assessed using the Kaplan-Meier (KM) Plotter database (http://kmplot.com/analysis/index.php?p=service&cancer=gastric), which consists of a pool of gene expression and clinical data (19). The median time to first progression (FP) was 18.3 months and the median overall survival (OS) was 28.9 months. Overall survival time was assessed. The patient samples were divided into two groups according to the median gene expression value (ECT2High/ECT2Low, 437 cases/438 cases). A KM survival plot was used to compare the two groups. The hazard ratio (HR) with 95% confidence intervals (CIs) and log rank P-values were calculated.
Statistical analysis
Data are presented as the mean ± standard error of the mean from three independent experiments and were analyzed with SPSS v.22.0 software (IBM, Corp.). Pearsons χ2 test and Fishers exact test were used to assess the association between ECT2 expression and clinicopathological characteristics of patients with gastric cancer. Unpaired Students t-test was used to assess the differences in ECT2, BUB1 and E2F7 expression levels between gastric cancer and control tissues. Spearmans correlation test was performed between BUB1 and ECT2 expression levels, and E2F7 and ECT2 expression levels, respectively. Survival analysis was performed using the KM method and the log-rank test was used to assess statistical significance between the curves. Univariate and multivariate survival analyses were performed using the Cox proportional hazards regression model. P<0.05 was considered to indicate a statistically significant difference.
Results
ECT2 expression is upregulated in human gastric cancer
Following a routine H&E staining, IHC was performed to detect and grade ECT2 expression in gastric cancer and paired normal tissue sections. ECT2 positive staining was detected mainly in the nucleus and cytoplasm of gastric cancer cells. Weak staining was observed in the adjacent normal tissues. ECT2 expression was identified in 81.5% (106/130) of the gastric carcinoma tissues and 36.1% (39/108) of the adjacent normal tissues. The level of ECT2 protein was significantly higher in the gastric carcinoma tissues compared with the adjacent tissues (IRS, cancer=3.12±2.00 vs. normal=0.98±1.56; P<0.01; Fig. 1A and B).
Association between clinicopathological characteristics and ECT2 positivity in gastric cancer
The clinicopathological significance of ECT2 expression in gastric cancer was investigated. Analysis of the association between ECT2 expression levels (strong positivity vs. weak positivity/absent) and clinicopathological characteristics demonstrated that strong ECT2 positivity was significantly associated with advanced TNM stage (P<0.001) and higher pT stage (deeper tumor invasion; P=0.039). In the 130 gastric cancer cases assessed, high ECT2 expression was not associated with age, sex, tumor localization, pN stage and pM stage (all P>0.05; Table I; Table SI).
 | Table I.Correlation between ECT2 positivity and clinicopathological characteristics of patients with gastric cancer (n=130). |
A significant association between strong ECT2 positivity and histological patterns of gastric cancer was not established (Table I; Table SI); however, ECT2 expression was closely associated with the histological differentiation degree (Fig. 1C). ECT2 expression escalated from well differentiated (WD), to moderately differentiated (MD) and to poorly differentiated (PD) variants (IRS, PD=4.06±1.87 vs. WD=2.16±2.06 or MD=2.64±1.83; P<0.01; Fig. 1C).
ECT2 mRNA upregulation in public datasets of gastric cancer
To further investigate the pathological role of ECT2 in the progression of gastric cancer, the ECT2 expression pattern in gastric cancer samples based on transcriptomic data from the GEO database was assessed. Consistent with IHC analysis, ECT2 mRNA expression levels were significantly increased in primary gastric adenocarcinoma tissues compared with surrounding normal tissues in the GSE13861 dataset (P<0.0001; Fig. 2A). Similar trends were observed in comparisons between paired samples of gastric carcinoma and adjacent normal tissues in the GSE29272 dataset (P<0.0001; Fig. 2B), advanced gastric carcinoma and adjacent normal tissues in the GSE51575 dataset (P<0.0001; Fig. 2C), and gastric cancer tissues and adjacent normal tissues in the GSE65801 dataset (P<0.0001; Fig. 2D). Collectively, these results indicate that ECT2 upregulation may play an important role in the malignant progression of human gastric cancer.
ECT2 upregulation predicts poor clinical outcome
The association between ECT2 expression and the prognosis of patients with gastric cancer was assessed using the KM plot database. High ECT2 expression was significantly associated with a shorter survival time in patients with gastric cancer, stratified according to ECT2 expression levels. The median survival time for ECT2High patients was 16 months, which was significantly shorter than the 26 months observed for ECT2Low patients (P=0.0017; Fig. 3A). Survival analysis using KM curves was performed to verify these results. The results demonstrated that patients with gastric cancer, with low ECT2 expression exhibited a significantly longer overall survival time than those with high ECT2 expression (P=0.015; median OS, 74.360 vs. 50.430 months; Fig. 3B).
To determine whether ECT2 is an independent prognostic factor for the survival of patients with gastric cancer, univariate and multivariate Cox regression analyses were performed. As presented in Table II, the univariate analysis suggested that ECT2 was significantly associated with overall survival time in patients with gastric cancer [P=0.017; HR (95% CI), 1.905 (1.122-3.233)]. Tumor location (body, whole and multiple), histology (PD of adenocarcinoma, signet ring cell, mucinous adenocarcinoma) and TNM stage were all associated with overall survival time in patients with gastric cancer (all P<0.05). Multivariate analysis further demonstrated that high ECT2 expression was a significant independent prognostic marker for patients with gastric cancer [P=0.001; HR (95% CI), 3.105 (1.567-6.153)]. Taken together, these results suggest that ECT2 may serve as a prognostic biomarker for patients with gastric cancer.
| Table II.Prognostic factors associated with overall survival as determined by univariate and multivariate analyses. |
Gastric tumors with higher ECT2 expression levels possess transcriptional traits of CSCs
To determine the ECT2-associated cellular processes and signaling pathways in gastric cancer, GSEA was performed using transcriptome data from TCGA. GSEA demonstrated highly significant enrichment of breast-cancer-progenitor-related genes in gastric cancer samples with higher ECT2 expression (Fig. 4A). Notably, BUB1 mitotic checkpoint serine/threonine kinase and E2F7, two genes previously reported to account for CSC functionality (20–22), were strongly enriched in ECT2High gastric cancer samples (Fig. 4B). A significant correlation was identified between ECT2 and BUB1 mRNA expression levels (r=0.63; P<0.0001; Fig. 5A), and between ECT2 and E2F7 mRNA expression levels in gastric cancer tissues (r=0.45; P<0.0001; Fig. 5B).
BUB1 and E2F7 IHC staining was performed in gastric cancer samples to verify these findings. The results demonstrated that BUB1 protein expression was significantly higher in gastric cancer tissues compared with adjacent normal tissues (IRS, cancer=4.61±1.89 vs. normal=1.94±0.89; P<0.01; Fig. 6A and B). Similarly, E2F7 protein expression was significantly higher in gastric cancer tissues compared with adjacent normal tissues (IRS, cancer=4.01±1.75 vs. normal=1.72±0.86; P<0.01; Fig. 6A and C). Collectively, these results suggest that gastric tumors with high ECT2 expression levels may possess transcriptional traits of CSCs.
Discussion
Gastric cancer remains the third leading cause of cancer-associated mortality worldwide (4). CSCs are implicated in different types of cancer, including gastric cancer (1). The identification of gastric CSCs has improved understanding of the molecular and cellular etiology of gastric cancer, and may aid the development of effective treatments. Experimentally, CSCs are characterized by their capacity for tumor propagation (2). CSCs are resistant to chemotherapy and radiotherapy and possess a quiescent nature (23). Thus, these cells play an important role in cancer recurrence (24). As a result, identification of specific gastric CSCs and the detection of their expression level will lead to the development of novel methods for the diagnosis and treatment of gastric cancer, which can further improve the survival rate of patients with the disease.
The results of the present study demonstrated that ECT2 expression was upregulated in gastric cancer tissues compared with adjacent normal tissues. This result was further verified based on the transcriptomic data from several independent clinical datasets. Consistent with the results of IHC analysis, ECT2 mRNA expression levels were significantly increased in gastric cancer tissues compared with adjacent normal tissues, suggesting that ECT2 upregulation may serve an important role in the malignant progression of human gastric cancer.
The present study also investigated the biological implications of ECT2 upregulation using GSEA. BUB1 and E2F7 upregulation have previously been demonstrated to play important roles in essential cellular processes, such as cell proliferation (25–27). It is speculated that BUB1 and E2F7 may be associated with transcriptional features of CSCs (20–22). A previous study revealed that BUB1 depletion using shRNAs reduces cancer stem cell potential of the MDA-MB-231 breast cancer cell line, resulting in inhibited formation of xenografts in immunocompromised mice (20). In addition, overexpression of E2F7 significantly enhanced the spheroid formation and growth rate of HepG2 and Huh7 cells (hepatocellular carcinoma cell lines), and also decreased their apoptosis (28). GSEA analysis indicated that ECT2 expression was notably associated with the transcriptional program of CSCs, with co-staining of BUB1 and E2F7 in gastric cancer tissues confirmed by IHC analysis.
The present study is not without limitations. First, only IHC analysis was performed to determine ECT2 protein expression, additional methods such as western blotting should be considered in future studies. However, IHC can simultaneously evaluate tissue expression localization, as well as morphology in cancer tissues, and thus is the preferred approach in analysis of clinical samples. Furthermore, future studies will focus on in vitro experiments to better understand the molecular mechanisms underlying ECT2 function in gastric cancer.
Carcinoembryonic antigen and cancer antigen 19-9 serve as the standard biomarkers for the diagnosis of gastric cancer; however, their use in clinical practice is limited due to low diagnostic sensitivity (29). Although emerging candidate biomarkers, such as microRNA and DNA methylation products have been extensively studied, several challenges hinder their application in a clinical setting (30). The results of the present study demonstrated that strong ECT2 positivity was significantly associated with advanced TNM stage and deeper tumor invasion. Furthermore, high ECT2 expression levels were associated with a shorter overall survival time. Thus, ECT2 expression may serve as an independent prognostic marker for the overall survival time of patients with gastric cancer. Similar results were reported by previous studies that demonstrated the prognostic value of ECT2 for gastric cancer (31,32). Taken together, the results of the present study suggest that upregulation of ECT2 predicts unfavorable clinical outcomes of patients with gastric cancer. Thus, ECT2 may serve as a potential prognostic marker and therapeutic target for the management of gastric cancer.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
The present study was funded by the National Natural Science Foundation of China (grant nos. 81803183, 81804066, 81904178 and 81873073), the Key Scientific Research Foundation of Department of Science and Technology of Sichuan Province (grant no. 19ZX0161Z090116002), the Project of Sichuan Provincial Administration of TCM (grant no. 2018QN022), the Science and Technology Developmental Foundation of the Hospital of Chengdu University of TCM (grant nos. 19TS03, 19LW05 and 19LW06), the ‘Xing-lin Scholars’ Project of Chengdu University of Traditional Chinese Medicine (grant no. QNXZ2019017) and ‘Hundred Talents Program’ of the Hospital of Chengdu University of Traditional Chinese Medicine (grant nos. 20-Q03, 20-Q05 and 20-Q18).
Availability of data and materials
The datasets used during the present study are available from the corresponding author upon reasonable request.
Authors contributions
JHZ and SZC conceived and designed the present study. TLY and YG collected and prepared the clinical samples. XC and YW performed H&E staining and confirmed pathological diagnosis. JHZ and DYG performed IHC and clinicopathological characteristics analyses. SZC acquired, interpreted and analyzed the GEO and GSEA data. DYG and SZC drafted the initial manuscript and critically revised it for important intellectual content. All authors have read and approved the final manuscript to be published.
Ethics approval and consent to participate
This retrospective study was approved by the Institutional Review Board of the Teaching Hospital of Chengdu University of TCM (Chengdu, China) (approval no. 2018KL-023) and written informed consent was provided by all patients prior to the study start.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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