Extracellular matrix-based gene signature for predicting prognosis in colon cancer and immune microenvironment
• A signature consisting of four extracellular matrix (ECM) genes (CXCL13, CXCL14, SFRP5, and THBS4) was selected to construct a prognostic model in TCGA colon cancer cohort and validated in three independent cohorts.
What is known and what is new?
• Cancer-associated ECM plays a vital role in progression, expansion, and prognosis of various malignancies.
• This study identified a novel ECM-related gene signature in colon cancer to provide clues for survival and immunotherapy.
What is the implication, and what should change now?
• This study highlights the prognostic value of ECM-related genes in colon cancer and reveals an ECM-related prognostic signature to further improve the prognosis prediction of patients with colon cancer. Our findings provide a basis for better understanding the vital roles of these genes in immune microenvironment organization and indicate the potential clinical implications of these genes in colon cancer.
According to the global cancer statistics, colon cancer (CC) is the third most diagnosed malignancy and the fourth leading cause of cancer-related death worldwide (1). There are more than 2.2 million new CC cases and 1.1 million CC-related deaths predicted by 2030 (2). In recent years, diverse novel surgeries and specific treatments significantly reduce the mortality of CC patients than before (3,4). However, in terms of the insidious onset and invasive rapid-progression, patients with CC were usually diagnosed at advanced stages, which thereby could result in missing the optimal therapeutic opportunity (5-7).
Due to the high clinical heterogeneity of CC, the conventional clinical features including the current American Joint Committee on Cancer (AJCC) staging, Tumor, Nodal Involvement, Metastasis (TNM) staging, and grade are insufficient for accurately predicting individualized prognosis (8-11). Therefore, it is essential to establish novel and robust prognostic biomarker signatures to identify the molecular changes that can reliably estimate clinical outcomes of CC patients, which would have tremendous value of guiding appropriate individualized clinical managements and treatments for CC patients. Much effort has been devoted to determining how the changes of distinct gene signatures such as ferroptosis-related, RNA binding proteins (RBPs)-related, immune-related gene signatures can be utilized to predict CC prognosis recently (12-14). The extracellular matrix (ECM) is composed of complex network of non-cellular components (CCs) of tissue, including glycoproteins, collagens, and proteoglycans, that provides both essential structural and biomedical supports for its cellular constituents, which can regulate the development and maintain tissue homeostasis (15-18). As a major component of the tumor microenvironment, previous studies have reflected the important roles of the ECM in regulating cell proliferation, migration, and apoptosis (17,19,20). Therefore, the cancer-associated ECM is recognized as an important feature of a tumor. Moreover, cancer-associating ECM actively contributes to the histopathology and behavior of tumors (21,22). For instance, Slattery et al. found that expression of matrix remodeling genes such as matrix metalloproteinases (MMPs) predicts a poor prognosis for breast cancer patients (23). Boyd et al. identified increased mammographic density associating with elevated collagen deposition, correlates with an higher risk of developing breast cancer (24). In CC, a study from Stenzinger and colleagues showed that high extracellular matrix metalloproteinases inducer (EMMPRIN) expression was strongly and independently associated with poor prognosis (25). Recently, the consensus molecular subtypes (CMSs) established by Guinney and colleagues were considered the most reliable classification system available for CC. This system divides CC into four subtypes (CMS1-CMS4) with distinguishing features. Among them, CMS4, the mesenchymal type, shows poor prognosis. It is characterized by the activation of transforming growth factor-β (TGF-β) signaling, angiogenesis, and ECM remodeling pathways (26). Although this classification system has been recognized as a critical step forward in distinguishing subtypes of CC, the utilization of this approach for individual patient prognostication has been hampered as analysis of thousands of genes is required. Therefore, simpler approaches such as gene signature-based prognostic risk models are urgently needed to aid in clinical decision making. In view of the association between ECM remodeling pathway activation and the poor outcomes of CMS4 subtype in CC patients, herein, we focus our efforts on developing an ECM-based prognostic signature for patient prognosis.
In this study, we mainly focused on investigating the prognostic implications of ECM-related genes in CC by analyzing publicly available datasets including The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The pipeline of the study is illustrated in Figure 1. Firstly, by taking advantage of differential expression analysis and univariate Cox regression analysis, twelve ECM-related genes with notable prognostic value in CC were identified. Next, a novel four genes ECM-based gene signature for risk stratification and overall survival prediction of CC was constructed by using LASSO-penalized multivariate Cox regression. Then the association between the risk signature and clinical features was studied. Importantly, we also validated the prognostic performance of the ECM-based individualized signature in another independent dataset. Furthermore, a novel nomogram based on ECM-related gene signature and independent clinical factors was developed to predict 1-, 3- and 5-year overall survival probability for patients with CC. We also compared the immune cell infiltration level of high- and low-risk groups in both training and validation cohorts. Finally, the Human Protein Atlas (HPA) database was utilized to verify the expression level of proteins. We present the following article in accordance with the TRIPOD reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-22-2036/rc).
CC datasets and ECM-related genes
In order to develop an ECM-based prognostic prediction model for patients with CC, we downloaded the RNA-seq transcriptome data and associated clinical data of CC patients from the public database Cancer Genome Atlas (TCGA-COAD) (https://portal.gdc.cancer.gov/) and GEO database (https://www.ncbi.nlm.nih.gov/geo). COAD cohort (n=366) was used as training cohort, GSE38832 (n=119), GSE39582 (n=556), GSE29621 (n=65) and combined dataset (n=740) were used as validation cohorts (27). ECM-related genes were obtained from the Molecular Signatures Database (MSigDB, version 6.2, https://www.gsea-msigdb.org/gsea/msigdb). Briefly, we downloaded the gene list encoding ECM and ECM-associated proteins from MsigDB (https://cdn.amegroups.cn/static/public/tcr-22-2036-1.xlsx). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013).
Identification of differentially expressed genes (DEGs)
The “limma” R package for RNA-seq transcriptome data was employed to do differential expression analysis to identify DEGs between tumor and normal samples in the training COAD cohort (28). The genes that meet the defined criteria: |logFC| >1 and FDR P<0.05, were considered as DEGs.
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analysis
We applied “clusterProfiler” package to perform GO enrichment and KEGG analysis for comprehensively analyzing the biological functions of differentially expressed ECM-related genes (29). GO analysis terms include cell component (CC), molecular function (MF) and biological process (BP). FDR P value <0.05 was a filter criteria.
Construction and verification of prediction model
Normalized RNA-seq data and clinical data of training cohort including overall survival time and overall survival status were utilized as the input of the univariate Cox regression analysis. The candidate DEGs with notable prognostic value were then identified based on the P value (P<0.05). Thereafter, to avoid overfitting the model, we employed “glmnet” package to do the least absolute shrinkage and selection operator (LASSO) Cox regression analysis (30) to further screen the prognostic genes. Cox proportional hazard regression was employed to determine the optimal prognostic genes of the model (31). The formula for the gene signature is as follows: risk score = Ʃ (βi * Exp.i) (i = the number of prognostic genes, βi represents the coefficient of gene i, and Exp.i represents expression level of gene i). Finally, CC patients were divided into high- and low-risk groups based on the median risk score of the patients in each dataset.
In order to evaluate the prognostic performance of the established ECM-related signature, Kaplan-Meier survival curve analyses was used to determine whether a significant difference in overall survival time was existed between high- and low-risk groups using the “survival” and “survminer” R packages (32,33). The log-rank test (P<0.05) was utilized to determine the statistic difference. Furthermore, the predictive power of the ECM-based prognostic signature was assessed using time-dependent receiver-operating characteristic (ROC) curve analysis with “timeROC” and “survival” R packages (33,34). In addition, the validation cohort was applied for confirming the prediction ability of the prediction model.
Nomogram development and validation
Based on the multivariate Cox proportional hazard regression analysis, the “rms” R package was utilized to build a nomogram for predicting the survival of COAD patients in 1, 3, 5 years by combining the ECM-related risk model and clinical factors of age and stage (35). Then we used calibration curves and time-dependent ROC to evaluate the predictive power of the developed nomogram.
Immune cell infiltration analysis
We estimated the immune cell infiltration level of both training and validation cohorts utilizing the online CIBERSORT tool. CIBERSORT is a widely used tool for characterizing the immune cell composition of different tissues through the gene expression profile (36).
Protein expression validation
Immunohistochemical staining images of the gene expression in both CC tissues and normal tissues were extracted from the HPA.
In this study, R (4.0.1) was performed for data analysis. If not specified, a two-sided P value or adjusted P value less than 0.05 was considered statistically significant.
Identification of differentially expressed ECM-related genes
By analyzing RNA-seq data of 471 COAD samples and 41 normal colon tissues from the TCGA database, we first identified 1,026 ECM-related gene expression data. Then the “limma” R package was utilized to analyze the differentially expressed ECM-related genes between the tumor and normal samples. We followed (|logFC| >1 and FDR P value <0.05) as the criteria to obtain 89 up-regulated genes and 105 down-regulated ECM-related genes (Figure 2A,2B and https://cdn.amegroups.cn/static/public/tcr-22-2036-2.xlsx).
Functional enrichment analysis of differentially expressed ECM-related genes
To explore and better understand the functions and BPs of these identified differentially expressed ECM-related genes, we took advantage of KEGG analysis and GO analysis. KEGG analysis results showed that differentially expressed ECM-related genes mainly enriched in functional categories such as cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway, ECM-receptor interaction, TNF and NF-kappa B signaling pathway (Figure 2C). In the GO analysis, differentially expressed ECM-related genes were mainly enriched in the BP including ECM organization, cellular response to chemokine, chemokine-mediated signaling pathway, leukocyte and neutrophil chemotaxis (Figure 2D). In terms of CC, the identified differentially expressed ECM-related genes were mainly related to collagen-containing ECM, collagen trimer and endoplasmic reticulum lumen (Figure 2E). In addition, MF analysis showed differentially expressed ECM-related genes enriched in receptor ligand activity and cytokine activity (Figure 2F).
Establishment of prognostic gene signature
In order to comprehensively analyze the prognostic value of differentially expressed ECM-related genes in CC, we first performed univariate Cox regression analysis on them. The results showed that twelve ECM genes (CCL11, CXCL1, CXCL13, CXCL14, CXCL8, ITLN1, MMP1, MMP10, MMP3, SFRP5, THBS4, and VEGFA) were significantly related to the prognosis of CC patients with P value <0.05 (Figure 3A). Then, we conducted LASSO regression to avoid overfitting the model (Figure S1A,S1B). Eight ECM-related genes (CCL11, CXCL13, CXCL14, ITLN1, MMP1, MMP3, SFRP5, and THBS4) were selected as candidates for the next analysis according to the optimal value of the model. Subsequently, in order to evaluate their roles as independent prognostic factors, we performed multivariate Cox regression analysis on the eight candidate ECM-related. Finally, we identified 4 ECM-related genes (CXCL13, CXCL14, SFRP5 and THBS4) as potential prognostic signature (Figure 3B). The risk score was then calculated for each CC patient based on the following formula: -0.51575 * the expression levels of CXCL13 + (−0.24950) * the expression levels of CXCL14 + 0.43928 * the expression levels of SFRP5 + (−0.31070) * the expression level of THBS4.
Verification of accuracy of four ECM-related gene signature in CC
Next, we verified the accuracy of our four ECM-related genes signature model in the training cohort (COAD) and four validation cohorts (GSE33882, GSE39582, GSE29621 and their combination). The individual risk score of each CC patient was determined based on the ECM-related genes signature model. All CC patients were then divided into high- and low-risk group in the light of the median value of risk score of each cohort (Figure 4A-4E). Patients were ranked from low to high according to their risk scores, and we found the scatter plot indicated that the survival rate of low-risk patients was much higher than that of high-risk patients (Figure 4A-4E). In the scatter plots, red dots represent the dead patients, and blue dots represent the alive patients. The heat maps displayed the difference between expression levels of four ECM-related genes in high- and low-risk groups (Figure 4A-4E).
Furthermore, we performed Kaplan–Meier survival curve analyses and demonstrated that patients in the high-risk group exhibited a significantly worse overall survival than those in the low-risk group of both the training (log-rank P<0.001; Figure 5A) and validation cohorts (GSE38832: P=0.00058; GSE39582: P=0.019; GSE29621: P=0.044; combined: 0.0012; Figure 5B-5E). In addition, the areas under the curve (AUC) of the ROC curves for predicting 1-, 3-, and 5-year OS of CC were 0.826, 0.764, and 0.755 for the training cohort, respectively (Figure 5A). The AUCs for 1-, 3-, and 5-year OS were 0.685, 0.736, and 0.748 in the GSE38832 cohort, respectively (Figure 5B). The AUCs for 1-, 3-, and 5-year OS were 0.562, 0.564, and 0.576 in the GSE39582 cohort, respectively (Figure 5C). The AUCs for 1-, 3-, and 5-year OS were 0.731, 0.672, and 0.634 in the GSE29621 cohort, respectively (Figure 5D). When we combined the three GEO validation cohorts together, the AUCs for 1-, 3-, and 5-year OS were 0.609, 0.599, and 0.602, respectively (Figure 5E). Taken together, our four ECM-related gene signature can distinguish the prognosis of CC patients.
Association between the risk score and clinical features of CC patients
We next investigated the association between the ECM-related risk score and different clinical features. By comparing the risk score distribution of clinical features (Figure 6A-6F), we found that there were significant differences in AJCC stage, T stage, N stage, and M stage (P<0.05; Figure 6C-6F). Importantly, we also found CMS4 patients had significantly higher risk scores than non-CMS4 group (Figure 6G). In addition, based on risk score, age, gender, AJCC stage, T stage, N stage, and M stage could be grouped into high- and low-risk groups, and Kaplan-Meier analysis showed that there were significant prognostic differences between two groups except (P<0.05) for T1-T2 stage (P>0.05; Figure 6H-6T). Moreover, the ECM-related risk score remained effective at distinguishing survival when adjusting to CMSs (P>0.05; Figure 6U,6V). These results further suggested that the ECM-based risk score model had good predictive ability in terms of different clinical features.
ECM-related gene prognostic signature is an independent prognostic factor
Next, to determine whether ECM-related gene prognostic signature is an independent predictor for the survival of CC patients, we applied both univariate and multivariate Cox regression analysis on clinical features (age, gender, AJCC stage, T stage, N stage and M stage) and risk score of patients in training cohort. Univariate analysis showed that age (P=0.01), AJCC stage III-IV (P=0.01), T3-T4 stage (P=0.032), N1-N2 stage (P=0.024), M1-MX stage (P=0.01), and risk score (P<0.0001) were significantly correlated with OS in training set (Figure 7A). Subsequent multivariate analysis further showed that age (P=0.008), AJCC stage III-IV (P=0.028), and risk score (P<0.0001) had significant correlation with overall survival in CC patients (Figure 7B). These results suggest that ECM-related gene prognostic signature is a significant independent factor affecting the prognosis of CC patients.
Construction of a nomogram
Then we developed a nomogram integrating multiple prognostic factors (age, stage, and risk score) to evaluate the 1-, 3- and 5-year overall survival probability of CC patients in training cohort (Figure 7C). The results showed that risk score had the greatest influence on overall survival prediction. In Figure 7D, ROC curve analysis exhibited that the 1-year AUC value of the ECM-related risk score model was 0.825, remarkably higher than the clinical factors including the AJCC TNM stage (AUC =0.691), patients’ age (AUC =0.676), T stage (AUC =0.650), N stage (AUC =0.676) and M stage (AUC =0.619) (Figure 7D). Importantly, when we comprehensively conducted the ROC analysis based on the risk score combined with clinical features (AJCC TNM stage and age), the ROC curve was notably higher than each alone (AUC =0.904). Meanwhile, we found the calibration curve also manifested a satisfactory agreement between predictive and actual observations at the probabilities of 1-, 3- and 5-year overall survival (Figure 7E). Together, these results indicate that the 1-, 3-, 5-year overall survival rate of patients with CC could be accurately predicted by the nomogram with risk score and it provides valuable insights for individualized clinical treatment of CC patients.
Correlation of the risk score with tumor-infiltrating immune cells
The GO analysis results in Figure 2 showed the differentially expressed ECM-related genes mainly focused on regulation of ECM organization, immune cells chemotaxis, and chemokine-mediated signaling pathway, which provided us with an important hint that the differences between high- and low-risk groups were related to immune response. Therefore, we attempted to analyze the relationship between the risk score and tumor immune microenvironment. By applying the online CIBERSORT tool to the RNA-seq data of both training and validation cohorts, the relative proportions of 22 immune cell subsets of CC were acquired. In Figure 8A,8B, we displayed the abundance of the 22 infiltrative immune cells in training and validation cohorts (GSE38832) in heat maps. Subsequently, as shown in the box plots (Figure 8C,8D), the infiltration levels of memory B cells, M0 macrophage, activated mast cells, monocytes, resting memory CD4+ T cells, and regulatory T cells were significantly higher in high-risk group compared to that in low-risk group. On the contrary, we found the infiltration levels of M1, M2 microphage, plasmas cells, activated memory CD4+ T cells, CD8+ T cells, follicular helper T cells, and gamma delta T cells were significantly higher in the low-risk group.
Verification of prognostic ECM genes
In order to explore the expression patterns of the proteins encoded by the 4 prognostic genes in our ECM-related signature, we then queried the HPA that provided representative immunohistochemistry images in normal and CC tissues (Figure 9A-9C). The results demonstrated that the expression of CXCL13, CXCL14 and THBS4 was decreased in CC tissues. For the remaining one SFRP5, the immunohistochemistry images are not currently available.
CC is one of the most common aggressive malignant tumors of the digestive tract and the third most common malignant tumor worldwide. Due to the lack of effective diagnostic markers and molecular targeted therapies, the prognosis of CC patients remains poor. By taking advantage of rapid development of omics sequencing technologies, researchers can investigate the underlying molecular mechanisms of CC progression (27,37-39). Moreover, transcriptome profiling of colon tumors provided valuable information for researchers to develop biomarkers-based risk score models to promote the prognosis of CC (12-14,40,41). The ECM, which can convey specific signals to cells, thereby can regulate several critical processes including immune cell migration, immune cell activation, proliferation, and their differentiation (19,42,43). In the occurrence and progression of various tumors, ECM can be altered, which has attracted extensive attention in recent years (17,19,20). Some studies of various cancers prognosis based on the ECM have been reported recently. For instance, Zhang and colleagues reported an ECM-based signature (consisting of CST1, NELL2, ADAMTSL4, and ANGPTL7) associated with immune microenvironment could be used to predict the prognosis and therapeutic responses of patients with oesophageal squamous cell carcinoma (44). Pang et al. identified SPP1 as an ECM signature for metastatic castration-resistant prostate cancer (45). Bergamaschi et al. found that an ECM signature (consisting of MARCO, PUNC, and SPARC) could identify breast cancer subgroups with different clinical outcome (46). In addition, recent CMSs classification system characterized CMS4 subtype with the activation of several critical pathways including ECM remodeling pathway (26). Therefore, in this study, our efforts have led to develop a clinically translatable ECM-based gene signature for risk stratification and overall survival prediction in CC patients and investigate its important prognostic implications through comprehensive bioinformatics methods.
In the present study, we successfully identified and validated a novel ECM-based gene signature (consisting of CXCL13, CXCL14, SFRP5, and THBS4) that could be used to effectively predict the overall survival of CC patients for the first time. The Kaplan-Meier curves indicated that patients in the high-risk group had a significant shorter survival time than those in the low-risk group of both training and validation cohorts, which preliminarily provided some evidenced of the validity of the model predicting the risk of CC. In addition, the time-dependent ROC curves of training cohort presented a relatively high prognostic prediction value, with the AUC of 0.755, 0.764 and 0.826 at 1, 3 and 5 years, respectively. The AUC in validation cohort also indicated the excellent performance of this risk model, with the AUC of 0.658, 0.736 and 0.748 at 1, 3 and 5 years, respectively. Moreover, the result of multivariate Cox regression analysis showed that our ECM-based signature was the most independent risk factor after adjustment for clinical features, such as age and AJCC stage. Importantly, when we stratified the patients according to the different pathological clinical features, the signature remained a robust prognostic tool. The nomogram combined the known risk factors (age and AJCC stage), and risk score was then developed to predict the overall survival probability of 1, 3, and 5 years of CC, whose prognosis efficacy was notably prior to the single factor (age, AJCC stage and risk score only). In summary, our data suggests that the ECM-based risk model is an excellent signature for helping predict the prognosis of CC patients.
CXCL13 and CXCL14 belong to chemokines family, many studies have revealed their important function of tumor proliferation and metastasis. For instance, previous studies showed the important role of CXCL13 and chemokine receptor 5 (CXCR5) signaling axis in the occurrence and development of various human cancers (47-50). In terms of its role in CC, Zhu et al. identified it may promote the growth, migration, and invasion of CC cells via the PI3K/AKT pathway (51). CXCL14, an orphan member of the CXC chemokine subfamily, has also been reported to be associated with tumor progression and metastasis (52-55). Previous studies showed that the expression of CXCL14 mRNA and its protein were ubiquitously detected in normal tissues but were absent in tumor cell lines and in primary tumors (56,57). In terms of the role of CXCL14 in CC, a study from Yi Zhang’s group indicated the absence of CXCL14 contributed to the cancer metastasis that then causes poor outcomes of patients with CC (58). Zeng et al. found CXCL14 might be a potential novel prognostic factor to predict the cancer recurrence and overall survival in CC patients (59). Our differential expression analysis also showed down-regulation of the CXCL13 and CXCL14 in the CC samples, which was consistent with these studies. SFRP5 is a secreted glycoprotein and one of five members of the Secreted Frizzled-Related Protein (SFRP) family. This family has been ascertained as modulators of the canonical Wnt-signaling pathway, down-regulation of them can lead to aberrant activation of Wnt-signaling pathway and then induce tumorigenesis (60,61). For instance, SFRP1 downregulation has been found in several human cancers including CC, which mainly because of the hypermethylation of SFRP1 promoter (62-64). In addition, SFRP2, SFRP4 and SFRP5 were also found to exert their roles in various human cancers (65-67). In consistent with these studies, our results showed a significant down-regulation of SFRP5 in the patients with CC in COAD dataset. Thrombospondin-4 (THBS4) is a secreted ECM protein and one of five members of the thrombospondin protein family. Previous studies showed that THBS4 could affect intracellular migration, adhesion, and attachment as well as proliferation under different conditions (68-71). Increasing studies support the important role of THBS4 in various cancers, such as gastrointestinal and prostate tumors (72,73). Taken together, the four ECM-related genes in our risk model might play essential roles in CC and are worthy of further investigations.
To further investigate the function of our model, we also performed KEGG and GO analysis of the differentially expressed ECM-related genes. Differentially expressed ECM-related genes were enriched in functional categories such as cytokine-cytokine receptor interaction, chemokine signaling pathway, IL-17 signaling pathway, ECM-receptor interaction, TNF and NF-kappa B signaling pathway in the KEGG analysis, while GO analysis revealed that these genes significantly enriched in ECM organization, response to chemokine, chemokine-mediated signaling pathway. These data suggest that the ECM signature-associated genes might affect cancer progression in CC patients through the immune processes and pathways above. The results of CIBERSORT indicated that the levels of immune cell infiltration were significantly associated with the risk score, such as B cells, macrophages, monocytes, plasma cells, CD4+ T cells, CD8+ T cells, Treg, etc. To some extent, these interesting results suggested that this novel ECM-based signature may play a critical role in tumor progression through the immune system. However, although our bioinformatics analysis are relatively comprehensive, further well-designed experiments are required to verify the exact crosstalk and mechanisms of the prediction.
In this study, we developed a four ECM-related gene prognostic signature which was able to discriminate high-risk CC patients from low-risk ones. Besides, we validated this signature in an independent dataset. We also provided important clues to further explore the function of the identified prognostic genes in this risk model and crosstalk between immune and ECM to further improve immunotherapy for CC.
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Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-22-2036/coif). The authors have no conflicts of interest to declare.
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