ACE2 is overexpressed in colon cancer tissues
We analysed the expression levels of ACE2 in colon cancer tissues using various independent bioinformatics databases and found that the expression levels of ACE2 mRNA, protein, and methylation were significantly increased in colon cancer samples. First, in the HPA database, we found that ACE2 expression differs among various types of human cancers, and it was highly expressed in colon cancer tissues (Fig. 1A). Second, we downloaded the relevant data from the Hong colorectal group through the Oncomine database for further analysis, which contained 70 colon cancer samples and 12 normal samples. It was found that the increase in expression level of ACE2 mRNA in the colon cancer samples was statistically significant (p<0.05, Fig. 1B). Differential ACE2 mRNA expression was further confirmed using the GEPIA database (Fig. 1C). From the GEO dataset (GSE1737), we compared 40 pairs of colon cancer and adjacent normal tissues and found that the ACE2 mRNA expression in tumour tissues was higher than that in adjacent tissues, which was statistically significant (Fig. 1D). Moreover, to further explore the expression levels of ACE2 protein and methylation in colon cancer tissues, the UALCAN website was used to find outstanding between 97 colon cancer samples and 100 normal samples (p<0.01, Fig. 2A). After grading and investigation, ACE2 expression was observed to be significantly elevated in stages 1 and 2 colon cancer samples compared with normal tissues (p<0.05, Fig. 2B). To further verify these observations, we excavated the immunohistochemical map from HPA, and found that ACE2 protein expression was positive in colon cancer, but negative in normal tissue (Fig. 2C). Finally, we explored the methylation level of ACE2 in colon cancer tissues using UALCAN, and the results suggested that the ACE2 methylation in colon cancer tissues was much lower than that in normal tissues (Fig. 2D). The grading samples also showed significant differences in methylation between stages 1, 2, and 3 colon cancer tissues and normal tissues (Fig. 2E). Moreover, by accessing the Cancer RNA-Seq Nexus (CRN) database, we found that ACE2 expression in the pathological stages (I-IV) of colon was higher than that observed in normal tissues (Table I). These results demonstrate that ACE2 is overexpressed in colon cancer tissues.
Table I The expression of ACE2 in TCGA Colon adenocarcinoma (COAD) RNA-seq dataset were analyzed by the Cancer RNASeq Nexus
|
ACE2 (Transcript ID: uc004cxb.2)
|
Colon adenocarcinoma
subset pair
|
Average
expression
in cancer
|
Average
expression
in normal
|
Cancer versus Normal
p-value
|
Colon adenocarcinoma––Stage I versus Normal (adjacent normal)
|
2.18
|
0.62
|
p <0.05
|
Colon adenocarcinoma––Stage II versus Normal (adjacent normal)
|
1.05
|
0.62
|
Colon adenocarcinoma––Stage IIA versus Normal (adjacent normal)
|
2.17
|
0.62
|
Colon adenocarcinoma––Stage III versus Normal (adjacent normal)
|
1.38
|
0.62
|
Colon adenocarcinoma––Stage IIIA versus Normal (adjacent normal)
|
2.45
|
0.62
|
Colon adenocarcinoma––Stage IIIB versus Normal (adjacent normal)
|
4.20
|
0.62
|
Colon adenocarcinoma––Stage IV versus Normal (adjacent normal)
|
1.80
|
0.62
|
Note: The Cancer RNASeq Nexus (CRN, http://syslab4.nchu.edu.tw/CRN) is an open resource for intuitive data exploration, providing coding-transcript/lncRNA expression profiles that was contained alternative splicing to support researchers generating new hypotheses in cancer research and personalized medicine.
Fig.1 Expression of ACE2 in different tumor tissues. (A) The expression levels of ACE2 in different cancer tissues were provided by HPA database. (B) The expression levels of ACE2 mRNA in colon cancer in Hong data sets was downloaded from oncomine database. (C)From the GEPIA database, the expression levels of ACE2 mRNA in colon cancer tissues was detected. (D)ACE2 mRNA expression levels in colon cancer tissues and paired normal tissues were downloaded from the GEO database.
Fig. 2 Expression of ACE2 protein and its methylation in colon cancer from UALCAN database. (A) The expression level of ACE2 protein in colon cancer tissues was compared with that in normal tissues. (B) Difference of ACE2 protein expression and tumor grade in colon cancer. (C) The immunohistochemical results of ACE2 in colon cancer and adjacent tissues were downloaded from HPA public bioinformatics website. (D) Methylation expression of ACE2 in colon cancer and normal tissues. (E) The association between the methylation level of ACE2 and the grade of colon cancer. (Among them, * represents significant difference, i.e, P < 0.05)
Correlation of ACE2 expression with clinicopathological features
The correlation between expression of ACE2 and clinicopathological features of patients with colon cancer was assessed using clinical data from the TCGA database. These data included age, sex, T stage, N stage, M stage, TNM stage, and clinical stage, which showed that ACE2 expression is positively correlated with age and T stage (p=0.003 and p=0.024, respectively), while it was negatively correlated with sex, N stage, M stage, and clinical stage (p>0.05) (Table II). From the univariate analysis, a multivariate analysis was performed and confirmed that ACE2 expression was significantly correlated with age, T stage, and clinical stage. The correlation between ACE2 and clinicopathological features in colon cancer samples should be clarified in future studies (Table III).
Table II Relationship between ACE2 expression and clinicpathological parameters in colon cancer
Parameter
|
|
Number
|
ACE2 mRNA expression
|
P value
|
Low(n=272)
|
High(n=167)
|
Age
|
<=80
|
370
|
218
|
152
|
0.003
|
>80
|
69
|
54
|
15
|
|
Gender
|
Male
|
231
|
140
|
91
|
0.539
|
Female
|
208
|
132
|
76
|
|
T stage
|
T1+T2+T3
|
385
|
231
|
154
|
0.024
|
T4
|
54
|
41
|
13
|
|
N stage
|
N0+N1
|
360
|
220
|
140
|
0.435
|
N2
|
79
|
52
|
27
|
|
M stage
|
Mx+M0
|
375
|
232
|
143
|
0.542
|
M1
|
64
|
40
|
24
|
|
Pathologic stage
|
Stage Ⅰ
|
74
|
44
|
30
|
0.628
|
Stage Ⅱ+Ⅲ+Ⅳ
|
365
|
228
|
137
|
|
Table III Multivariate Cox regression analysis of ACE2 and clinic pathological charateristics
Covariate
|
HR
|
95% CI for HR
|
P value
|
Age (<=80 vs >80)
|
0.405
|
0.219-0.750
|
0.04*
|
Gender (Male vs Female)
|
1.104
|
0.743-1.640
|
0.625
|
T (T1+T2+T3 vs T4)
|
0.537
|
0.346-0.833
|
0.006*
|
N (N0+N1 vs N2)
|
0.729
|
0.480-1.107
|
0.138
|
M (Mx+M0 vs M1)
|
0.709
|
0.433-1.160
|
0.171
|
Stage ( StageⅠvs StageⅡ+Ⅲ+Ⅳ)
|
1.642
|
1.032-2.611
|
0.036*
|
From the GEO database, we downloaded a microarray dataset associated with chemotherapy to further determine the biological role of ACE2 in the treatment of patients with colon cancer. From the dataset of GSE56496, it was found that rosemary diterpenes have an anti-tumour effect in colon cancer SW620 cell lines, and expression of ACE2 correlated with the dose of rosemary diterpenes (p=0.004) [35] (Fig. 3A). The dataset from GSE34299 demonstrated that colon 205RC PLX4720 cell lines presented high ACE2 expression, and the expression levels differed in different colon cancer cell lines. The differences among cell lines were statistically significant (p<0.05) [36] (Fig. 3B). Finally, we found from MethSurv, a survival analysis website, that a high expression of ACE2 correlated with a shorter survival time (p=0.05) (Fig. 3C).
Fig. 3 Analysis of ACE2 gene on the treatment of colon cancer from GEO database. (A) Dose effect of supercritical rosemary extract on SW620 colon cancer cells downloaded from GEO database. (B) Relationship between acquired resistance to BRAF inhibitors and expression of ACE2 in colon cancer cell lines. (C) To explore the relationship between ACE2 expression and survival time in colon cancer using by MethSurv database.
Analysis of the co-expression network and pathway enrichment of ACE2
From the above-mentioned general analysis of ACE2 expression, we further explored the biological functions of ACE2. From the cBioportal database, we downloaded ACE2-related co-expressed genes, performed primary screening based on the condition of Log|FC|>0.7 and p<0.05, and identified 217 differentially expressed genes (Supplementary Table 2, Additional File 2). We conducted a corresponding PPI network using the STRING site and Cytoscape software, and found that the trefoil factor 3 (TFF3) gene had the largest relevance. At the same time, the RAS signalling pathway was found to be the most correlated pathway using the functional annotation tool of DAVID for KEGG pathway enrichment analysis (Fig. 4B; Supplementary Table 3, Additional File 3). Furthermore, we used the WebGestalt tool to perform GO biological analysis of these 217 differentially expressed genes and obtained the following results. On biological process analysis, we found that biological regulation and the metabolic process were strongly enriched in these co-expressed genes, and on molecular function analysis, the main related process turned to protein binding. As for the cellular component, the membrane was mainly enriched (Fig. 4C).
Fig. 4 Analysis of ACE2 co-expression network. (A) Using STRING database, DAVID website and Cytoscape software to construct the network of co-expression gene with ACE2. (B) The KEGG enrichment pathway of co-expressed genes was analyzed by DAVID biological website, and bubble diagram was drawn by R language package. (C)The analysis of biological processes, molecular functions and cell components is from WebGestalt website.
Correlation between ACE2 expression and immune infiltration in colon cancer
To investigate the association between ACE2 and the immune response in the tumour microenvironment (TME), TIMER provided high-throughput immune cell infiltration data. Significant correlations were found between expression of ACE2 and the infiltrating levels of immune cells. Among them, CD8+ T cells (r=-0.207, p=5.38e-04) and neutrophils (r=-0.15, p=1.27e-02) showed a negative correlation, while CD4+ T cells (r=0.14, p=2.02e-02) showed a positive correlation (Fig. 5). These results suggest the potential inhibitory effect of ACE2 expression on immune infiltration in colon cancer.
Correlations with immune signature markers of typical immune infiltrating cells were further characterised using the TIMER database. We used signature TAM, monocyte, M1 macrophage and M2 macrophage markers to evaluate their association with ACE2 expression, and the results are illustrated in Fig. 6. Although there was no significant association between ACE2 expression and the TAM gene marker CCL2 (p>0.05), wihle the TAM gene marker CD68 and IL10 show significant correlations (all p<0.05, Fig. 6A). We also found that ACE2 expression significantly correlated with monocyte (CD86, C3AR1, and CSF1R), M1 macrophage (IRF5, PTGS2, and NOS2), and M2 macrophage (MS4A4A, CD163, and VSIG4) expressions (all p<0.05, Fig. 6B-D). Therefore, ACE2 may participate in restraining the monocyte-macrophage system and regulating macrophage polarisation. These results indicate that ACE2 plays an important role in the immune response in TME by affecting immune cell infiltration in colon cancer.
Fig. 5 Correlation between the expression of ACE2 and the level of immune infiltration in colon cancer. (A) There was a positive correlation between the expression of ACE2 and the immune infiltration of CD8 + T cells in colon cancer. (B) The expression of ACE2 was positively correlated with the immune infiltration of CD4 + T cells. (C) The infiltration of Neutrophil cells was negatively correlated with the expression of ACE2.
Fig. 6 Correlation between ACE2 expression and tumor markers of macrophages, monocytes, M1 macrophages, M2 macrophages. (A) Correlation between ACE2 expression and tumor associated gene markers CCL2, CD68 and L10 in macrophages. (B) ACE2 expression is associated with CD86、C3AR1 and CSF1, which are gene markers related to mononuclear . (C) ACE2 expression is associated with RF5, PTGS2 and NOS2, which are gene markers related to M1 macrophages. (D) ACE2 expression is associated with MS4A4a、CD163A and VSIG4, which are gene markers related to M2 macrophages.