Genomic alterations of ACE2 in LUAD
We then used the DriverDBv3 tool to determine the mutation rate and CNVs distribution of ACE2 and their correlations with LUAD. Data on the distribution of variants may help to further study the role of ACE2 in acute lung injury and lung function [19]. Fig. 1 A shows the mutation rate of ACE2 and its protein positions in LUAD. We found that the ACE2 protein has the highest mutation rate at positions 201-242. In contrast, the mutation rate of the ACE2 protein at 483-523 was the lowest. Additionally, a moderate mutation frequency occurs at the protein sites of 0-40, 80-121, 282-322, 644-684, and 765-805. The CNVs squares indicate the CNVs gain or loss of ACE2 in LUAD (Fig. 1B). We found the CNVs distribution mainly included gain, loss, none, and normal, and was positively correlated with ACE2 expression in LUAD (cor = 0.149, p = 0.00075). Among them, ACE2 expression was higher in copy number loss than in copy number gain.
ACE2 protein network analyses
Data from STRING were applied to determine the proteins interacting with ACE2 and the results are shown in Fig. 2. The following ten proteins were found to interact with ACE2: Angiotensinogen (AGT), Renin (REN), Neprilysin (MME), Dipeptidyl peptidase 4 (DPP4), Lysosomal Pro-X carboxypeptidase (PRCP), Meprin A subunit alpha (MEP1A), Type-1 angiotensin II receptor (AGTR1), Meprin A subunit beta (MEP1B), Xaa-Pro aminopeptidase 2 (XPNPEP2), and Type-2 angiotensin II receptor (AGTR2), and their correlation scores were 0.991, 0.950, 0.950, 0.942, 0.924, 0.915, 0.904, 0.880, 0.876, and 0.858, respectively.
The enrichment analyses of ACE2
To further explore the regulators of ACE2 in LUAD, we next statistically analyzed the significant GO enrichment terms and KEGG pathway of the identified genes via the Enrichr online database (Fig. 3, Table S2). The biological processes of these proteins were mainly involved in the regulation of systemic arterial blood pressure by renin-angiotensin (GO: 0003081), angiotensin maturation (GO: 0002003), and regulation of angiotensin levels in the blood (GO: 0002002). Regarding molecular functions, these proteins were mainly involved in the dipeptidyl-peptidase activity (GO: 0008239), aminopeptidase activity (GO: 0004177), and exopeptidase activity (GO: 0008238). The cell component analysis of these proteins showed that they were significantly enriched in invadopodium (GO: 0071437), azurophil granule membrane (GO: 0035577), and ficolin-1-rich granule membrane (GO: 0101003). Moreover, KEGG pathway analysis showed enrichment in the renin-angiotensin system, protein digestion and absorption, and renin secretion.
Relationships between ACE2 expression and clinical-pathological parameters of patients with LUAD
The goal of our study was to gain insights into the interaction between ACE2 expression and the clinical-pathological parameters of patients with LUAD (Table S1). To accomplish this, we first investigated ACE2 expression based on sample types, As shown in Fig. 4 A, the expression of ACE2 in primary samples was significantly higher than that in normal tissues (p = 2.16E-8). An analysis of individual cancer status showed that stage 1, stage 2, and stage 3 cancer tissues had significantly higher expression than that in normal tissues (normal-vs-stage 1: p = 1.81E-10; normal-vs-stage 2: p = 1.26E-03; normal-vs-stage 3: p = 2.48E-03), However, there was no significant difference between stage 4 and normal tissues (p > 0.05) (Fig. 4 B).
In comparing the patient’s race (Fig. 4 C), we found that Caucasians and Asians with cancer had significantly higher ACE2 expression than normal control individuals (normal-vs-Caucasian: p = 1.55E-05; normal-vs-Asian: p = 4.57E-02). However, there was no significant difference in the expression of ACE2 among Caucasians, Asians, and African Americans (p > 0.05).
In addition, we analyzed the relationship between ACE2 expression and patient’s age (Fig. 4 D). Notably, we found that the expression of ACE2 in patients aged 61-80 years was significantly higher than that in patients aged 21-40 years (p = 7.23E-04), the expression of ACE2 in patients aged 81-100 years was significantly higher than that in patients aged 21-40 years (p = 3.68E-02), and the expression of ACE2 in patients aged 61-80 years was significantly higher than that in patients aged 41-60 years (p = 1.60E-03). The results help explain why older people are more susceptible to SARS-CoV2.
Next, we investigated whether there was a difference between the expression of ACE2 and the patients’ gender. As shown in Fig. 4 E, ACE2 expression was higher in both male and female cancer patients than that in the normal group (normal-vs-male: p = 6.17E-04; normal-vs-female: p = 1.45E-09), but no significant difference was found between ACE2 expression and sexes (male-vs-female: p > 0.05).
Smoking is the most important risk factor for lung cancer [20]. The relationship between the expression of ACE2 and smoking in LUAD remains to be studied. Here, we focused on ACE2 expression according to patient’s smoking habits (Fig. 4 F), including non-smoker, smoker, reformed smoker 1 (< 15 years), and reformed smoker 1 (> 15 years). Regarding the smoking habits of LUAD patients, the expression levels of ACE2 in patients with all conditions were higher than those in normal controls, but we found that there were no significant differences between patients' smoking habits and the expression of ACE2 (p > 0.01). Therefore, we speculate that the expression of ACE2 may not be related to the smoking habits of patients.
Subsequently, it is worth noting the relationship between ACE2 expression and node metastasis status (N0: no regional lymph node metastasis; N1: metastases in 1 to 3 axillary lymph nodes; N2: metastases in 4 to 9 axillary lymph nodes; and N3: metastases in 10 or more axillary lymph nodes). As shown in Fig. 4 G, we found that there was no significant difference between ACE2 expression and node metastasis status (p > 0.05).
Based on histological subtypes, the data showed that ACE2 expression was highest in lung clear cell adenocarcinoma (Clear Cell), but there was no significant difference compared with the normal group (p > 0.05). We found high ACE2 expression in the lung adenocarcinoma-not otherwise specified (NOS) and lung adenocarcinoma mixed subtype compared to normal controls in this cancer, with greater statistically significant (normal-vs-NOS: p = 4.38E-10; normal-vs-mixed: p = 2.46E-03) (Fig. 4 H). We also found that the expression of ACE2 was higher in lung mucinous adenocarcinoma compared to NOS in LUAD tumors (p = 7.67E-09).
Taken together, ACE2 expression in the lung increased with age, we further analyzed the expression of ACE2 in patient’s age by immunohistochemistry were shown in Fig. 5. The expression level of ACE2 was quantified by scoring the intensity of staining, including negative (-) and weak (+) staining, moderate (++) and strong (+++) staining. We found that ACE2 mainly localized in the plasma membrane and cytoplasm. ACE2 showed moderate expression in patients aged 81-100 years samples, and patients aged 61-80 years samples showed weak expression. However, the expression of ACE2 in patients aged 21-40 years and 41-60 years was negative.
ACE2 expression in other cancers
To study the expression of ACE2 in other cancers and whether it is related to the patient’ s age. We examined the difference in ACE2 expression between tumor and adjacent normal tissues by using GEPIA (Fig. 6 A). We found that ACE2 was also highly expressed in COAD, KIRP, PAAD, READ, and STAD compared to normal tissues. These results suggested that the transcription level of ACE2 was cancer type-specific (p < 0.05). However, there was no significant difference in the expression of ACE2 among patients aged 21-40 years, 41-60 years, 61-80 years, and 81-100 years (Fig. 6 B).
SNVs, CNVs, and pathway activity of hub proteins in LUAD
To further understand the SNVs, CNV, and pathway activity of these proteins, we performed the analysis with GSCALite (Fig. 7 A-C). The SNVs module presented the SNVs frequency and variant types of these genes in LUAD. We found that the SNVs frequencies of MME, XPNPEP2, DPP4, AGTR1, and ACE2 were in the top five, and are 19 %, 16 %, 14 %, 12 %, and 11 %, respectively. Among them, the variant types of ACE2 were missense mutations. In the CNVs module, the main copy number variants of ACE2 include heterozygous amplification and heterozygous deletion.
We then determined the pathway activity of these genes (Fig. 7 D-F). The pathways involved are apoptosis, cell cycle, DNA damage response, EMT, hormone AR, hormone ER, PI3K/AKT, RAS/MAPK, RTK (receptor tyrosine kinase), and TSC/mTOR. The results showed that RTK was activated by DPP4 and ACE2. The EMT pathway was mainly activated by AGTR1, DPP4, MME, and XPNPEP2. However, the cell cycle was mainly inhibited by AGTR1, DPP4, and PRCP. Besides, we found that the Hormone AR pathway was mainly inhibited by DPP4, AGTR1, XPNPEP2, MME, MEP1A, and ACE2.