ACE2 maybe serve as a prognostic biomarker in breast invasive carcinoma

Abstract Background Breast cancer is a frequently occurring malignant tumor in women. Angiotensin‐converting enzyme 2 (ACE2) is widely expressed in most organs; however, the association of ACE2 with prognosis and immune infiltration in breast invasive carcinoma (BRCA) remains elusive. Methods We explored the expression level and prognostic value of ACE2 in patients with BRCA using a series of online bioinformatics analysis databases encompassing Oncomine, UALCAN, Kaplan–Meier plotter, TIMER, LinkedOmics, and GEO. qRT‐PCR was performed to verify our findings. Results Angiotensin‐converting enzyme 2 mRNA and protein expression levels were decreased in BRCA tissues, and patients with low ACE2 expression levels had a poor prognosis. DNA promoter methylation of ACE2 significantly downregulated ACE2 expression in BRCA, while the expression of this protein was positively linked to immune infiltration of B cells, CD8+ and CD4+ T cells, neutrophils, and dendritic cells in BRCA tissues. The high expression level of ACE2 in enriched basophils, CD8+ T cells, and type‐2 helper T cells, which showed decreasing levels, indicated a better prognosis for BRCA. Enrichment analyses revealed that NF‐κB, IL‐17, and TNF signaling pathways were highly correlated to ACE2 in BRCA. Verification study revealed that downregulation of ACE2 was associated with a better prognosis in BRCA. Univariate and multivariate analysis confirmed ACE2 expression and clinical stage as independent prognostic factors for breast cancer. Conclusions Angiotensin‐converting enzyme 2 may be a potential prognostic biomarker and target for BRCA. Nevertheless, future investigations are needed for validating our findings and promoting the clinical application of ACE2 in BRCA.


| INTRODUC TI ON
Breast cancer mostly occurs in women and is associated with high mortality. Classically, the prognosis of breast cancer correlates with age, stage, tumor type, tumor grade, and lymphovascular status. 1 According to Global Cancer Statistics 2018, breast cancer accounted for 11.6% of cases, being the second-largest commonly diagnosed cancer. 2 BRCA1 and BRCA2 genes are tumor suppressor genes that help repair DNA damage to suppress breast cancer tumorigenesis.
People with mutations in BRCA genes are vulnerable to breast cancer.
Statistics show that BRCA1 and BRCA2 mutations could increase the susceptibility of 80-year-olds individuals by 72% and 69%, respectively. 3 The treatment options available for noninvasive and invasive Most organs, including lungs, intestines, heart, arteries, and kidneys, as well as the female reproductive system, express ACE2. 6 Furthermore, in the cardiovascular system, ACE2 can protect the heart and relax blood vessels. ACE2 also plays important roles in the renal and reproductive system. 7 ACE2 deficiency results in hypertension, diabetes, and several cardiovascular diseases. 8 Recently, ACE2 has been reported to protect breast cancer patients. One possible mechanism of ACE2 to suppress the carcinogenesis of breast cancer can be the inhibition of the VEGFa/VEGFR2/ERK pathway. 9 However, the complete mechanism of how ACE2 acts in breast cancer, especially BRCA, remains unclear.
In this work, we evaluated ACE2 mRNA and protein expression, as well as promoter methylation levels using the Oncomine and UALCAN databases. BRCA survival analysis was performed in the Kaplan-Meier plotter database (KMPD). The relationship between ACE2 expression and immune infiltration and immune cell gene biomarkers was explored in Tumor Immune Assessment Resource (TIMER) database. Furthermore, Gene Set Enriching Analysis (GSEA) was utilized to perform the GO and KEGG enrichment analyses using the LinkedOmics database.

| Oncomine
Oncomine contains 715 datasets and 86,733 samples, being the most comprehensive online cancer gene chips database worldwide. 10 The expression of ACE2 in BRCA and other tumors was studied in Oncomine (https://www.oncom ine.org/resou rce/main. html). The threshold was a p-value of 1E-4, a twofold change, and a top 10% of the gene ranking.

| UALCAN
UALCAN is a comprehensive database providing diverse in-depth analysis of TCGA expression data (http://ualcan.path.uab.edu/ cgi-bin/ualca n-res.pl). 11 The TCGA BRCA dataset (N = 537) in the "Expression Analysis" module in TCGA was adopted for assessing ACE2 mRNA levels in various subgroups of BRCA patients.

| Kaplan-Meier plotter (KM-plotter)
Kaplan-Meier plotters analyze the influence of various genes on the survival of patients with certain types of cancers, including breast, ovarian, lung, and gastric cancer (https://kmplot.com/ analy sis/). 12 The TCGA BRCA dataset was obtained by selecting "breast cancer" from the Pan-cancer RNA-seq module. We analyzed the association of ACE2 expression with overall survival (OS) and relapse-free survival (RFS) in BRCA patients (n = 1090) using a KM survival plot. Furthermore, the correlation of ACE2 expression and the RFS of BRCA patients with different clinicopathological features was analyzed. Patients were split based on "automatically select the best cut-off point," and the follow-up threshold covered all patients.

| Tumor Immune Assessment Resource (TIMER)
Tumor immune assessment resource tool is used for investigating correlations between immune infiltration and TCGA tumor (https:// cistr ome.shiny apps.io/timer/). 13 We used TIMER database Gene and SCNA modules to evaluate the correlation between ACE2 expression and immune infiltration and gene biomarkers of immune cells using the TCGA BRCA dataset (N = 537).

| LinkedOmics
The LinkedOmics database is an online platform that helps analyze 32 types of cancer and 11,158 patients from TCGA (http://www. linke domics.org/login.php) 14 . Differentially expressed genes that correlated to ACE2 expression were determined by the LinkFinder module (n = 1093). The Spearman test was used to analyze the results statistically. In the LinkInterpreter module, GSEA (Gene Set Enriching Analysis) tool was utilized for carrying out GO (CC, BP, and MF), KEGG pathway, kinase-target enrichment, miRNA-target enrichment, and transcription factor enrichment analyses. The rank criteria were based on p-value from LinkFinder results. The minimal gene size was 3 and 500 simulations were tested.

| Clinical specimens
After obtaining informed consent from patients of Enze Hospital, Taizhou Enze Medical Center (Group), breast cancer tissues and pairnormal breast tissues (n = 52) of these patients were used for the study. Our study was approved and supported by the ethics committee of Enze Hospital, Taizhou Enze Medical Center (Group). None of the patients received local or systemic treatment preoperatively.
Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. The 2 −ΔΔCt method was used to calculate fold-changes.
The difference in the expression of ACE2 and the prognosis of ACE2 in breast cancer were explored with Student's t test and Kaplan-Meier analysis.

| ACE2 expression in BRCA
We initially analyzed the Oncomine database to study differential ACE2 expression between various cancers and normal tissues. From the results, an abnormal expression level of ACE2 was observed in breast, colorectal, renal, and lung cancers, as well as sarcoma. For breast cancer, ACE2 expression increased particularly in Invasive Breast Carcinoma Stroma and decreased in IDC and ILC ( Figure 1A).
Then, we evaluated the transcription and protein expression level of ACE2 in BRCA using the Oncomine and UALCAN databases.
Both databases showed that samples from BRCA patients had

| ACE2 served as a potential prognostic biomarker in BRCA
By using KMPD, we investigated the effect of ACE2 expression on RFS and OS of BRCA patients. BRCA patients with elevated ACE2 expression had a better prognosis, but the OS rate was not affected ( Figure 5A,B). Further univariate and multivariate analysis revealed that ACE2 expression, pT stage, pN stage, and pM stage  Table 1).

| ACE2 was related to immune infiltration in BRCA
Substantial studies have reported that ACE2 plays key roles in immunity. 15 Therefore, we adopted the TIMER database for exploring the link between ACE2 expression and immune infiltration level in BRCA. From the results, we observed that ACE2 expression levels

| Enrichment analysis of ACE2 in BRCA
According to the mRNA sequencing data of 1093 TCGA BRCA patients from LinkedOmics, we discovered that 8628 genes were positively correlated while 5817 genes were negatively correlated with ACE2 expression (Figure 7A-C). show the top 50 genes positively and negatively correlated with ACE2, respectively. GO enrichment analysis of ACE2 using GSEA revealed that terms related to adaptive immune response, regulation of innate immune response, cell chemotaxis, cell-substrate junction, and receptor-ligand activity were enriched ( Figure 7D-F). Moreover, according to KEGG enrichment analysis, ACE2 was demonstrated to be involved in NF-κB, IL-17, and TNF signaling pathways ( Figure 7G).

| ACE2-associated kinase, miRNA, or transcription factor targets in BRCA
Given the significance of ACE2 in BRCA, the kinase, miRNA, and transcription factor networks of ACE2 in BRCA were further

| Validation of the expression and prognostic value of ACE2 in breast cancer
qRT-PCR was performed to verify the expression and prognostic value of ACE2 in breast cancer using the clinical tissues from Taizhou First People's Hospital (N = 52). As shown in Figure 8A, ACE2 was downregulated in breast cancer versus normal tissues (p < 0.001).
Moreover, OS analysis indicated a better prognosis in breast cancer patients with high ACE2 expression (p < 0.001, Figure 8B). As shown in Figure 8C,D, the univariate and multivariate analysis also demonstrated ACE2 expression and clinical stage as independent prognostic factors for breast cancer. These data further confirm the prognostic value of ACE2 in BRCA.

| DISCUSS ION
Recent studies have reported that ACE2 levels negatively correlate with tumor grade and the ability to metastasize. 16 In UCEC (Uterine Corpus Endometrial Carcinoma) and KIRP (Kidney Renal Papillary Cell Carcinoma), ACE2 expression shows a positive correlation with the prognosis, accompanied by elevated immune cell infiltration levels. 17 As for SARS-CoV-2 infection, ACE2 together with TMPRSS2 acts as a receptor for host cell entry. 18 In this study, Oncomine and UALCAN databases were used to assess ACE2 expression levels. RFS and OS survival analyses were performed in the KMPD. We also explored the interaction between From the results, we observed that ACE2 expression levels decreased in breast cancer, especially BRCA. ACE2 may serve as a probable prognosis factor for BRCA, partly by affecting immune infiltration. ACE2 performs both positive and negative roles in cancer therapies. Low ACE2 expression level is commonly a sign of cancer presence along with diabetes. 19 In several cancers such as pancreatic, lung, colon, and breast cancers, ACE2 inhibits metastasis and angiogenesis. 20 Specifically, in hepatocellular carcinoma (HCC) patients, increased ACE2 expression reflects a longer survival time compared to those with decreased ACE2 expression, which implies the prognosis role of ACE2 in HCC. 21 Likewise, ACE2 has been pointed out as   IL-17 cytokines family can activate the production of NF-κB, IL8, IL6, TNFα, and IL1b, contributing to various inflammatory diseases and cancers. 28 Recently, another study shows that high expression of IL17B and its receptor IL17RB relates to poor prognosis of breast cancer patients. 29 Moreover, Protein kinase C α (PKCα) has been reported to contribute to the metastasis and aggravation of breast cancer. Specifically, high expression levels of PKCα and FOX2 are found in TNBC tumors. 30 Activation of PKCα is reported to enhance the internalization of HER2. 31 In breast cancer patients, miR-23A is involved in the regulation of autophagy pathways, implying a future potential therapeutic target and biomarker. 32 To explore the reasons for reduced ACE2 expression in BRCA, we investigated the DNA methylation level of ACE2 in BRCA.
Usually, high expression levels of ACE2 in tumors, including colon adenocarcinoma (COAD), Kidney renal papillary cell carcinoma (KIRP), pancreatic cancer (PAAD), rectal adenocarcinoma (READ), and lung adenocarcinoma (LUAD), are always accompanied by low DNA methylation level of ACE2. 33 Surprisingly, in our study, the ACE2 expression level was found to decrease after DNA promoter methylation. ACE2 may be inactivated and downregulated due to hypermethylation, which may partly explain the decreased ACE2 expression in BRCA.
Our study had a few limitations. First, most analyses were performed at the mRNA level but not at the protein and gene level.
Furthermore, our results need to be validated by performing in vivo and in vitro experiments. In addition, histochemistry staining should be performed to verify the expression of ACE2 in BRCA.

| CON CLUS ION
In conclusion, ACE2 expression declined in BRCA, which to some extent resulted from ACE2 methylation. ACE2 exhibited good prognostic value and was associated with better RFS and immune infiltration in BRCA. Thus, ACE2 may serve as a potential biomarker and therapeutic target for BRCA.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The analyzed datasets generated during the study are available from the corresponding author on reasonable request.